Hepatitis B, a common viral infection, affecting millions of people every year, leads to disability and death. It has become one of the alarming diseases in the world. Vaccination is the best possible way to prevent this deadly viral infection and its consequences. Unaffordability of the mass vaccination program due to low health budgets especially in developing countries have demanded the economical, effective and easily available vaccine production against hepatitis B virus. The expression of subunit vaccines and recombinant proteins in plants is a convenient, safe and potentially economical platform technology. Hence, development of a plant-based vaccine could be promising. Therefore, the present investigation focused on expression and large- scale production of hepatitis B surface antigen (HBsAg) in Coleus forskohlii for the development of vaccine. Eight transformed C. forskohlii plants were generated via Agrobacterium -mediated transformation method. The integration of 681bp of HBsAg gene into the plant genome was confirmed using PCR. SDS-PAGE showed the presence of ~48kDa dimer and ~24kDa monomer form of HBsAg protein and the expression of recombinant protein was further confirmed by western blot. C. forskohlii expressed HBs Ag was recorded 260µg/g leaf fresh weight as measured by ELISA. Transformed plants of HBsAg showed the accumulation of Virus Like Particles of 22 nm size using transmission electron microscopy. This study offers a great potential for the large -scale production of hepatitis B vaccine in C. forskohlii which provides a strategy for contributing a means to achieve global immunization for the hepatitis B prevention and eradication.
Rekha presented on edible vaccines. Edible vaccines involve introducing genes from pathogens into plants to produce antigens. When consumed, these plant-based vaccines stimulate mucosal and systemic immunity. Various pathogens have been expressed in plants, including ETEC, cholera, Norwalk virus, hepatitis B, and measles. Advantages are oral delivery, lower costs, and stability without refrigeration. Challenges include ensuring consistent dosage and developing tolerance. Many organizations in India are researching edible vaccine candidates for diseases like malaria, tuberculosis, dengue, and hepatitis E.
Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modified foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine technology. Various technical obstacles, regulatory and non-scientific challenges, though all seem surmountable, need to be overcome. This review attempts to discuss the current status and future of this new preventive modality.
I reviewed several manuscripts, books, grants and project proposals. This is one of the paper I reviewed recently published in Plant Biotechnology Journal
This document discusses optimal strategies for inducing broadly neutralizing antibodies (bnAbs) against highly mutable pathogens like HIV. It presents a computational model of affinity maturation in the presence of multiple variant antigens. The model suggests there is an optimal level of "frustration" between conflicting selection forces from different antigens that maximizes the probability of evolving bnAbs. An intermediate level of frustration allows for cross-reactivity while avoiding overspecialization. The results provide guidance for rational vaccine design and reveal principles at the intersection of immunology and evolutionary biology.
This document summarizes several research grant proposals awarded by the International Centre for Genetic Engineering and Biotechnology (ICGEB) in 2019. The proposals cover a wide range of topics related to agriculture, health and biotechnology:
1) A proposal to study ferroptosis-like cell death in plants which occurs in response to heat stress and could lead to new approaches for crop protection during climate change.
2) Engineering vitamin A-rich orange eggplant which could help reduce vitamin A deficiency in developing countries.
3) Defining the soybean leaf microbiome to search for disease-suppressing bacteria against Asian Soybean Rust, a major threat to Brazilian soybean production.
4) Develop
Syngulon - Breakout session Synthetic Biology June 10, 2022.pdfSyngulon
This document summarizes a presentation given by Dr. Philippe Gabant on the applications of synthetic biology. The presentation discusses Syngulon's work developing bacteriocins and tuning microbiota to address antimicrobial resistance and contamination issues. It provides an overview of Syngulon's technologies, markets in biopharma, cosmetics and more. It also discusses their PARAGEN collection of bacteriocin genes and peptides, and how they are applying synthetic biology to expand this collection. The goal is to balance and control microbial life through developing alternatives to antibiotics.
The word "vaccine" originates from the Latin word “vacca”, meaning “cow” a virus (cowpox) which manly effect the cow. which Edward Jenner demonstrated in 1798 could prevent smallpox in humans.
BIOTECHNOLOGY 3RD ICA ASSIGNMENT BY ALOK KUMAR.pptxAlok Kumar
The document discusses different types of vaccines and how plant-based vaccines are produced. It describes the process of producing plant vaccines which involves selecting an antigen-encoding gene and plant host, inserting the gene into the plant, and harvesting and processing the plant tissue to purify or consume the vaccine. Plant vaccines stimulate both mucosal and systemic immunity. Examples of plant vaccines discussed include those against bacteria, viruses, parasites, and SARS-CoV-2.
Rekha presented on edible vaccines. Edible vaccines involve introducing genes from pathogens into plants to produce antigens. When consumed, these plant-based vaccines stimulate mucosal and systemic immunity. Various pathogens have been expressed in plants, including ETEC, cholera, Norwalk virus, hepatitis B, and measles. Advantages are oral delivery, lower costs, and stability without refrigeration. Challenges include ensuring consistent dosage and developing tolerance. Many organizations in India are researching edible vaccine candidates for diseases like malaria, tuberculosis, dengue, and hepatitis E.
Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modified foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine technology. Various technical obstacles, regulatory and non-scientific challenges, though all seem surmountable, need to be overcome. This review attempts to discuss the current status and future of this new preventive modality.
I reviewed several manuscripts, books, grants and project proposals. This is one of the paper I reviewed recently published in Plant Biotechnology Journal
This document discusses optimal strategies for inducing broadly neutralizing antibodies (bnAbs) against highly mutable pathogens like HIV. It presents a computational model of affinity maturation in the presence of multiple variant antigens. The model suggests there is an optimal level of "frustration" between conflicting selection forces from different antigens that maximizes the probability of evolving bnAbs. An intermediate level of frustration allows for cross-reactivity while avoiding overspecialization. The results provide guidance for rational vaccine design and reveal principles at the intersection of immunology and evolutionary biology.
This document summarizes several research grant proposals awarded by the International Centre for Genetic Engineering and Biotechnology (ICGEB) in 2019. The proposals cover a wide range of topics related to agriculture, health and biotechnology:
1) A proposal to study ferroptosis-like cell death in plants which occurs in response to heat stress and could lead to new approaches for crop protection during climate change.
2) Engineering vitamin A-rich orange eggplant which could help reduce vitamin A deficiency in developing countries.
3) Defining the soybean leaf microbiome to search for disease-suppressing bacteria against Asian Soybean Rust, a major threat to Brazilian soybean production.
4) Develop
Syngulon - Breakout session Synthetic Biology June 10, 2022.pdfSyngulon
This document summarizes a presentation given by Dr. Philippe Gabant on the applications of synthetic biology. The presentation discusses Syngulon's work developing bacteriocins and tuning microbiota to address antimicrobial resistance and contamination issues. It provides an overview of Syngulon's technologies, markets in biopharma, cosmetics and more. It also discusses their PARAGEN collection of bacteriocin genes and peptides, and how they are applying synthetic biology to expand this collection. The goal is to balance and control microbial life through developing alternatives to antibiotics.
The word "vaccine" originates from the Latin word “vacca”, meaning “cow” a virus (cowpox) which manly effect the cow. which Edward Jenner demonstrated in 1798 could prevent smallpox in humans.
BIOTECHNOLOGY 3RD ICA ASSIGNMENT BY ALOK KUMAR.pptxAlok Kumar
The document discusses different types of vaccines and how plant-based vaccines are produced. It describes the process of producing plant vaccines which involves selecting an antigen-encoding gene and plant host, inserting the gene into the plant, and harvesting and processing the plant tissue to purify or consume the vaccine. Plant vaccines stimulate both mucosal and systemic immunity. Examples of plant vaccines discussed include those against bacteria, viruses, parasites, and SARS-CoV-2.
1.72±0.25
1.88±0.35
1.72±0.15
15.01±10.50
17.01±24.16
20.07±1.57
21.12±8.41
23.01±1.12
25.12±5.56
27.21±25.37
28.88±0.97
33.94±0.97
The study examined the immune response to the hepatitis B virus vaccine in vaccinated groups. ELISA assays were used to detect hepatitis B surface antigen and antibodies in serum samples before and after vaccination.
1. Edible vaccines offer a cost-effective way to deliver vaccines orally by incorporating genes encoding antigens into plants. This allows for vaccines to be stored and administered more easily compared to traditional vaccines.
2. Edible vaccines stimulate both mucosal and systemic immunity by producing antigens when consumed. Various foods like bananas, potatoes, and tomatoes are being investigated for use as edible vaccines.
3. Edible vaccines overcome many issues with traditional vaccines like cold storage and transportation requirements. However, challenges remain around standardizing dosage and ensuring plant stability of the encoded antigens. Future applications of edible vaccines show promise for diseases like cancer, birth control, and autoimmune disorders.
This document discusses strategies for developing recombinant vaccines, including bacterial and viral vectors. It notes that recombinant vaccines using bacterial vectors like BCG can elicit both antibody and cellular immune responses, and have been used to develop vaccines against pathogens like HIV, tuberculosis, and pertussis. Viral vectors like adenovirus are also promising and have been used in HIV vaccine development, though trials of Ad5-based vaccines showed they were not protective and increased risk of HIV infection in people with prior Ad5 immunity. Overall recombinant vaccines show potential but challenges remain in eliciting the right immune response against intracellular pathogens.
Edible vaccines are plant-based vaccines that are given orally. They were first tested on humans in 1997 using potatoes to deliver vaccines. Since then, many plant species like tobacco, tomatoes, and potatoes have been genetically engineered to produce vaccines for diseases like hepatitis B, cholera, norovirus, and rabies. Recent research has focused on developing edible vaccines for insects like honeybees and livestock like pigs. Advantages are low-cost production and inducing mucosal immunity through oral administration but challenges include variable dosage and immunotolerance.
1. Edible vaccines work by introducing genes into food crops so that they produce vaccine antigens, which then stimulate an immune response when the plant is eaten.
2. Genes can be inserted through techniques like particle bombardment or Agrobacterium transformation, then the plant is grown and eaten to deliver the vaccine.
3. Clinical trials have explored edible vaccines for diseases like cholera, hepatitis B, rabies, and norovirus with some success in animal models.
This document describes research into developing production and purification processes for a recombinant fragment of pneumococcal surface protein A (rfPspA3) in Escherichia coli. Fed-batch fermentation was performed using chemically defined medium with either glucose or glycerol as the carbon source. While glycerol reduced acetate production, cell growth and rfPspA3 production were higher with glucose. Downstream processing included cell disruption, clarification, and a multi-step chromatography purification process. Anion exchange chromatography prior to metal affinity chromatography gave better purification results than the reverse order. The optimized process achieved over 90% purity of rfPspA3 from both carbon sources.
This document provides an overview of edible vaccines. It discusses how edible vaccines are produced by introducing genes encoding vaccine antigens into edible plants using transformation methods. The document outlines various plant species used for edible vaccines like tomatoes, rice, maize, potatoes, and tobacco. It discusses factors affecting the efficacy of edible vaccines and provides examples of edible vaccine research for diseases like malaria, measles, hepatitis B, norovirus, and Alzheimer's disease. The conclusion states that edible vaccines could improve vaccination programs in developing countries by reducing costs and need for cold storage.
1. This study analyzed 20,027 pneumococcal genome sequences from multiple countries and clustered them into 621 lineages called Global Pneumococcal Sequence Clusters (GPSCs).
2. Thirty-five dominant GPSCs contained over 100 isolates each. Both vaccine and non-vaccine serotypes were found in 22 of these dominant GPSCs before PCV introduction, indicating potential for serotype replacement.
3. Increased resistance to penicillin and multiple drug classes was found in some dominant GPSCs. The country of isolation predicted the antibiotic resistance patterns in most dominant GPSCs.
