| Objective To investigate the effects of BamHI A rightward open-reading frame 1(BARF1)gene on proliferation of gastric epithelial cells,so as to understand its roles in the EBV-associated gastric carcinoma (EBVaGC).Methods(1)The specific primers were designed according to the sequence of EBV BARF1 in GenBank to amplify the open reading frame cDNA by RT-PCR.The PCR products were ligated into pGEM-T Vector carrying ampicillin resistance gene and transformed into competent E.coli DH5α. The positive clones were identificated by double restriction enzyme digestion and sequencing.The BARF1 gene fragment was directly inserted into eukaryotic expression vector pSG5 and confirmed by double restriction enzyme digestion.The recombinant plasmid was transformed into E.coli DH5αand the positive clone was selected by Amplicillin fastness and was identificated by double restriction enzyme digestion.(2)The recombinant eukaryotic expression vector pSG5-BARF1 was transfected into gastric carcinoma cell line SGC-7901 by Lipofectamine 2000,and the expression of BARF1 was confirmed by RT-PCR.The speed of cell proliferation was analyzed by cell counting.Results (1)A 696bp of BARF1 ORF was obtained through RT-PCR and successfully inserted eukaryotic expression vector pSG5,and pSG5-BARF1 was proved to be correct by restriction analysis and sequencing.(2)BARF1 expression was confirmed by RT-PCR in pSG5-BARF1 transfected gastric carcinoma cell line SGC-7901.(3)The promotion of cell proliferation of BARF1 was demonstrated by cell counting.Conclusion(1)We have successfully cloned the BARF1 gene of Epstein-Barr virus.(2)We have constructed the eukaryotic expression vector pSG5-BARF1.(3)BARF1 mRNA has been expressed in the pSG5-BARF1 transfected gastric carcinoma cell line SGC-7901.(4)BARF1 expression promotes the proliferation of SGC-7901.
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