Chen Ai-jun, Guo Jian-qiang, Yao Li-hong, Cheng Cong-sheng, Liu Xiao-yu, Fu Jin-qi, Xu Peng-wei, Zhang Zhi-qing
State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2011 Aug;25(4):254-7.
To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.
M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.
The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.
These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.
构建表达人H5N1流感病毒M1蛋白的大肠杆菌载体，为检测人H5N1流感病毒及研究M1蛋白的生物学功能提供有用工具。
以流感病毒基因片段7为模板，通过PCR扩增M1基因片段，并将其亚克隆到pQE80-L载体中。将重组质粒pQE80-L/M1转化至大肠杆菌BL21(DE3)菌株。用异丙基-β-D-硫代半乳糖苷诱导M1表达。用Ni2+亲和层析法纯化带有多组氨酸标签的重组M1蛋白。用纯化的M1蛋白免疫小鼠以制备抗M1抗体。
重组M1蛋白能被抗H5N1亚型流感病毒抗血清识别，在免疫动物中引发特异性抗体。
这些结果证实我们成功构建了表达人H5N1流感病毒M1蛋白的大肠杆菌载体。
