BY ABHILASHA SHUKLA & N.

RACHANA

CONTENT
INSULIN (INTRODUCTION) PRODUCTION OF REC.INSULIN HUMAN GROWTH HORMONE(Hgh)

PRODUCTION OF REC.Hgh
RECOMBINANT VACCINE RECOMBINANT HEPATITIS B VACCINE

RECOMBINANT VACCINIA VIRUS VACCINE

INSULIN
Hormone Insulin is produced by the Beta cells of

islets of langerhans. Human insulin contains 51 Amino Acids , arranged in 2 polypeptide chains. Chain-A has 21 amino acids while Chain-B has 30 amino acids. Both are held together by Di-sulphide bonds. Occurrence of Diabetes is due to insufficient Insuline.

PRODUCTION OF RECOMBINANT INSULIN

In the early years, insulin isolated and purified from

the pancreases of pigs and cows was used for the treatment of Diabetics Production of Insulin by recombinant DNA Technology started in late 1970s. Basic technique consisted of inserting human insulin gene and the promoter gene of lac operon on to the plasmid of E. coli. In 1986, Eli Lilly company received approval to market Human Insulin under trade name Humulin.

TECHNIQUE FOR RECOMBINANT INSULIN PRODUCTION.

Recombinant Insulin is produced by using E. coli

bacteria. The genes for Insulin A chain and B chain are separately inserted to the plasmids of two different E. coli cultures. The lac operon system is used for the expression of both the genes. Presence of lactose in the culture medium induces synthesis of Insulin A and B chains. Insulin chains are isolated and joined together to form a complete human Insulin

HUMAN GROWTH HORMONE

INTRODUCTION:

Growth hormone is produced by the pituitary gland. Growth hormone regulates overall body growth by increasing the cellular uptake of Amino Acids, and Protein synthesis. Insufficient human growth hormone in childrens leads to pituitary dwarfism.

PRODUCTION OF RECOMBINANT hGH

hGH is a protein comprised of 191 amino acid.

During its synthesis in the body hGH is tagged with a

signal peptide(with 26 amino acid). The signal peptide is removed during secretion to release the active hGH. Signal peptide interrupts hGH production by recombinanant technology. cDNA encoding signal peptide can be cut to solve these problems.

Continue.

Continued. Base sequence in cDNA encoding signal peptide (26 Amino Acids) + neighbouring 24 Amino Acids = Total 50 Amino Acids; cut by R.E.( ECoRI) Now a gene (cDNA) for 24 Amino Acid sequence of hGH is freshly synthesized and ligated to the remaining hGH cDNA. It is attached to a vector and inserted into E.coli bacterium. In this manner hGH can be produced by DNA technology.

RECOMBINANT VACCINES
INTRODUCTON:
Recombinant/purified protein vaccines are based on the concept that humoral immune responses mounted to an infection are often targeted toward specific localized regions on the surface of protein antigens known as epitopes. The recombinant proteins for vaccination are produced by expressing these immunogenic proteins using heterologous expression systems.
The advancement of genomics, proteomics and biotechnology provide us the opportunity to develop safe and more effective vaccines

Vaccination is the most cost-effective method of reducing animal suffering and economic losses due to infectious diseases in animals

SUBUNIT VACCINES
SUBUNIT VACCINES ARE DEFINED AS THOSE CONTAINING ONE OR MORE PURE OR SEMI-PURE ANTIGENS

INTRODUCTION:Hepatitis B is a widespread disease in man.

It affects liver causing chronic hepatitis, Cirrhosis and Liver cancer.

Hepatitis B virus is a 42 nm particle, called Dane particle. It consists of core containing a viral genome(DNA) surrounded by a

phospholipids envelope carrying surface antigens.

Production Of Recombinant Hepatitis B Vaccine

Hepatitis B vaccine is produced by cloning HbsAg gene in yeast cell(Saccharomyces

Cerevisiae).
Gene for HbsAg is inserted which is linked to alcohol dehydrogenase promoter. These plasmids are then transferred and cultured. The cells grown in Tryptophan, free

medium are selected and cloned.

HbsAg gene is expressed to produce 2nm sized particles similar to those found in

patients infected with Hepatitis B.

The subunit HbsAg as 22nm particles can be isolated and used to immunize individuals

against Hepatitis B.

Vaccines Against Viruses-Vaccinia Virus

INTRODUCTION: Vaccinia virus is basically the vaccine that was originally used by

Jenner for the eradication of Small pox.

Vaccinia virus contains a Double-Stranded DNA.

Genome of the virus can accommodate stretches of foreign DNA

which can be expressed along with the viral gene .

Advantage with this vaccine is that it stimulates B-Lymphocytes

& T-Lymphocytes.

Production of Recombinant Vaccinia Viruses

It is a two steps process:1.

Assembly of plasmid insertion vector:

Fresh vaccinia (cowpox) viruses are processed to release their DNAs. Genes from Hepatitis B virus, Herpes simplex virus and influenza virus are added and inserted into vaccinia virus genome. These DNA clusters are cloned in E-Colli to produce plasmid insertion vectors. The recombinant plasmids are isolated and purified and serve as plasmid insertion vectors.

2.

Production of recombinant Vaccinia virus:

The animal cells are infected with plasmid insertion vectors and normal vaccinia viruses. As the viral replication occurs, plasmids are taken upto produce recombinant vaccinia viruses.

Continued..
The plasmid insertion vectors incorporates its

genes into vaccinia virus genome at a place that encodes for the enzyme Thymidine Kinase(TK). This the recombinant viruses have lost their ability to produce TK gene due to which it is easy to select recombined vaccinia viruses that lack TK gene. These viruses are less infectious than the normal viruses. The recombinant vaccinia viruses, release from the cultered animal cells,can be successfully used as vaccines.

Advantages: Antigens that closely resemble natural antigens can be produced.

The Virus can replicate in host cell. This enables the amplification of

the antigens for their action on B-Lymphocytes & T-Lymphocytes.

There is possibility of vaccinating several diseases with one

recombinant Vaccinia virus.

REFERENCE
BIOTECHNOLOGY by U. Satyanarayana
IMMUNOLOGY by KUBY www.virology.ws

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