流感病毒两种反向遗传学操作系统的初步构建

发布时间：2018-11-21 12:06

【摘要】：目的构建依赖辅助病毒和宿主细胞内合成病毒核糖核蛋白复合体(vRNPs)的反向遗传学系统,利用上述两系统表达绿色荧光蛋白(EGFP)。方法将EGFP cDNA精确插入人RNA聚合酶I启动子和小鼠终止子序列之间,构建RNA POLI系统,此系统被克隆入pBluescript II sk(+),获得重组质粒pBluescript II sk(+)-tI-EGFP-PIh;基于辅助病毒H1N1,pBluescript II sk(+)-tI-EGFP-PIh转染MDCK细胞,观察荧光。来源于甲型流感病毒H1N1分离株A/Puerto Rico/8/34的PB2、PB1、PA、NP 4个基因片段cDNA克隆入载体pcDNA3.1(+),获得pcDNA3.1-PB2、pcDNA3.1-PB1、pcDNA3.1-PA、pcDNA3.1-NP,将pBluescript II sk(+)-tI-EGFP-PIh与4个表达质粒共转染293T细胞,观察荧光。结果所构建的载体均经菌液PCR及测序验证正确;两种转染体系均可观察到绿色荧光。结论所构建的依赖辅助病毒和宿主细胞内合成vRNPs的反向遗传学系统均可使绿色荧光蛋白表达。
[Abstract]:Objective to construct a reverse genetic system for the synthesis of viral ribonucleoprotein complex (vRNPs) by co-dependent virus and host cells, and to express green fluorescent protein (EGFP). Using these two systems. Methods EGFP cDNA was inserted precisely between human RNA polymerase I promoter and mouse Terminator, and the RNA POLI system was constructed. The system was cloned into pBluescript II sk (), to obtain the recombinant plasmid pBluescript II sk () tI-EGFP-PIh;. II sk ()-tI-EGFP-PIh was transfected into MDCK cells based on H1-1 pBluescript II sk ()-tI-EGFP-PIh and fluorescence was observed. CDNA cloning of four PB2,PB1,PA,NP fragments from H1N1 isolate A/Puerto Rico/8/34 of Influenza A virus into pcDNA3.1 (), to obtain pcDNA3.1-PB2,pcDNA3.1-PB1,pcDNA3.1-PA,pcDNA3.1-NP, PBluescript II sk ()-tI-EGFP-PIh and four expression plasmids were co-transfected into 293T cells to observe the fluorescence. Results the constructed vectors were proved to be correct by PCR and sequencing, and the green fluorescence could be observed in the two transfection systems. Conclusion the constructed reverse genetic system of co-virus and host cell vRNPs synthesis can make green fluorescent protein express.
【作者单位】：暨南大学药学院;深圳市疾病预防控制中心;中山大学公共卫生学院;
【基金】：深圳市科委基础研究项目(JCYJ20140410164811662)资助~~
【分类号】：R373.13

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