中东呼吸综合征冠状病毒N蛋白的原核表达、纯化和鉴定  

Prokaryotic expression, purification and identification of the N protein of middle east respiratory syndrome coronavirus

作　　者：王延群 郝春绪 邓瑶 杨扬 赵莉 沈晓玲[2] 谭文杰 

机构地区：[1]中国疾病预防与控制中心病毒病预防控制所,北京102206 [2]内蒙古医科大学基础医学院微生物学教研室,呼和浩特010110

出　　处：《中华微生物学和免疫学杂志》2014年第10期764-769,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的：对中东呼吸综合征冠状病毒（ middle east respiratory syndrome coronavirus， MERS-CoV）的核衣壳蛋白（ nucleocapsid，N）进行原核表达、纯化和鉴定，了解其免疫原性。方法通过人工合成N基因，构建重组质粒pET30a-MERS-CoV-N，然后转化E．coli BL21（DE3），利用 IPTG进行诱导表达，并优化确定表达条件；采用镍柱亲和层析纯化，SDS-PAGE和Western blot验证。结果原核表达成功并纯化获得与预期大小（46＆#215;103）一致的高纯度可溶性N蛋白。 Western blot表明重组蛋白可与免疫阳性兔血清及His标签抗体发生特异性反应，但与北京地区2008年收集的30份成人血清无反应。结论 MERS-CoV的N蛋白成功在原核系统中实现表达并纯化、鉴定，为MERS-CoV的免疫学诊断和免疫原性研究奠定了基础。Objective To express the N protein of middle east respiratory syndrome coronavirus （ MERS-CoV） in prokaryotic expression system and evaluate the immunogenicity of the purified recombinant N protein.Methods The gene encoding N protein of MERS-CoV was synthesized and cloned into the vector pET30a to construct the recombinant expression plasmid pET30a-MERS-CoV-N.The transformed E.coli BL21 （ DE3） strains carrying expression plasmid were induced by IPTG to express N protein.The expressed protein was purified by using affinity chromatography.SDS-PAGE and Western blot assays were used to iden-tify the expressed N protein and evaluate its immunogenicity.Results The recombinant N protein was suc-cessfully expressed in soluble form with the size of 46＆#215;103 .The results of Western blot assay showed that the recombinant N protein could specifically react with rabbit serum samples positive for antibodies against N protein B-cell epitope peptide and mouse anti-His tag antibodies.No positive band against N protein was found when primary antibody was used with thirty adult serum samples from Beijing in 2008.Conclusion N protein of MERS-CoV was successfully expressed in prokaryotic expression system.The successful expres-sion of N protein laid the foundation for immunological diagnosis of N protein of MERS-CoV and researches on its immunogenicity.

关 键 词：中东呼吸综合征冠状病毒 核衣壳蛋白 原核表达 纯化 鉴定 

分 类 号：R373[医药卫生—病原生物学]