Objective        To construct and identify a lentiviral expression vector containing the human tumor necrosis factor receptor associated death domain protein (TRADD) gene and to determine the effect of the recombinant lentivirus on the proliferation of fibroblasts derived from hypertrophic scars.        Methods        The TRADD specific fragment was amplified by PCR and cloned into the EcoRⅠsite of the lentiviral vector pLVX-EGF

P-3FLAG-Puro. The recombinant plasmid was obtained and transformed into DH5α competent cells

and then identified by PCR using colonies directly as templates. The positive recombinant clones were detected by DNA sequencing analysis. Consequently

recombinant lentiviruses were produced after the 293FT packing cells were co-transfected with pLVX-TRADD-EGFP-3FLAG-Puro and lentiviral packaging plasmids

while titer of virus was detected by real-time PCR and the expression level of TRADD-GFP-FLag fusion protein was analyzed by Western blotting. MTT assay was used to test the effect of recombinant lentiviruses on the proliferation of hypertrophic scar fibroblasts.        Results        A 1 200-bp strap was obtained from positive clone of colony PCR amplification production after gel electrophoresis

while DNA sequencing confirmed that the positive clone was consistent with that in GenBank. The green fluorescence was observed in 293FT cells under fluorescence microscopy after transfected with recombinat lentiviral vector. Real-time PCR showed the titer of the virus was 3.22×10

8

IU/ml

and Western blotting demonstrated that fusion protein was effectively expressed in 293FT cells. MTT assay indicated that recombinant lentiviruses exerted inhibitory effect on the proliferation of hypertrophic scar fibroblasts.       Conclusion       The recombinant lentiviral vector pLVX-TRADD-EGFP-3FLAG is constructed successfully

and it has inhibitory effect on the proliferation of hypertrophic scar fibroblasts.