HIV-1 Vif与ElonginC蛋白在大肠杆菌中的共表达

目的在大肠杆菌中共表达HIV-1Vif与ElonginC蛋白。方法PCR扩增ElonginC基因全长DNA片段,克隆入pMDT-easy载体,经NdeⅠ/BamHⅠ双酶切后,与质粒pRSETB连接,构建质粒pRSETB-ElonginC。将质粒pMRI-Vif转化大肠杆菌BL21(DE3),获得表达菌株,再将此菌株制备成感受态细胞,用pRSETB-ElonginC质粒转化该细胞,经1mmol/L IPTG诱导,表达产物经SDS-PAGE及Western blot鉴定。结果质粒pRSETB-ElonginC经NdeⅠ/BamHⅠ双酶切,可切出约3000和400bp的2条片段,测序证明质粒构建正确。在大肠杆菌中共表达了Vif与ElonginC蛋白,该蛋白具有良好的抗原特异性。结论在大肠杆菌中共表达了Vif与ElonginC蛋白。

Coexpression of Vif and ElonginC Proteins of HIV-1 in E. Coli

Objective To coexpress Vif and ElonginC proteins of HIV-1 in E. coli. Methods Amplify the full-length of DNA encoding ElonginC protein by PCR and clone into plasmid pMDT-easy. Digest the constructed recombinant plasmid with NdeⅠ/ BamHⅠ, and insert the obtained target gene into plasmid pRSETB to construct recombinant plasmid pRSETB-ElonginC. Transform recombinant plasmid pMRI-Vif constructed previously to E. coli BL21(DE3), and prepare the obtained recombinant E. coli into competent cells. Transform recombinant plasmid pRSETB-ElonginC to the prepared competent cells for expression under induction of 1 mmol / L IPTG. Identify the expressed product by SDS-PAGE and Western blot. Results Two gene fragments, at lengths of about 3 000 and about 400 bp respectively, were obtained by digestion of pRSETB-ElonginC with Nde Ⅰ/ BamHⅠ, which proved that the recombinant plasmid was constructed correctly. SDS-PAGE and Western blot proved that both Vif and ElonginC proteins were expressed and showed good antigenic specificity. Conclusion Vif and ElonginC proteins were coexpressed in E. coli.