人BTLA-Fc融合蛋白的制备及其生物学活性的研究  

Preparation of human BTLA-Fc fusion protein and study of its biological function

作　　者：朱耿超[1,2] 黄子逸[1,2] 曹丽娟[1,2] 陈永井[1,2] 王雪峰[1,2] 张学光[1,2] 

机构地区：[1]苏州大学医学生物技术研究所 [2]苏州大学基础医学与生物科学学院,苏州215006

出　　处：《现代免疫学》2011年第5期363-367,共5页Current Immunology

基　　金：国家自然科学基金资助项目(30600548);苏州大学博士基金资助项目(Q4134801);江苏省青蓝工程资助

摘　　要：为建立CHO/BTLA-Fc基因转染细胞株,使之能稳定分泌BTLA-Fc融合蛋白,采用PCR法从本单位构建的pEGZ-Term/B7-H1-Fc重组质粒中扩增人IgG1(Fc)恒定区,Xho I、BamH I双酶切后与真核表达载体pIRES2-EGFP连接为重组质粒pIRES2-EGFP-Fc,同时PCR法从pEGZ-Term/BTLA重组质粒中获得人BTLA胞外段基因片段,用Nhe I、Xho I双酶切插入上述pIRES2-EGFP-Fc构建重组载体pIRES2-EGFP/BTLA-Fc。脂质体法以该重组载体转染鼠CHO细胞,G418筛选并亚克隆化。Protein G亲和层析柱对上清中的融合蛋白进行纯化,Western blot作定性分析。CCK-8检测BTLA-Fc融合蛋白对抗人CD3单抗激发的T淋巴细胞增殖的影响。结果表明,基因转染CHO细胞培养上清中表达的BTLA-Fc融合蛋白能与L929/HVEM细胞有效结合,纯化的融合蛋白经Western blot鉴定有清晰的目的条带,而且BTLA-Fc融合蛋白对T细胞的体外增殖具有抑制作用。To establish the CHO/BTLA-Fc gene transfected cell line expressing human BTLA-Fc fusion protein encoding human IgG1（Fc） was amplified by PCR from recombinant vector pEGZ-Term/B7-H1-Fc and then the target fragment was inserted to eukaryotic expression vector pIRES2-EGFP after being digested with Xho I,BamH I.The extracellular grne of BTLA was amplified by PCR from recombinant vector pEGZ-Term/BTLA,inserted into recombinant vector pIRES2-EGFP-Fc after being digested with Nhe I,Xho I.The recombinant vector was transfected into CHO cells with LipofectAMINETM 2000,and the cells were further selected with G418.The expression of BTLA-Fc fusion protein was analysed by flow cytometry and the BTLA-Fc fusion protein was purified by affinity chromatography and analysed qualitatively via Western blot assay.Its influence on process of T lymphocyte proliferation was detected by using CCK-8.It was demonstrated that the stable secretion of human BTLA-Fc fusion protein from the transfected cell line was identified by flow cytometry,the fusion protein could combine with mouse anti-human BTLA monoclonal antibody and inhibit proliferation of T cells in vitro.The CHO/BTLA-Fc cell line stably expressing human BTLA-Fc protein has been thus obtained and the fusion protein can inhibit the proliferation of T lymphocytes in vitro.

关 键 词：B、T淋巴细胞衰减子(BTLA) 融合蛋白 单纯疱疹病毒侵入介质(HVEM) T细胞 

分 类 号：R392.12[医药卫生—免疫学]