Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.
Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR
Basic principles of genetic engineeringSteffi Thomas
Basic principles of genetic engineering, Recombinant DNA, Genetically Modified organism (GMO), Tools used in genetic engineering, restriction endonuclease, DNA ligase, cloning vector, process of genetic engineering, applications of genetic engineering (in animals, plants, human), production of insulin by rDNA technology, gene therapy, possible hazards of genetic engineering
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
Basic principles of genetic engineeringSteffi Thomas
Basic principles of genetic engineering, Recombinant DNA, Genetically Modified organism (GMO), Tools used in genetic engineering, restriction endonuclease, DNA ligase, cloning vector, process of genetic engineering, applications of genetic engineering (in animals, plants, human), production of insulin by rDNA technology, gene therapy, possible hazards of genetic engineering
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
Applications of rdna technology in medicinesAdarsh Patil
Applications of R-DNA Technology in medicines:
Introduction Steps involved in recombinant technology:
DNA fragments coding for proteins of interest are synthesized chemically or isolated from an organism.
These DNA fragments are inserted into an endonuclease cleavage site of the vector that does not inactivate any gene that is required for the vector’s maintenance and selective marker.
The recombinant DNA molecules are then introduced into a host to replicate using the replication origin of the vector.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
A vaccine is a biological preparation that improves immunity to a particular disease.
In the edible vaccine, Transgenic plants are used as vaccine production systems.
The genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunologic properties.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Study of cloning vectors and recombinant dna technologySteffi Thomas
Study of cloning vectors, restriction endonuclease and DNA ligase, Recombinant DNA technology, Application of genetic engineering in medicine, Application of rDNA technology and genetic engineering in the production of interferons, Vaccines-hepatitis-B, Hormones-Insulin, Brief introduction to PCR
UNIT 6 Fermentation technology, Fermenters, Study of Media, types of fermenta...Shyam Bass
UNIT-6 6th Sem B.Pharma Pharmaceutical Biotechnology-
Following slides include-
Fermentation technology and biotechnological products :
Fermentation methods and general requirements
Study of media
Equipment
Sterilization methods
Aeration process
Stirring
large scale production fermenter design and its various controls
BY- SHYAM BASS
UNIT 4 Microbial genetics:Transformation,Transduction,Conjugation,Plasmids an...Shyam Bass
(6th Sem B.Pharma Pharmaceutical Biotechnology)
Microbial genetics:
• Transformation,
• Transduction,
• Conjugation,
• Plasmids and transposons,
• Study of the production of - Penicillins, Citric acid, Vitamin B12, Glutamic acid,
Griseofulvin,
• Blood Products: Collection, Processing, and Storage of whole human blood,Dried
human plasma, Plasma substitutes
BY- SHYAM BASS
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.
Applications of rdna technology in medicinesAdarsh Patil
Applications of R-DNA Technology in medicines:
Introduction Steps involved in recombinant technology:
DNA fragments coding for proteins of interest are synthesized chemically or isolated from an organism.
These DNA fragments are inserted into an endonuclease cleavage site of the vector that does not inactivate any gene that is required for the vector’s maintenance and selective marker.
The recombinant DNA molecules are then introduced into a host to replicate using the replication origin of the vector.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
A vaccine is a biological preparation that improves immunity to a particular disease.
In the edible vaccine, Transgenic plants are used as vaccine production systems.
The genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunologic properties.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Study of cloning vectors and recombinant dna technologySteffi Thomas
Study of cloning vectors, restriction endonuclease and DNA ligase, Recombinant DNA technology, Application of genetic engineering in medicine, Application of rDNA technology and genetic engineering in the production of interferons, Vaccines-hepatitis-B, Hormones-Insulin, Brief introduction to PCR
UNIT 6 Fermentation technology, Fermenters, Study of Media, types of fermenta...Shyam Bass
UNIT-6 6th Sem B.Pharma Pharmaceutical Biotechnology-
Following slides include-
Fermentation technology and biotechnological products :
Fermentation methods and general requirements
Study of media
Equipment
Sterilization methods
Aeration process
Stirring
large scale production fermenter design and its various controls
BY- SHYAM BASS
UNIT 4 Microbial genetics:Transformation,Transduction,Conjugation,Plasmids an...Shyam Bass
(6th Sem B.Pharma Pharmaceutical Biotechnology)
Microbial genetics:
• Transformation,
• Transduction,
• Conjugation,
• Plasmids and transposons,
• Study of the production of - Penicillins, Citric acid, Vitamin B12, Glutamic acid,
Griseofulvin,
• Blood Products: Collection, Processing, and Storage of whole human blood,Dried
human plasma, Plasma substitutes
BY- SHYAM BASS
This presentation is all about biotechnology. It is about the basic aspects of Biotechnology and covers a lot of topics under biotechnology, recombinant DNA technology. This is specifically for the HSC students of Mumbai. I hope that it helps.
