Expression Of HGH In Siklworm Pupae And The Study On Its Bioactivity

hGH is synthesized and stored by somatotroph cell within the anterior pituitary gland. The physiological actions of hGH are pleiotropic and involve multiple organs and physiological systems. hGH exerts many metabolic effects that persist throught life. In this work the recombinant plasmid pET28a-hGH was constructed at first, and His6-tagged rhGH was expressed in E. coli by being induced with 1mM IPTG. The non-active rhGH fusion protein was purified as antigen to immunize rabbits. After four-time immunization, the rabbit anti-hGH serum was collected and purified by (NH₄)₂SO₄. Employing the analyzing way of ELISA, we concluded that the effect of the rabbit anti-hGH was 1:25600. Western blotting was used to identify the specificity of anti-hGH polyclonal antibody. As a result, the antibody could bind specially with rhGH fusion protein and hGH polypeptides. The preparation of efficient anti-hGH polyclonal antibody was the base of deeper work on hGH.The insect-baculovirus expression system is widely used for its high expression of active exogenous proteins, parallel with the important system of prokaryocyte, yeast and mammal cells. Here the recombinant plasmid pBacPAK-hGH and linearized genomic DNA of baculovirus BmBacPAK6 was recocombinated homeologously in BmN cells and recombinant baculovius was generated. The recombiant virus vBmhGH was purified by plaque assay and blue-white assay for 4 cycles. Southern blotting identified that the hGH gene was inserted the genomic DNA of baculovirus successfully. The titer of vBmhGH was confirmed to be 6.0×108 PFU/ml by the way of TCID50. The BmN cells were infected with vBmhGH, the rhGH protein was produced and the peak expression of 52μg/106 cells was appeared at 72 h post-infection. Then the silkworm pupae was infected with vBmhGH and the rhGH protein was identified by Western blotting in the hemolymph of pupae. Results of ELISA showed that the peak production of rhGH was about 300μg/pupa at 120 h post-infection. About 180μg rhGH was purified from silkworm pupae by ways of gel chromatography, immunoprecipitation and revered phase chromatography (RPC) from 1 ml pupae hemolymph. Experiments of colony formation demonstrated that the rhGH from pupae could stimulate the proliferation of K562 human erythroleukemia cells and the protein was active in vitro hypophysectomized male rats were gavaged with rhGH protein preparation. As a result, the body weight gain (BWG) was improved, so did the parameters of liver weight and tibia epiphyseal width, which meant that the rhGH preparation was active in vivo. Results of the acute toxicity and long-term toxicity pre-clinic experiments showed that the rhGH capsule had no distinct side effects, which means that the rhGH is a great potential protein drug to treat cretinism, Alzheimer's disease, and other diseases caused by hGH. Studies on anti-oxidation in animal and life-delaying in fruit flies illustrated that the rhGH decreased the lipofuscin content in heart and increased the SOD specific activity of liver, and lengthened the life-span of fruit flies. Therefore, the rhGH capsule has the effect of anti-aging, and it is a potential anti-aging protein drug. In a conclusion, it is promising to widen the application area of rhGH, and develop drugs and functional foods based on rhGH preparation from silkworm pupae.