Application of r DNA technology and genetic engineering in the products

Recombinant DNA (rDNA) is widely used in biotechnology, medicine and research. Proteins and other products that
result from the use of rDNA technology are found in essentially every pharmacy, doctor’s or veterinarian’s office,
medical testing laboratory, and biological research laboratory. Organisms that have been manipulated using
recombinant DNA technology, and products derived from those organisms have found their way into many medicines.
Biochemical products of recombinant DNA technology in medicine and research include: human recombinant insulin,
growth hormone, blood clotting factors, hepatitis B vaccine, and diagnosis of HIV infection.

INTERFERONS
Interferons (IFNs) are proteins made and released by host cells in response to the presence of pathogens such as
viruses, bacteria, parasites or tumor cells. They allow for communication between cells to trigger the protective
defenses of the immune system that eradicate pathogens or tumors.
IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after their ability to
"interfere" with viral replication within host cells. IFNs have other functions: they activate immune cells, such as
natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen
presentation to T lymphocytes; and they increase the ability of uninfected host cells to resist new infection by virus.
Certain symptoms, such as aching muscles and fever, are related to the production of IFNs during infection.
Functions
All interferons share several common effects; they are antiviral agents and can fight tumors. As an infected cell dies
from a cytolytic virus, viral particles are released that can infect nearby cells. However, the infected cell can warn
neighboring cells of a viral presence by releasing interferon. The neighboring cells, in response to interferon, produce
large amounts of an enzyme known as protein kinase R(PKR). This enzyme phosphorylates a protein known as eIF-2
in response to new viral infections; the phosphorylated eIF-2 forms an inactive complex with another protein, called
eIF2B, to reduce protein synthesis within the cell. Another cellular enzyme, RNAse L— also induced following PKR
activation—destroys RNA within the cells to further reduce protein synthesis of both viral and host genes. Inhibited
protein synthesis destroys both the virus and infected host cells. In addition, interferons induce production of hundreds
of other proteins—known collectively as interferon-stimulated genes (ISGs)—that have roles in combating viruses.
They also limit viral spread by increasing p53 activity, which kills virus-infected cells by promoting apoptosis. The
effect of IFN on p53 is also linked to its protective role against certain cancers.
Interferons, such as interferon gamma, directly activate other immune cells, such as macrophages and natural killer
cells. Interferons can inflame the tongue and cause dysfunction in taste bud cells, restructuring or killing taste buds
entirely.
About ten distinct IFNs have been identified in mammals; seven of these have been described for humans. They are
typically divided among three IFN classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all IFN
classes are very important for fighting viral infections.
Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor
(IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β and
IFN-ω.
Types of Interferon:

• alpha (leukocyte interferon): (α-IFN) produced by virus infected leukocytes

• beta (fibroblast interferon): (β-IFN) produced by virus infected fibroblasts or epithelial cells

• gamma (immune interferon): (γ-IFN) produced by certain activated T cells & NK cells
Production of interferons by genetic engineering:
• 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 harvesting, the E. coli cells are homogenized and the inclusion bodies recovered via centrifugation. After
solubilization 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 resented as a 30 mg/ml solution in glass vials and displays a shelf- life of 24 months when stored at 2–8°C`.
 Vaccines- hepatitis- B
Vaccine Types: There are several different types of vaccines. Each type is designed to teach your immune system
how to fight off certain kinds of germs — and the serious diseases they cause. When scientists create vaccines, they
consider:
• How your immune system responds to the germ
• Who needs to be vaccinated against the germ
• The best technology or approach to create the vaccine
Based on a number of these factors, scientists decide which type of vaccine they will make. There are 4 main types of
vaccines:
• Live-attenuated vaccines
• Inactivated vaccines
• Subunit, recombinant, polysaccharide, and conjugate vaccines
• Toxoid vaccines
Live-attenuated vaccines: Live vaccines use a weakened (or attenuated) form of the germ that causes a disease.
Because these vaccines are so similar to the natural infection that they help prevent, they create a strong and long-
lasting immune response. Just 1 or 2 doses of most live vaccines can give you a lifetime of protection against a germ
and the disease it causes. But live vaccines also have some limitations. For example:
• Because they contain a small amount of the weakened live virus, some people should talk to their health care
provider before receiving them, such as people with weakened immune systems, long-term health problems,
or people who’ve had an organ transplant.
• They need to be kept cool, so they don’t travel well. That means they can’t be used in countries with limited
access to refrigerators.
Live vaccines are used to protect against:

