Preparation of Recombinant Retrovirus siRNA of BclGL Gene and Its Effect on Jurkat T Cell Proliferation

Objective: To prepare the recombinant retrovirus siRNA of BclGL gene and to determine the effect of the retrovirus on Jurkat cell proliferation.Methods: Three sequences corresponding to human BclGL genes were designed on Ambion'S Website.Three complementary DNA fragments were synthesized and cloned into the sites of BamHⅠand Hind Ⅲ of the pSilencer 5.1-H1 retrovirus vector for constructing the recombinant retrovirus plasmid.Then these plasmids expressing the 3 diffe-rent siRNAs were packaged through PT67 cells in order to prepare the recombinant retrovirus expressing siRNA of BclGL gene.Retrovirus serum was collected and used to directly transfect Jurkat cells.After puromycin screening,cells with stable expression of siRNA were cultured.The silencing effects of BclGL gene in these three groups were determined by real time RT-PCR and Western blot assay.Proliferation of Jurkat cells were detected by CCK-8 assays.Results: Three complementary DNA fragments were successfully cloned into pSilencer 5.1~H1 retrovirus vector separately.The siRNA expressed by recombinant retrovirus effectively interfered BclGL gene expression in Jurkat cells.CCK-8 assay showed that proliferations of BclGL-interfered cells were increased.Conclusion: The recombinant human BclGL siRNA expression retrovirus has been prepared successfully and the downregulation of BclGL expression could increase the proliferation of Jurkat cells in vitro,which is the basis for further study of molecular functions of BclGL.