This document provides an overview of live bacterial vaccines, focusing on attenuated pathogens and food-related lactic acid bacteria used as vaccine vehicles. It discusses the advantages of live bacterial vaccines, including their mimicry of natural infection and intrinsic adjuvant properties. However, it also outlines some safety concerns, such as the risk of reversion to virulence for attenuated pathogens. The document reviews several examples of attenuated pathogens currently being developed as vaccines, and food-related lactic acid bacteria that have been studied as potential vaccine delivery vehicles due to their generally recognized as safe status. It concludes that while live bacterial vaccines show promise, very few have reached the market due to challenges in developing safe and effective candidates.
The document discusses the production of genetically engineered vaccines. It describes how the gene for an antigen from a pathogen can be inserted into a plasmid or virus vector, which is then used to produce the antigen and elicit an immune response. Recombinant DNA technology allows specific antigens to be included in vaccines, making production cheaper and vaccines safer. Examples discussed include the first genetically engineered vaccine for hepatitis B, which expressed the viral surface antigen in yeast cells. Genetically engineered vaccines hold promise as a future tool in disease prevention.
This document describes engineering Mycobacterium smegmatis to produce polyhydroxyalkanoate (PHA) beads displaying mycobacterial antigens for use as a tuberculosis vaccine. M. smegmatis was engineered to express three enzymes involved in PHA production and a fusion protein linking PHA synthase to the TB antigens Ag85A and ESAT-6. This resulted in PHA beads displaying the antigens on their surface. Mice were vaccinated with the engineered PHA beads and they induced a cellular immune response against the mycobacterial antigens, demonstrating their potential as a TB vaccine.
This document summarizes the generation and characterization of novel recombinant BCG vaccine candidates expressing perfringolysin O (Pfo) and overexpressing key Mycobacterium tuberculosis antigens. A strain called AERAS-401 was generated by integrating a gene for mutant PfoAG137Q into the BCG genome under control of the Ag85B promoter. This strain secreted active Pfo and was safe in mice. A second strain called AFRO-1 incorporated a plasmid overexpressing Ag85A, Ag85B and TB10.4 antigens into AERAS-401. Mice and guinea pigs vaccinated with AFRO-1 showed enhanced immune responses compared to parental BCG. Mice vaccinated with AF
Bacteriophage therapy for antimicrobial resistant and biofilm forming [Autosa...kamal shrestha
This document discusses bacteriophage therapy as a potential treatment for antibiotic-resistant and biofilm-forming bacteria. It provides background on antibiotic resistance and biofilms, how they form and confer resistance. Bacteriophages are introduced as viruses that infect and replicate within bacteria. The history of bacteriophage therapy is covered, along with its advantages over antibiotics in being non-toxic and specifically targeting bacteria. Recent advances aim to improve efficacy, such as using cocktails of phages with broader host ranges or genetically modifying phages. Overall, the document argues that bacteriophage therapy shows promise as an alternative to antibiotics for resistant bacterial infections.
G. Poste presenting at Exponential Medicine 2016 Conference (San Diego)Stephanie Calderone
This document discusses accelerating global vaccine production to combat new pandemic threats. It outlines several past pandemics and resurgent viral threats in recent decades. It argues that current vaccine production methods are too slow and that computational design and chemical synthesis could enable faster, larger-scale production of synthetic epitope-based vaccines. This would allow expanding global protection against threats like a novel "Agent X". The document reviews computational and machine learning approaches to predict immunogenic epitopes and design cross-protective pan-vaccines. It advocates converting vaccine technology from biological to chemical production to meet global needs during a pandemic.
BACTERIOPHAGES_ A biocontrol strategy against bacterial phytopathogens; Credi...ramjithnp1999
BACTERIOPHAGES_ A biocontrol strategy against bacterial phytopathogens; Credit Seminar- RAMJITH N P (93_Ag_PP_2022-M)- Faculty of Agriculture, SKUAST-K, Wadura, Sopore
Vaccine (L. vacca = cow) is a preparation/suspension or extract of dead/attenuated (weakened) germs of a disease which on inoculation (injection) into a healthy person provides temporary/permanent active/passive immunity by inducing antibodies formation.
Thus antibody provoking agents are called vaccines.
This document summarizes the potential of RNA interference (RNAi) technology for crop improvement. It discusses how RNAi was discovered through early experiments in plants in the 1990s. The mechanism of RNAi involves long double-stranded RNA being cleaved by an enzyme called Dicer into small interfering RNAs (siRNAs) that are incorporated into a protein complex called RISC that targets and degrades complementary mRNAs, preventing gene expression. The document outlines several successful applications of RNAi technology for increasing biotic stress tolerance in crops against viruses, bacteria, fungi and insects by silencing key pathogen genes. It also discusses using RNAi to modify other crop traits like nutritional quality and abiotic stress resistance.
Scientists develop a new sars co v-2 vaccine with the successful experience o...DoriaFang
Researchers developed a new SARS-CoV-2 vaccine based on the successful hepatitis B vaccine platform. They used yeast to express the receptor binding domain protein of the coronavirus and supplemented it with a new adjuvant. Testing in monkeys showed the vaccine reduced virus shedding and symptoms when exposed to SARS-CoV-2. The vaccine aims to help meet global vaccination needs and may be effective against emerging variants.
Engineering pathogen resistance in crop plants current trends and future pros...UdayenduL10
This document summarizes engineering pathogen resistance in crop plants through genetic engineering techniques. It discusses current trends in developing disease resistance including directly interfering with pathogen virulence factors, regulating natural host defense responses, and pathogen mimicry. Transgenic crops with resistance to fungi, viruses and bacteria are still a small proportion of commercialized GM crops which are dominated by herbicide and insect resistance traits. Future work needs a better understanding of plant-pathogen interactions and developing durable resistance strategies. While transgenic resistance has been successful for viruses in papaya, socioeconomic acceptance of GM crops remains a challenge.
A critical study on wild species of Solanum distributed in Dibrugarh District...Open Access Research Paper
The genus Solanum L. is the largest genus in Solanaceae family, with around 1250 species worldwide. The present study aims to characterize the morphological and anatomical account of six wild species of Solanum namely, S. nigrum L., S. americanum Mill., S. viarum Dunal, S. torvum Sw., S. indicum L., and S. aethiopicum L. collected from the district. Variation in stomatal index and stomatal density were observed among the species. The stomatal index varies from 42.85% in S. viarum Dunal to 35.29% in S. americanum Mill. Similarly, stomatal density varies from 71.33mm2 in S. nigrum L. to 40.76mm2 in S. torvum Sw. and S. viarum Dunal respectively. Considering 47 morphological traits and 6 OTUs), a dendrogram was constructed by using a cluster analysis software SPSS 26.0 version which shows S. nigrum L. and S. americanum Mill. as the most similar having standard Euclidean distance 6 and S. nigrum L. and S. viarum Dunal as the least similar having standard Euclidean distance 171. Ethnobotanically, out of the six studied species five species S. nigrum L., S. americanum Mill., S. torvum Sw., S. indicum L. and S. aethiopicum L. were used as vegetables. All the six Solanum species are also used to treat problems such as fever, cold and cough, skin diseases, stomach aches, asthma, gastric, burns etc.
Estimating the direct use values of mangrove ecosystem: A case in Panguil Bay...Open Access Research Paper
Valuing the mangrove ecosystem poses some challenges as it is underrated by the policymakers and in some cases, it is excluded as inputs to decision making. This study aimed to estimate the direct use values of the mangrove ecosystem in Panguil Bay in 2020. A mixed method of research was applied: quantitative techniques employed surveys involving 924 respondents to get the direct use values of the mangrove ecosystem; qualitative techniques made use of key informant interviews and direct observation. The direct use values of the mangrove ecosystem were obtained by getting the volumes of goods and services extracted from it multiplied by the market price less the operation costs. Results revealed that while respondents lived in mangrove swamps, their mangrove utilization was tempered by the strict implementation of the no-cutting of trees policy in Panguil Bay. Mainly uprooted mangroves or fallen branches battered by strong winds were utilized for house construction and repair. Only 0.32% of the respondents utilized mangroves for the construction of their houses with an estimated total direct use value of Php11,599.20 (USD 31.98) and only 0.22% used mangroves for the repair of their houses with an estimated total direct use value of Php P1,401.57 (USD 0.22). The total estimated direct use value of the fishery resources (Metapenaeus endeavouri and Scylla serrata) was sizable at Php456.96 (USD 9.14) per fishing day. An important implication is to strictly implement appropriate policies for the mangrove ecosystem to be a rich nursery for fishery resources.
More Related Content
Similar to Expression of Hepatitis B Surface Antigen in Coleus forskohlii
1.72±0.25
1.88±0.35
1.72±0.15
15.01±10.50
17.01±24.16
20.07±1.57
21.12±8.41
23.01±1.12
25.12±5.56
27.21±25.37
28.88±0.97
33.94±0.97
The study examined the immune response to the hepatitis B virus vaccine in vaccinated groups. ELISA assays were used to detect hepatitis B surface antigen and antibodies in serum samples before and after vaccination.
1. Edible vaccines offer a cost-effective way to deliver vaccines orally by incorporating genes encoding antigens into plants. This allows for vaccines to be stored and administered more easily compared to traditional vaccines.
2. Edible vaccines stimulate both mucosal and systemic immunity by producing antigens when consumed. Various foods like bananas, potatoes, and tomatoes are being investigated for use as edible vaccines.
3. Edible vaccines overcome many issues with traditional vaccines like cold storage and transportation requirements. However, challenges remain around standardizing dosage and ensuring plant stability of the encoded antigens. Future applications of edible vaccines show promise for diseases like cancer, birth control, and autoimmune disorders.
This document discusses strategies for developing recombinant vaccines, including bacterial and viral vectors. It notes that recombinant vaccines using bacterial vectors like BCG can elicit both antibody and cellular immune responses, and have been used to develop vaccines against pathogens like HIV, tuberculosis, and pertussis. Viral vectors like adenovirus are also promising and have been used in HIV vaccine development, though trials of Ad5-based vaccines showed they were not protective and increased risk of HIV infection in people with prior Ad5 immunity. Overall recombinant vaccines show potential but challenges remain in eliciting the right immune response against intracellular pathogens.
Edible vaccines are plant-based vaccines that are given orally. They were first tested on humans in 1997 using potatoes to deliver vaccines. Since then, many plant species like tobacco, tomatoes, and potatoes have been genetically engineered to produce vaccines for diseases like hepatitis B, cholera, norovirus, and rabies. Recent research has focused on developing edible vaccines for insects like honeybees and livestock like pigs. Advantages are low-cost production and inducing mucosal immunity through oral administration but challenges include variable dosage and immunotolerance.
1. Edible vaccines work by introducing genes into food crops so that they produce vaccine antigens, which then stimulate an immune response when the plant is eaten.
2. Genes can be inserted through techniques like particle bombardment or Agrobacterium transformation, then the plant is grown and eaten to deliver the vaccine.
3. Clinical trials have explored edible vaccines for diseases like cholera, hepatitis B, rabies, and norovirus with some success in animal models.
This document describes research into developing production and purification processes for a recombinant fragment of pneumococcal surface protein A (rfPspA3) in Escherichia coli. Fed-batch fermentation was performed using chemically defined medium with either glucose or glycerol as the carbon source. While glycerol reduced acetate production, cell growth and rfPspA3 production were higher with glucose. Downstream processing included cell disruption, clarification, and a multi-step chromatography purification process. Anion exchange chromatography prior to metal affinity chromatography gave better purification results than the reverse order. The optimized process achieved over 90% purity of rfPspA3 from both carbon sources.
