含3C蛋白酶切位点的人CD226胞膜外区Ig融合蛋白的真核表达及应用

目的：构建含3C蛋白酶酶切位点的人CD226(PTA1)分子胞膜外区Ig融合蛋白编码基因的真核表达载体，并进行表达和初步鉴定。方法：将人CD226分子的胞膜外区基因，克隆入含3C蛋白酶靶序列的Ig真核表达载体p-3c—Ig中。测序证实后，转染COS7细胞并进行瞬时表达。表达产物经亲和层析件纯化，并通过免疫荧光染色及3C蛋白酶酶切反应进行鉴定结果：经表达和纯化获得CD226胞膜外区的Ig融合蛋白。该融合蛋白可与表达于ECV304细胞表面的CD226配体有效结合。同时，融合蛋白的Fc段亦经3C蛋白酶切除，从而获得CD226胞膜外区的真核表达分子。结论：成功地获得了含有3C蛋白酶酶切位点的人CD226分子胞膜外区Ig真核表达产物，为进一步对CD226分子进行结构和功能研究，以及X线结晶衍射提供了重要条件。

Eukaryotic expression and application of human CD226 Ig fusion protein containing 3C protease-restricted site

AIM: To construct, express and characterize the eukaryotic expression vector of encoding human CD226 (PTA1) extracellular region Ig fusion protein gene containing 3C protease restricted site. METHODS: The gene fragment encoding extracellular region of human CD226 was cloned into the eukaryotic expression vector p 3C Ig containing 3C protease restricted site and human Ig Fc fragment gene. After sequencing, the vector was transfected into COS7 cells, and the expressed molecule was purified by affinity chromatography. Finally, the product was characterized by immunofluorescent staining and 3C protease digestion. RESULTS: After expression and purification, the Ig fusion protein could bind effectively to the CD226 ligand expressed on ECV304 cells. The Fc fragment could also be cut off by 3C protease, so that the CD226 extracellular fragment was obtained. CONCLUSION: The extracellular region of human CD226 Ig fusion protein with 3C protease restricted site is expressed successfully in COS 7 cells, which lays the foundation for the structural and functional study of this molecule.