PCP-2胞外区/Fc融合蛋白的真核表达、纯化及其生物学活性鉴定(英文)

Expression,purification and characterization of PCP-2EC/Fc fusion protein in mammal cells

目的:构建PCP 2胞外区(PCP 2EC)与人免疫球蛋白IgG Fc融合蛋白表达载体,在哺乳动物细胞中表达并纯化PCP 2EC/Fc蛋白,研究其在神经细胞黏附中发挥的作用。方法:以PBLIISK PCP 2为模板扩增PCP 2EC片段,定向插入真核表达载体pIGplus;重组质粒分别转染COS 7细胞和293细胞,可溶性表达PCP 2EC/Fc融合蛋白,并通过金葡菌蛋白A特异性纯化;以此融合蛋白作为基质,观察原代培养神经元的黏附情况。结果: 成功构建PCP 2EC/Fc融合蛋白表达载体,表达载体的构建与预期设计相符;进一步在哺乳动物细胞中表达并纯化PCP 2EC/Fc蛋白;PCP 2EC/Fc蛋白可以促进原代培养神经元的黏附作用。结论:本研究成功地构建了PCP 2EC/Fc融合蛋白真核表达载体,获得有活性的PCP 2 胞外区可溶性分子,初步研究发现其在神经细胞黏附中发挥促进作用,为后续神经及其他领域中的功能研究奠定基础。

Objective: To construct the extracellular region of PCP-2(PCP-2EC) and the immunoglobin IgG Fc fusi on protein expression vector,and then express and purify the soluble PCP-2EC/Fc fusion protein for the study of its function in neuronal adhesion.Methods: PCP-2 extracellular region was amplified and cloned into an expression vector pIGplus containing human IgG Fc; PCP-2EC/Fc fusion protein was expressed by COS-7 and 293 cells transfected by the constructed plasmid and purified by protein A.The purified fusion protein was used as substrate to study its function in neuronal adhesion.Results:PCP-2 extracellular region was cloned into IgG Fc expression vector successfully; PCP-2EC/Fc fusion protein was expressed and purified in mammal cells; and the purified fusion protein promoted neuronal adhesion.Conclusion:PCP-2EC/Fc fusion protein expression system is successfully constructed and the purified fusion protein can promote neuronal adhesion.These results lay a foundation for the research on the PCP-2 function in neuronal adhesion and the further functional study in the nervous system and other fields.