This document provides an overview of edible vaccines. It discusses how edible vaccines are produced by introducing genes encoding vaccine antigens into edible plants using transformation methods. The document outlines various plant species used for edible vaccines like tomatoes, rice, maize, potatoes, and tobacco. It discusses factors affecting the efficacy of edible vaccines and provides examples of edible vaccine research for diseases like malaria, measles, hepatitis B, norovirus, and Alzheimer's disease. The conclusion states that edible vaccines could improve vaccination programs in developing countries by reducing costs and need for cold storage.
1. This study analyzed 20,027 pneumococcal genome sequences from multiple countries and clustered them into 621 lineages called Global Pneumococcal Sequence Clusters (GPSCs).
2. Thirty-five dominant GPSCs contained over 100 isolates each. Both vaccine and non-vaccine serotypes were found in 22 of these dominant GPSCs before PCV introduction, indicating potential for serotype replacement.
3. Increased resistance to penicillin and multiple drug classes was found in some dominant GPSCs. The country of isolation predicted the antibiotic resistance patterns in most dominant GPSCs.
This document provides an overview of live bacterial vaccines, focusing on attenuated pathogens and food-related lactic acid bacteria used as vaccine vehicles. It discusses the advantages of live bacterial vaccines, including their mimicry of natural infection and intrinsic adjuvant properties. However, it also outlines some safety concerns, such as the risk of reversion to virulence for attenuated pathogens. The document reviews several examples of attenuated pathogens currently being developed as vaccines, and food-related lactic acid bacteria that have been studied as potential vaccine delivery vehicles due to their generally recognized as safe status. It concludes that while live bacterial vaccines show promise, very few have reached the market due to challenges in developing safe and effective candidates.
The document discusses the production of genetically engineered vaccines. It describes how the gene for an antigen from a pathogen can be inserted into a plasmid or virus vector, which is then used to produce the antigen and elicit an immune response. Recombinant DNA technology allows specific antigens to be included in vaccines, making production cheaper and vaccines safer. Examples discussed include the first genetically engineered vaccine for hepatitis B, which expressed the viral surface antigen in yeast cells. Genetically engineered vaccines hold promise as a future tool in disease prevention.
This document describes engineering Mycobacterium smegmatis to produce polyhydroxyalkanoate (PHA) beads displaying mycobacterial antigens for use as a tuberculosis vaccine. M. smegmatis was engineered to express three enzymes involved in PHA production and a fusion protein linking PHA synthase to the TB antigens Ag85A and ESAT-6. This resulted in PHA beads displaying the antigens on their surface. Mice were vaccinated with the engineered PHA beads and they induced a cellular immune response against the mycobacterial antigens, demonstrating their potential as a TB vaccine.
This document summarizes the generation and characterization of novel recombinant BCG vaccine candidates expressing perfringolysin O (Pfo) and overexpressing key Mycobacterium tuberculosis antigens. A strain called AERAS-401 was generated by integrating a gene for mutant PfoAG137Q into the BCG genome under control of the Ag85B promoter. This strain secreted active Pfo and was safe in mice. A second strain called AFRO-1 incorporated a plasmid overexpressing Ag85A, Ag85B and TB10.4 antigens into AERAS-401. Mice and guinea pigs vaccinated with AFRO-1 showed enhanced immune responses compared to parental BCG. Mice vaccinated with AF
Bacteriophage therapy for antimicrobial resistant and biofilm forming [Autosa...kamal shrestha
This document discusses bacteriophage therapy as a potential treatment for antibiotic-resistant and biofilm-forming bacteria. It provides background on antibiotic resistance and biofilms, how they form and confer resistance. Bacteriophages are introduced as viruses that infect and replicate within bacteria. The history of bacteriophage therapy is covered, along with its advantages over antibiotics in being non-toxic and specifically targeting bacteria. Recent advances aim to improve efficacy, such as using cocktails of phages with broader host ranges or genetically modifying phages. Overall, the document argues that bacteriophage therapy shows promise as an alternative to antibiotics for resistant bacterial infections.
G. Poste presenting at Exponential Medicine 2016 Conference (San Diego)Stephanie Calderone
This document discusses accelerating global vaccine production to combat new pandemic threats. It outlines several past pandemics and resurgent viral threats in recent decades. It argues that current vaccine production methods are too slow and that computational design and chemical synthesis could enable faster, larger-scale production of synthetic epitope-based vaccines. This would allow expanding global protection against threats like a novel "Agent X". The document reviews computational and machine learning approaches to predict immunogenic epitopes and design cross-protective pan-vaccines. It advocates converting vaccine technology from biological to chemical production to meet global needs during a pandemic.
BACTERIOPHAGES_ A biocontrol strategy against bacterial phytopathogens; Credi...ramjithnp1999
BACTERIOPHAGES_ A biocontrol strategy against bacterial phytopathogens; Credit Seminar- RAMJITH N P (93_Ag_PP_2022-M)- Faculty of Agriculture, SKUAST-K, Wadura, Sopore
Vaccine (L. vacca = cow) is a preparation/suspension or extract of dead/attenuated (weakened) germs of a disease which on inoculation (injection) into a healthy person provides temporary/permanent active/passive immunity by inducing antibodies formation.
Thus antibody provoking agents are called vaccines.
This document summarizes the potential of RNA interference (RNAi) technology for crop improvement. It discusses how RNAi was discovered through early experiments in plants in the 1990s. The mechanism of RNAi involves long double-stranded RNA being cleaved by an enzyme called Dicer into small interfering RNAs (siRNAs) that are incorporated into a protein complex called RISC that targets and degrades complementary mRNAs, preventing gene expression. The document outlines several successful applications of RNAi technology for increasing biotic stress tolerance in crops against viruses, bacteria, fungi and insects by silencing key pathogen genes. It also discusses using RNAi to modify other crop traits like nutritional quality and abiotic stress resistance.
Scientists develop a new sars co v-2 vaccine with the successful experience o...DoriaFang
Researchers developed a new SARS-CoV-2 vaccine based on the successful hepatitis B vaccine platform. They used yeast to express the receptor binding domain protein of the coronavirus and supplemented it with a new adjuvant. Testing in monkeys showed the vaccine reduced virus shedding and symptoms when exposed to SARS-CoV-2. The vaccine aims to help meet global vaccination needs and may be effective against emerging variants.
Engineering pathogen resistance in crop plants current trends and future pros...UdayenduL10
This document summarizes engineering pathogen resistance in crop plants through genetic engineering techniques. It discusses current trends in developing disease resistance including directly interfering with pathogen virulence factors, regulating natural host defense responses, and pathogen mimicry. Transgenic crops with resistance to fungi, viruses and bacteria are still a small proportion of commercialized GM crops which are dominated by herbicide and insect resistance traits. Future work needs a better understanding of plant-pathogen interactions and developing durable resistance strategies. While transgenic resistance has been successful for viruses in papaya, socioeconomic acceptance of GM crops remains a challenge.
A critical study on wild species of Solanum distributed in Dibrugarh District...Open Access Research Paper
The genus Solanum L. is the largest genus in Solanaceae family, with around 1250 species worldwide. The present study aims to characterize the morphological and anatomical account of six wild species of Solanum namely, S. nigrum L., S. americanum Mill., S. viarum Dunal, S. torvum Sw., S. indicum L., and S. aethiopicum L. collected from the district. Variation in stomatal index and stomatal density were observed among the species. The stomatal index varies from 42.85% in S. viarum Dunal to 35.29% in S. americanum Mill. Similarly, stomatal density varies from 71.33mm2 in S. nigrum L. to 40.76mm2 in S. torvum Sw. and S. viarum Dunal respectively. Considering 47 morphological traits and 6 OTUs), a dendrogram was constructed by using a cluster analysis software SPSS 26.0 version which shows S. nigrum L. and S. americanum Mill. as the most similar having standard Euclidean distance 6 and S. nigrum L. and S. viarum Dunal as the least similar having standard Euclidean distance 171. Ethnobotanically, out of the six studied species five species S. nigrum L., S. americanum Mill., S. torvum Sw., S. indicum L. and S. aethiopicum L. were used as vegetables. All the six Solanum species are also used to treat problems such as fever, cold and cough, skin diseases, stomach aches, asthma, gastric, burns etc.
Estimating the direct use values of mangrove ecosystem: A case in Panguil Bay...Open Access Research Paper
Valuing the mangrove ecosystem poses some challenges as it is underrated by the policymakers and in some cases, it is excluded as inputs to decision making. This study aimed to estimate the direct use values of the mangrove ecosystem in Panguil Bay in 2020. A mixed method of research was applied: quantitative techniques employed surveys involving 924 respondents to get the direct use values of the mangrove ecosystem; qualitative techniques made use of key informant interviews and direct observation. The direct use values of the mangrove ecosystem were obtained by getting the volumes of goods and services extracted from it multiplied by the market price less the operation costs. Results revealed that while respondents lived in mangrove swamps, their mangrove utilization was tempered by the strict implementation of the no-cutting of trees policy in Panguil Bay. Mainly uprooted mangroves or fallen branches battered by strong winds were utilized for house construction and repair. Only 0.32% of the respondents utilized mangroves for the construction of their houses with an estimated total direct use value of Php11,599.20 (USD 31.98) and only 0.22% used mangroves for the repair of their houses with an estimated total direct use value of Php P1,401.57 (USD 0.22). The total estimated direct use value of the fishery resources (Metapenaeus endeavouri and Scylla serrata) was sizable at Php456.96 (USD 9.14) per fishing day. An important implication is to strictly implement appropriate policies for the mangrove ecosystem to be a rich nursery for fishery resources.
Where are they now: Native trees in the City of Malolos, Province of Bulacan,...Open Access Research Paper
This study focuses on tracing the presence of native trees where the names of the barangays were derived. Also included is the assessment of the knowledge and attitudes of locals about the native trees and their attitude towards willingness to support possible conservation of trees. Tree inventory was conducted to determine the presence of native trees in the selected areas. Residents’ knowledge of the native status of common tree species and related topics such as their location in the city were explored through a survey. 194 individual tree species identified and 62 of which are native trees. Only 5 Diospyros blancoi (Kamagong or Mabolo tree) were observed in Barangay Mabolo, while there is no presence of the native trees namely Dillenia philippinensis (Katmon), Nauclea orientalis L. (Bangkal), Glochidion rubrum Blume (Bagnang-pula) and Afzelia rhomboidea (Balayong/Tindalo) in the other selected barangays. Most common native species observed is Adonidia merrilii (Manila Palm Tree). Overall knowledge was high wherein correct identification of native trees was higher than that for non-native trees. Generally, positive attitudes were expressed by most of the respondents towards native trees and were in favor of planting more native trees.
Floristic diversity in a unique ecosystem of Burkina Faso: The case of the Ko...Open Access Research Paper
The objective of this study is to provide a better knowledge of the flora of a classified forest relic and its capacity to preserve biodiversity. A forest inventory on 28 plots for woody species and 11 plots for herbaceous species was carried out in the Kou Classified Forest (KCF). The results show that the flora of the KCF is rich in 207 species belonging to 62 families and 165 genera. It includes 121 woody species and 85 herbaceous species. The most represented family is the Fabaceae 38 taxa. The diversity indices show that the woody and herbaceous flora is diverse, with respectively 13.61 and 7.85 for Margalef’s absolute species richness, 3.36 and 3.80 for Shannon-Weaver’s diversity index (H’), 0.93 and 0.97 for Simpson’s dominance (D’); 0.74 and 0.92 for Piélou’s equitability index (E). A total of 36 special-status species and 87.86% rare species (rarity index >80%) were recorded. Structural parameters such as basal area (15.73 ± 2.86 m2/ha), density (456 ± 51 ft/ha), average herbaceous cover (22% ± 4%) and Weibull’s form factor (C=0.7) show good ecological health and stability of the vegetation with a high regeneration potential of 1280 seedlings/ha and a regeneration rate of 236.37%. This ecosystem is of major importance in the conservation of phytodiversity in Burkina Faso.
