摘要
目的构建含有人B7H4基因的重组逆转录病毒载体,获得稳定表达B7H4基因的L929细胞并初步研究其对T细胞的共信号作用及可能机制。方法从含人B7H4基因的cDNA序列FLJ22418中,采用PCR扩增出B7H4全长基因,双酶切装入逆转录病毒载体pEGZHATerm,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B7H4蛋白的L929细胞株;采用免疫荧光标记和流式细胞仪分析免疫分子的表达、3HTdR掺入检测细胞增殖以及ELISA测定细胞因子的水平。结果构建了含人B7H4基因的重组逆转录病毒载体和获得含有B7H4基因的重组病毒,筛选获得能稳定高表达人B7H4蛋白的L929转基因细胞;该转基因细胞对T细胞体外具有抑制增殖、活化和细胞因子分泌的作用。结论构建了稳定表达人B7H4蛋白的细胞株,B7H4分子在体外通过抑制T细胞分泌IL2和促进其凋亡作用可显著地下调T细胞功能的效应。
Objective To construct recombinant retmvirus vector carrying human B7-H4 cDNA, which can be stably expressed in mammal cell line L929, and to study the costimulatory effect and mechanism to T lymphocyte. Methods The full-length human B7-H4 gene was amphfied by PCR from cDNA FLJ22418 that contains human B7-H4. Digested with the restriction endonucleases Pst Ⅰ and BamH Ⅰ , the B7-H4 gene was inserted into retrovims vector pEGZ-HA-Term. The recombinant vector together with its two helper vires vectors were co-transfected into the package cell 293T with LipofectAMINE 2000^TM . The supernatant of 293T was used to infect L929 cells. L929 cells stably expressing B7-H4 protein were selected in the presence of Zeocin （500μg/ml）. Then the biological function of B7-H4 transfected cells was studied by FCM analysis of cell phenotypes, ^3H-TdR incorporation and ELISA. Results The full-length B7-H4 gene was successfully cloned and the recombinant retrovirus vector carrying B7-H4 gene for expression was constructed. The selected L929 cells stably expressed human BT-H4 protein on the membrane of cells. The transfected cells have an inhibitory effect on the growth, activation and cytokine secretion of T cells. Conclusion B7-H4 protein may play a role in negative regulation of T cell response in vitro by downregulating production of IL-2 as well as prompting apoptosis.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第8期609-613,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金重点资助项目(No.300330540)
江苏省临床医学重点实验室资助项目
