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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。
Objective To construct a reverse genetics system that relies on helper viruses and synthetic viral ribosomal protein complexes (vRNPs) in host cells and express green fluorescent protein (EGFP) using these two systems. Methods The RNA POLI system was constructed by inserting the EGFP cDNA between human RNA polymerase I promoter and mouse terminator. The system was cloned into pBluescript II sk (+) to obtain recombinant plasmid pBluescript II sk (+) - tI -EGFP-PIh; MDCK cells were transfected with helper virus H1N1 and pBluescript II sk (+) - tI-EGFP-PIh to observe the fluorescence. The 4 cDNA fragments of PB2, PB1, PA and NP from A / Puerto Rico / 8/34 of influenza A virus H1N1 isolate were cloned into pcDNA3.1 (+) vector to obtain pcDNA3.1-PB2 and pcDNA3.1 293T cells were co-transfected with pBluescript II sk (+) - tI-EGFP-PIh and four expression plasmids respectively. Fluorescence was observed. Results The constructed vectors were verified by bacterial PCR and sequencing. Green fluorescence was observed in both transfection systems. Conclusion The constructed reverse genetics system that relies on helper virus and vRNPs synthesized in host cells can express green fluorescent protein.