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Cloning of survivin- an inhibitor of apoptosis proteins and expressed in pichia pastoris system
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- Author:
- No author available
- Journal Title:
- CANCER RESEARCH AND CLINIC
- Issue:
- 5
- DOI:
- 10.3760/cma.j.issn.1006-9801.2005.05.005
- Key Word:
- Survivin;凋亡抑制;Pichia pastoris表达;肿瘤
Abstract： 目的克隆凋亡抑制因子Survivin基因,利用甲醇酵母Pichia pastoris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(4 mg/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,48 h的表达量最高.结论用Pichia pastoris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.
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