PRODUCTION OF HB VACCINE AND INTERFERONS BY rDNA - Copy.pptx

APPLICATION of RECOMBINANAT DNA TECHNOLOGY : IN THE PRODUCTION OF HEPATITIS B VACCINE ,INSULIN and INTERFERON

1. NAGPUR COLLEGE OF PHARMACY ( Hingna Road, Wanadongri, Nagpur, Maharashtra 441110) APPLICATION of RECOMBINANAT DNA TECHNOLOGY : IN THE PRODUCTION OF HEPATITIS B VACCINE ,INSULIN and INTERFERON PRESENTED BY MS KARISHMA DUHIJOD (M.PHARMA QA) Assistant professor

2. HEPATITIS B : Hepatitis B is a virus affects liver causing chronic hepatitis, C irrhosis and liver cancer. It has a 42 nm particle, called Dane particle. lt consists of a core containing a viral genome (DNA) surrounded by a phospholipid envelope carrying surface antigens. When hepatitis B virus become Infectious produced Dane particles with 22 nm sized particles, contain surface antigens which are more immunogenic and trigger the immune response which is used in diagnostics test.

3. STRUCTURE OF HEPATITIS – B

4. Step 1 : Isolate HBsAg Step 2 : pMA 56(Yeast Vector )linked with ADH(Promoter) Step 3 :Restricted Endonuclease (E- coRI ) Step 4: Ligase Step 5 :HBsAg gene Step 6 : Transform into yeast cell Step 7 : Provide a culture(Tryptophan) or growing Step 8 : Culture cells are selected Step 9 : Lysis yeast cells Step 8 : Purify HBs gene ( 22nm particles) PRODUCTION OF HEPATITS B - VACCINE

5. The subunit HBsAg as 22 nm particles can be isolated and used to immunize individuals against hepatitis B. Work: vaccine contains HBsAg which identifies foreign substance . Hepatitis B – first Recombinant vaccine( Recombivax and Engerix-0). Safe to use, very effective and produces no allergic reactions. Administered three doses over a period of six months.

6. Insulin : The hormone insulin produced by the β -cells of islets of Langerhans of pancreas. Human insulin contains 51 amino acids, forming two polypeptide chains. The chain A has 21 amino acids while B has 30 amino acid Early years : insulin isolated and purified from the pancreases of pigs and cows was used for the treatment of diabetics, but there is some differences found in Amino acids in the structure of animal insulin compared to human insulin. Which causes some allergic infections . Animals are also have to be sacrificed.

7. Production of recombinant insulin 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 (consisting of inducer gene, promoter gene, operator gene and structural gene Z lor B-galactosidase)s used for expression of both the genes. The presence of lactose in the culture medium induces the synthesis of insulin A and B chains in separate cultures. The so formed insulin chains can be isolated, purified and joined together to give a full-pledged human insulin.

8. Insulin : first ever pharmaceutical product of recombinant DNA technology administered to humans. The procedure employed for the synthesis of two insulin chains A and B . After injecting the insulin, the plasma concentration of insulin rises slowly, And for this reason, insulin injection has to be taken at last 15 min before a meal , cause hyperinsulinemia.

9. second generation insulins by site directed mutagenesis and protein engineering. It can be constructed with hexamers ( by modified amino acid residues at position 29 and 30 of the B-chain of insulin) for faster dissociation called Insulin Lispro which can be injected immediately before a meal . The second-generation recombinant proteins are termed as muteins .

10. INTERFERON An viral substance , and first line of defense against viral attacks. Origin – interference (on viral replication). Discovered : Alick lsaacs and Jean Lindemann (1957) . group of more than 20 substances (MW between 20,000-30,000 Daltons. ) All the interferons are glycoproteins.

11. On the basis of their structure and function ,(3 types) Interferon-α(IFN- α) Interferon-β(IFN- β) Interferon-γ(IFN-γ) MECHANISM OF ACTION Virus enters the cell its releases the nucleic acid into cellular cytoplasm , and stimulate the host DNA to produce the Interferons ,it get absorbed to adjacent cells . Here, interferon stimulate the Cells DNA to produce an antiviral enzymes (protein), so formed protein inhibit the viral replication and protects the cells, by binding to mRNA virus and inhibit their Protein Synthesis.

12. PRODUCTION OF INTERFERON Step 1 : Isolate I nterferon mRNA Step 2: Synthesis of C omplementary DNA (cDNA) Reverse transcription S tep 3: Suitable vector ( plasmid) RE(cleavage) RE(cleavage) Step 4 : DNA Fragment Vector Fragment (DNA Ligase) Step 5 : Insertion of DNA into a vector Step 6 : rDNA molecule formed Step 7 : Introduced into host cell (cultural medium )for replication Step 8 : INTERFERON(Multiple copies)

13. but Bacterial host was less used because most of the interferons are glycoproteins in nature and bacteria do not possess glycosylation of proteins. Production interferons by yeasts : The yeast Saccharomyces cerevisiae (s uitable) for the Production of recombinant Interferons, because the yeast possess the mechanism to carry out glycosylation of proteins. The DNA sequence (human interferon) can be attached to the yeast alcohol dehydrogenase gene in a plasmid and introduced into 4 yeast cells. The yield of interferons is several fold higher compared to E- coli. (But it is possible to avoid the glycosylation step)

14. Production of hybrid interferons : using E-coli Genes of IFN-α 2 and IFN-α 3 are used , digested by restricted endonuclease into fragments and Joined by using DNA ligase to generate hybrid gene. ADVANTAGE : different interferons with different antiviral activities can be combined to produce a more efficient interferon.

15. APPLICATIONS : in various Viral diseases like leukemia, kaposis sarcoma, bladder cancer, head and neck cancer, renal cell carcinoma, skin cancer and multiple myeloma. The other disease employing interferon therapy are AIDS, multiple sclerosis genital warts, hepatitis C , herpes zoster etc. Interferons are also employed in the treatment of common colds and influenza(Nasal spray ).