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Expression of hPD-L1-Ig fusion protein in yeast system and its inhibitory effects on T cells
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Abstract:
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 51Cr 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.
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Project Supported:
Supported by the Major National Basic Research Development Program of China（No.2003CB515503）
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