P-selectin糖蛋白配体-1的基因克隆及其Ig融合蛋白的表达和纯化

Cloning of P-selectin glycoprotein ligand-1 gene and expression and purification of its Ig fusion protein

目的:克隆P-selectin糖蛋白配体-1的基因,表达和纯化其Ig融合蛋白。方法:RT-PCR法克隆出P-选择素糖蛋白配体-1(CD162)cDNA,构建真核表达载体,DEAD-Dextran法转染COS7细胞,上清经纯化后,分别使用WesternBlot和流式细胞仪鉴定CD162-hIgFc融合蛋白的性质和活性。结果:RT-PCR克隆出801bp的CD-162cDNA片段,插入载体构建出pIg-CD162,将其转染COS7细胞后获得CD162-hIgFc融合蛋白,分子量为74KD。WesternBlot证实其为CD162-hIgFc融合蛋白,流式细胞仪证明获得的CD162-hIgFc融合蛋白能够识别血小板表面的CD62P。结论:本研究成功制备了CD162-hIgFc融合蛋白,为进一步探讨P选择素与其配体CD162之间的作用打下坚实的基础。

Objective To Clone P-selectin glycoprotein ligand-1 （CD162）gene, as well as to express and purify its Ig fusion protein. Methods The CD162 cDNA was cloned by RT-PCR. The CD162 cDNA fragment was then insert into eukaryotic expression vector. The expression vector was then transfected into COS7 cells by DEAD- Dextran method. The supernatant was harvested and the CD162-hIgFc fusion protein was identified by Western Blotting. The biological activity of CD162-hIgFc was assayed by flow cytometry. Results A 801 bp cDNA fragment of CD162 was cloned out. Expression vector plg-CD162 was therefore constructed and transfected into COS7 cells. The CD162-higFc fusion protein was harvested and purified from the supernatant. Western blotting confirmed the fusion protein identity. Flow cytometry assay showed that the CD162-hIgFc fusion protein was able to identify the CD 62P on the surface of platelets. Conclusion In this study, the CD162-hlgFc fusion protein was expressed and purified,therefore,a solid foundation was built for the further study the interaction of P selectin and its ligand CD162.