Recombinant Human Growth Hormone (hGH)
The cloned genes are utilized commercially in various fields such as pharmaceuticals, industry, agriculture, pollution control, medical science.
The production of medically useful human peptides and proteins (e.g. human growth hormones, insulin, somatostanin and interferon) are of much importance.
The pituitary gland of humans produces growth hormones that regulate the growth and development. However, in children stunded growth occurs due to deficiency of the hormone which is called pituitary dwarfism. Such children are regularly treated with growth hormone which is procured from the pituitary glands of deceased persons. The injection of hGH has been found effective in children.
Now, the hGH is available as recombinant protein. The hGH-coding DNA sequence is linked with the bacterial signal sequence of E. coli. The hGH is secreted into periplasmic space of bacterial cell by the signal peptides where from the protein is purified. The hGH lacks terminal methionine, hence it is called met- less hGH.
In the USA the recombinant growth hormone is extensively used for farm animals for increased milk production and meat. But for the safety of food produced such methods may lead ethical problems.
Insulin is a peptide hormone secreted by the Islets of Langerhans of pancreas. It catabolizes glucose in blood. Insulin is a boon for the diabetics whose normal function for sugar metabolism generally fails. However, diabetes affects a significant percentage of world population. The diabetics take daily injection of insulin for its control. Previously insulin for injection had been isolated from the pancreas of cows, pigs etc. It was quite effective for diabetic but some patients developed antibodies against insulin as it was antigen because insulin of human and animals has antigenic differences.
Insulin consists of two short polypeptide chains: A (21 amino acid long) and B (30 amino acid long). These two chains are linked by two sulfide bridges. These two peptides are connected by a third peptide chain-C (35 amino acid long). The precursor of insulin is pre-proinsulin which is about 109 amino acids long. The structure of pre- proinsulin is- NH2- (peptide) ÃŸ-chain- (peptide C)-A chain-COOH. For the production of mature insulin molecule post translational proteolysis of pre hormone is essential.
Vaccine for Hepatitis B virus:
For the production of recombinant vaccines, genes for desired antigens are identified and cloned into suitable vectors. The vectors are introduced into suitable hosts for expression. Production of recombinant vaccine through this method has several advantages.
Recombinant vaccine for HBV was produced by cloning HBsAg gene of the virus in yeast cells. The yeast system has its complex membrane and ability of secreting glycosylate protein. This has made it possible to build an autonomously replicating plasmid containing HBsAg gene near the yeast alcohol dehydrogenase (ADH) I promoter. The HbsAg gene contains 6 bp long sequence preceding the AUG that synthesizes N- terminal methionine. This I sjoined to ADH promoter cloned in the yeast vector PMA- 56. The recombinant plasmid is inserted into yeast cells. The transformed yeast cells are multiplied in tryptophan- free medium. The transformed cells are selected. The cloned yeast cells are cultured for expression of HBsAg gene. This inserted gene sequence expresses and produces particles similar to the 22 Âµm particle of HBV as these particles are produced in serum of HBV proteins. The expressed HBsAg particles have similarity in structure and immunogenicity with those from HBV- infected cells of patients. Its high immunogenicity has made it possible to market the recombinant product as vaccine against HBV infection.
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