Classification of Clove sizes as planting material to the bulb yield of Garli...Open Access Research Paper
Garlic is one of the highly valued crops in the Philippines. However, low production yield is the main constraint, specifically in the native varieties that could not satisfy the demand. Among the limiting factors are the use of unsuitable clove size as planting materials. The results revealed that clove sizes significantly influenced the growth of garlic. Large clove size and extra-large clove size obtained average plant vigor with ratings of 5.83 and 6.33, respectively. Significant differences were also found in both fresh and dry bulb weights, with the largest clove size yielding the heaviest weights at 19.36g and 16.67g, respectively. Moreover, large and extra-large clove sizes produced the highest number of cloves per bulb with an average of 19.87 and 19.33 respectively. However, no significant differences were observed in yield per plant and yield per hectare. Consequently, large clove sizes employed as planting material increased the vigor, bulb weights, and the number of cloves with no significant effect on the yield. The study showed that planting large clove sizes (2.0-2.50g) is more promising as planting materials of native varieties like Ilocos white.
Exploring indigenous practices in Northern Philippines and their environmenta...Open Access Research Paper
This study aims to explore the policy implications of documented indigenous practices in vernacular housing in Northern Philippines towards sustainable green architecture. The research is grounded on the need to address the growing concern for environmental degradation and how indigenous knowledge can be used as a solution. The study utilized qualitative research methods including interviews, observations, and archival analysis to gather data. Results reveal that traditional housing practices of the Itawes, Ibanag, and Malaweg promote sustainable architecture, which emphasizes the use of locally sourced materials, passive cooling and ventilation systems, and climate adaptive practices. However, these practices are threatened by modernization, lack of policies and regulations, and limited awareness of their environmental and cultural significance. This study underscores the need for policy reforms that support the integration of traditional practices into contemporary sustainable architecture. The documentation of indigenous practices also serves as a resource for educators and policymakers in developing sustainable, culturally sensitive designs that address environmental challenges.
Water sources and management practices among the household residents of Baran...Open Access Research Paper
The purpose of this study is to determine the water sources and management practices among the household residents of Barangay Labuyo, Tangub City, Misamis Occidental. A total of 204 household respondents were randomly interviewed using a survey questionnaire. Data on water management practices were measured using a 5-point Likert scale. One-way ANOVA and Pearson correlation were further used to determine the differences and relationships between demographic profiles and management practices. Results show that pipe water supply from the main source (68%) was the primary water source used among household residents. In terms of sex, females often practice water management on the water sources with a weighted mean of 3.42. While respondents with age 68 years and older exhibit a higher degree (weighted mean=3.63) of involvement in water conservation measures. And respondents with college degree have applied their in-depth comprehension and knowledge on water conservation with a weighted mean of 3.48. The study also revealed a significant difference in the management practices between two sexes, among all ages and levels of educational attainment with p values <0.05. The association between demographic profiles with management practices further presents a significant relationship. Generally, the demographic profile (sex, age, and educational attainment) has a weak positive relationship towards management practices with values, r = 0.26, p = 0.00021; r = 0.34, p = <0.05; and r = 0.26, p = 0.00014, respectively. Thus, results suggest the need for enhancing community awareness on sustainable water source management and putting it into practice consequently reducing inadequacy of water supply in the area.
The modification of an existing product or the formulation of a new product to fill a newly identified market niche or customer need are both examples of product development. This study generally developed and conducted the formulation of aramang baked products enriched with malunggay conducted by the researchers. Specifically, it answered the acceptability level in terms of taste, texture, flavor, odor, and color also the overall acceptability of enriched aramang baked products. The study used the frequency distribution for evaluators to determine the acceptability of enriched aramang baked products enriched with malunggay. As per sensory evaluation conducted by the researchers, it was proven that aramang baked products enriched with malunggay was acceptable in terms of Odor, Taste, Flavor, Color, and Texture. Based on the results of sensory evaluation of enriched aramang baked products proven that three (3) treatments were all highly acceptable in terms of variable Odor, Taste, Flavor, Color and Textures conducted by the researchers.
Microbiological assessment of air quality of selected locations within Verita...Open Access Research Paper
The study investigated the microbiological quality of indoor and outdoor air of certain locations – the chapel, basement, classroom, hostel, as well as the old and new microbiology laboratories in Veritas University, Abuja. The settle plate technique using open Petri dishes containing different culture media was employed to collect samples daily for 5 weeks at 7 days intervals. Standard microbiological methods were employed for the identification of bacterial and fungal isolates. The bacterial counts ranged from 1.90×106 to 5.3×106 and 2.90 x 106 to 6.20 x 106 for indoor and outdoor air while the fungal counts ranged from 2.30 x106 to 3.70 x 106 and 2.10 x 106 to 4.40 x 106 also for indoor and outdoor air respectively. The bacterial isolates were identified to include Bacillus species and Staphylococcus aureus with percentage occurrence of 44.0% and 56.0% respectively. The results obtained also showed the occurrence of three major fungal species namely Aspergillus sp (60.0%), Rhodotolura sp (5.0%), and Rhizopus sp (35.00%). The bacterial isolate, Staphylococcus aureus (56.0%) was shown to be the most predominant airborne bacteria while Aspergillus sp (60.0%) was the most frequently isolated fungal species. The 95% confidence level statistical analysis showed a significant difference between the indoor and outdoor air microbial load of the selected locations. Data generated underline the usefulness of monitoring the air quality of the selected locations because the contamination of indoor and outdoor habitats can cause health problems and even an increase in human mortality.
Kinetic studies on malachite green dye adsorption from aqueous solutions by A...Open Access Research Paper
Water polluted by dyestuffs compounds is a global threat to health and the environment; accordingly, we prepared a green novel sorbent chemical and Physical system from an algae, chitosan and chitosan nanoparticle and impregnated with algae with chitosan nanocomposite for the sorption of Malachite green dye from water. The algae with chitosan nanocomposite by a simple method and used as a recyclable and effective adsorbent for the removal of malachite green dye from aqueous solutions. Algae, chitosan, chitosan nanoparticle and algae with chitosan nanocomposite were characterized using different physicochemical methods. The functional groups and chemical compounds found in algae, chitosan, chitosan algae, chitosan nanoparticle, and chitosan nanoparticle with algae were identified using FTIR, SEM, and TGADTA/DTG techniques. The optimal adsorption conditions, different dosages, pH and Temperature the amount of algae with chitosan nanocomposite were determined. At optimized conditions and the batch equilibrium studies more than 99% of the dye was removed. The adsorption process data matched well kinetics showed that the reaction order for dye varied with pseudo-first order and pseudo-second order. Furthermore, the maximum adsorption capacity of the algae with chitosan nanocomposite toward malachite green dye reached as high as 15.5mg/g, respectively. Finally, multiple times reusing of algae with chitosan nanocomposite and removing dye from a real wastewater has made it a promising and attractive option for further practical applications.
Effects of Covid-19 pandemic on commodity price volatility and the welfare of...Open Access Research Paper
The evolving uncertainty of the emergence of the COVID-19 pandemic has adversely impacted some commodity prices, the welfare of farming households, and the economic growth and development of the country. The study is aimed at assessing the effects of Covid -19 pandemic on commodity price volatility and the welfare of farming households in Nigeria. The study was based on secondary data from (2010- 2022, and forecasts from 2023 -2026), collected from the Central Bank of Nigeria publication, IMF World economic outlook, and the World Bank report. Time- response graph and Autoregressive Moving Average (ARMA) was used to analyze the trend of the inflation rate, and T-test statistics, were used to test the relationship between the real income of farming household before and during the pandemic. The result showed that prices of some commodities doubled after the pandemic. There is a significant difference between the real income of farming households before and during the pandemic. The study recommends that a price control mechanism should be put in place to manage the affordability of goods and services in the market because this has a direct effect on the growth and development of the country.
Effect of foliar application of water soluble fertilizer on growth, yield and...Open Access Research Paper
The tomato is the one of the most famous crops in Pakistan. It is used and consumed as fresh as well as in processed form. Its botanical name is Solanum lycopersicon Mill. The proposed study had been conducted in the year 2017-2018 in order to find the best combination of water soluble NPK fertilizers as compared to control. Five different types of water soluble fertilizers were collected from different sources and were applied at 5% concentration during the entire growing period of the tomato crop. The experiment was designed using Randomized Complete Block Design (RCBD) with five treatments and three replications of each. Different vegetative, reproductive and bio-chemical parameters were recorded and analyzed statistically at 5% level of significance. The treatments were compared using LSD test. It was concluded that different NPK water soluble fertilizers showed variation in physical and bio chemical parameters in tomato plants as compared to control. The plants showed variation in plant height, No. of fruit per plant, fruit yield per hectare, Individual fruit weight, fruit weight per plant, No. of diseased fruit per plant, date of first harvest, Fruit color Fruit length (cm), fruit size (cm), fruit firmness, total NPK contents, vitamincmg Chlorophyll concentration, pH, (TSS), Electricity conductivity (EC) and Titratable acidity Among all the water soluble treatments, the T3 treatment (WSF 20:20:20) yielded the comparatively better results as compared to other WSF treatments. So it was concluded that T3 was the recommended water soluble fertilizer for tomato.
Yield response of aman rice to transplanting geometry and seedlings per hill ...Open Access Research Paper
In a condition of limited scope of horizontal yield expansion, rice yield can be increased by efficient utilization of land through proper transplanting arrangement with maximum number of plant population unit-1 area. Thus, the present experiment was conducted in the farmer’s field at Batiaghata upazila of Khulna district, Bangladesh during T. aman season (Jul-Nov) to evaluate the effect of transplanting geometry and number of seedlings hill-1 on growth and yield of aman rice (Binadhan-7, a short duration variety of its’ early harvest can create opportunity to cultivate winter crops in this region). The experiment had four types of transplanting geometry (single row rectangular system, single row triangular system, double row rectangular system and double row triangular system) and three levels of seedling hill-1 (3, 4 and 5 seedlings) with three replications. The results showed that individually transplanting geometry or number of seedlings hill-1 had substantial influence on yield attributes and yield but their interaction effect had non-significant influence on almost all measured parameters except number of tillers and effective tillers hill-1. The maximum grain yield (5.6 t ha-1) was achieved from double row triangular system yet the highest plant height, effective tillers hill-1, grain panicle-1, 1000 grain weight and straw yield were obtained from single row triangular system. In case of seeding hill-1, 3 seedlings hill-1 produced the highest grain yield (5.33 t ha-1) plant height, effective tillers hill-1, grain panicle-1, 1000 grain weight and straw yield. It can be concluded that double row transplanting geometry with 3 seedlings hill-1 can utilize the land efficiently for grain yield and be recommended for cultivation of Binadhan-7 in the coastal region of south-western Bangladesh.
Evaluation of lead and arsenic content of Azardirachta indica seed oil and Ci...Open Access Research Paper
There is an increase preference for plant-based repellents due to their effectiveness, environmentally friendliness and biodegradable nature. It is therefore necessary to ascertain the safety of these repellents by analysing their heavy metal content. This study has shown that lead and arsenic content of Azadirachta indica seed oil cream and Citrus sinensis peel oil cream as mosquito repellent is insignificant and therefore very safe for use according to the Ghana Standard Authority specification. These results provide new insight into the safety of these natural mosquito repellents.
Determination of hydroxy methyl furfural concentration in honey using ultra v...Open Access Research Paper
This paper aimed to determine the concentration of hydroxyl methyl furfural (HMF) using UV-visible spectroscopy to assess the quality of honey. The honey samples were collected from three honeys productive temperature zones: temperate, sub-tropical and tropical. Following the procedure of white method, the concentration of HMF of temperate, sub-tropical and tropical zone honey are found to be 11.18 ± 0.052mg/kg, 24.95± 0.119mg/kg, and 56.94±0.366mg/kg respectively. There is statistically significance differences between the groups in HMF concentration at 95% confidence level (p<0.05). All the samples are found to have HMF value less than the maximum concentration of HMF in honey set by standard controlling international organizations, which shows good quality of the honey in the study areas.
