To construct overexpression lentiviral-based vector carrying rat B7x gene,B7x gene precursor sequences amplified by polymerse chain reaction（PCR） were ligated with pLVTHM to generate pLVTHM-B7x gene expression lenti...To construct overexpression lentiviral-based vector carrying rat B7x gene,B7x gene precursor sequences amplified by polymerse chain reaction（PCR） were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector.The positive clones were selected to be submitted to DNA sequencing.HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene.The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells.The B7x protein levels were detected by Western blot analysis in HEK293T cells.The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed.Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL.It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.展开更多
基金Supported by the National Natural Science Foundation of China(No.30870354)the Fund of Jilin Provincial Science and Technology Services,China(Nos.20070720,200805120,20090732)the Jilin Provincial Development and Reformation Committee Fund,China(No.2009Y042J12314)
文摘To construct overexpression lentiviral-based vector carrying rat B7x gene,B7x gene precursor sequences amplified by polymerse chain reaction（PCR） were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector.The positive clones were selected to be submitted to DNA sequencing.HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene.The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells.The B7x protein levels were detected by Western blot analysis in HEK293T cells.The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed.Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL.It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.
