SARS-CoV、HCoV-229E和OC43核衣壳蛋白的克隆表达及抗原相关性分析  

Cloning, expressing and antigenicity analysis of nucleocapsid proteins of SARS-CoV, HCoV-229E and OC43

作　　者：车小燕[1] 廖志勇[1] 王压娣[1] 丘立文[1] 温坤[1] 潘玉先[1] 徐华[1] 梅亚波[1] 郝卫[1] 丁彦青[2] 

机构地区：[1]南方医科大学珠江医院中心实验室,广州510282 [2]南方医科大学病理学教研室,广州510282

出　　处：《中华微生物学和免疫学杂志》2005年第9期711-715,共5页Chinese Journal of Microbiology and Immunology

基　　金：广东省防治非典型肺炎科技攻关项目(200380)

摘　　要：目的克隆表达SARS冠状病毒(SARS-CoV)和人冠状病毒(HCoV-229E、HCoV-OC43)核衣壳(N)蛋白,并对其重组蛋白的抗原相关性进行探讨。方法RT-PCR扩增SARS-CoV、HCoV-229E和HCoV-OC43的N基因全长序列,克隆到原核表达载体pQE30,IPTG诱导表达重组蛋白,用Westernblot和免疫荧光鉴定。结果获得SARS-CoV、HCoV-229E和HCoV-OC43His-N融合蛋白的相对分子质量(Mr)分别约为47×103、44×103、50×103,与相应预测值相符,Westernblot和免疫荧光证实,3种融合蛋白仅与相应的免疫血清特异性结合,3种蛋白的抗原性相互间无交叉反应。结论获得具有免疫原性的SARS-CoV、HCoV-229E和HCoV-OC43的核衣壳融合蛋白,其3种N蛋白抗原性在免疫动物血清中不存在交叉反应。Objective To obtain a recombinant nucleocapsid（N） protein of SARS-CoV, HCoV-229E and HCoV-OC43, respectively and to study antigenic relationships of N proteins between SARS-CoV and human coronaviruses 229E and OC43. Methods The genes encoding the full-length of N proteins from SARS-CoV, HCoV-229E and HCoV-OC43 were amplified by RT-PCR and cloned into the prokaryotie expression vector pQE30. The His6-tagged N proteins were expressed in the M15 strain and further purified with affinity chromatography. The antigenicity of N proteins was analyzed by Western blot and Immunofluorescenee assay. Results The N genes of 1281, 1182 and 1359 bp from SARS-CoV, HCoV-229E and HCoV-OC43, respectively were amplified with their corresponding primer pairs. The recombinant plasmids were sequenced, and they were all in frame with sequences matching those for the N genes of the three eoronaviruses. The expressed recombinant His6-tagged N proteins were identified by Western blot assay with anti-His tag monoclonal antibody. The immunoreactive protein bands with expeeted sizes were 47 kDa, 44 kDa and 50 kDa from SARS-CoV, HCoV-229E and HCoV-OC43, respectively. The nucleocapsid proteins of SARS-CoV, HCoV-229E and HCoV-OC43 strongly and specifically reacted with the virus specific rabbit sermn and with the nucleoprotein specific murine serum. No cross-reactivity was found among the nucleocapsid proteins of SARS-CoV, HCoV-229E and HCoV-OC43. Conclusion The immunogenic nucleocapsid recombinant proteins from SARS-CoV, HCoV-229E and HCoV-OC43 were obtained. There was no antigenic relationship among the three N proteins.

关 键 词：冠状病毒 核衣壳蛋白 抗原性 冠状病毒(SARS-CoV) HCoV-229E 核衣壳蛋白 抗原相关性 克隆表达 相关性分析 OC43 Western RT-PCR扩增 

分 类 号：R392[医药卫生—免疫学]