人PD1基因真核表达载体构建与鉴定

Construction and Identification of Eukaryotic Expression Vector of Human Programmed Death Receptor 1

目的:构建含EGFP标签的人程序性死亡受体(hPD-1)的真核表达载体,为后续基因转移实验奠定基础。方法:PCR扩增hPD-1基因编码框,将PD-1和pEGFP-N1质粒双酶切后进行连接转化,构建重组载体pEGFP-N1-PD-1,酶切重组质粒并测序鉴定,将构建好的重组质粒转染HEK293T细胞,并提取蛋白进行Western blot检测。结果:重组真核表达载体pEGFP-N1-PD-1经限制性内切酶分析,双酶切之后的条带与理论值相符,测序结果未见碱基变异,提取转染后细胞的总蛋白进行Western blot可以检测到目标条带。结论:成功构建了pEGFP-N1-PD-1真核表达载体,并且能够在293T细胞中正常表达,为后续研究PD-1基因的功能奠定基础。

Objective: To construct a eukaryotic expression vector containing human EGFP-tagged human programmed death receptor (hPD-1), which lays a foundation for subsequent gene transfer experiments. Methods: The hPD-1 gene coding frame was amplified by PCR. The PD-1 and pEGFP-N1 plasmids were digested and ligated, and the recombinant vector pEGFP-N1-PD-1 was constructed. The recombinant plasmid was digested and sequenced for identifying. The recombinant plasmid was transfected into HEK293T cells, and the protein was extracted for Western blot analysis. Results: The recombinant eukaryotic expression vector pEGFP-N1-PD-1 was analyzed by restriction endonuclease. The bands after double digestion were consistent with the theoretical values. No nucleotide variation was observed in the sequencing results. The total protein of the transfected cells was extracted. Western blot can detect the target band. Conclusion: The eukaryotic expression vector pEGFP-N1-PD-1 was successfully constructed and expressed in 293T cells, which laid a foundation for the subsequent study of the function of PD-1 gene.