论文部分内容阅读
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 Survivin gene, an inhibitor of apoptosis, and express Survivin protein using Pichia pastoris eukaryotic expression system. Methods Survivin gene was amplified by PCR after Survivin specific primers were amplified and sequenced. Survivin gene with the correct sequence was recombined with the secretion expression vector pPic9k. The recombinant plasmids were linearized by electroporation and transferred into yeast host strain GS115 / His -, were screened with G418 / YPD plates at various concentrations. The positive clones were identified by PCR. The expressed proteins were induced by BMMY containing 1% methanol and the expression products were detected by ELISA. Results The Survivin gene was cloned in the same way as the gene sequence of Survivin in GenBank without any mutation. The eukaryotic expression vector pPic9k-Survivin was constructed and transformed into yeast GS115 / His-. ) G418 positive clones (GS115 / His +) 6 and identified by PCR method; the expression of Survivin with time, the highest expression 48h. Conclusion The method of expressing Survivin protein by Pichia pastoris eukaryotic expression system is feasible. Survivin protein can be overexpressed if conditions are further optimized, which provides an important experimental basis for further study on the biological function of Survivin and its role in tumorigenesis.