人VSIG4-Fc融合蛋白的表达及其生物学活性的研究  

Expression of human VSIG4-Fc fusion protein and characterization of its bioactivity

作　　者：张世杰[1] 王蕾[2] 王月颖[1] 李道明[1] 朱守兵[1] 蒋玉平[1] 蒋靓[1] 张学光[1] 顾宗江[1] 

机构地区：[1]苏州大学医学生物技术研究所 [2]苏州大学附属第一医院心内科,苏州215007

出　　处：《现代免疫学》2009年第3期208-212,共5页Current Immunology

摘　　要：为获取人VSIG4-Fc融合蛋白,并研究其对T细胞的调节效应。首先采用PCR获取人VSIG4基因的胞外段序列及人IgG1Fc恒定区序列,将两者顺次连接插入逆转录病毒载体,以293T为包装细胞,制备含病毒颗粒的培养上清,反复感染CHO细胞,Zeocin筛选能稳定分泌VSIG4-Fc蛋白的基因转染细胞并以RT-PCR、Dot-blot及Western blot等鉴定。以MTT和ELISA分别检测VSIG4-Fc融合蛋白对T细胞增殖及IL-2分泌的影响。结果成功获取了CHO基因转染细胞,该细胞能稳定分泌VSIG4-Fc蛋白,纯化后的VSIG4-Fc蛋白能与T细胞表面未知受体结合,并具有体外抑制T细胞增殖、活化和IL-2分泌的作用。To obtain human VSIG4-Fc fusion protein in eukaryotic cells and to investigate its regulatory effects and mechanism on T lymphocyte activation. The genes coding extracellular domain of human VSIG4 and the Fc fragment of human IgG1 were amplified by PCR, and they were inserted into retrovirus vector pEGZ-Term to construct the recombinant vector pGEZ-Term- VSIG4 Fc. The package cell 293T transfeced by the recombinant vectors and its two helper virus vectors was engaged to produce the intact virus granules which was used to infect CHO cells. CHO cells stably expressing VSIG4-Fe protein were selected in the presence of Zeocin, and then analyzed by RT-PCR, Dot blot and Western blot assays. The functions of VSIG4-Fc in T cell proliferation and IL-2 secretion of T cells were studied by MTT and ELISA respectively. In the end, the tranfected CHO was obtained successfully, and the selected CHO cells stably expressed VSIG4-Fc fusion protein that could bind to unknown receptor on T cells. Also, the VSIG4-Fc fusion protein showed inhibitory effects on the activation, proliferation and IL-2 secretion of T cells.

关 键 词：VSIG4 逆转录病毒 融合蛋白 共刺激分子 

分 类 号：R392.12[医药卫生—免疫学]