Construction and expression of a recombinant eukaryotic vector containing testis-specific gene T490 and EGFP genes

Objective To construct a over-expression plasmid T490-EGFP,which contains a enhanced green fluorescent protein(EGFP) report gene and a testis-specific gene T490,and detect the expression of T490 in Hela cells. Methods Gene T490 was amplified by RT-PCR from a adult Balb/C mice testes,and then it's product was cloned to eukaryotic expression vector pEGFP-N1 to form a fusion gene T490-EGFP,then this fusion gene was inserted into pcDNA3.1(-) vector to form pcDNA3.1-T490-EGFP.The recombinant vector pcDNA3.1-T490-EGFP was transfected into Hela cells with Lipofectamine 2000.The expression of T490-EGFP fusion protein was observed under fluorescent microscope and mRNA expression of T490 was detected by RT-PCR.Resulets cDNA of T490 was obtained by RT- PCR.The T490-EGFP fusion gene was successful cloned into the pcDNA3.1(-) vector which was confirmed by DNA sequencing and PCR.Green fluorescence was very strong in transfected Hela cells with T490-EGFP after 24h transfection. and mRNA overexpression of T490 was also found in transfected Hela cells.Conclusion Recombinant plasmid pcDNA3.1(-)-T490-EGFP was successfully constructed and T490 gene was expressed strongly in transfected Hela cells.These results might provide a tool for biological function analysisof the testis-specific gene T490.