Improving the viability of probiotics by encapsulation methods for developmen...Open Access Research Paper
The popularity of functional foods among scientists and common people has been increasing day by day. Awareness and modernization make the consumer think better regarding food and nutrition. Now a day’s individual knows very well about the relation between food consumption and disease prevalence. Humans have a diversity of microbes in the gut that together form the gut microflora. Probiotics are the health-promoting live microbial cells improve host health through gut and brain connection and fighting against harmful bacteria. Bifidobacterium and Lactobacillus are the two bacterial genera which are considered to be probiotic. These good bacteria are facing challenges of viability. There are so many factors such as sensitivity to heat, pH, acidity, osmotic effect, mechanical shear, chemical components, freezing and storage time as well which affects the viability of probiotics in the dairy food matrix as well as in the gut. Multiple efforts have been done in the past and ongoing in present for these beneficial microbial population stability until their destination in the gut. One of a useful technique known as microencapsulation makes the probiotic effective in the diversified conditions and maintain these microbe’s community to the optimum level for achieving targeted benefits. Dairy products are found to be an ideal vehicle for probiotic incorporation. It has been seen that the encapsulated microbial cells show higher viability than the free cells in different processing and storage conditions as well as against bile salts in the gut. They make the food functional when incorporated, without affecting the product sensory characteristics.
Microbial characterisation and identification, and potability of River Kuywa ...Open Access Research Paper
Water contamination is one of the major causes of water borne diseases worldwide. In Kenya, approximately 43% of people lack access to potable water due to human contamination. River Kuywa water is currently experiencing contamination due to human activities. Its water is widely used for domestic, agricultural, industrial and recreational purposes. This study aimed at characterizing bacteria and fungi in river Kuywa water. Water samples were randomly collected from four sites of the river: site A (Matisi), site B (Ngwelo), site C (Nzoia water pump) and site D (Chalicha), during the dry season (January-March 2018) and wet season (April-July 2018) and were transported to Maseno University Microbiology and plant pathology laboratory for analysis. The characterization and identification of bacteria and fungi were carried out using standard microbiological techniques. Nine bacterial genera and three fungi were identified from Kuywa river water. Clostridium spp., Staphylococcus spp., Enterobacter spp., Streptococcus spp., E. coli, Klebsiella spp., Shigella spp., Proteus spp. and Salmonella spp. Fungi were Fusarium oxysporum, Aspergillus flavus complex and Penicillium species. Wet season recorded highest bacterial and fungal counts (6.61-7.66 and 3.83-6.75cfu/ml) respectively. The results indicated that the river Kuywa water is polluted and therefore unsafe for human consumption before treatment. It is therefore recommended that the communities to ensure that they boil water especially for drinking.
High histological grade breast cancer morphological evaluation on mammogram u...Open Access Research Paper
To evaluate the high-grade breast cancer morphological complexity on mammogram. We conducted a retrospective study using an open source data got from figshare repository. These anonymized data were collected and used for a study approved by the institutional review board. Cranio-Caudal and Medio-lateral mammograms and their tumor segmented images from 66 patients subdivided in two groups high histological grade (n=23) low-grade (low and intermediate, n=41). From breast cancer image segmentation, we extracted fractal dimension using Fraclac, plugin of ImageJ software based on box-counting method. For our analysis we used comparatively the fractal dimension from cranio-caudal (CC) and medio-lateral (MLO) images. We summarized the fractal dimension of our cohort using boxplot and performed the Wilcoxon non-parametric statistic for fractal dimension comparison of two groups (High-grade and low-grade). There was not difference between CC (mean ± std= 1.1583±0.067) andmLO (mean ± std =1.1551±0.055) breast cancer fractal dimension. For the high-grade differentiation, CC andmLO images fractal dimension were contributed respectively at a little difference but without statistically difference (P value=0.438 and 0.435). High-grade fractal dimensions mean were respectively 1.142±0.044 and 1.144±0.075 for CC andmLO images against 1.166±0.050 and 1.160±0.057 for low-grade. It had been recorded a lower mean value of fractal dimension for high-grade breast cancer without statistically significant. This finding shows that the high-grade breast cancer tends to have a regular shape.
Characterization of inflammatory syndrome in smokers, from C-Reactive protein...Open Access Research Paper
A local inflammatory syndrome is characterized by a classic semiological tetrad: pain, swelling, redness and heat. These signs are easily observed when the inflammation concerns the skin or an adjacent tissue. Throughout this study, the aim was to characterize chronic inflammation in smokers using two parameters, rate of erythrocytes sedimentation (RES) and C – reactive protein (CRP). Our study was done on a sample of 35 smoking subjects, composed of men and women. The CRP measurement o was carried out using a CRP-Latex agglutination test which detects only serum CRP levels around 6mg/L. The technique used for the RES measurement is that of Westergreen. From the results, we observed that 31% of our sample presented a positive CRP and a high RES against 40% having regular CRP and RES. The gender of the subject did not play a role in the results obtained. On the other hand, a significant difference (p = 0.031) in CRP was observed between subjects with normal RES and those with high RES. Therefore, these results make it difficult to confirm that RES and CRP can be used as reliable markers for the characterization of inflammation linked to smoking.
Prevalence of diarrhea among severely malnourished children admitted in Gover...Open Access Research Paper
Mortality rate of children under the age of five has reduced worldwide, but still the probability of a child dying before the age of five is greatest in underdeveloped countries. Pakistan reports child mortality rates in same bracket as other South Asian countries due to malnutrition and diarrhea. To determine the prevalence and factors associated in children less than five years of age a cross sectional study was conducted with mothers whose children were admitted in pediatric government Hospital, Lahore. A convenient sample of 101 children (6-59 months, 53 males and 48 females) suffering from malnutrition and diarrhea were selected from hospital. Data about socio demographic, anthropometric, clinical and dietary variables were collected and analyzed by using SPSS version 16. The results showed that mean age of patients was 19.36 ± 10.5 months. The illiteracy rate among mother and father of patients was 94.1% and 69.3% respectively. 94.1% of the patients were breastfed while 47.5% of the patients were on bottle feed with breast milk. 66.3% patients families has very low-income rate while 33.7% were satisfactory. 40.6% patients were not vaccinated against immunization. Out of 101 patients, 39.6% of the patients had diarrhea while 6.9% of the patients had chronic diarrhea. The prevalence rate of diarrhea (39%) was less among children who were younger than 18 months as compared to those who were above 18 months (40.5%). The most significant factors that caused the incidence of diarrhea in children was form of water storage system, complementary feeding practices, and hand wash cleaning materials. This study concluded that government, nongovernmental organizations and families living with children and mothers could cooperate on strategies to minimize the risks of the diarrhea among children less than five years of age.
THE SACRIFICE HOW PRO-PALESTINE PROTESTS STUDENTS ARE SACRIFICING TO CHANGE T...indexPub
The recent surge in pro-Palestine student activism has prompted significant responses from universities, ranging from negotiations and divestment commitments to increased transparency about investments in companies supporting the war on Gaza. This activism has led to the cessation of student encampments but also highlighted the substantial sacrifices made by students, including academic disruptions and personal risks. The primary drivers of these protests are poor university administration, lack of transparency, and inadequate communication between officials and students. This study examines the profound emotional, psychological, and professional impacts on students engaged in pro-Palestine protests, focusing on Generation Z's (Gen-Z) activism dynamics. This paper explores the significant sacrifices made by these students and even the professors supporting the pro-Palestine movement, with a focus on recent global movements. Through an in-depth analysis of printed and electronic media, the study examines the impacts of these sacrifices on the academic and personal lives of those involved. The paper highlights examples from various universities, demonstrating student activism's long-term and short-term effects, including disciplinary actions, social backlash, and career implications. The researchers also explore the broader implications of student sacrifices. The findings reveal that these sacrifices are driven by a profound commitment to justice and human rights, and are influenced by the increasing availability of information, peer interactions, and personal convictions. The study also discusses the broader implications of this activism, comparing it to historical precedents and assessing its potential to influence policy and public opinion. The emotional and psychological toll on student activists is significant, but their sense of purpose and community support mitigates some of these challenges. However, the researchers call for acknowledging the broader Impact of these sacrifices on the future global movement of FreePalestine.
Elevate Your Nonprofit's Online Presence_ A Guide to Effective SEO Strategies...TechSoup
Whether you're new to SEO or looking to refine your existing strategies, this webinar will provide you with actionable insights and practical tips to elevate your nonprofit's online presence.
How to Setup Default Value for a Field in Odoo 17Celine George
In Odoo, we can set a default value for a field during the creation of a record for a model. We have many methods in odoo for setting a default value to the field.
Geography as a Discipline Chapter 1 __ Class 11 Geography NCERT _ Class Notes...
Expression of Hepatitis B Surface Antigen in Coleus forskohlii
1. Int. J. Biomol. Biomed.
Guleria et al
29
RESEARCH PAPER OPEN ACCESS
Expression of Hepatitis B Surface Antigen in Coleus forskohlii
Neha Guleria*1
, Paramanahally Hanumanthe Gowda Ramanjini Gowda1
,
Satish Kumar Kariyaiah1
, Renu Pasricha2
1
Department of Plant Biotechnology, University of Agricultural Sciences, GKVK, Bangalore, India
2
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
Key words: Agrobacterium, Coleus forskohlii, HBs Ag, VLPs, Western blot.
Article Published: 17 July 29, 2016
Abstract
Hepatitis B, a common viral infection, affecting millions of people every year, leads to disability and death. It has
become one of the alarming diseases in the world. Vaccination is the best possible way to prevent this deadly viral
infection and its consequences. Unaffordability of the mass vaccination program due to low health budgets
especially in developing countries have demanded the economical, effective and easily available vaccine
production against hepatitis B virus. The expression of subunit vaccines and recombinant proteins in plants is a
convenient, safe and potentially economical platform technology. Hence, development of a plant-based vaccine
could be promising. Therefore, the present investigation focused on expression and large- scale production of
hepatitis B surface antigen (HBsAg) in Coleus forskohlii for the development of vaccine. Eight transformed C.
forskohlii plants were generated via Agrobacterium -mediated transformation method. The integration of 681bp
of HBsAg gene into the plant genome was confirmed using PCR. SDS-PAGE showed the presence of ~48kDa
dimer and ~24kDa monomer form of HBsAg protein and the expression of recombinant protein was further
confirmed by western blot. C. forskohlii expressed HBs Ag was recorded 260µg/g leaf fresh weight as measured
by ELISA. Transformed plants of HBsAg showed the accumulation of Virus Like Particles of 22 nm size using
transmission electron microscopy. This study offers a great potential for the large -scale production of hepatitis B
vaccine in C. forskohlii which provides a strategy for contributing a means to achieve global immunization for the
hepatitis B prevention and eradication.
*Corresponding Author: Neha Guleria neha.gul5@gmail.com
International Journal of Biomolecules and Biomedicine (IJBB)
ISSN: 2221-1063 (Print), 2222-503X (Online)
http://www.innspub.net
Vol. 5, No. 1, p. 29-39, 2016
2. Int. J. Biomol. Biomed.
Guleria et al
30
Introduction
Hepatitis B virus (HBV) causes transient and chronic
infection of the liver ultimately leads to untreatable
liver cancer (Yogendra et al., 2009). In the HBV-
infected people, the virus persists for the rest of their
lives and can be passed on to others (Goldstein et al.,
2005). HBV infection has a worldwide distribution. It
is estimated that more than 2 billion people have
been infected. Of these, approximately 400 million
are chronically infected and at risk of serious illness
and death from cirrhosis and hepatocellular
carcinoma (HCC), diseases that are estimated to
cause 650 000–780 000 deaths each year worldwide
(WHO, 2015). Therefore, Hepatitis B has become a
global public health problem.Vaccination against this
virus was introduced over 25 years ago (Czyzet al.,
2014), a proven strategy to control HBV infection.
