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，51Cr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55 000，含量为120 μg/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 hPDL1Ig fusion protein in the Pichia pastoris expression system, and to investigate the biological activity of the protein. Methods: The hPDL1IgG4 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 SDSPAGE and Western blotting. The binding ability of hPDL1Ig fusion protein to PD1 receptor was examined by ELISA. The inhibitory effects of hPDL1Ig 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 pPIC9KPDL1IgG4 was successfully constructed, and hPDL1IgG4 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. PDL1Ig fusion protein had high affinity with PD1 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 hPDL1IgG4 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.

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