人可诱导的共刺激分子转基因细胞的构建及其对B细胞产生抗体的影响

Construction of the Transfected Cell Line Expressing the Human Inducible Co-stimulator(ICOS) Gene and Effects of the ICOS on the Antibodies Production

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 retrovirus vectors carrying human ICOS gene,which can be stably expressed in cell line L929,and to study the effects of the ICOS on the antibodies production from PWM-driven B cells . Methods The total RNA was isolated from tonsil cells ,and the ICOS gene was amplified by RT-PCR ,digested with restriction endonuclease Pst I and BamH I ,and inserted into retrovirus vector pGEZ-Term. The recombinant vector together with its two helper virus vectors was cotransfected into the package cell 293T with Lipfectamine 2000. Then the supernatant of the 293T cell was used to infect L929 cells . After 72 hours cultivation ,the L929 cell ,and the stably expressed the ICOS molecules were screened in the presence of Zeocin,which was then analyzed by PCR and FCM. The effects of the ICOS on the antibodies production from PWM-driven B cells were analyzed by ELISA. Results It was observed that the full-length of ICOS gene was successfully cloned ,and the recombinant retrovirus vector carrying the ICOS gene was constructed. The screened L929 cell clone could stably express the human ICOS gene on the cell membrane. ICOS could efficiently increase antibodies production from B cells in the in vitro PWM cultivation system. Conclusion ICOS,a newly identified member of CD28 superfamily,plays a capital role in immune responses.