整合H1亚型流感病毒血凝素蛋白伪型病毒的构建

为构建包装含有H1亚型流感病毒HA蛋白的伪型病毒,本研究将人工合成的H1N1流感病毒(A/Califorma/04/2009株)血凝素(Hemagglutinin,HA)基因连接至真核表达载体pcDNA3.1,该重组质粒与表达逆转录病毒相关元件的骨架质粒pHIT111及pHIT60共转染人胚胎肾细胞293T,构建了以鼠白血病病毒为核心、包装含有HA蛋白的伪型病毒.通过对伪病毒感染细胞中LacZ报告基因表达产物的检测,证明伪病毒可以感染MDCK细胞;同时其感染过程可被流感病毒免疫后的小鼠阳性血清所阻断,表明该伪型病毒可模拟野生型病毒完成对宿主细胞的感染过程.本研究所构建的伪病毒系统为研究H1亚型流感病毒HA蛋白抗原特性及新型中和抗体检测方法的建立提供了理想的工具.

Characterization of retroviral pseudotypes with influenza virus hemagglutinin of H1 subtype

In order to develop influenza hemagglutinin(HA) protein-pseudotyped retroviral vector system,the HA gene of an H1 subtype influenza strain(A/Califorina/04/2009) was synthesized and inserted into eukaryotic expression vector pcDNA3.1.The HA-expressing recombinant plasmid was co-transfected into human embryonic kidney(HEK) 293T cells with two murine leukemia virus(MuLV) based retroviral vectors(pHIT60,encoding MuLV Gag-Pol;pHIT111,encoding an MuLV genome with a β-galactosidase reporter gene) for the pseudotyped virus rescue.Results indicated that the β-galactosidase was detected in the pseudotyped virus infected Madin-darby canine kidney(MDCK) cells and the virus infection was able to be neutralized by the influenza virus-specific serum,suggesting that the HA-pseudotyped viral particles mimiced the surface of influenza virions.The pseudotyped virus reported here presents a useful and safe surrogate for the study of antigenic properties of HA protein of H1N1 influenza virus,and for the development of pseudotyped virus-based neutralization assay.