人PD-1分子基因克隆及其在COS-7细胞中表达的研究

目的：克隆人PD—1分子的编码序列cDNA，将其重组进真核表达载体中，并表达于COS—7细胞。方法：从活化的人T细胞cDNA文库中以PCR方法克隆编码人PD—l的编码cDNA序列，将其克隆进真核表达载体pcDNA3．1( )，构建成重组表达载体。并测序证实。用脂质体法转染COS—7细胞，流式细胞仪检测PD—1分子的膜表达。结果：PCR方法扩增出—880bp左右的基因片段，插入pcDNA3．1( )载体后构建成重组表达质粒，经测序证实扩增的片段为人PD—l编码序列。将重组质粒转染COS—7细胞，流式细胞仪检测显示有21．10％的细胞表达人PD—1分子。结论：本研究为进一步研究PD—1分子在免疫耐受、自身免疫性疾病中的作用奠定了基础。

Cloning of human PD-1 molecule and its expression on COS-7 cells

Objective: To clone full-length human PD-1 encoding sequence and express functional PD-1 molecule on the COS-7 cells. Methods: PCR technique was used to clone the full-length DNA encoding PD-1 molecule from human activated T cell cDNA library .The PCR product was digested and inserted into pcDNA3.1(+) vector and sequenced. The right recombinant was transfected into COS-7 cell by lipofectamine reagent. The expressions of PD-1 molecule on the membrance of COS-7 cells were detected with FACS. Results: A fragment about 880 bp was cloned from human T cell cDNA library, and was subsequently digested and cloned into pcDNA3.1(+) vector. Sequence analysis demonstrated that the fragment was common with published PD-1 encoding sequence. Twenty one point one percent cells were detected for expressing human PD-1 molecules. Conclusion: This study makes it possible to further investigate the role of PD-1 molecules in immune tolerance and autoimmune disease.