凋亡抑制因子Survivin基因的克隆及其在Pichia pastoris中的表达

Cloning of survivin-an inhibitor of apoptosis proteins and expressed in pichia pastoris system

目的克隆凋亡抑制因子Survivin基因,利用甲醇酵母Pichiapastoris真核表达系统表达Survivin蛋白。方法利用Survivin特异引物,通过PCR方法扩增人Survivin基因后进行测序,将序列正确的Survivin基因与分泌表达载体pPic9k重组,重组体酶切线性化后用电穿孔法转入酵母宿主菌GS115/His-中,以不同浓度G418/YPD平板筛选,经PCR扩增鉴定阳性克隆,再用含1%甲醇的培养基BMMY诱导表达蛋白,ELISA法检测表达产物。结果克隆的人Survivin基因与GenBank中的Survivin基因序列完全一致,无突变;构建了真核表达载体pPic9k-Survivin,并转入酵母菌GS115/His-后,筛选出了抗高浓度(4mg/mL)G418的阳性克隆(GS115/His+)6个,并用PCR法进行了鉴定;Survivin的表达量与时间有关,48h的表达量最高。结论用Pichiapastoris真核表达系统表达Survivin蛋白的方法可行,若进一步优化条件,可大量表达Survivin蛋白,为进一步研究Survivin的生物学功能及其在肿瘤发生发展中的作用提供了重要的实验基础。

Objective To clone human survivin gene and express survivin-an inhibitor of apoptosis proteins in Pichia pastoris eukaryotic expression system. Methods Full length survivin gene was amplified by PCR using survivin specific primers. The verified survivin gene by sequencing was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear survivin gene was introduced into GSllS/His^- cells by electroporation. The transformants were transferred onto YPD plates that contained different concentrations of G418 for screening the positive clones. The integrated survivin gene in positive clones was confirmed by PCR. Selected transformants were cultured in BMMY medium with 1% methanol for inducing the expression of survivin protein. The expressed survivin protein was analyzed by ELISA. Results The cloned human survivin sequence was the same as that in the GeneBank. The recombinant pPic9k-survivin was constructed in Picha pastoris eukaryotic expression vector. After the linear digestion, the recombinant vector was introduced into GS115/His^-cells, the 6 positive clones against G418 （4 mg/mL） were obtained and confirmed by PCR; the highest survivin protein was expressed when expressed for two days in 1% methanol BMMY medium. Conclusions Improved survivin protein yield may be reached by modifying the experimental conditions. This will help to further study the biological functions of survivin and its roles in tumor developments.