PD-L1-Ig融合蛋白的酵母表达及其对T细胞的抑制作用  

Expression of hPD-L1-Ig fusion protein in yeast system and its inhibitory effects on T cells

作　　者：张意[1] 吴聪[1] 蒋应明[1] 龙瑶[1] 陈国友[1] 

机构地区：[1]第二军医大学免疫学研究所,上海200433

出　　处：《中国肿瘤生物治疗杂志》2009年第5期464-468,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展计划(973计划)资助项目(No.2003CB515503)~~

摘　　要：目的:利用毕赤酵母表达系统表达PD-L1-Ig融合蛋白,检测该融合蛋白的生物学活性。方法:化学合成hPD-L1-IgG4融合基因,构建携带该基因的酵母表达载体,转化GS115酵母菌株后分泌表达融合蛋白,亲和层析和离子交换层析法纯化,SDS-PAGE和Western blotting分析鉴定。ELISA法检测融合蛋白与受体PD-1的结合能力,混合淋巴细胞反应检测融合蛋白对T细胞功能的抑制能力,51Cr稀释法检测其对CTL杀伤人结肠癌SW480细胞效应的抑制作用。结果:成功构建酵母表达载体pPIC9K-PD-L1-IgG4,转化菌株分泌表达PD-L1-IgG4融合蛋白,相对分子质量为55000,含量为120μg/ml。发酵菌株,纯化制备融合蛋白。融合蛋白与受体PD-1具有良好的结合能力,能够显著抑制T细胞增殖、活化(P<0.01),并抑制CTL对结肠癌细胞的杀伤(P<0.01)。结论:成功制备了酵母表达PD-L1-IgG4融合蛋白,该融合蛋白具有良好的生物学活性,为深入研究其在肿瘤免疫应答中的调控效应奠定了良好基础。Objective：To express hPDL1Ig fusion protein in the Pichia pastoris expression system, and to investigate the biological activity of the protein. Methods： The hPDL1IgG4 fusion gene was chemically synthesized and cloned into Pichia yeast expression vector. The recombinant vector was then transfected into GS115 Pichia yeast strain, and the fusion protein was purified by affinity chromatograph and ion exchange method before further identified by SDSPAGE and Western blotting. The binding ability of hPDL1Ig fusion protein to PD1 receptor was examined by ELISA. The inhibitory effects of hPDL1Ig fusion protein on T cells and on cytotoxicity of CTL against colon cancer SW480 cells were examined by MLR and 51Cr release assay, respectively. Results： The expression vector pPIC9KPDL1IgG4 was successfully constructed, and hPDL1IgG4 fusion protein was secreted by GS115 Pichia yeast strain, with the molecular weight being about 55 000 and the concentration being 120 μg/ml. The fusion protein was purified using fermentation strain. PDL1Ig fusion protein had high affinity with PD1 receptor; it also significantly inhibited the proliferation and activation of T cells （P〈0.01） and the cytotoxicity of CTL against colon tumor cells （P〈0.01）. Conclusion： The hPDL1IgG4 fusion protein has been successfully prepared, and it has active biological functions, which lays a foundation for further investigating its regulatory effect on tumor immune response.

关 键 词：PD-L1 融合蛋白 酵母表达系统 PD-1 T细胞 结肠癌细胞 

分 类 号：R392.1[医药卫生—免疫学] R730.3[医药卫生—基础医学]