重组HIV-1 CN54 Nef抗原的制备及鉴定

目的原核表达HIV-1CN54调控蛋白Nef,并进行纯化、复性及鉴定。方法将CN54 Nef基因克隆至pThio-HisA载体,构建非融合表达载体,转化大肠杆菌BL21 Codonplus-RIL,IPTG诱导表达,通过尿素梯度洗涤包涵体纯化Nef蛋白,透析复性。Western blot检测目的蛋白的特异性。结果Nef蛋白以包涵体的形式表达,表达量占菌体总蛋白的40%以上。用尿素洗涤法可直接纯化Nef,纯度达到90%以上。Nef蛋白透析后溶解于PBS缓冲液中。Western blot检测显示了良好的特异性。结论HIV-1 CN54 Nef抗原蛋白可在大肠杆菌中高效表达,采用的纯化和复性Nef蛋白的工艺简便、快捷,为开发针对Nef基因的HIV-1疫苗奠定了基础。

Preparation and Identification of Recombinant HIV-1 CN54 Nef Antigen

Objective To express the regulatory protein Nef of HIV-1 CN54 strain in prokaryotic cells,and purify,re-naturalize and identify the expressed product.Methods Clone CN54 Nef gene into vector pThioHisA and transform the constructed recombinant plasmid to E.coli BL21 Codonplus-RIL for expression under induction of IPTG.Purify the expressed Nef protein by gradient washing of inclusion body with different concentrations of urea,then re-naturalize by dialysis and test for specificity by Western blot.ResultsThe expressed Nef protein,in a form of inclusion body,contained more than 40% of total somatic protein and reached a purity of more than 90% after purification by urea gradient washing.After dialysis,Nef protein was dissolved in PBS.Western blot proved good specificity of expressed Nef protein.Conclusion HIV-1 CN54 Nef antigen was highly expressed in E.coli,the procedure for purification and re-naturalization of Nef protein was simple and rapid.The study laid a foundation of developing HIV-1 DNA vaccine.