重组人乳头瘤病毒16型E6蛋白的表达、纯化和动物免疫效果分析

目的 诱导表达人乳头瘤病毒16型(HPV16)E6蛋白,制备可溶性蛋白,并通过动物免疫进行免疫效果评估.方法 采用IPTG诱导pQE30-HPV16E6/BL21(DE3)重组菌株,表达产物经SDS-PAGE和Western blot分析鉴定.提取包涵体进行变性处理,变性蛋白经Ni2+金属螯合层析纯化,将纯化产物用复性透析方法处理以获取可溶性蛋白.免疫Bal B/c小鼠,检测血清抗体、CD4+/CD8+和IFN-γ.结果 诱导后重组菌有相对分子量18 000蛋白质表达,以不溶性包涵体为主要表达方式.目的蛋白可与HPV16E6多克隆抗体识别,纯化和透析处理后得到可溶性的蛋白.小鼠免疫后血清抗体滴度升高,淋巴细胞CD4+/CD8+升高,IFN-γ未见升高.结论 成功表达了HPV16E6蛋白,同时处理包涵体并制备了可溶性目的蛋白.目的蛋白可刺激小鼠体内产生有效的免疫反应,该蛋白的成功制备为患者血清抗体的检测和肿瘤细胞杀伤的免疫研究奠定了坚实基础.

Expression,purification and immunization assay of human papillomavirus type 16 E6 protein in E.coli

Objective To induce prokaryotie-expressed HPV16 E6 protein,produce soluble protein and is evaluate these protein by animal immunization.Methods The recombinant strains,pQE30-HPV16E6/BL21(DE3)were induced with IPTG.The protein product Was analyzed by SDS-PAGE and Western blot.HPV16E6 protein was purified from insoluble inclusion bodies by degeneration and Ni2+ affinity chromatogra-phy treatment.The soluble protein Was obtained from purified protein through degeneration and dialysis.By immunizing BAL B/c mice,the sera antibody,CO4+/CO8+ and ZNF-γ of the mice immunized were defee-ted.Results A Mr 18 000 band Was observed in SDS-PAGE.The specificity of target protein Was con-firmed by HPV16E6 polyelonal antibody.Soluble protein was obtained from inclusion bodies treated.There is no change in INF-γ detection. Both era antibody fiter and CD4+/CD8+ both increased. Conclusion HPV16E6 Was expressed successfully.The soluble protein,produced from inclusion bodies,induced the ef-feetive immunization reaction in Bal B/c mice.The production of HPV16 E6 protein has established founda-tion for further research of sera antibody detection in patients and tumor CTL immunology study.