论文部分内容阅读
目的 :构建含 3C蛋白酶酶切位点的人CD2 2 6 (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CD2 2 6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柱纯化 ,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CD2 2 6胞膜外区的Ig融合蛋白。该融合蛋白可与表达于ECV30 4细胞表面的CD2 2 6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CD2 2 6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CD2 2 6分子胞膜外区Ig真核表达产物 ,为进一步对CD2 2 6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
OBJECTIVE: To construct an eukaryotic expression vector encoding the extracellular Ig fusion protein of human CD2 2 6 (PTA1) containing 3C protease restriction sites and to express and identify it. Methods: The extracellular domain of human CD2 2 6 gene was cloned into Ig eukaryotic expression vector p 3C Ig containing 3C protease target sequence. After sequencing confirmed, COS7 cells were transfected and transiently expressed. The expressed product was purified by affinity chromatography and identified by immunofluorescence staining and 3C protease digestion. Results: Ig fusion protein of CD2 2 6 extracellular domain was obtained by expression and purification. This fusion protein binds efficiently to the CD2 2 6 ligand expressed on the surface of ECV304 cells. At the same time, Fc fragment of the fusion protein was also excised by 3C protease to obtain eukaryotic expression molecules in the extracellular domain of CD2 2 6. CONCLUSION: The eukaryotic expression product of Ig in human extracellular domain of CD226 containing 3C protease cleavage site was successfully obtained, which is important for the further study on the structure and function of CD226 and the X-ray crystallography condition