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  

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

作　　者：商庆龙[1] 王燕[1] 陈思佳[1] 李承刚[1] 韩聪[1] 邵迪[1] 李茉[1] 谷鸿喜[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室,黑龙江省普通高校病原生物学重点实验室,150081

出　　处：《国际免疫学杂志》2008年第4期244-247,共4页International Journal of Immunology

基　　金：黑龙江省科技攻关项目（GB02C111）;哈尔滨市科委重大攻关项目（2004AA3CS176-1）;黑龙江省卫生厅资助项目（2005-12）;黑龙江省教育厅资助项目（10553046）

摘　　要：目的诱导表达人乳头瘤病毒16型（HPVl6）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-HPVl6E6／BL21（DE3）重组菌株，表达产物经SDS-PAGE和Westernblot分析鉴定。提取包涵体进行变性处理，变性蛋白经Ni^2＋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BalB／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18000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。Objective To induce prokaryotic-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 chromatography 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 defected. Results A Mr 18 000 band was observed in SDS-PAGE. The specificity of target protein was confirmed by HPyl6E6 polyclonal antibody. Soluble protein was obtained from inclusion bodies treated. There is no change in INF-γ detection. Both era antibody titer and CD4^＋/CD8^＋ both increased. Conclusion HPV16E6 was expressed successfully. The soluble protein, produced from inclusion bodies, induced the effective immunization reaction in Bal B/c mice. The production of HPV16 E6 protein has established foundation for further research of sera antibody detection in patients and tumor CTL immunology study.

关 键 词：人乳头瘤病毒16型 E6蛋白 纯化 免疫 

分 类 号：R737[医药卫生—肿瘤]