Biotechnology is the utilization of biology to figure out problems and develop beneficial products. The most important area of biotechnology is the manufacturing of therapeutic proteins and other drugs via genetic engineering.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
Embracing GenAI - A Strategic ImperativePeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
Genetic engineering principle, tools, techniques, types and application
1. GENETIC ENGINEERING
Principle, Tools, Techniques,
Types and Application
Presented by:
Mr. TARUN KAPOOR
Assistant Professor,
Shri Ram College of Pharmacy, Karnal
2. Contents
• Genetic Engineering: An Introduction & Basic principles
• Tools of Genetic Engineering (Enzymes and transformation factors)
Cloning vectors, restriction endonucleases and DNA ligase
• Methods of Gene transfer
• Methods of DNA amplification (Gene Cloning and PCR)
• Recombinant DNA technology & its Application
• Recombinant Pharmaceuticals: An Overview
• Production of genetic engineering products (Interferon, Vaccines-
hepatitis- B, Insulin)
• Brief introduction to PCR: Principle, Advantages and Applications
3. Genetic Engineering
• Genetic Engineering involves manipulation of genetic
material, is also called Recombinant DNA technology or
Gene Cloning.
• Genetic recombination technology consists of the breakage
and joining of DNA molecules.
• Genetic engineering primarily involves the manipulation of
genetic material ( DNA) to achieve the desire goal in pre
determined way.
• Other terms are Recombinant DNA technology, Gene
manipulation, Gene cloning, Genetic modifications.
4. Basic principle of Genetic
Engineering
• DNA fragment of interest is obtained by cleaving
chromosomes by Restriction endonuclease.
• Cloning vector is cleaved with Restriction
endonuclease.
• Fragments are ligated to the prepared cloning vector.
• Recombinant vector DNA is introduced into the host
cell
• Propagation (cloning) produces many copies of
recombinant DNA
• The gene is extracted and harvested the product
8. Restriction Endonucleases (REs)
Used as mol. scissors to cut DNA –DNA at specific
DNA sequences
To generate a set of smaller fragments
Recognize specific DNA sequences called
“palindrome” (restriction sites) Example : EcoRI
recognizes sequence cuts the Phosphodiesterase
bonds of the DNA on both the strands
11. Applications of RE
1) Sequencing of DNA
2) Cloning of DNA
3) Antenatal diagnosis of inherited disorders
4) DNA finger printing (having forensic applications)
5) For Southern blot technique (for detecting the
presence of a particular base sequence in the sample
DNA)
12. DNA ligase
– Joins two DNA molecules or fragments.
DNA Polymerases
– Synthesis of DNA using DNA template
Reverse transcriptase
– Enzyme found in retroviruses that makes
DNA copy, using RNA as template
13. Transformation factors
• Into the DNA of the vector a foreign DNA can be
inserted, integrated/incorporated.
• Use : For amplification by cloning and for gene
therapy.
• Plasmid, Bacteriophage, Cosmid, Yeast
14. Cloning Vectors
• Vectors are the DNA molecule, which can carry A
foreign DNA fragment to be cloned. The are self
replicating in an appropriate host cell.
• The most important vectors are Plasmids,
Bacteriophages & Cosmid.
• An ideal characteristics of an vector is should be
small in size with endonuclease site.
15. Plasmid
A small, circular, dsDNA present in bacteria
Confer antibiotics resistance against the bacteria
Many copies of plasmid in a bacterium
Replicate independent of the bacterial DNA.
16. Cosmid: Cosmid are vectors posses the characteristic of
both plasmid and bacteriophage.
Can carry larger DNA fragments
Cosmid can be constructed by adding a fragment of
DNA to plasmid.
17. • Yeast Artificial Chromosomes (YAC) is a synthetic
DNA that can accept large fragment (particular human
DNA). It is possible to clone large DNA pieces by
using YAC.
• Bacteriophage: It is a virus that can infect bacteria.
18. Methods of gene Transfer
• Transformation
• Transduction
• Electroporation
• Conjugation
• Microinjection
• Liposome mediated
gene transfer
20. • Electroporation: Application of high voltage electrical field
to cells
• Direct transfer:
– Micro injection
– Particle bombardement
• Liposome mediated gene transfer
21.
