| 摘要: |
| 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. |
| 关键词: PCP胞外区、IgG免疫球蛋白类,Fc、融合蛋白、蛋白纯化 |
| DOI： |
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| 基金项目: |
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| Expression, purification and characterization of PCP-2EC/Fc fusion protein in mammal cells |
| 张鹏,鄢和新,王红阳 |
| (第二军医大学东方肝胆外科研究所信号转导研究实验室,上海,200438) |
| Abstract: |
| 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: PCP2 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. |
| Key words: PCP 2 extracellular demain IgG immunoglobulins,Fc fusion protein protein purification |
