(国家食品安全风险评估中心 卫生部食品安全风险评估重点实验室,北京 100021)
王佳慧 女 助理研究员 研究方向为食品卫生E-mail:wangjiahui@cfsa.net.cn通信作者:┣┣(中)通信作者┫┫江涛 男 研究员 研究方向为食品卫生E-mail:jiangtao001@cfsa.net.cn
北京市自然科学基金(No.5141002)
(Key Laboratory of Food Safety Risk Assessment of Ministry of Health,China National Center for Food Safety Risk Assessment,Beijing 100021,China)
通过杆状病毒表达系统,获取高纯度GII.3型诺如病毒重组衣壳蛋白,为制备抗GII.3型诺如病毒单克隆和多克隆抗体提供免疫原。方法 将GII.3型诺如病毒衣壳蛋白基因片段修饰后插入pHTA表达载体中,经测序鉴定,将鉴定正确的重组质粒转化到MAX Efficiency DH10BacTM感受态细胞中,获取表达杆粒并转染SF9细胞,表达GII.3型诺如病毒重组衣壳蛋白。重组蛋白用Ni-NTA His蛋白亲和柱纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blot)方法鉴定。结果 SDS-PAGE和Western blot结果表达了分子量约为60 kDa的重组蛋白,动物试验表明重组蛋白具有较好的免疫原性,为制备抗GII.3型诺如病毒单克隆抗体和多克隆抗体、建立相应的免疫学检测方法奠定了基础。结论 构建了GII.3型诺如病毒衣壳蛋白表达载体,并获得GII.3型诺如病毒重组衣壳蛋白。
To express the Norovirus GII.3 capsid protein by gene recombination. Methods The Norovirus GII.3 capsid protein gene was modified and inserted into the pHTA plasmid. The recombinant plasmid was transformed into the MAX Efficiency DH10BacTM competent cell after sequencing and the expression plasmid was obtained. The plasmid was transformed into SF9 cell, and the recombinant capsid protein was expressed. The recombinant capsid protein was purified by Ni-NTA His affinity chromatography purification column followed by identification with sodium dodecyl sufate polyacrylamide gel electrophoresis and Western blot. Results The recombinant capsid protein of about 60 kDa was expressed. It had good immunogenicity, which was verified by animal experiments. The recombinant capsid protein made it possible to prepare monoclonal antibody against Norovirus GII.3 and to develop the immunology detection method.Conclusion The Norovirus GII.3 capsid protein expression plasmid was constructed and the recombinant capsid protein was expressed.
王佳慧,李凤琴,李楠,韩春卉,江涛. GII.3型诺如病毒重组衣壳蛋白的表达和纯化鉴定[J].中国食品卫生杂志,2017,29(1):9-13.
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