• Measles, mumps, rubella (MMR combined • Smallpox

vaccine) • Chickenpox
• Rotavirus • Yellow feve
Inactivated vaccines: Inactivated vaccines use the killed version of the germ that causes a disease. Inactivated
vaccines usually don’t provide immunity (protection) that’s as strong as live vaccines. So, you may need several doses
over time (booster shots) in order to get ongoing immunity against diseases. Inactivated vaccines are used to protect
against:
• Hepatitis A • Polio (shot only)
• Flu (shot only) • Rabies
Subunit, recombinant, polysaccharide, and conjugate vaccines: Subunit, recombinant, polysaccharide, and
conjugate vaccines use specific pieces of the germ — like its protein, sugar, or capsid (a casing around the germ).
Because these vaccines use only specific pieces of the germ, they give a very strong immune response that’s targeted
to key parts of the germ. They can also be used on almost everyone who needs them, including people with weakened
immune systems and long-term health problems. One limitation of these vaccines is that you may need booster shots
to get ongoing protection against diseases. These vaccines are used to protect against:
• Hib (Haemophilus influenzae type b) disease • Pneumococcal disease
• Hepatitis B • Meningococcal disease
• HPV (Human papillomavirus) • Shingles
• Whooping cough (part of the DTaP combined
vaccine)
Toxoid vaccines: Toxoid vaccines use a toxin (harmful product) made by the germ that causes a disease. They create
immunity to the parts of the germ that cause a disease instead of the germ itself. That means the immune response is
targeted to the toxin instead of the whole germ. Like some other types of vaccines, you may need booster shots to get
ongoing protection against diseases. Toxoid vaccines are used to protect against:

• Diphtheria
• Tetanus
 RECOMBINANT VACCINES

Biotechnology sector has also played its part in developing vaccines against certain diseases. Such vaccine which
makes use of recombinant DNA technology is known as recombinant vaccines. It is also known as subunit vaccines.
DNA vaccines: DNA vaccination is a technique for protecting an organism against disease by injecting it with
genetically engineered DNA to produce an immunological response. Nucleic acid vaccines are still experimental, and
have been applied to a number of viral, bacterial and parasitic models of disease, as well as to several tumor models.
DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range
of immune response types. Here the gene encoding for immunogenic protein is isolated and used to produce
recombinant DNA which acts as vaccine to be injected into the individual.
Steps involved: Production of recombinant vaccines involves the following steps:
(i) First and foremost, protein which is crucial to the growth and development of the causative organism be identified.
(ii) The corresponding gene is then isolated applying various techniques.
(iii) This gene is then integrated into a suitable expression vector to produce a recombinant DNA.
(iv) This rDNA is used as vaccines or is introduce into another host organism to produce proteins which acts as
vaccines.

Hepatitis B Vaccine: This vaccine gives protection against the hepatitis B virus, which is a major cause of serious
liver disease, including liver cancer and cirrhosis. The individual hepatitis B vaccine can be given at the same time as
other vaccines such as the PCV, hepatitis A, MMR, pre-school booster and other travel vaccines. The vaccines should
be given at a separate site, preferably in a different arm or leg. The vaccine does not contain any live viruses, and
cannot cause hepatitis B disease.
General features of nucleic acid of Hepatitis B Virus: Plasma of human has been detected to have varying amount
of HB antigens. Three types of viral coat proteins are recognized to be antigenic –
• viral surface antigen (HBsAg)

• viral core antigen (HBcAg)

• the e-antigen (HBeAg)

