Construction，identification And Expression Of PNL-hHGF-EGFP Lentivirus Vector

Objectives:To construct the pNL–EGFP lentiviral vector system containing Humanfull-length HGF cDNA. RT-PCR、ELISA technology was taken to detect the mRNAand protein expression of human hepatocyte growth factor gene in293T cells,whichcan provide a basia for further study of the fuction of the human hepatocyte growthfactor.Methods:1. The cDNA fragment of hHGF gene containing SRα promoter and polyA tailfrom pUC-SRα-HGF was released by restriction endonuclease and cloned tolentivirus vector of pNL-EGFP. The Recombinant lentiviral vector was desinated aspNL-hHGF-EGFP.2. The recombinant lentiviral vector pNL-hHGF-EGFP was transfected to293Tcells. RT-PCR、ELISA technology was taken to detect the expression of humanhepatocyte growth factor gene in293T cells.3. The recombinant lentiviral vector pNL-hHGF-EGFP and packaging plasmidsof pHELPER and pVSVG were co-transfected into293T cells. The supernatant wascollected after48hours and viral particles were recovered by ultra-filtration andconcentration. The titer of lentivirus was detected.Results:1. The hHGF gene fragments were successfully constructed into pNL-EGFPlentivirus vector.Through electrophoresis and restriction enzyme digestion showed that the reconstructed plasmid was consistent with the theoretical fragment.Thesequence result was exactly the same with Genebank.2. After293T cells were transfected with the pNL-hHGF-EGFP vector the greenfluorescent protein was visiable.The RT-PCR analysis confirmed the hHGF genemRNA which can express at24hours.The hHGF protein was52.72±13.09ng/ml at48hour.3. After293T cells were co-transfected with the three plasmids the titer oflentivirus was1.5×106TU/ml.Conclusions:1. pNL-hHGF-EGFP lentivirus vector was successfully construceted.2. The recombinant lentiviral vector pNL-hHGF-EGFP is a functional expressi-on system.3.Obtain a higher titer which could be appiled to further experiments in vivo andin vitro.