关键词: 猪表皮生长因子, 毕赤酵母, 分泌表达, 生物活性

Abstract: Porcine epidermal growth factor (pEGF) can stimulate dormant primordial follicle into active follicle and stimulate growth of gastrointestinal (GI) tract and repair the GI tract in pigs, it may enhance productivity in sows and reduce the stress of early-weaned piglets, necessitating the development of recombinatant pEGF. 【Method】According to the codon usage preference of pichia pastoris, a pEGF gene was synthesized., a PCR was performed to obtain pEGF-6-His fragment using the synthetic pEGF gene as template. Then pEGF-6-His fragment was cloned into plasmid pPIC9. The resulting plasmid pPIC9-pEGF was linearized and transformed into pichia pastoris by electroporation, multicopy strains were screened out by dot blotting analysis, and the target protein in the medium supernatants was purified by His-tag affinity column. The purified protein was analyzed by Western bloting using anti His-tag monoclonal antibody, amino-terminal sequencing, and in vitro bioactivity assay. 【Results】The stronger hybridization signal of the transformant in dot bloting, the more obvious target protein band from this transformant was observed in Tricine-SDS-PAGE. An immunobloting band was observed in the western bloting analysis, and N-terminal amino-acid sequencing showed that purified products contained two peptides. The N-terminal sequence of one peptide was the same as the sequence of the nature pEGF; and the other peptide was Glu-Ala -pEGF. Finally, the expressed pEGF fusion protein could significantly increase the proliferation of BALB/c 3T3 cells. 【Conclusion】These results indicated that a recombinant strain which can effectively and stably produce recombinatant porcine epidermal growth factor with high biaoactivity was obtained.

Key words: Porcine epidermal growth factor, Pichia pastoris, Secreting expression, Bioactivity