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目的构建含人可诱导共刺激分子(ICOS)基因的重组逆转录病毒载体,获得稳定表达人ICOS基因的L929细胞株,并初步研究ICOS对B细胞产生抗体的影响。方法采用RT-PCR方法从扁桃体激活的T细胞总RNA中扩增出ICOS cDNA,双酶切装入逆转录病毒载体pGEZ-Term,与辅助病毒载体脂质体法共转染包装细胞293T,用其培养上清感染L929细胞72h后,经Zeocin筛选出稳定表达ICOS分子的L929细胞株;采用RT-PCR和流式细胞仪分析ICOS的分子表达,ELISA法研究ICOS信号对B细胞体外产生抗体的作用。结果成功地构建了含ICOS基因的重组逆转录病毒载体;筛选获得能稳定高表达人ICOS分子的L929转基因细胞,ICOS在PWM体外培养体系中,能有效地促进B细胞生长及IgG的分泌。结论 ICOS是CD28家族的新成员,在机体免疫应答过程中起着重要的作用。
Objective To construct recombinant retroviral vector containing human inducible costimulatory molecule (ICOS) gene and obtain L929 cell line stably expressing human ICOS gene, and to study the effect of ICOS on antibody production by B cell. Methods ICOS cDNA was amplified by RT-PCR from total T lymphocytes stimulated by tonsil. The recombinant plasmid was subcloned into retroviral vector pGEZ-Term and co-transfected into 293T cells with helper virus vector. The culture supernatant infected L929 cells for 72h, Zeocin screen stable expression of ICOS molecules L929 cell line; RT-PCR and flow cytometry ICOS molecular expression, ELISA study of ICOS signal to B cells in vitro antibody effect. Results Recombinant retroviral vector containing ICOS gene was constructed successfully. L929 transgenic cells stably expressing human ICOS gene were screened out. ICOS can effectively promote B cell growth and IgG secretion in PWM in vitro culture system. Conclusion ICOS is a new member of CD28 family and plays an important role in the process of immune response.