HIV-1 Vif基因的克隆与原核表达

目的克隆HIV-1Vif基因编码区序列,并在原核细胞中表达。方法构建原核表达载体pMRI,将Vif基因序列克隆至该载体中,转化大肠杆菌BL21(DE3),经IPTG诱导,菌体裂解后获得特异性表达蛋白,用组氨酸标签特异性亲和树脂分离纯化该蛋白,并经SDS-PAGE及Westernblot鉴定。结果表达质粒pMRI-Vif经双酶切,可见约600bp的Vif基因片段,测序结果证明质粒构建正确。在大肠杆菌BL21(DE3)中高效表达了可溶性Vif蛋白,纯化后经凝胶自动扫描分析,纯度达85%以上,蛋白浓度约为1g/L。纯化产物具有良好的抗原特异性。结论在大肠杆菌中高效表达了可溶性Vif蛋白。

Cloning and Prokaryotic Expression of HIV-1 Vif Gene

Objective To clone the sequence of coding region of HIV-1 Vif(virus infectivity factor) gene and express in prokaryotic cells. Methods Amplify Vif gene by PCR and clone to prokaryotic expression vector pMRI. Transform the constructed recombinant plasmid pMRI-Vif to E.coli BL21(DE3) for expression under induction of IPTG. Purify the expressed protein by Ni2+-NTA affinity chromatography and identify by SDS-PAGE and Western blot. Results The restriction map of pMRI-Vif showed the target gene fragment at a length of about 600 bp. Sequencing result proved that the recombinant plasmid was successfully constructed. The expressed Vif protein reached a purity of more than 85% and a protein concentration of about 1 g/L. The purified protein showed good antigen specificity. Conclusion Soluble Vif protein was successfully expressed in E. coli.