The Screening Of Pichia Pastoris GSll5 Strains With Highly Effective Expression Of Human Insulin-like Growth Factor I

Human Insulin-like Growth Factor I (huIGF-I) is a polypeptide hormone, which possess biological activity of stimulating growth and mutoses and has similar metabolic function of insulin, HuIGF-I plays an important role in treating diabetes, osteoporosis, desease of catablism, Laron syndrome and dwarfism, etc. Expression of huIGF-I in Pichia pastoris GS115 was studied and the Strains with highly expression of human Insulin-like Growth Factor I was screened out in this study. First, cloning vector wais constructed: The gene encoding human insulin-like growth factor-I was modified. The modified gene was ligated to pGEMT-T easy vector and sequenced. The second, the expression vector was constructed: One yeast integration plasmids were constructed as following: pGAPZ-huIGF-I, which were screened with antibiotic zeocin and identificated by dot blot. The third, the recombinant strains were constructed: the recombinant plasmids were transformed to strain Pichia pastoris GS115. Recombinant yeast strains were screened by using zeocin , PCR, SDS-PAGE identification. Finally, recombinant strains of highly effective expression were isolated: The recombinant strains (el and cl) of highly expression were isolated by SDS-PAGE. The expression level of el and cl reach to 32. 0 mg / L and 40. 5 mg / L, respectively. Then the protein in the supernatant of the culture of these strains was purified by chromatography. Results of western blot indicated that recombinant protein could react with antibodies against anti-IGF-I. The supernatant of the culture of these strains had biological activity that promoted proliferation of cell. So the highly expression strains were isolated and gotten in this study.