摘要
目的:SARS冠状病毒(SARS-CoV)核衣壳蛋白(N蛋白)在杆状病毒-昆虫系统的表达,研究其抗原性。方法:利用PCR方法扩增SARS-NP全长基因,使用BamHⅠ和SalⅠ限制性内切酶定向插入Bac-to-Bac系统的pFastBac HTC载体,构建重组质粒转化DH10Bac细胞,获得重组杆粒DNA后转染Sf9细胞,在High Five细胞中表达SARS-NP;表达产物经NiNTA亲和层析纯化及免疫印迹和ELISA方法验证抗原性。结果:成功构建pFastBac HTC-SARS-NP重组质粒,并在High Five细胞高效表达,获得相对分子量约48 kD的重组蛋白质。经验证,重组蛋白能被SARS-CoV NP的特异性单抗(8E1A17)和兔免疫血清识别,能与SARS患者血清反应,与健康人血清、人冠状病毒229E型和OC43型的杂交瘤培养上清、相对应的兔免疫血清、患者血清均无交叉反应,具良好抗原反应性。结论:SARS-NP成功表达于杆状病毒-昆虫系统,具较强抗原反应性,初步认为其与229E型和OC43型感染血清无交叉反应性。
Objective：To express SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus Expression System and analyze the antigenicity of the recombinant protein.Methods： The SARS-CoV nucleocapsid gene was amplified by PCR.The PCR product digested with BamHⅠand SalⅠrestriction endonucleases was cloned into vector pFastBac HTC of Bac-to-Bac Baculovirus expression system.Recombinant plasmid was transformed DH 10Bac cells to obtain the recombinant Bacmid DNA.Recombinant Bacmid DNA was transferred into Sf9 cells which were inducted to express the recombinant protein in High Five cells.After purified by Ni affinity chroma-tography ,the antigenicity of the recombinant protein was analyzed by Western blot and ELISA.Results：Recombinant plasmid was con-structed successfully.The recombinant protein with the relative molecular mass of 48 kD was efficiently expressed in High Five cells and purified successfully by Ni affinity chromatography.Western blot and ELISA analysis showed that the recombinant protein could be spe -cifically recognized by the monoclonal antibody to SARS-CoV N protein and immune serum from rabbits ,respectively.The recombinant protein can specifically reacted with serum from SARS patients ,not with serum from healthy persons and patients infected with hCoV-229 E and hCoV-OC43.Conclusion： SARS-CoV nucleocapsid protein has been expressed successfully in the Bac-to-Bac Baculovirus Expression System ,and obtained good antigenicity.It is preliminary deemed that it can＆#39;t reacted with serum from patients infected with hCoV-229E and hCoV-OC43.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2014年第6期721-725,共5页
Chinese Journal of Immunology
基金
国家重大科技专项课题(2012ZX10004-213)
广东省高等学校珠江学者岗位计划资助项目(2009GDUPS)
