人B7-H3基因转染及其生物学功能的研究

Construction of Human B7-H3 Transfected Cell Line and the Investigation of Its Bioactivity in vitro

B7 H3是新近发现的B7家族新成员。为探讨其体外生物学功能及单克隆抗体的研制构建了B7 H3基因转染细胞。利用PCR的方法扩增出B7 H3基因 ,继而插入逆转录病毒载体pGEZ Term ,重组逆转录病毒载体与两个辅助病毒载体脂质体法共转染包装细胞 2 93T ,经用含有完整病毒颗粒的 2 93T细胞的培养上清感染L92 9细胞 ,Zeocin筛选获得B7 H3的基因转染细胞。RT PCR、Westernblot和流式细胞仪表型分析等方法鉴定表明 ,B7 H3/L92 9基因转染细胞能稳定表达人B7 H3蛋白。继而基因转染细胞对T细胞体外培养试验显示该基因转染细胞与T细胞共培养可抑制其体外增殖。ELISA法分析表明该基因转染细胞抑制活化T细胞IFN γ及IL 10的分泌。CD2 8信号可以逆转B7 H3对活化T细胞的抑制效应。综上结果证实 ,B7 H3对体外活化的T细胞具有负性调控作用。

To explore the biological functions of human B7-H3, a novel member of B7 superfamily, and to prepare its monoclonal antibody, the gene encoding the human B7-H3 protein was obtained by PCR. Digested with EcoR I and BamH I, the PCR product was cloned into corresponding region of pGEZ-Term vector. The recombinant plasmid together with its two helper virus vectors was cotranstected into the package cell 293T with LipfectAMINE 2000. Then the supernatant of 293T was used to infect L929 cells. Selected by zeocin after transfection, a stable cell line B7-H3/L929 expressing the human B7-H3 was established. The expression stability and efficiency of the target molecule was identified by RT-PCR, Western blotting and flow cytometry analysis. In the presence of anti-CD3 mAb, B7-H3/L929 was evidenced to have a potency to inhibit activated T cell proliferation and IFN-γ, IL-10 secrefion in vitro, but the inhibitory effect could be reversed by the signal of CD28. In conclusion, B7-H3 has a down-regulatory characteristic to activated T cells in vitro.