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目的:构建重组人pSG5-Fc-hPD-1嵌合基因质粒,并在真核细胞进行表达,得到分泌性的sPD-1-Fc融合蛋白。为PD-1的生物学活性研究奠定基础。方法:从GenBank里查到人PD-1胞外区的基因序列,设计特异性引物,以人淋巴细胞cDNA为模板PCR扩增得到人PD-1胞外区片段。以pSG5-Fc真核表达质粒为载体,构建得到重组质粒pSG5-Fc-hPD-1。脂质体瞬时转染猴肾成纤维细胞(Cos-7),收取72 h的细胞上清。用Western blot鉴定,并与原核表达的人PD-L1蛋白免疫共沉淀检测生物学活性。结果:通过EcoRⅠ/BamHⅠ双酶切及测序证实构建的重组质粒pSG5-Fc-hPD-1正确。重组质粒在Cos-7细胞中高效表达并分泌融合蛋白sPD-1-Fc到细胞上清,应用Western blot测定到上清中的融合蛋白,且得到的sPD-1-Fc融合蛋白具有与PD-L1结合的生物学活性。结论:通过基因重组方法在真核细胞里得到高效分泌型表达的sPD-1-Fc重组蛋白,为研究PD-1生物学活性奠定了实验基础。
OBJECTIVE: To construct a recombinant plasmid pSG5-Fc-hPD-1 chimeric gene and express it in eukaryotic cells to obtain secreted sPD-1-Fc fusion protein. The study laid the foundation for the biological activity of PD-1. Methods: The gene sequence of human PD-1 extracellular region was found from GenBank, and specific primers were designed. Human PD-1 extracellular region was amplified by PCR using human lymphocyte cDNA as a template. The pSG5-Fc eukaryotic expression plasmid was used as a vector to construct the recombinant plasmid pSG5-Fc-hPD-1. Liposomes were transiently transfected into monkey kidney fibroblasts (Cos-7) and cell supernatants were harvested for 72 h. Western blot was used to detect the biological activity of the human PD-L1 protein co-immunoprecipitation. Results: The recombinant plasmid pSG5-Fc-hPD-1 was confirmed by EcoRⅠ / BamHⅠ double digestion and sequencing. Recombinant plasmids efficiently expressed and secreted the fusion protein sPD-1-Fc into the supernatant of Cos-7 cells. The fusion protein in the supernatant was determined by Western blot. The obtained sPD-1-Fc fusion protein showed high affinity with PD- L1 binding biological activity. Conclusion: The sPD-1-Fc recombinant protein, secreted and expressed efficiently in eukaryotic cells by gene recombination, laid the experimental foundation for studying the biological activity of PD-1.