EB病毒LMP1 C端重组蛋白多克隆抗体的制备和鉴定  

Expression of EBV LMP1 C-terminal protein and preparation of its polyclonal antibody

作　　者：毛珊珊[1] 王路得 周萌 吕开绩 朱进顺 Kamara Saidu 叶晓鲜 张丽芳[1] MAO Shanshan;WANG Lude;ZHOU Meng;LYU Kaiji;ZHU Jinshun;KAMARA Saidu;YE Xiaoxian;ZHANG Lifang(Institute of Molecular and Immunology,Department of Medical Microbiology and Immunology,Wenzhou Medical University,Wenzhou,32503)

机构地区：[1]温州医科大学分子病毒与免疫研究所病原生物与免疫学系,浙江温州325035

出　　处：《温州医科大学学报》2018年第7期469-473,共5页Journal of Wenzhou Medical University

基　　金：国家自然科学基金资助项目(81172463;81372447)

摘　　要：目的:制备EB病毒(EBV)潜伏膜蛋白1(LMP1)C端(187～386)的原核表达蛋白及其多克隆抗体并检验其生物学特性。方法:选择LMP1C端的基因并进行原核密码子优化,全基因合成pET21a(+)/EBVLMP1C端重组质粒,并转入E.coliBL21(DE3)感受态细菌进行诱导表达重组蛋白,利用His标签提取纯化重组蛋白并经SDS-PAGE及Westernblot分析鉴定。将纯化的LMP1C端重组蛋白免疫日本大耳白兔,获得多克隆抗体,分别采用ELISA、Westernblot及免疫荧光方法对兔多克隆抗体进行分析。结果:EBVLMP1C端(187～386)重组蛋白可经原核表达系统表达;通过免疫兔获得了特异性的多克隆Ig G抗体;制备的兔多克隆抗体可特异性结合并识别LMP1C端蛋白。结论:EBVLMP1C端重组蛋白免疫原性较强,制备的兔多克隆抗体效价高、特异性强。Objective： To prepare EBV LMP1 C-terminal（187-386 aa） protein by prokaryotic expression system and polyclonal antibody against it and verify its biological character. Methods： A prokaryotic vector p ET21 a（＋）/EBV LMP1 C-terminal（187-386 aa） was constructed and transformed into E.coli BL21（DE3）, and then the recombinant protein was induced by IPTG and purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis demonstrated the purified LMP1 C-terminal protein was expressed correctly. To obtain the polyclonal antibody, the rabbit was immunized with the purified LMP1 C-terminal recombinant protein. An analysis of polyclonal antibody was made by ELISA, Western blot and immunofluorescence technique. Results：The recombinant protein of LMP1 C-terminal was successfully expressed and purified by the prokaryotic expression system. The polyclonal antibody was obtained by immunizing the rabbits with the recombinant protein. Conclusion： The recombinant protein of LMP1 C-terminal has strong immunogenicity and the polyclonal antibody of LMP1 C-terminal has high titer and specificity.

关 键 词：EB病毒 LMP1蛋白 多克隆抗体 

分 类 号：R373.1[医药卫生—病原生物学]