22. Types of DNA amplification
Cloning
• In vivo method using bacteria
• Used to amplify longer segments of DNA
• Suitable for large scale protein production
Polymerase Chain Reaction (PCR)
• In vitro method using DNA polymerase
• Shorter segments of DNA can be amplified
• Shorter time for amplifying DNA fragments
23. DNAAmplification
• Production of many identical copies of a DNA
fragment of interest.
• Uses
1) Further DNA analysis
2) For large-scale genetic expression
3) Protein production
24. Cloning
• Production of an identical copy of either DNA or a cell
or an organism is called cloning.
• It is of 2 Types:
1. Molecular cloning: Production of identical DNA
molecules (i.e., identical in base-sequence)
2. Somatic cloning: Production of cells or
organisms with identical genetic makeup
25. • Genetic recombination is exchange of information between
two DNA segments within same species. But artificially
when a gene of one species is transferred to another living
organism, it is called recombinant DNA technology or
genetic engineering.
• rDNA: Production of a unique DNA molecule by joining
together two or more DNA fragments which are derived
from different biological sources.
• rDNA technology: A series of procedures used to
recombine DNA segments. Under certain conditions, a
recombinant DNA molecule can enter into cell & replicate.
26. History of rDNA technology
• Recombinant DNA technology is one of the
recent advances in biotechnology, which was
developed by two scientists named Boyer and
Cohen in 1973.
27. Applications of rDNA Technology
1. Large scale production of human proteins by genetically
engineered bacteria.
–Recombinant human insulin
–Recombinant human growth hormone
–Recombinant blood clotting factors
–Recombinant hepatitis B vaccine
–Cytokines and growth factors (IF, IL)
–Monoclonal antibodies
–Recombinant enzymes
–Recombinant HIV protein for ELISA testing
–Albumin, fibrinolytic and thrombolytic agents
28.
29. 2) Basic research – understanding structure and functions of
DNA & proteins (Human Genome Project)
3) Gene therapy for genetic diseases
4) Food production
5) Plant: Genetically modified corn
6) Forensic applications
7) Genetically modified organisms are called transgenic
organisms. Mice (Study human immunity), Chicken
(Resistant to infection), Cows (Increase milk & leaner
meat)
8) Applications in ecology: Recombinant Bacteria which can
be engineered to “eat” oil spills.
30. Recombinant Pharmaceuticals
• Human Insulin
• Human Growth Hormone
• Human blood clotting factors
• Vaccines
• Monoclonal Antibodies
• Interferons
• Antibiotics & other secondary metabolites
31. Human Insulin
• Insulin is a hormone produced by β-islets of Langerhans of
pancreas. It was discovered by sir Edward Sharpey Schafer
(1916) while studying Islets of Langerhans.
• People who do not produce the necessary amount of insulin
have diabetes.
• Chemically, insulin is protein consist of 51 amino acids, 30
construct polypeptide chain B and 21 amino acids construct
polypeptide chain A and both chains linked by disulfide
bond.
32.
33. • Modify E.coli cells to produce insulin; performed by Genentech
in 1978
• Prior, bovine and porcine insulin used but induced immunogenic
reactions. Also, there were many purification and contamination
hassles. To overcome these problems, researchers inserted human
insulin genes into a suitable vector (E.coli).
• First, scientists synthesized genes for the two insulin A & B
chains. Then inserted into plasmids. The genes were inserted in
such a way that the insulin & B-galactosidase residues would be
separated by a methionine residue. This is so that the insulin A &
B chains can be separated easily by adding cyanogen bromide.
34. Producing Recombinant Insulin
• The vector was then transformed into E.coli cells.
• Once inside the bacteria, the genes were
"switched-on" by the bacteria to translate the
code into either the "A" chain or the "B" chain
proteins found in insulin
• The purified insulin A and B chains were then
attached to each other by disulphide bond
formation under laboratory conditions
35.
36. Hepatitis B Vaccine
• Hepatitis B virus (HBV) is common infectious diseases. WHO
estimates that there are 285 million chronic carriers of HBV
worldwide.
• Hepatitis B is 50 to 100 times more infectious than AIDS. Hepatitis B
is irritation and swelling (inflammation) of liver due to infection with
hepatitis B virus (HBV). Other types include: Hepatitis A, C and D.
• It produces several chronic liver disorders such as Liver cirrhosis and
primary liver cancer.
• Hepatitis B Recombinant Vaccine: It’s a novel and significant
developed vaccine which is produced from the antigenic proteins of
Hepatitis B virus by recombinant process that duplicates the chemical
messages and secreted factors (Interleukin-2) for the communication
and activity of immune cells.
