Application of Biotechnological Tools in Human Health Care

1. Advanced Biotechnological tools for Human
health care
Presentated By:-
Dr. Shiv Om Pratap Dixit
Head of Department
Department of Biotechnology
SRGI, Jhansi
Email: drshivompratap@gmail.com
Review Article

2. Contents:
 Introduction
 Principle of Gene Expression/Manufacturing of
desired products
 Applications of Biotechnology for Human Health Care

3. Introduction
 Biotechnology means, ‘the utilization of Biological and Engineering
principles to enhance researches and productivity by using
Microorganisms and their derivatives or modifying products for Human
welfare.
 Advancements of Biotechnological tools have been widely employing for
the laboratory researches, industrial productions, medicines,
pharmaceuticals, healthcare, agriculture, animal husbandry, environment
and food industry etc.
 Biotech companies are making pharmaceutical at molecular level by
‘Recombinant DNA technology’, which entails genetic manipulation of
cells through Genetic engineering principles.

4.  The ‘Hybridoma Technology’ is a technique, used for the production of
Monoclonal antibody as a ‘Magic bullet’ in the treatment of several
diseases.
 Biotech companies have been designing and producing safer vaccines by
various manipulated Organisms (GMOs), which are transformed
through Genetic Engineering by minimizing the risks of infections.
 These products of many Pharmaceutical companies are widely using for
the prevention, diagnosis or treatment of many types of diseases have
been made through Biotechnological aspects.
Introduction Continue…….

5. Introduction Continue…….
 Pharmaceutical companies are widely utilizing ‘Biotechnological tools and
techniques’ of for the manufacturing of drugs, pharmacogenomics-agents, genetic
testing and disease diagnosis etc.
 Study of Genetic variation underlying differential responses to drugs.
 The more important benefit of pharmaceutical biotechnology leads in the form of
better ‘Vaccinal’ immunization’.

7. Biotechnological Pathway to Regulate the Gene Expression:
The Central Dogma Molecular bases of Life
replication

8. Recombinant Technology: A basic tools for the production of Industrial
Products through Biotechnological
Aspects
The Insertion of a desired
gene in to a ‘Vector
Molecules’ followed by
Ligation and Transfection in
to host cell for the Gene
Expression to obtained
desired product is known
‘RDT’.
For exp. Production of
Metabolites, Pharmaceutical
product, Insulin, HCG,
Proteins, Transgenic animals
& plants, recombinant or
Vaccine s etc.

9. Applications of Biotechnology for Human Health Care
1. Molecular marker based disease diagnosis
 These are most efficient tools of disease diagnosis on molecular level,
to identify the presence or absence of a particular nucleotide
sequences of a Pathogen through PCR amplifications with immense
reliability.
 Recent advances of molecular tools have opened up a new horizons
for the assessment of genetic variability as well as disease diagnosis at
the DNA level.
 These Molecular markers are; Microsatellites or STRs, RFLPs,
VNTRs consist a short repeat of nucleotide sequences of genome
from coding or non coding regions.

10. PCR: A Novel Technique of
Determination Pathogenic
Nucleotides Sequences

11. Molecular markers based disease diagnosis
Continuous ……
 The efficiency of Molecular markers has increased in multifold through
advanced marker, Single Nucleotide Polymorphism (SNPs) for
the identification of a particular disease upon the basis of presence or
absence of a particular single nucleotide of a pathogen.
 The polymorphism of SNPs can provide efficient confirmation of Cancer,
Genetic disease, Breast cancer, Diabetes, Hemophilia etc. immediately
through PCR amplifications.
 The ‘DNA chip’ technology, modern method to screen SNPs
hybridization in the current laboratory diagnosis methods at molecular
label for the potent confirmation of a Pathogen in the host.

12. Molecular markers based disease diagnosis Continue…..
Molecular marker system requires prior Genomic knowledge of a pathogen
and a specific RNA primer designed by using Gene Tool, Gene Map
through the use of NCBI, US, Chinese data base resources available till
date.

13. 2. Gene Therapy
 The advancements of Molecular Biology and Biochemistry have
enhanced the genetic basis of a disease inheritance.
 It is a replacement of a defective gene with efficient gene to overcome the
genetic disorders.
 The new gene insert, that encodes specific proteins overcome several
deficiencies, responsible for multiple disorders after Gene Expression.
 Gene therapy primarily involves selection and improvement of desired
gene followed by insertion for efficient gene expression of desired
proteins/metabolites.