The current subunit vaccines e.g. Shanvac B, Engerix
B, Genevac-B, Biovac-B etc. produced in Pichia
pastoris (methylotrophic yeast) which express the
major surface antigen of the Hepatitis B virus, is used
for vaccination. But HBV is still responsible for
significant morbidity and mortality in poor countries
(Kong et al., 2001). The situation is deteriorating, also
in developing countries due to limited budget for
health care as large- scale vaccination programs are
required (Smith et al., 2003). Hence, there is a need
for large-scale production and use of relatively cheap
and effective hepatitis B vaccine.
Earlier, the majority of work was focused on the
production of recombinant proteins and subunit
vaccines in prokaryotes, mainly in Escherichia coli
because it offers low cost and short production
timescale (Ma et al., 2003). With the time, limitations
of prokaryotes came into light such as low product
yield and lack of post-translational modifications.
Therefore, then researchers turned to eukaryotic
systems: yeast, mammalian cells, insects and
transgenic animals. These systems were also proven
inefficient in terms of cost, production, safety,
authenticity and scalability (Balen and Rasol, 2007;
Ahmad, 2014). The plants obtained in recent decades
synthesize great number of valuable proteins such as
human serum proteins, growth regulators, antibodies,
vaccines, industrial enzymes, biopolymers, and
reagents for molecular biology and biochemistry.
Plant cells possess enzymatic systems of post-
translational modification, which are necessary for
the assembly of synthesized monomeric proteins of
vaccine into immunogenic multimers (Rukavtsova et
al., 2015). The target antigens causing active immune
response can also be synthesized in plant cells (Mason
and Arntzen, 1995). Thus, now the plants have
received the most attention in molecular pharming
community for the production of subunit vaccines.
The expression of subunit vaccines and recombinant
proteins in plants has emerged as a convenient, safe
and potentially economical platforms technology
(Thomas et al., 2011; Xu et al., 2012). Similarly,
several studies have revealed that plant expression
system produce many biologically active complex
human proteins (Merle et al., 2002; Peeters et al.,
2001). Therefore, with the time extensive technology
advancements for glycan modification makes plants
the most versatile platform for the production of
recombinant protein and subunit vaccines.
Hepatitis B surface antigen (HBsAg) is a transmembrane
protein consisting of S, M and L-HBsAg. All HBsAg
proteins are encoded within a single reading frame and
contain a common S domain. The S-HBsAg contains
only the S domain and form strongly immunogenic
determinant (Pniewski, 2013; Rybicki, 2014). The
hydrophobic S-HBsAg carries all the necessary inform-
ation for membrane translocation, the component of
virus envelope and assembled into immunogenic
structures known as Virus Like Particles (VLPs).
These particles are self- assembled protein structures,
devoid of viral DNA, unable to replicate and non-
infectious (Hyun et al., 2014). It has been known that
the lipid molecules closely associated with S-HBsAg
are responsible for the antigenicity, protein
conformation and stability of the VLPs (Jadwiga et
al., 2014). VLPs possess repetitive high-density
displays of viral surface proteins and are potentially
effective in eliciting strong humoral and cellular
immune responses. Hence, these characteristics
provide a great platform for VLPs as an effective
vaccine candidate (Natasha et al., 2012).
3. Int. J. Biomol. Biomed.
Guleria et al
31
In 1986, Federal Drug Administration (USA)
approved the first recombinant protein-based
Hepatitis B vaccine for humans. It is based on a
recombinant HBV surface antigen (HBsAg), which
upon production in yeast or mammalian cells forms
22-nm spherical VLPs (Greiner et al., 2012). Similarly
the structure authenticity and function of plant
produced VLPs is first explained by Mason et al., in
1992 who studied expression and production of
HBsAg VLPs in tobacco. The same group studied the
immunogenic response of VLPs produced in
transgenic potato on mice. The study emphasized the
importance and effectiveness of VLP vaccine.
Therefore, it would be safe to say that VLPs can
represent one of the most exciting new technologies
to rapidly produce effective vaccines with long lasting
protection (Rybicki, 2014).
The present report is the first study on the expression
of HBsAg in C. forskohlii for the development of
injection HBsAg vaccine. C. forskohlii is animportant
medicinal herb in Indian Ayurvedic medicine
considered endangered medicinal plant, the C.
forskohlii, a member of the Lamiaceae family is
valued for the production of labdane diterpenoid
forskolin (produced only in roots) from its tuberous
roots used for relief of cough, eczema, skin infections,
heart failures and certain type of cancers (Boby and
Bagyaraj, 2003).
The novelty and priority of the research is that C.
forskohlii is a potential candidate to produce vaccine,
as this crop is vegetatively propagated with high
biomass production and can be grown in the field
without any chances of genetic contamination (does
not set seeds), so it offers good platform to produce
novel compounds. It means, large quantities of
recombinant protein can be produced quite rapidly,
thereby significantly reducing the cost of production.
When we have a plant which does not carry threat of
gene pollution then biosafety laws of any given
country can accommodate a programme to grow
recombinant protein producing plant (Lou et al.,
2007).
Material and methods
Strains and Plasmids
Plant binary vector pHB118 contained HBsAg gene
under the control of CaMV 35S promoter was kindly
supplied by Dr. Hugh S. Mason, Arizona Biodesign
Institute USA and further maintained in
Agrobacterium tumefaciens strain LBA4404 in our
laboratory. The construct contains npt-II as selection
marker gene (Fig. 1.).
Fig. 1.Structure of plasmid pHB118. The HBsAg
coding region lies downstream of CaMV 35S
promoter and is followed by the nopaline synthase
(NOS) terminator.
Plant Regeneration and Transformation studies
As a pre-requisite for plant transformation studies, a
regeneration protocol of C. forskohlii was standar-
dized. Different combinations of Benzyl Amino Purine
(BAP) and Naphthalene Acetic Acid (NAA) were used
along with MS salts (Sigma-Aldrich, USA). The leaf
explants were kept under running tap water to
remove adhering dust particles and surface sterilized
with 0.8% (w/v) bavistin and 0.1% (w/v) mercuric
chloride for 1-2 minutes, followed by 3-4 washing
with sterile distilled water, under aseptic conditions.
After four rinses in sterile distilled water, leaves were
trimmed into pieces of about 1 cm2and then
inoculated onto culture media prepared using
Murashige and Skoog,1962 salts, added with MS
micronutrients, vitamins, 3% (w/v) sucrose 0.8% agar
and supplemented with different concentration of
BAP (0.2 & 0.3 mg /L) along with NAA (0.4,0.5, 0.6,
0.7 & 0.8 mg/L). The pH of the media was adjusted to
5.8 with 0.1 N NaOH and autoclaved at 121°C and 16
pounds/square inch for 15 min.
4. Int. J. Biomol. Biomed.
Guleria et al
32
The cultures were maintained at 25 ± 2°C in the
growth chamber under a 16 h photoperiod using
white fluorescent tubes. All cultures were kept for 4
weeks without subculture to fresh medium.
Regenerated shoots were sub-cultured in every 15
days of interval and rhizogenesis was obtained on
same media.
The pHB118 plant binary vector carrying HBsAg gene
was mobilised into Agrobacterium tumefaciens
LBA4404 strain which was then further used to
transform C. forskohlii using leaf discs. The
Agrobacterium culture was grown in yeast extract
peptone (YEP) broth media at 28°C and 200 rpm
speed for overnight. Further, the grown culture was
centrifuged at 6000 rpm for 5 minutes at room
temperature and pellet was suspended in half
strength MS broth. The OD600 of Agrobacterium
suspension maintained was 0.75 and then used for
transformation experiment. The co-cultivation was
done for two days and then explants were washed
with cefatoxime 250 mg/L dissolved in autoclaved
distilled water to remove the Agrobacterium overg-
rowth. The transformed shoots were selected on
standardized regeneration MS medium containing
kanamycin 50mg/L and cefatoxime250mg/L. Shoots
were rooted on the same medium and transplanted
into the cocopeat: sand (1:1) pots and well maintained
in the greenhouse.
Genomic DNA isolation and PCR analysis
Plant genomic DNA was isolated by Cetyl Trimethyl
Ammonium Bromide (CTAB) method from young
leaves of transformed plants and also from the non-
transformed plant as a control (Doyle and Doyle,
1987). The integration of the HBsAg gene was
confirmed by PCR using a pair of gene specific
primers 5' GCG AATT CATGGA GAG CAC AAC ATC
AGGA TTC3' forward and 5'TGAAGCTTT CAAATGTA
TACCCAAAGACAAAAG3' reverse. The genomic DNA
(100 ng) from each transformed plant was used as a
template DNA. The amplification was done using PCR
machine model Rcorbett research CG1-96.
The PCR programme was set at 94°C for 5 min
followed by 30 cycles of 94°C for 1 min, 58°C for 1
min and 72°C for 1.5 min, with a final extension at
72°C for 10 min. The PCR products were analysed by
electrophoresis on a 1.0% agarose gel.
SDS-PAGE and Western blot
Total protein from both transformed and non-
transformed C. forskohlii plants were isolated using a
protein extraction buffer (pH 7.8, 2M K2HPO4, 0.5M
EDTA, Triton X-100, 80% Glycerol, 1.0M DTT). The
total protein was isolated from 1 g of young leaves
with 2 ml of 1X protein extraction buffer. The samples
were centrifuged at 10,000 rpm for 10 min at 4°C.
The crude protein collected was partially purified by
using 10kDacentricon column (Pall Corporation). The
crude sample (2ml) was pipetted out into sample
reservoir of centricon and centrifuged at 10,000 rpm
for 30 min at 4°C. The desired protein sample was
collected into a sterile Eppendorf (2ml) from the
sample reservoir.
Protein expressed by transformed plants carrying
HBsAg gene was detected by 12% Sodium Dodecyl
Sulfate – Poly Acrylamide Gel Electrophoresis (SDS-
PAGE) using discontinuous buffer system followed by
Western immunoblot as per standard procedures
(Sambrook et al., 1989).
The protein ladder (Pure-gene, Genetix) was used as
marker. The membrane was probed consecutively
with a primary antibody (mouse anti-hepatitis B
surface antigen, Invitrogen, 1:1000 dilution) and
secondary antibody (goat anti-mouse IgG horseradish
peroxidase conjugate, Invi-trogen, 1: 20,000
dilution). The protein produced by non-transgenic
plant was used as a negative control and commercial
vaccine (Shanvac B, manufactured by Shantha
Biotechins, India) as a positive control.
ELISA
The protein samples containing expressed HBsAg
were detected by ELISA and quantified with standard
curve established by serial dilutions of bovine serum
albumin (BSA) standards.
5. Int. J. Biomol. Biomed.
Guleria et al
33
The partially purified protein samples of transgenic C.
forskohlii were used as an antigen source for ELISA.
They were incubated in blocking solution for 1 h at
37°C, followed by incubation with the primary(mouse
anti-hepatitis B surface antigen, Invitrogen, 1:1000
dilution) and secondary antibody (goat anti-mouse
IgG horseradish peroxidase conjugate, Invitrogen, 1:
20,000 dilution) for 1 h each at 37 °C. The washing
steps were performed after every incubation with 1X
PBST (phosphate buffer saline tween -20) buffer for
ten minutes. Detection involved 0.4 mg/mL of
chrom-ogen tetra methyl benzidine liquid substrate
system (Sigma-Aldrich, USA).
The blue coloration was deve-loped in all the HBsAg
transformed plant samples. The absorbance was
recorded at 450 nm. The nega-tive control (protein
extracted from non-transformed plants) and positive
control (commercial vaccine) were used.
