【摘要】 为获得人4IgB7-H3-Fc融合蛋白,研究其对T细胞的共刺激作用,采用PCR技术分别从pMD18-T/4IgB7-H3和pMD18-T/hIgG1(Fc)重组载体中扩增出人4IgB7-H3胞外段基因和人IgG1重链Fc恒定区基因,通过RT-PCR技术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经脂质体法共转染包装细胞293T,收集含有完整病毒颗粒的293T细胞的培养上清反复感染L929细胞,利用Zeocin筛选获得能稳定分泌4IgB7-H3-Fc融合蛋白的基因转染细胞。基因转染细胞经无血清培养后,收集的上清用Dot blot检测,超滤浓缩并经Protein G柱纯化,再用SDS-PAGE、Western blot鉴定。以4IgB7-H3-Fc蛋白检测活化T细胞表面未知受体的表达,通过MTT方法分析4IgB7-H3-Fc融合蛋白对T细胞的共刺激作用。结果显示,成功地构建了pEGZ-Term/4IgB7-H3-Fc重组逆转录病毒载体,经转染包装细胞293T后,筛选获得能稳定分泌4IgB7-H3-Fc蛋白的L929基因转染细胞株;纯化后的4IgB7-H3-Fc蛋白能够与活化T细胞表面结合,并对T细胞有显著的促增殖作用。为探讨人4IgB7-H3-Fc融合蛋白对T细胞的共刺激作用和寻找其未知受体奠定良好的物质基础。更多还原

【Abstract】 As a novel member of B7 family,the function of B7-H3 is not so clear and has some disputes.In addition human B7-H3 has two isoforms(2IgB7-H3 and 4IgB7-H3).In order to study the biological function of 4IgB7-H3,the expression of the 4IgB7-H3-Fc fusion protein was studied.The gene encoding the 4IgB7-H3 extracellular domain and human IgG1(Fc) constant region were amplified from pMD18-T/4IgB7-H3 and pMD18-T/hIgG1Fc vectors by PCR.After RT-PCR,the fusion gene was inserted into retrovirus vector pEGZ-Term,and the recombinant vector together with its helper virus vectors was cotransfected 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 cell line secreting 4IgB7-H3-Fc fusion protein stably was established.The unkown receptor of 4IgB7-H3 on activaton T cells was detected by the fusion protein with FACS,and the costimulatory effection to T cell was measured by MTT.The results showed that the transfected L929 cell line secreting 4IgB7-H3-Fc fusion protein was constructed successfully,and the activation of T cells could be enhanced by 4IgB7-H3-Fc in vitro.This construction of cells stably secreting human 4IgB7-H3-Fc fusion protein and the purified protein will contribute to further research on the functions of B7-H3.更多还原