14.  The human Gene therapy trials for deficiencies of TNFs, ADA, SCID, Ils,
multi drug resistance, growth hormones, Phenylalanine hydroxylase,
hemophilia (IX) etc. have been completed on animals/humans
Ashi DeSilva : Enzyme deficiency
No Immune System

15. 3. Disease Diagnosis
 The presence of a pathogen like; bacteria, virus and fungi causing
disease, can be detected by identifying and confirming foreign genes
inside host.
 The nucleic acid DNA/RNA is a genetic material of almost pathogens having
characteristic information to contribute in the identification of a specific
pathogen.
 These are the most reliable and potent Biotechnological tools rather than
traditional and indirect symptoms based diagnostic methods.
 For the laboratory diagnosis of a pathogenic, DNA sequences information is
essential.
 Nucleic acid Hybridization and DNA probing are widely used for the
detection of infectious; Tuberculosis, Malaria, Aids, Human Papilloma virus and
genetic diseases like: Cystic fibrosis, SCID, Alzheimer’s disease etc.

16. 4. Medical forensics
 It is also known as ‘DNA fingerprinting’ or DNA profiling, in the
practice of modern medical forensics.
 A minute amount of DNA from blood stains, secretary body fluids,
hairs, skin etc.
 This method involves DNA sample, Restriction Fragments Length
polymorphism (RFLPs), electrophoresis, blotting and
Hybridization techniques for the detection of a culprits.
 DNA profiling has wide range of applications, related to Medical
forensics like; identification of criminals, settlements of paternity
disputes , use of immigration tests cases and disputes etc.

17. 5. Pharmaceutical Products
Certain proteins could have pharmacological properties which can be
utilized as a pharmaceutical agent to cure many human diseases;
infectious viral, bacterial diseases and common diseases such as
Diabetes, Cancer etc.
 However, availability of such therapeutic agents was very limited due to
their cost and old conventional methods of processing and poor yields as
used earlier.
 These were also associated with several complications like; dose, mode of
administration, and harmful side effects to the humans.
The common pharmaceutical products which are produced through
Biotechnological aspects on commercial scales are as follows-

18. biotechbuddy.googlepages.com
Target
Cell
Therapeutic
Protein
Transgene
Nucleus
Production Strategies of Therapeutic
Proteins

19. Pharmaceutical Products Continue…….
a) Recombinants Proteins:
 The advent tools of Biotechnology; Recombinant DNA Technology
(RDT), Genetic Engineering, Gene Expression study have opened a
new vista to pharmaceutical industry for Human health care.
 This RDT was started in late seventies (1970) to produce specific
proteins which open a new horizon for Pharmaceutical researches.
 Certain Pharmaceutical proteins can be used as therapeutic agents and
are listed in the Table No.1, which are approved by US Food and Drug
Administration agency.

20. S. N Recombinant Protein Disease
1. Hemoglobin, Erythroproietin Anamia
2. Interleukins-I receptor Asthama
3. Tissue plasminogen activator (tPA),
Urokinase
Blood Clots
4. Epidermal Growth Factors Burns
5. Interferons, Tumor Necrosis Factor,
Interleukins,
Macrophages activating Factors
Cancer
6. Insulin, Insulin Like Growth factor Diabetes
7. Choronic Gonadotropic Hormones Female Infertility
8. Superdioxide dimutase Freeradical
Damage
9. Growth Hormone, Somatotropin-C Growth disorder
10. Heart Attacks Prourokinase
11. Hepatitis –B Vaccines Hepatitis
12. ILs, B-Cells, growth factors Immune disorders
13. Erythropoietin Kidney disorders
14. Interferon Multi sclerosis
15. Calcitonin Osteomalacia
16. Endorphins andenkephalins Pain
17. Adrenocorticotropic hormones Rheumatic disease
18. Urogestron Ulcer
19. Interferons Viral infections
Table No. 1. List of Pharmaceutical
Proteins

21. Activase (tissue plasminogen activator) heart attack
Humulin (Insulin) diabetes
Engerix-B (Hepatitis B Vaccine) hepatitis B infection
Epogen (erythropoietin) anemia
Herceptin Breast Cancer
Reopro cardiac ischemia
Benefix hemophilia
Rituxan non-Hodgkin’s
lymphoma
Medicinal Applications -Approved Drugs Products

22. b) Second Generation Therapeutic Proteins
A class of Proteins which are produced by Protein engineering through
‘Site directed mutagenesis’ generally known as ‘Muteins’.
These proteins have changes in their amino acids sequences through
Protein Engineering to modify a protein with regards to its stability,
solubility, specificity, substrate affinity, pharmacokinetics etc.
These Proteins found most efficient and reliable for the purpose of human
health care e.g. Insulinlespro, Alteplase etc.

23. c) Insulin
Amongst the earliest uses of biotechnology in pharmaceutical
manufacturing, is ‘Recombinant Technology’ to produce human insulin,
Escherichia coli by Genentech in 1978.
Insulin Protein having a artificial ‘genes’ for each of two protein chains
(A& B ) that comprise the insulin molecule on assembling .
The artificial gene was inserted into plasmids
having multiple genes and resistances genes .