Transmission Electron Microscopy
Young leaves of transformed and non- transformed
plants were excised, cut into 1-mm squares, with razer
blade, and fixed in 2% paraformaldehyde + 2%
glutaraldehyde in 0.1 M sodium phosphate buffer,
pH6.8, for 0.5h at 23°C followed by 1 h at 4°C. The
tissue was post-fixed in 2% osmium tetraoxide at
23°C for 1h, washed and dehydrated in a graduated
ethanol series at 4 0C, 1:1 ethanol and 100% ethanol.
The tissue was infiltrated with spurr’s resin at 23°C
and cured at 60°C overnight. Sections of about 60nm
in thickness were obtained with an ultra RMC
microtome.
The sections were viewed on a FeiTecnai T12 Biotwin
(Philips, Eindhoven, The Netherland) at 100KV.
Statistical Analysis
Each treatment of C. forskohlii regeneration experi-
ment had 5 replicates with 15 explants and all the
experiments repeated twice to see the reproducibility
of the results. The data were reported as mean
standard error and means were analysed by Analysis
of variance (one way ANOVA) and significant
differences between means were compared by least
significant difference (LSD).
Results and discussion
Regeneration and transformation of HBsAg gene in
C. forskohlii
Plant regeneration protocol of C. forskohlii was
standardized using leaf explant. The optimized media
for regeneration was found to be MS supplemented
with 2.00 mg/L BAP along with 0.5 mg/L NAA
(Table1).
The regenerated shoots were found to be healthy with
well-developed root system (Fig. 2A., B.& C.). In the
development of any regeneration protocol, growth
hormone combination and choice of explant play a
major role.
In the present study, combination of BAP (2.0 mg/L)
and NAA (0.5 mg/L) proved to be the best. These
results are in consistent with the data obtained earlier
in our laboratory by Guleria and Gowda, 2015; Khin
et al., 2006 reported the combination involving high
concentration of cytokinin and low concentration of
auxin promote adventitious shoot formation in C.
forskohlii leaf explant.
Table 1.Effect of BAP and NAA on shoot regeneration from leaf explants of Coleus forskohlii.
MS media
with(mg/L)
BAP NAA
Frequency
of callus
regenerated
into shoots
*(%)
No. of
shoots per
explant *
Average No.
of days
taken for
shoot
initiation
Shooting
observation
Average
No. of days
taken for
rooting
Root
number/sho
ot*
Root Characters
2.0 0.4 60.4c 14.8b 35 Short, healthy 20 5.2b Thin, longer
2.0 0.5 93.4a 35a 30 Long, Healthy 14 8.8a Thick, longer, healthy
2.0 0.6 57.8d 10.2c 42 Long, vitrified, weak 22 4.4c Thin, shorter
2.0 0.7 77.2b 14.6c 45 Short, not healthy 25 4.0c Thin, shorter
2.0 0.8 53e 10c 49 Short, vitrified 29 1.8d Long, thin
15 explants cultured per treatment.
*Different letters within the column indicate significant differences according to LSD (P≤0.05) following ANOVA.
6. Int. J. Biomol. Biomed.
Guleria et al
34
Fig. 2. Standardization of regeneration of C. forskohlii
from leaf explants (BAP 2.0mg/L+NAA 0.5mg/L). (A)
Adventitious shoots (B) Well developed rooting system
(C) Hardened transformed plant (D) Transformed C.
forskohlii plant in greenhouse.
In transformation studies, 500 leaf explants were co-
cultivated with Agrobacterium tumefaciens strain
LBA4404 containing pHB118 plasmid and eight
transformed plants of C. forskohlii were generated
(Fig. 2D.). Genetic transformation in plants mediated
via Agrobacterium tumefaciens has been known as
most common and reliable method (De la Riva et al.,
1998). It has the advantage for allowing stable
integration of defined gene into the plant genome that
generally results in single copy number, fewer
rearrangements and more stability expression over
generations than other transfection methods (Hu et
al., 2003). Therefore, micropropagation combined
with Agrobacterium transformation, provides a
method for routine genetic transformation of many
important medicinal plant species (Khan et al.,
2009).
Polymerase Chain Reaction (PCR) Analysis
The integration of HBsAg gene into plant genome was
confirmed by using PCR amplification. Presence of
expected amplicon size of 681bp in all transformed
samples, confirmed the integration of HBsAg gene
whereas there was no amplification in non-
transformed samples (Fig. 3.). The plasmid pHB118
which contained HBsAg gene showed amplicon size of
681bp used as positive control. When a gene is
transformed into plant cells/organisms, PCR is a
quick and simple technique to detect the presence of
the gene in the transformed plant.
The present results are in line with the study where
expression of HBsAg studied in tobacco Yogendra et
al. (2009, 2013) and in banana (Elkholy et al. 2009).
This confirms the stable integration of gene of
interest and successful transformation. However
tobacco as a model crop could not be used for
commercial production. Banana had lot of problems
like less accumulation of protein, long duration of the
crop, difficult to regenerate. Hence C. forskohlii has
better advantage over other crops.
Fig. 3. PCR analysis of HBsAg gene into the trans-
formed C. forskohlii using gene specific primers. Lane
M: Marker (100bp); lane 1-8 transformed plant
samples with amplicon size of 681bp; lane 9: non-
transformed plant sample (negative control); lane 10:
plasmid pHB118 (positive control).
Expression of HBsAg in C. forskohlii leaves
Total protein was isolated from young leaves of
transformed C. forskohlii plant and partially purified
using 10kDa centricon. Further, the protein was
estimated by ELISA using standard curve. The
protein concentration was found to be 260µg/g of
fresh leaf weight, whereas Kapusta et al., 1999
recorded 5.5ng/g of fresh weight in lettuce leaf, Li et
al., 2011 found HBsAg protein 100.36 ng/g of fresh
weight in leaves and 127.54 ng/g of fresh weight in
fruits of cherry tomato, Gao et al., in 2003 reported
HBsAg expression level at 300ng/g fresh weight of
leaves and 10ng/g fresh weight of fruit in cherry
tomato,0.01% total soluble HBsAg protein in tobacco
leaf was recorded by Mason et al.,1992 and Ehsani et
al.,1997 observed 0.008% total soluble HBsAg
protein in potato leaf. Rukavtsova et al., 2015 has
recorded HBsAg protein in tubers of various potato
lines upto 1 µg/g of wet weight.
7. Int. J. Biomol. Biomed.
Guleria et al
35
In our study rHBsAg protein expressed at higher level
than other reports. The possible explanation for this
higher expression level could be the use of different
plant host system. As C. forskohlii is vegetatively
propagated (does not set seeds) and produce large
biomass 2.5 tons per hectare, the expressed protein
can be isolated at large scale. Therefore, the
recombinant protein expressed in the transformed C.
forskohlii plants could be sufficient enough to make a
vaccine economical. The ELISA analysis also suggests
that C. forskohlii expressed rHBsAg protein has an
excellent immune-oreactivity. The non-transformed
C. forskohlii did not express any recombinant protein
that react with specific antibody of HBsAg.
The partially purified HBsAg protein samples were
subjected to 12% SDS-PAGE analysis. The results
showed the expression of protein of interest of ~48
kDa size which is dimer form of HBsAg protein.
Monomer form ~24kDa was also reported (Fig. 4A.).
Further, westernblot was performed to confirm the
integrity of the antigen/protein expressed in
transformed plants under denaturing conditions. P.
pastoris produced commercial vaccine was used as
positive control. C. forskohlii produced HBsAg
protein showed a band size of ~48 kDa (dimer) and
~24 kDa (monomer) but not in non-transformed
plants (Fig. 4B.). A typical western blot relies upon a
protein samples containing target protein that can be
detected by antibodies. Therefore, antibodies specific
to HBsAg protein made a complex via specific binding
of antibodies to proteins immobilized on a membrane
and expressed a dimeric form of ~48kDa suggesting
the formation of disulphide bond and strong
immunogenicity.
The results are in line with previous studies on plant
derived HBsAg showed that the HBsAg expressed in
plant system accumulated as dimeric form with
strong immunogenic behavior (Sojikul et al., 2003;
Zhong and Mason, 2004; Zhong et al., 2008).
Mishiro et al., 1980 and Wampler et al.,1985 also
showed that the dimeric form of HBsAg retain all
antigenic determinants and full immunogenicity in
comparison to monomeric form of HBsAg.
Therefore, correct folding of plant expressed protein
is necessary for full biologically active protein.
Fig. 4. SDS-PAGE and western immunoblot analysis
of recombinant HBsAg protein expressed in leaf of C.
forskohlii. (A) Lane M: Protein ladder; lane 1: protein
sample of non-transformed plant (control); lane 2:
protein samples of transformed plants expressing
both dimeric (~ 48 kDa) and monomeric (~24 kDa)
form of HBsAg. (B) Western immunoblot analysis.
Lane M: Protein ladder; lane 1: P. pastoris derived
commercial vaccine (positive control); lane 2: protein
sample of non-transformed plant (negative control).
lane 3: protein samples of transformed plants
confirmed both dimeric (~ 48 kDa) and monomeric
(~24 kDa) forms of HBsAg.
rHBsAg assembled into VLPs
The rHBsAg viral proteins have the ability to
assemble into highly immunogenic virion-like
structures without genetic material (Zhong et al.,
2005). Such particles are known as virus like
particles. It had been reported that HBsAg gets
assembled into virus like particle with diameter of 22
nm and stabilized by multiple disulphide bonds
(Jadwiga et al., 2014).
8. Int. J. Biomol. Biomed.
Guleria et al
36
The cells of HBsAg transformed and non-transformed
C. forskohlii plants harboured unusual membrane
bound vesicles in the cytoplasm (Fig. 5B.). These
vesicles contained circular structures ranging in
diameter 13 to 40 nm. Max-imum number of particles
were in range of 15 and 29 nm diameter. Therefore,
the average diameter was found to be 22 nm in size
indicated by arrows (Fig. 5C.). Microscopic
examination of non-transformed cells did not show
any vesicles containing circular structures (Fig. 5A.).
VLPs of different diameters ranging from 13 to 40 nm
and average of 22nm were observed in the
transformed C. forskohlii plant cells which were
evident in case of HBsAg expression in tobacco
(Mason et al.,1992). The formation of HBsAg VLPs
has also been observed in potato (Richter et al.,
2000) and in soyabean (Smith et al., 2002). Thus, the
present work showed the correct folding of C.
forskohlii expressed HBsAg. Thus, plant derived
rHBsAg antigen appears to possess all the necessary
antigenic properties which are considered as major
aspects of any effective vaccine.
Fig. 5.TEM of Non-transformed & HBsAg expressing
C. forskohlii: (A) Thin section of non-transformed C.
forskohlii cells (Bar=0.5µm) (B) Thin section of
transformed C. forskohlii cells (Bar=0.5µm. (C)
Enlarged image of membrane bound vesicles, 22nm
VLPs indicated by arrows. (Bar=100nm).
The number of VLP vaccines are in process and
available in the market (Dwarakaprasad and Rao,
2013). When a person is injected with the VLPs, the
immune system will mimic VLPs as authentic virus
and ultimately give strong immune response for the
production of antibodies, which are recognized
readily by the immune system and presents viral
antigen in a more authentic conformation.
Furthermore, the compact highly ordered structures
of VLPs may provide resistance to degradative
enzymes in the gut; the particulate nature of VLPs
allows them to be efficiently sampled by the ‘M’ cells
of gut epithelium that transport antigens across the
mucosal barrier (Santi et al., 2006).
The potency of VLPs can elicit a protective response
at lower doses of antigen, which significantly reduces
vaccine cost (Roldao et al., 2010). VLPs have
therefore shown dramatic effectiveness as a vaccine
candidate.
