人HVEM-Fc融合蛋白的制备及其生物学特性

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

为建立CHO/HVEM-Fc基因转染细胞株,使之能稳定分泌HVEM-Fc融合蛋白,采用常规PCR法分别从本单位已构建的重组载体pGEZ-Term/B7-H1-Fc和pIRES2-EGFP/HVEM上扩增人IgG1(Fc)恒定区和人HVEM胞外段编码基因,XhoⅠ和Bam HI双酶切人IgG1(Fc)恒定区及真核表达载体pIRES2-EGFP,将两种酶切的目的产物连接为pIRES2-EGFP/Fc,同时将获得的人HVEM胞外段基因片段经XhoⅠ和NheⅠ酶切后插入上述pIRES2-EGFP/Fc构建成pIRES2-EGFP/HVEM-Fc重组载体,通过脂质体转染仓鼠卵巢癌CHO细胞,培养基中添加G418长期筛选并将分泌目的蛋白的基因转染细胞株亚克隆化。流式细胞术检测HVEM-Fc融合蛋白的表达,收集含融合蛋白的基因转染细胞培养上清,Protein G亲和层析柱对其进行纯化,Western blot定性分析。MTT检测HVEM-Fc融合蛋白体外对抗人CD3单抗联合基因转染细胞L929/LIGHT刺激的T淋巴细胞增殖的影响。结果表明,基因转染细胞CHO/HVEM-Fc分泌的HVEM-Fc融合蛋白能与L929/LIGHT细胞有效结合,纯化后的蛋白经Western blot鉴定出现特异性清晰条带,并且HVEM-Fc融合蛋白对L929/LIGHT协同刺激的T细胞体外增殖具有抑制作用。

To establish CHO cells secreting human HVEM-Fc fusion protein and analyze its co-stimulatory effect on T cells, the gene fragment encoding IgG1 （Fc） and that encoding the extracel[ular domain of human HVEM were respectively amplified by PCR from pGEZ-Term/ B7 H1-Fc and plRES2-EGFP/HVEM. Subsequently, the gene fragment encoding IgG1 （Fc） was inserted into eukaryotic expression vector plRES2-EGFP after being digested with Xho I and BarnH I to construct a recombi- nant vector plRES2-EGFP/Fc. Further, the gene fragment encoding extracellular domain of human HVEM was linked to pIRES2 EGFP/Fc through digestion with Xho I and Nhe I to establish pIRES2-EGFP/HVEM-Fc. The recombinant vector plRES2 EGFP/HVEM-Fc was transfected into CHO cells with LipofectAMINETM2000. The expression of HVEM-Fc fusion protein was analyzed by flow cytometry. The HVEM-Fc fusion protein was purified by affinity chromatography and analyzed qualitatively via Western blot assay. Its influence on T lymphocyte proliferation was detected by MTT method. Stable secretion of human HVEM-Fc fusion protein from the transfected cell line was identified by flow cytometry. The fusion protein could combine with mouse anti-human HVEM monoclonal antibody and partially inhibit in vitro T cell proliferation induced by anti- CD3 mAb in the presence of L929/LIGHT.