24. Production of Insulin on Commercial
level
Insulin molecules

25. d) Growth hormone
In 1979, Genentech produced human growth hormone (HCG) by
inserting DNA encodes ,into a plasmid.
E. coli bacteria was used for the Gene expression through
‘Recombinant DNA technology’.
The gene fragment having total 551 base pairs was inserted into the
plasmid was prepared by reverse transcription of the mRNA found in
Pituitary glands by to produce human growth hormone HGH on the
industrial scale.

26. e). Vaccines
These are inactive/attenuated, purified lived or dead pathogenic
metabolites, toxins, toxiccoids, proteins, polysaccharides which can
generates Immune responses for prolonged immunity as Vaccinal
immunization..
 These are of mainly three types-
• Dead Bacteria or inactivated viruses
• Live non virulent or attenuated strains
• Viral fragments or Bacterial molecules as sub unit vaccines
 These vaccines also known as conventional vaccines are widely accepted
for
many diseases; Small pox, Chicken pox, Cholera, Typhoid, Tuberculosis,
Poliomyetis, etc.

27. f ) Recombinant Vaccines
 Vaccination is a preventive mode of immunization through modern
generation of vaccines with the addition of manipulated genes to
produced specific proteins or metabolite as vaccines.
 There are several modern strategies to establish a novel recombinant
vaccines. These are of two types-
1) Sub-Unit Recombinant Vaccines: These are the components of
pathogenic organisms including proteins, peptides and DNA
particles.
2) Attenuated Recombinant Vaccines: These are Genetically
Modified Organisms (GMOs) like bacteria and virus which are
modified in such a way that they could only produce
immune responses but not diseases.

28. S. No. Disease Types Pathogenic Organisms
1. Acute respiratory Disease Viral Influenza A&B
2. AIDS Viral HIV
3. Liver damage Viral Virecella
4. Genital Ulcer Viral Herpes Virus
5. Cholera Bacterial Vibrio Cholere
6. Diarrhea Bacterial E. coli
7. Tetanus Bacterial Clostridium Tetany
8. Leprosy Bacterial Mycobacterium Leprae
9. Pneumonia Bacterial S. Pneumonia
10. Tuberculosis Bacterial Mycobacterium tuberculosis
11. Filariasis Parasites Wuchereria banfrofti
12. Malaria Parasites Plasmodium species
13. Sleeping Sickness parasites Trypanosoma species
Table No.2. Recombinant Vaccines which developed or developing to human health care.
Recombinant Vaccines
continue……..

29. g) DNA Vaccines
This is a novel approach of a potent immunization through direct
administration of genetic material DNA/RNA in the host to evoke
immune responses.
These DNA vaccines can be administered through; nasal spray,
intramuscular injection, gene gun transfer to reproduce immune responses
for efficient immunization.
There are some disadvantages of DNA vaccines; Cancer, alteration in
gene expression and problem in the post translational modifications may
take place.

30. Micro- projectile: Partical Gun
Instrument
Gene Expression of desire DNA Fragment
Strategies to Design a DNA Vaccine

31. h) Monoclonal Antibodies (MAbs)
 These are ‘Immunoglobulins’ found epitope specific, developed by
Georg Kohler and C. Milstein in 1975 through ‘Hybridoma
Technology’.
 These MAbs are the results of a fusion between B-lymphocytes and
Myeloma cells followed by HAT selection, De-novo synthesis, Salvage
pathways.
 These MAbs having wide acceptability for both diagnostic; Cancer,
bacterial infections AIDS as well as therapeutic agents, catalytic
proteins, autoantibody fingerprinting.
 These MAbs also can be utilized as a ‘Magic Bullet’ to treat potent

32. Monoclonal Antibodies (MAbs): Principle for the Production of Mabs
through ‘Hybridoma technology’

33. Production of Mabs through
‘Hybridoma technology’

34. i ) Assisted Reproductive Technology
 The manipulations in the reproduction in animals and human are
collectively considered under Assisted Reproductive Technology (ART)
for increasing the rate of reproduction of a desired species with the
advances of Biotechnology.
 All mammals having the capability to produce millions of sperms to
fertilize limited number of female ovum having complexity due to many
reasons, known as ‘Infertility’.
 The ART having multiple prospective of artificial insemination, embryo
transfer, In-vitro fertilization, embryo cloning in-contrasting traditional
breeding strategies.

35.  There are certain reasons of male and female infertility; Oligospermia,
Azoospermia, tubal and unfunctional ovaries, non functional uterus, and
idiopathic infertility, respectively which can be overcome through ‘In-
vitro Fertilization’ (IVF) applications for the human health care.
Reproductive Cloning
For Exp. Test Tube Babies for the
overcome of Male & Female
infertility
Assisted Reproductive Technology Continue ……………….

