論文ID: ess21242
Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).
