携带绿色荧光蛋白基因重组猫泛白细胞减少症病毒的拯救

Rescue of Recombinant Feline Panleukopenia Virus Carrying GFP Gene

本研究旨在构建携带绿色荧光蛋白(green fluorescent protein,GFP)的重组猫泛白细胞减少症病毒(feline panleukopenia virus,FPV),以便更快速有效的检测中和抗体。参照GenBank中GFP基因序列设计特异性引物,应用PCR技术扩增获得携带AgeⅠ和NheⅠ酶切位点的GFP基因,并对pFPV质粒进行定点突变,从而获得AgeⅠ和NheⅠ酶切位点,然后用AgeⅠ和NheⅠ同时双酶切pFPV和GFP基因,经4℃过夜连接、转化,成功构建携带GFP基因的重组猫泛白细胞减少症病毒质粒pFPV-GFP,应用脂质体转染法将重组质粒pFPV-GFP导入猫肾细胞(F81),利用GFP独特的发光机制可在荧光显微镜下对标记的重组病毒rFPV-GFP进行细胞内活体观察,利用Western blotting鉴定重组病毒rFPV-GFP中GFP的表达情况。结果显示,重组质粒pFPV-GFP转染F81细胞24 h时可观察到绿色荧光蛋白,且随着转染时间的延长观察到的绿色荧光蛋白数量增多,说明重组质粒pFPV-GFP成功转染到F81细胞中,Western blotting鉴定出GFP在重组病毒rFPV-GFP中稳定表达。本研究通过在pFPV上成功插入GFP并拯救出病毒,说明pFPV上可以插入外源基因,为其他外源基因在pFPV上的插入奠定基础,同时GFP的插入为FPV的研究提供了更加有利的研究工具,为FPV的基础研究以及中和抗体的快速检测奠定基础。

The purpose of this study was to construct a recombinant feline panleukopenia virus(FPV)carrying green fluorescent protein(GFP)in order to detect neutralizing antibody more quickly and effectively.GFP specific primers were designed by referring to the GFP gene sequence in GenBank,and the GFP gene carrying AgeⅠand NheⅠdigestion sites was amplified by PCR,and plasmid pFPV preserved in laboratory was subjected to site-directed mutagenesis to obtain the AgeⅠand NheⅠrestriction sites.pFPV and GFP genes were double-digested with AgeⅠand NheⅠ,and ligated at 4℃overnight and transformed to obtain the recombinant plasmid pFPV-GFP.The recombinant plasmid pFPV-GFP was successfully transfected into feline kidney cells(F81)by Lipofection 3000.Using the unique luminescence mechanism of GFP,the labeled recombinant virus rFPV-GFP could be observed in vitro under fluorescence microscope,and Western blotting was used to identify the expression of GFP in the recombinant virus rFPV-GFP.The results showed that green fluorescent protein could be observed when the recombinant plasmid pFPV-GFP was transfected into F81 cells for 24 hours,and with the extension of transfection time,the amount of green fluorescent protein was increased,indicating that the recombinant plasmid pFPV-GFP was successfully transfected into F81 cells and GFP was stably expressed in the recombinant virus rFPV-GFP.In this study,GFP was successfully inserted into pFPV and rescued recombinant virus,indicating that foreign genes could be inserted into pFPV,laying a foundation for the insertion of other foreign genes into pFPV.At the same time,GFP insertion provided a more favorable research tool for the research of FPV,which laid the foundation for the basic research of FPV and the rapid detection of neutralization antibodies.