36. 7. Transgenic Animals
 These are the advancements of Biotechnology to improve our ‘live stock’
which are important resources of our daily need, also known as
Genetically Modified Organisms (GMOs) to obtained desired traits of
economic importance for human welfare.
 Transgenic animals mostly suited as ‘Bioreactors’ due to its capabilities
to produce metabolites, hormones, enzymes, pharmaceuticals and the
study of researches, gene expression analysis.
 The term ‘Transgenesis’ is; insert the gene of interest, inside the host for
the expression of desired proteins/enzymes/hormones to determine the
desired traits of importance.

37.  These transgenic animals having better productive traits, pharmaceutical agents,
metabolites having great significance.
The following steps are used for the production of transgenic animals-
manipulation of desired gene, selection of an efficient viral vectors,
recombinant technology, gene expression analysis and confirmation of
‘Transgene’.
 For exp transgenic ‘mice’ is widely used for the laboratory purposes.
Transgenic Animals Continue …….

38. Transgenic livestock: a Novel Approaches for the ‘Molecular F
By using as a ‘Bioreactor’

39. 8. Molecular Farming: Resent Advances for the Human Health Care
 The modern approaches to synthesize specific metabolites, growth
regulators, growth promoters in-vivo by using transgenic animals as a
‘Bioreactor’ directly to the human consumptions for the better nutritional
health care.
 The desired gene found responsible for the production of desired
metabolites introduced inside the female at the embryonic stage and they
secreted in their milk after their expression.
 The transgenic cow and goat are currently being used for the production of
interferon, tumor necrosis factor (TNFs) for the multiple purposes for
better immunity.

40. 9. Animal Cell Culture: A boon for the Pharmaceutical Researches
This is the most important area of today’s research having great potential
of researches as well as commercial production for the health care and
human welfare through cell and tissue culture techniques.
Generally, more valuable products are commercially produced through
cell culture like; vaccines, monoclonal antibodies, pharmaceuticals
agents, drugs, researches of cancer, genetic manipulation, study of
gene expression and drug testing etc.
Animal cell culture is a specific in-vitro culture conducted under the
hygienic conditions by using nutrients, growth regulators and promoters
for better output.

41. In the modern time, several valuable pharmacological agents having great
medicinal importance have been produced, using animal cell culture or
genetically engineered cells.
1. Requirements of Animal Cell Culture:
a) Substrate for cell growth like; plastics , glasses, Palladium followed
by substrate treatments by using purified fibrinectin or collagens, gelatin
or poly D-lysine, UV light for sterilization purposes.
b) Culture media: Natural and synthetic media (Serum media)
c) Sterilization : Glass wares, equipments and culture media.
2. Equipments for Animal Cell Culture:
Laminar air flow, Co2 incubator, Centrifuge and inverted microscope.
3. Establishment of Cell Culture:
The cell culture is maintained as a Primary culture and Secondary culture
like Finite cell lines; IMR 90 from human lungs and Hela from the human
epithelial cells.
Animal Cell Culture: A boon for the Pharmaceutical Researches
Continue……..

42. Animal Cell Culture: A boon for the Pharmaceutical Researches
Continue……..
Role of Animal Cell Culture in ‘Testing of Drugs’:
Animal cell culture has a significant role in testing of
pharmacological agents without any social ethics regarding the
animals.
Earlier many animals had to suffer or even got dead due to strong
chemical nature and side effects of these active pharmacological
drugs. With the help of animal cell culture, the drugs efficacy can be
done safely on animal cells without harming the animal lives such as;
Tissue Plasminogen Activator (tPA), commercial insulin (Humulin),
erythropoietin and blood clotting factors on commercial scale.

43. 10. Pregnancy Kits
 These are the instant methods to detect pregnancies by using
Biotechnological tools.
 These kits having a ‘Substrate’ with oxidative enzymes to oxidized
‘Human Chorionic Growth hormone’ (HCG) found in the urine of
females to initiate Placental development for embryo attachment with
uterine wall.
 Now if the urine sample has HCG than it will be oxidized by Substrate
enzyme to produce a specific color, as an indication of positive sign of
pregnancy.
 It can be detected through Pregnancy kits, by taking 1-3 drops of urine in
the given space in the Pregnancy Kits as a sample for testing.

44. 11. Transplantation or Grafting
 Earlier, autografting, xenografting and allografting methods were
employed, during the surgery for transplantation.
 These methods have more complications and minimal rate of success due
to rejections through blood groups and antigenic determinant sites, genesis
etc.
 Animal cell culture is playing an important role in transplantation by
producing artificial skin from the same genesis under laboratory conditions
by using biotechnological aspects.
 The cells/tissues are taken from the patient and grow artificially and
transplanted in the host with larger successive rate.

45. 45