贵州医科大学学报

2009, v.34;No.138(03) 250-254

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人BclG_L基因siRNA逆转录病毒的制备及其对T淋巴细胞增殖的影响
Preparation of Recombinant Retrovirus siRNA of BclGL Gene and Its Effect on Jurkat T Cell Proliferation

罗娜;郭晟;杨承英;费蕾;吴胜昔;陈永文;吴玉章;郝飞;
LUO Na1,GUO Sheng2,YANG Chengying2,FEI Lei2,WU Shengxi2,CHEN Yongwen2,WU Yuzhang2,HAO Fei1 (1.Deparment of Dermatology,Xinqiao Affiliated Hospital of The Third Military Medical University,Chongqing 400038,China;2.PLA Institut of Immunology,The Third Military Medical University,Chongqing 400038,China;)

摘要(Abstract):

目的:制备表达针对BclGL基因的siRNA逆转录病毒,研究其对体外培养Jurkat细胞中BclGL基因的沉默效应及Jurkat细胞增殖的影响。方法:利用siRNA预测软件获取3条siRNA编码序列,插入pSilencer5.1-H1构建重组逆转录病毒siRNA表达质粒,脂质体法转染PT67细胞包装重组逆转录病毒pSilencer 5.1-H1-BclGL,体外感染Jurkat细胞,嘌呤霉素筛选病毒感染阳性细胞克隆,实时定量PCR及Western验证干扰效率;CCK-8法体外检测BclGL基因沉默后Jurkat细胞的增殖能力。结果:酶切及测序结果证实,表达siRNA的逆转录病毒构建成功,重组病毒表达的siRNA能有效干扰Jurkat细胞内BclGL基因的表达;BclGL表达下调可增强Jurkat细胞的增殖能力ela。结论:成功制备了表达人BclGL基因siRNA的重组逆转录病毒,并证实其对Jurkat细胞的生长抑制效应。
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.

关键词(KeyWords): BclGL基因;病毒;siRNA;T淋巴细胞;逆转录聚合酶链反应
BclGL gene;viruses;siRNA;T-lymphocytes;reverse transcriptase polymerase chain reaction

Abstract:

Keywords:

基金项目(Foundation): 国家自然基金面上项目(30671886)

作者(Author): 罗娜;郭晟;杨承英;费蕾;吴胜昔;陈永文;吴玉章;郝飞;
LUO Na1,GUO Sheng2,YANG Chengying2,FEI Lei2,WU Shengxi2,CHEN Yongwen2,WU Yuzhang2,HAO Fei1 (1.Deparment of Dermatology,Xinqiao Affiliated Hospital of The Third Military Medical University,Chongqing 400038,China;2.PLA Institut of Immunology,The Third Military Medical University,Chongqing 400038,China;)

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DOI: 10.19367/j.cnki.1000-2707.2009.03.005

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