猪表皮生长因子在毕赤酵母中的表达及鉴定

Expression and Characterization of Porcine Epidermal Growth Factor in Pichia pastoris

【目的】猪表皮生长因子(pEGF)具有刺激静止期猪卵母细胞的成熟以及刺激仔猪胃肠上皮细胞成熟的功能,从而可以提高母猪繁殖率和降低断奶仔猪的应激。因此开发猪表皮生长因子具有重要意义。【方法】用人工合成的pEGF基因为模板,用PCR扩增获得pEGF-6His片段,将其克隆到载体pPIC9构建重组质粒pPIC9-pEGF并电转化毕赤酵母。用斑点杂交法筛选多拷贝整合转化子即基因工程菌,用Ni-NTA亲和层析法纯化目的蛋白,纯化产物用抗His-tag的单克隆抗体进行蛋白质免疫印迹、氨基端测序鉴定、体外生物活性检测。【结果】斑点杂交法筛选中信号越强的转化子,甲醇诱导表达的目的蛋白含量越高。免疫印迹中可以观察到印迹条带。氨基端氨基酸测序发现纯化的表达产物含有2条肽链,其N-端15个氨基酸的序列分别与天然的pEGF以及Glu-Ala-pEGF的序列相同。体外生物活性检测结果表明,纯化的表达产物对BALB/c3T3细胞具有显著的增殖作用。【结论】本研究获得了能高效表达具有生物学活性的重组猪表皮生长因子的基因工程菌。

[Objective] 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 and it may enhance productivity of sows and reduce the stress of early-weaned piglets, so it is necessary to develop recombinant pEGF. [Method] According to the codon usage preference ofpichia pastoris, a pEGF gene was synthesized and the 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. [Result] 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 natural 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 recombinant porcine epidermal growth factor with high biaoactivity was obtained.