Recombinant Protein (Hepatitis B): In July 1986, a recombinant hepatitis B vaccine was licensed in the United
States. This vaccine built on the knowledge that heat-inactivated serum containing hepatitis B virus (HBV)
and hepatitis B surface antigen (HBsAg) was not infectious, but was immunogenic and partially protective against
subsequent exposure to HBV. HBsAg was the component that conferred protection to HBV on immunization.
General steps for Recombinant Hepatitis B Vaccine production: Production of these genes (HBsAg, or “S” gene)
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 (HBsAg, or “S” gene) is isolated from the HB virus by normal 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.
4. This recombinant DNA, containing the target gene, is introduced into a yeast cell (Saccharomyces cerevisiae)
forming the recombinant yeast cell.
5. The recombinant yeast cell multiplies in the fermentation tank (medium used in this process is a complex
fermentation medium that consists of an extract of yeast, soy peptone, dextrose, amino acids, and mineral salts )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.
8. HBsAg are combined with preserving agent and other ingredients and bottled. Now it is ready for vaccination in
humans.
Hepatitis B vaccines are sterile suspensions for intramuscular injection. The vaccine is supplied in four formulations:
pediatric, adolescent/high-risk infant, adult, and dialysis.
 Hormones- Insulin

Insulin is a hormone that regulates the amount of glucose (sugar) in the blood and is required for the body to function
normally. Insulin is produced by cells in the pancreas, called the islets of Langerhans. These cells continuously release
a small amount of insulin into the body, but they release surges of the hormone in response to a rise in the blood
glucose level. Insulin is synthesized in significant quantities only in beta cells in the pancreas. Since it is a protein or a
polypeptide structure it is synthesized like most other proteins via transcription and translation of DNA into mRNA
and amino acid chains or polypeptide chains. Thereafter the protein undergoes structural changes to achieve its final
form.

Insulin's structure varies slightly between species of animal. Both porcine (from pigs) and bovine (from cows) insulin
are similar to human insulin but porcine insulin resembles human insulin more closely.

Synthetic human insulin was the first golden molecule of the biotech industry and the direct result of recombinant
DNA technology. Currently, millions of diabetics worldwide use synthetic insulin to regulate their blood sugar levels.
Synthetic insulin is made in both bacteria and yeast. Human insulin is the name which describes synthetic insulin
which is laboratory grown to mimic the insulin in humans. Human insulin was developed through the 1960s and 1970s
and approved for pharmaceutical use in 1982.Before human insulin was developed animal insulin, usually a purified
form of porcine (pork) insulin, was used. Human insulin is laboratory created by growing insulin proteins within E-
coli bacteria (Escherichia coli).

Human insulin is available in two forms, a short acting (regular) form and intermediate acting (NPH) form. NPH
(Neutral Protamine Haledon) insulin, also known as isophane insulin, is a suspension meaning that the insulin vial
should be rolled or repeatedly turned upside down to ensure the solution is uniformly cloudy. Some examples of
human insulin:

• Regular (short acting): Humulin S, Actrapid, Insuman Rapid

• NPH (intermediate acting): Humulin I, Insuman basal, Insulatard

• Premixed human insulins: Humulin M2, M3 and M5, Insuman Comb 15, 25 and 50

Although bovine and porcine insulin are similar to human insulin, their composition is slightly different.
Consequently, a number of patients' immune systems produce antibodies against it, neutralizing its actions and
resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin,
were fears of long-term complications ensuing from the regular injection of a foreign substance, as well as a projected
decline in the production of animal derived insulin. These factors led researchers to consider synthesizing Humulin by
inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce insulin that is chemically identical
to its naturally produced counterpart. This has been achieved using Recombinant DNA technology. This method is a
more reliable and sustainable method than extracting and purifying the abattoir by-product.

Human insulin is grown in the lab inside common bacteria. Escherichia coli is by far the most widely used type of
bacterium, but yeast is also used. Researchers need the human protein that produces insulin. Synthesizing human
insulin is a multi-step biochemical process that depends on basic recombinant DNA techniques and an understanding
of the insulin gene.

Method: The sequence that codes for proinsulin is inserted into the non-pathogenic E. coli bacteria. The bacteria go
through the fermentation process where it reproduces and produces proinsulin. Then the connecting sequence between
the A and B chains is spliced away with an enzyme and the resulting insulin is purified.