改进型反向基因操作系统的构建和流感病毒变异株的选育

Construction of an improved reverse genetics and generation of attenuated influenza virus

目的构建一个新型的反向基因操作系统,并利用该技术获得流感病毒变异株。方法用分子生物学方法把表达蛋白载体(pQBI50_BFP_fA1)和转录基因载体(pHH21)两个载体有机地整合,构建出既能表达病毒蛋白又能转录基因的单一载体(pPol_Ⅱ/Ⅰ);同时通过RT_PCR提取A/Ann Arbor/6/60病毒的cDNA,然后把8个病毒基因分别克隆到pPol_Ⅱ/Ⅰ载体;通过点变异和基因删除方法对M和NS基因进行改进;用细胞培养和细胞免疫染色法测定重组病毒的生物特征。结果成功构建了由8个小质粒组成的转染细胞能力较强的一个新型反向基因操作系统。同时在M和NS基因上加以分子生物学的改进,并获得2个对温度敏感和增殖力低的新毒株。结论一个改进型反向基因操作系统被成功建立,且利用这个系统拯救出2个流感变异株。这个系统不仅在流感病毒的分子基础研究而且将在减毒性活疫苗的研发上发挥重要作用。这2个流感变异株作为减毒性活疫苗的候选株具有较好的开发前景。

Objective To construct a novel reverse genetics, which is not only a core technology for influenza virus research, but also an important technique flat roof on the developing of attenuated Flu vaccine. Methods With a serial molecular biologic technology, it is constructed a single vector （ pPol-Ⅰ/Ⅱ ） which can express viral protein and transcribe viral RNA from combined two vectors pQB150-BFP-fA1 and pHH21, while as isolated cDNA from A/Ann Arbor/6/60 by a RT-PCR and then cloned eight fragment viral gene to the pPol-Ⅰ/Ⅱ vector. It is also improved M and NS genes by a site mutation and a gene deletion method. Finally, it is detected the biologic character by a cell culture and an immunostaining method. Results In this study, we have constructed a new reverse genetics system, which consist of eight small plasmids, so that it increased transfect activity to cells. On other hand, we further improved viral gene in M and NS genes, and then get two attenuated viruses by the reverse genetics. Conclusions An improved reverse genetics for influenza virus has been constructed, and further obtained two attenuated viruses by this system. The reverse genetics will play an important role on the basic research for influenza virus and the developing for Flu vaccine. The two attenuated viruses will have a better developing foreground for using as candidate strains of attenuated Flu live vaccine.