重组CC家族趋化因子RANTES的原核表达及趋化活性分析

摘要

目的深入研究趋化因子RANTES的结构与功能，为其进一步的应用研究打下基础。方法利用PCR技术扩增RANTES成熟蛋白的编码区序列，将其克隆到原核表达载体，经表达纯化得到重组蛋白质纯品，利用趋化小室法测趋化活性。结果成功扩增RANTES cDNA编码区序列，在大肠杆菌中成功表达重组RANTES蛋白，经纯化蛋白纯度达95％以上，对外周血淋巴细胞(PBL)，U937淋巴瘤细胞系，和HEK293-CCR4稳定株有趋化活性，对Jurkat细胞无明显趋化作用。结论原核表达的重组RANTES对多种细胞具有趋化活性，并建立了用培养细胞系检测趋化活性的模型，为下一步RANTES的结构与功能研究打下了基础。%Objective To further understand the structure and function of RANTES and provide basis for research rnfor application. Methods PCR product corresponding to region encoding mature RANTES protein was rncloned into E.coli expression vector;, purified recombinant protein was obtained by heparin affinity chrornmatography and its chemotactic activity was determined by Boyden chamber. Results The recombinant rnRANTES was expressed in E.coli with high efficiency. Purified protein showed chemotactic activity to pernripheral blood lymphocytes, U937 cells, and CCR4 stable-transfected HEK293 cells. Conclusion The purirnfled recombinant protein exerted chemotactic activity on PBL and cultured cell lines, and we have estabrnlished a experimental system for further study of structure and function of RANTES.