In conclusion, HBsAg protein expressed at higher
level (260µg/g) in C. forskohlii leading it to potential
candidate for the production of vaccines.
The plant derived HBsAg assembled into VLPs of
22nm size. These results demonstrated that the
rHBsAg VLPs expressed in C. forskohlii could provide
potential, promising, economic and large scale
materials for the development of diagnostic kit and
vaccine to prevent the HBV infection.
9. Int. J. Biomol. Biomed.
Guleria et al
37
Acknowledgements
The authors are highly thankful to Department of
Biotechnology (DBT-HUB programme), Ref No.
BT/INF/UAS Bangalore/2011, Government of India,
New Delhi, for giving financial assistance to this
project. We thank Dr. Mason H.S, Biodesign Institute
at Arizona State University, USA for providing the
pHB118 construct.
References
Ahmad K. 2014. Molecular farming: strategies,
expression systems and bio-safety considerations.
Czech Journal of Genetics and Plant Breeding 50,1-10.
Balen B, Rasol MK. 2007. N-Glycosylation of
Recombinant Therapeutic Glycoproteins in Plant
Systems. Food Technology and Biotechnology 45,1–10.
Boby BU, Bagyaraj DJ. 2003. Biological control of
root- rot of Coleus forskohlii Briq. using microbial
inoculants. World Journal of Microbiology and
Biotechnology19,175-180.
CzyzM, Dembczynski R, Marecik R, Wojas-
Turek J, Milczarek M, Pajtasz-Piasecka E,
Wietrzyk J, Pniewski T. 2014. Freeze-Drying of
Plant Tissue Containing HBV Surface Antigen for the
Oral Vaccine against Hepatitis B. [published online
ahead of print October 12, 2014]. BioMed Research
Internationaldoi:10.1155/2014/485689.
De la Riva GA, Cabrera GJ, Padron RV, Pardo
CA.1998. Agrobacterium tumefaciens: a natural tool
for plant transformation. Electronic Journal of
Biotechnology doi: 10.2225/Vol.1-issue 3.
Doyle JJ, Doyle JL.1987. A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.
Phytochemical Bulletin 19,11-15.
Dwarakaprasad AA, Rao S. 2013. Virus like particles
as vaccines. International Journal for Pharmaceutical
Research Scholars 2, 512-544.
Ehsani P, Khabiri A, Domansky NN.1997.
Polypeptides of hepatitis B surface antigen produced
in transgenic potato. Gene 190, 107–111.
ElkholySF, Roba MI, Ahmed B, Atef SS, Magdy
AM. 2009. Expression of Hepatitis B surface antigen
(HBsAg) gene in transgenic banana (Musa sp).
Arabian Journal of Biotechnology 12, 291-302.
Gao Y, Ma Y, Li M, Cheng T, Li SW, Zhang J,
Xia NS. 2003. Oral immunization of animals with
transgenic cherrytomatillo expressing HBsAg. World
Journal of Gastroenterology 9, 996–1002.
Goldstein ST, Zhou F, Hadler SC, Bell BP, Mast
EE, Margolis HS. 2005. A mathematical model to
estimate global hepatitis B disease burden and vacci-
nation impact. International Journal of Epidemi-ology
34, 1392-1339.
Greiner VJ, Ronzon F, Larquet E, Desbat B,
Esteves C, Bonvin J, Greco F, Manin C,
Klymchenko AS, Mely Y. 2012. The structure of
HBsAg particles is not modified upon their
adsorption on aluminium hydroxide gel. Vaccine 30,
5240–5245.
Guleria N, Gowda Ramanjini PH. 2015. An
efficient regeneration and genetic transformation
protocol of Coleus forskohlii using Biolistic gun.
International Journal of Agriculture, Environment
and Biotechnology 8, 227-235.
Hu I, Metz S, Chay C, Zhou HP, Biest N, Chen
G, Cheng M, Feng X, Radionenko M, Lu F, Fry
J. 2003. Agrobacterium- mediated large scale
transformation of wheat using glyphosate selection.
Plant Cell Reports 21,1010-1019.
Hyun SK, Jae HJ, Kyung JL, Kisung K. 2014. N-
Glycosylation Modification of Plant-Derived Virus-
Like Particles: An Application in Vaccines. [published
online ahead of print May 25, 2014]. BioMed Research
International doi.org/10.1155/2014/249519es.
Jadwiga C, Inga S, Ewa S. 2014. Virus-like particles
as vaccine .Acta Biochimica Polonica 61,531–539.
Kapusta J, Modelska A, Figlerowicz M,
Pniewski T, Letellier M, Lisowa O, Yusibov V,
Koprowski H, Plucienniczak A Legocki AB.
1999. A plant-derived edible vaccine against hepatitis
B virus. Federation of American Societies for Experi-
mental Biology 13,1796-1999.
10. Int. J. Biomol. Biomed.
Guleria et al
38
Khan MY, Saleh A, Vimal K, Rajkumar S. 2009.
Recent advances in medicinal plant biotechnology.
Indian Journal of Biotechnology 8, 9-22.
Khin ML, Gowda Ramanjini PH, Chandrashekar
S, Gowda TKS, Vasundhara M, Nagesha N,
Virupaksha GUP, Sreenevasa S, Manjunatha T.
2006. In vitro regeneration and transformation of
Coleus forskohlii with glucanase-chitinase gene. Journal
of Non-Timber Forest Products 13,193-197.
Kong Q, Richter L, Yang YF, Arntzen CJ, Mason
HS, Thanavala Y. 2001. Oral immunization with
hepatitis B surface antigen expressed in transgenic
plants. Proceedings of National Academy of Sciences
USA 98, 11539–11544.
Li T, Jing Kuan S, Zhao Hua L, Qing L. 2011.
Transformation of HBsAg (Hepatitis B Surface Antigen)
Gene into Tomato Mediated by Agrobacterium tumefa-
ciens. Czech Journal of Genetics and Plant Breeding 47,
69–77.
Lou XM, Yao QH, Zhang Z, Peng R H, Xiong SA,
Wang HK. 2007. Expression of the human hepatitis B
virus large surface antigen gene in transgenic tomato
plants. Clinical Vaccine Immunology 4, 464–469.
Ma JKC, Drake P, Christou P. 2003. The
production of recombinant pharmaceutical proteins
in plants. Nature Reviews Genetics 4, 794-805.
Mason HS, Arntzen CJ. 1995. Transgenic plants as
vaccine production systems. Trends in Biotechnology
13, 388–392.
Mason HS, Lam DM, Arntzen CJ. 1992.
Expression of hepatitis B surface antigen in
transgenic plants. Proceedings of National Academy
of Sciences USA 89, 11745–11749.
Merle C, Perret S, Lacour T, Jonval V,
Hudaverdian S, Garrone R, Ruggiero F, Theisen
M. 2002. Hydroxylated human homotrimeric collagen I
in Agrobacterium tumefaciens-mediated transient exp-
ression and in transgenic tobacco plant. Federation of
European Biochemical Societies Letters 515, 114–118.
Mishiro S, Imai M, Takahashi K, Machida A,
Gotanda T, Miyakawa Y, Mayumi M. 1980. A
49000-dalton polypeptide bearing all antigenic
determinants and full immunogenicity of 22-nm
hepatitis B surface antigen particles. Journal of
Immunology 124, 1589–1593.
Murashige J, Skoog F. 1962. A revised medium for
rapid growth and bioassays with tobacco cultures.
Plant Physiology 15, 473-497.
Natasha K, Stephen J. Streatfield, VY. 2012.
Virus-like particles as a highly efficient vaccine platform:
Diversity of targets and production systems and
advances in clinical development. Vaccine 31, 58-83.
Peeters K, De Wilde V, De Jaeger C, Angenon
GG, Depicker A.2001. Production of antibodies and
antibody fragments in plants. Vaccine 19, 2756–2761.
Pniewski T. The twenty year story of a plant-based
vaccine against hepatitis B: Stagnation or Promising
Prospects? International Journal of Molecular
Sciences 14, 1978-1998.
Richter LJ, Thanavala Y, Arntzen CJ, Mason
HS. 2000. Production of hepatitis B surface antigen
in transgenic plants for oral immunization. Nature
Biotechnology 18, 1167–1171.
Roldao A, Mellado MCM, Castilho LR, Carrondo
MJT, Alves PM. 2010. Virus like particles in vaccine
development. Expert Review of Vaccines 9, 1149-1176.
Rybicki EP. 2014. Plant based vaccines against
viruses. Virology Journal 11, 205.
RukavtsovaElena B, Natalya V Rudenko, Elena
N Puchko, Natalya SZakharchenko, YaroslavI
Buryanov. 2015. Study of the immunogenicity of
hepatitis B surface antigen synthesized in transgenic
potato plants with increased biosafety. Journal of
Biotechnology 203, 84–88.
11. Int. J. Biomol. Biomed.
Guleria et al
39
Sambrook J, Fritsch EF, Maniatis T. 1989.
Molecular Cloning: A Laboratory Manual (2nd
edition). Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
Santi L, Zhong H,Mason H. 2006. Virus like particles
productionin greenplants. Methods 40,66-76.
Smith ML, Mason HS, Shuler ML. 2002b.
Hepatitis B surface antigen (HBsAg) expression in
plant cell culture: kinetics of antigen accumulation in
batch culture and its intracellular form.
Biotechnology Bioengineering 80, 812–822.
Smith ML, Richter L, Arntzen CJ, Shuler ML,
Mason HS. 2003. Structural characterization of
plant-derived hepatitis B surface antigen employed in
oral immunization studies. Vaccine 21, 4011–4021.
Sojikul P, Buehner N, Mason HS. 2003. A plant
signal peptide–hepatitis B surface antigen fusion
protein with enhanced stability and immunogenicity
expressed in plant cells. Proceedings of National
Academy of Sciences USA 100, 2209–2214.
Thomas DR, Claire AP, Amrita M, Amanda
MW. 2011. Evolution of plant –made
pharmaceuticals. International Journal of Molecular
Sciences 12, 3220-3236.
Wampler DE, Lehman ED, Boger J, McAleer
WJ, Scolnick EM. 1985. Multiple chemical forms of
hepatitis B surface antigen produced in yeast.
Proceedings of National Academy of Sciences USA
82, 6830–6834.
WHO. 2015. Hepatitis B. Available at http://www.
who.int/mediacentre/factsheets/. March, 2015. Acce-
ssed on April 2, 2015.
Xu J, Dolan MC, Medrano G, Cramer CL,
Weathers PJ. 2012. Green factory: plants as
bioproduction platforms for recombinant proteins.
Biotechnology Advances 30, 1171-1184.
Yogendra KN, Ramanjini Gowda PH, Sandesh
HS, Raghavendra G, Asha VN, Ningaraju TM,
Deepak N. 2009. Production and Characterization
of Hepatitis B Recombinant Vaccine in Tobacco
(Nicotiana tobaccum cv. ‘Kanchun’).Transgenic Plant
Journal 3, 97-101.
YogendraKN, Raghavendra G, Ramanjini
Gowda PH. 2013. Stability and inheritance analysis
of transgenic tobacco expressing Hepatitis B surface
antigen. Australian Journal of Crop Sciences 7, 1010-
1015.
Zhong H, Galina E, Bryan J, Maloneyc NB,
Charles JA, Yasmin T, Mason HS. 2005. Virus-
like particle expression and assembly in plants:
hepatitis B and Norwalk viruses. Vaccine 23, 1851–
1858.
Zhong H, Kate Le P, Galina E, Yasmin T,
Mason HS. 2008. High-yield rapid production of
hepatitis B surface antigen in plant leaf by a viral
expression system. Plant Biotechnology Journal 6,
202–209.
Zhong H, Mason HS. 2004. Conformational
analysis of hepatitis B surface antigen fusions in an
Agrobacterium-mediated transient expression
system. Plant Biotechnology Journal 2, 241–249.