37. Production of recombinant HBV
Vaccine
Production of these genes is needed in order to get production of
vaccines on a large scale. A general procedure for the production of
recombinant Hepatitis B vaccines are described here-
1. HBs antigen producing gene is isolated from HB virus by isolation
process (cell lysis, protein denaturation, precipitation,
centrifugation and drying).
2. A plasmid DNA is extracted from a bacterium- E.coli and is cut
with restriction enzyme- Eco RI forming the plasmid vector.
3. The isolated HBs antigen producing gene is located and inserted
into the bacterial plasmid vector on forming the recombinant
DNA.
38. 4. This recombinant DNA, containing the target gene, is
introduced into a yeast cell forming the recombinant yeast
cell.
5. The recombinant yeast cell multiplies in the fermentation
tank and produces the HBs antigens.
6. After 48 hours, yeast cells are ruptured to free HBsAg. The
mixture is processed for extraction.
7. The HBs antigens are purified. HBsAg are combined with
preserving agent and other ingredients and bottled.
8. Now it is ready for vaccination in humans.
39.
40. INTERFERONS
Interferons (IFNs) were the first family of cytokines to be discovered.
In 1957 researchers observed that if susceptible animal cells were
exposed to a colonizing virus, these cells immediately become
resistant to attack by other viruses. This resistance was induced by a
substance secreted by virally-infected cells, which was named
‘interferon’ (IFN). Humans produce at least three distinct classes,
IFN-a, IFN-b and IFN-g.
Biological effects: Induction of cellular resistance to viral attack.
Regulation of most aspects of immune function. Regulation of
growth and differentiation of many cell types. Sustenance of early
phases of pregnancy in some animal species.
41. Production of Interferons
• A DNA sequence coding for the product was synthesized and
inserted into E. coli. The recombinant product accumulates
intracellularly as inclusion bodies.
• Large-scale manufacture entails an initial fermentation step. After
harvest, the E. coli cells are homogenized and the inclusion bodies
recovered via centrifugation.
• After solubilization and refolding, the interferon is purified to
homogeneity by a combination of chromatographic steps.
• The final product is formulated in the presence of a phosphate
buffer and sodium chloride.
• It is presented as a 30 mg/ml solution in glass vials and displays a
shelf- life of 24 months when stored at2–8°C.
42. Human
Fibroblast
Human Interferon
β Gene
Modified Human Interferon
β Gene
Plasmid
Restriction enzyme cut
Plasmid
E. Coli containing its Own
gene
E. Coli
containing
rDNA
Replicated E.
Coli
producing
IFN β
Purified
Interferon
1β
Packed and
Ready for Use
rDNA
43.
44. Polymerase Chain Reaction
• PCR is an in vitro technique for the amplification of DNA.
• Developed by Kary Mullis in the 1980s
• Much faster
• More sensitivemethod than cloning.
• Very little DNA sample is sufficient
• Can only amplify short segments of DNA
• Cannot be used for amplifying genes and for production of
proteins
45. Principle
• Double stranded DNA of interest is denatured to
separate strands
• Each strand is then allowed to hybridize with a
primer. The primer template duplex is used for
synthesis.
• Program thermocycler for times include three steps:
Denaturation, anneling and extension repeated again
and again to generate multiple forms of target DNA.
• The primer extension product synthesized in 1 cycle
serve as template for next cycle.
46. Procedure
• A mixture of DNA sample + dNTP + Primer + Enzyme
: Taq DNA polymerase
• Treatment of the mixture :1 cycle
94 – 95˚ C : Denaturation of DNA : 30 – 60 sec
52 – 54˚ C : Annealing of primers : 30 – 60 sec
72˚ C : Extension of the DNA : 1 min
47.
48. Types of PCR:
– Real-time PCR
– Nested PCR
– Inverse PCR
– Reverse transcription PCR
Advantages of PCR
1. Very little DNA sample is required
2. Amplification time is very short.
3. Amplification rate is high.
Uses: When insufficient DNA molecules are present in test
samples for DNA analytical techniques.
49. Applications of PCR
1. Diagnosis: Bacterial and viral diseases (TB, Hepatitis C, HIV)
2. Medicolegal/ Forensic cases: DNA amplification from hair,
saliva, semen and blood sample (DNA fingerprinting)
3. Diagnosis of genetic disorders: SCD, thalassemia, cystic fibrosis
4. Prenatal diagnosis of inherited disorders
5. Cancer detection:
i. To monitor abnormal cells present in treated patients.
ii. Identification of mutation in oncosuppressor
genes
5. Fossil studies: To study evolution by comparing the sequences in
the extinct and living organism