凋亡抑制蛋白Livin-α、Livin-β的原核表达及纯化

摘要

目的：对凋亡抑制蛋白Livin的两种异构体（ Livin-α和Livin-β）进行原核表达，对获得的Livin蛋白进行纯化。方法：以Hela细胞总RNA为模板，RT-PCR获得Livin-α和Livin-β基因全长cDNA序列，酶切后，插入原核表达载体pET32a（+）的多克隆位点，经酶切、PCR鉴定、测序后，构建Livin基因异构体表达载体pET32a（+）-Livin-α和pET32a（+）-Livin-β。将重组质粒转入表达菌株BL21，诱导表达，收集菌液，超声碎菌，取其上清和沉淀分别进行 SDS-PAGE电泳，并对获得的 Livin蛋白进行纯化。结果：成功获得Livin-α和Livin-β全长cDNA序列，克隆到原核表达载体pET32a（+）上后进行诱导表达，获得大小为55kD左右的可溶性融合蛋白，经纯化后，有效减少了杂蛋白的含量。结论：Livin基因异构体原核表达载体的构建及其融合蛋白的制备、纯化，为进一步将Livin基因的两种异构体蛋白应用于抗体制备以及各类肿瘤的临床辅助诊断研究奠定了基础。%Objective:To use the prokaryotic expression system to express the protein of Livin-αand Livin-β, and purify the protein of Livin-αand Livin-βby Ni-NTA Superflow system. Methods:Firstly,total RNA of Hela cell was extracted and the cDNA of Livin isoforms were coloned by RT-PCR. Then,the cDNA of Livin isoforms were inserted into pET32a( +)vector and the recombinant plasmids pET32a( +)-Livin-αand pET32a( +)-Livin-β were constructed. Finally,the Bacterium coli pET32a( +)-Livin-α/BL21 and pET32a( +)-Livin-β/BL21 were induced by IPTG and the expression of Livin-αand Livin-βprotein were analysed by SDS-PAGE,the protein of Livin-αand Livin-βwere purified by Ni-NTA Superflow system. Results:Full length cDNA of Livin-α and Livin-βwere cloned respectively and the fragment of Livin-αand Livin-βgenes were inserted into pET32a ( +)vector successfully. Fusion protein of Livin-α and Livin-β were expressed by pET32a( +)prokaryotic ex-pression system,the protein of Livin-α and Livin-β were purified successfully. Conclusion:The expression and purification of Livin-α and Livin-βprotein is essential to study the application prospect of Livin-αand Livin-βprotein in cancer diagnosis.