【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。更多还原

【Abstract】 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 prokaryotic 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 Immunofluorescence 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 coronaviruses. The expressed recombinant His6-tagged N proteins were identified by Western blot assay with anti-His tag monoclonal antibody. The immunoreactive protein bands with expected 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 serum 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.更多还原