新疆出血热病毒核蛋白多克隆抗体的制备及其免疫学评价  

Preparation of polyclonal antibody to nucleoprotein from Xinjiang hemorrhagic fever virus and its immunological evaluation

作　　者：刘东亮[1] 李阳[1] 赵晶[1] 吴婷[1] 孙素荣[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源与基因工程重点实验室,新疆乌鲁木齐830046

出　　处：《细胞与分子免疫学杂志》2013年第8期838-841,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81060129,30860255);新疆生物资源基因工程重点实验室开放课题(XJDX020l-2011-01)

摘　　要：目的原核细胞中表达、纯化新疆出血热病毒BA88166毒株核蛋白(NP)并制备及鉴定抗NP蛋白的多克隆抗体。方法采用逆转录-聚合酶链式反应(RT-PCR)扩增出BA88166毒株S基因的cDNA片段,将其构建到原核表达载体pET-32a上,形成重组原核表达质粒pET-88166S。构建好的质粒在大肠杆菌BL21(DE3)中进行诱导表达,经镍柱亲和层析法纯化NPHis融合蛋白,SDS-PAGE分析蛋白相对分子质量(M r)。用该纯化蛋白免疫新西兰白兔制备抗血清,ELISA和Western blot法检测血清效价和特异性。结果双酶切鉴定和DNA测序证实构建的pET-88166S重组表达载体正确,目的基因序列与GenBank中公布的序列一致,在E.coli BL21中表达的NP-His融合蛋白经SDS-PAGE分析,其M r约为66 000。ELISA检测抗体效价高于1∶25 600,蛋白免疫印迹实验结果表明抗体能特异性识别新疆出血热病毒YL04057毒株的NP蛋白及其截短蛋白。结论成功获得新疆出血热病毒NP-His融合蛋白,得到了特异性兔抗NP蛋白多克隆抗体。Objective To express and purify the nucleoprotein （NP） from Xinjiang hemorrhagic fever virus （XHFV） strain BA88166 in E. coil, and prepare and identify its polyclonal antibody. Methods The cDNA of S gene segment of BA88166 strain was amplified by RT-PCR and cloned into prokaryotic expression vector pET-32a to generate a recombinant plasmid named pET-88166S. The pET-88166S was transformed into E. coli BL21 （ DE3 ）. The NP-His fusion protein was induced by IPTG, purified by Ni-NTA purification system, and analyzed by SDS-PAGE. To prepare the antiserum, New Zealand white rabbits were immunized with the purified NP-His protein. The titer and specificity of the antiserum to NP were analyzed by ELISA and Western blotting, respectively. Results Restriction endonuclease analysis and DNA sequencing showed that the prokaryotic expression vector of pET-88166S was constructed successfully. NP-His fusion protein was expressed in E. coli BL21 （DE3） after IPTG induction and its relative molecular mass （Mr） was about 66 000. ELISA and Western blotting showed that the titers of the antisera were above 1：25 600, and that the antisera can specifically bind with the entire and truncated NP protein of XHFV strain YL04057. Conclusion NP-His fusion protein can be successfully expressed in E. coli and the specific anti-NP rabbit polyclonal antibody has been obtained, which will provide the basic information for the studies on the diagnosis, treatment and prevention of Xinjiang hemorrhagic fever.

关 键 词：新疆出血热病毒 核蛋白 原核表达 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学] R373.32[医药卫生—基础医学]