论文部分内容阅读
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 and to study its regulatory effect on T cells. First, the extracellular domain of human VSIG4 gene and the sequence of human IgG1 Fc constant region were obtained by PCR. The two sequences were inserted into the retroviral vector and the 293T was used as the packaging cell to prepare the culture supernatant containing the virus particles and repeatedly infect CHO cells , Zeocin was screened for stable transfected VSIG4-Fc gene transfected cells and identified by RT-PCR, Dot-blot and Western blot. The effects of VSIG4-Fc fusion protein on T cell proliferation and IL-2 secretion were detected by MTT and ELISA, respectively. Results The transfected CHO cells were successfully transfected with VSIG4-Fc protein. The purified VSIG4-Fc protein could bind to unknowns on T cells and inhibit the proliferation and activation of T cells and IL-2 Secretion of the role.