人B7-H4-Fc融合蛋白的表达及其应用研究

Study on the Preparation of B7-H4-Fc Fusion Protein and Its Application

B7-H4分子是近年发现的一个新的协同刺激分子，它通过抑制T细胞的增殖、细胞因子的分泌以及细胞周期的进行，进而下调T细胞的免疫功能。为获取人B7．H4IgG融合蛋白，研究其对T细胞的调节效应，采用PCR法分别从pEGZ—Term／PD—L1-Fc和含人B7．H4基因序列的重组质粒中扩增出人IgGFc恒定区基因及人B7＋H4基因的胞外段序列，将两者插入逆转录病毒载体pEGZ—Term中，构建pEGZ．Term／B7．H4一Fc逆转录病毒重组载体，用脂质体法与两个辅助病毒载体共转染293T包装细胞，用含病毒颗粒的培养上清反复感染CHO细胞。用Zeocin筛选能稳定分泌人B7-H4-Fc融合蛋白的基因转染细胞并亚克隆之，经大量培养增殖，裂解细胞后收集裂解上清用ProteinG柱纯化，再经Westernblot鉴定。以体外T细胞活化体系观察其对T细胞活化的抑制作用。结果表明，成功地构建了表达人B7-H4-Fc融合蛋门的重组逆转录病毒载体；获得的CHO／B7-H4-Fc细胞能稳定分泌人B7-H4-Fc融合蛋门，该融合蛋白可以有效地抑制T细胞的活化。

B7-H4 is a recently discovered co-stimulatory molecules,which can regulate down the T cells immune function by inhibiting T cell proliferation,secreting cytokine and interrupting cell cycle.In order to stably express human B7-H4-Fc fusion protein in eukaryon cells and to investigate the regulatory effect on T cells,extracellular domain of human B7-H4 gene was amplified by PCR from a recombinant plasmid containing human B7-H4 gene,and human IgG I （Fc） constant region was obtained through PCR from pEGZ-Term/PD-L1- Fc.The two amplified genes were inserted into the reverse transcriptase virus vector pEGZ-Term,and constructed the pEGZ-Term/B7-H4-Fc vector.The recombinant vector together with its 2 helper virus vector was cotransfected into the package cell 293T.The supernatant of 293T containing intact virus granules was used to infect CHO cells. The gene infected cells stably secreting human B7-H4-Fc fusion protein were selected by Zeocin,then subcloned and cultured.B7-H4-Fc fusion protein was purified by Protein G column from the gene infected cells lysis supernatant,and identified by Western blot.B7-H4-Fc fusion protein precoated on the 96 wells of plates was used to detect its influence on the T cells activated by agonistic anti-CD3 mAb and anti-CD28 mAbs.The results showed that recombinant retro virus vector carrying human B7-H4-Fc fusion gene was constructed successfully and CHO/ B7-H4-Fc cells stably secreting human B7-H4-Fc fusion protein were obtained and confirmed.The activation of T cells was obviously inhibited by B7-H4-Fc fusion protein in vitro.B7-H4-Fc fusion protein will contribute to further research on the function of B7-H4 molecule.