Construction And Expression Of The Vector With Multi-Copy HEGF Gene In Pichia Pastoris

Human epidermal growth factor(hEGF) was found in 1975 by Cohen and Gregory respectively. It is a small polypeptide composing with 53 amino acids. Three disulfurate bond in it make the structure very steady. hEGF possesses many biological activities, plays an important role in cell splinter, renewing skin wound, stomach and intestines ulceration and cornea scathe, targeting and then killing tumor cells with high density hEGF receptors.Expression of hegf gene in Pichia pastoris GS115 was studied and the Strains with highly expression of hEGF were screened out in this study.The works included: (1) Synthesized hegf gene from three fragments, connected using overlap extension PCR, then constructed cloned vector and sequenced; (2) Yeast expression vector pGAPZα-hegf was constructed and isoschizomers(AvaⅢand PstⅠ) were used to construct multi-copy expression vectors, including 2,3,4 multi-copy expression vectors, and pGAPZα-2hegf,pGAPZα-3hegf and pGAPZα-4hegf were successfully constructed , respectively. (3) Chemical and electroporation transformation method were used for transformation of P. Pichia, and recombinants were obtained by using zeocin screened, PCR identification and SDS-PAGE analysis. (4) Urea-SDS-PAGE and glycerol-SDS-PAGE gel systems were compared, and the urea-SDS-PAGE gel system was suitable to small polypeptide separation. (5) Each type of highly expression of hEGF Strain was screened by SDS-PAGE, and the yield were 25.2mg/l, 28.6 mg/l, 38.9 mg/l and 26.0 mg/l, about 35-40% of total secreted protein. (6) The strains were cultured, and the protein in the supernatant was purified by chromatography and the biological activities were analyzed. Results of western blot indicated that recombinant protein could react with antibodies against anti-hEGF. The results of MTT assay proved that the supernatant of the culture had biological activity that promoted proliferation of Balb-3T3 cell. The research was established a theory foundation for gene engineering drug and clinic application of hEGF.