人类CC3配体样蛋白1在果蝇Schneicier-2细胞的表达和活性分析  

Expression and function assay of human CC ligand 3-like protein 1 in Drosophila Schneider-2 cells

作　　者：徐斌[1] 石英[1] 李俊红[1] 张薇[1] 吴昊[1] 陈德喜[1] 

机构地区：[1]首都医科大学附属北京佑安医院感染科,北京100069

出　　处：《中华传染病杂志》2007年第9期538-542,共5页Chinese Journal of Infectious Diseases

基　　金：北京市科委科技计划重大项目(D0906003040591)

摘　　要：目的对HIV-1辅助受体趋化因子受体5(CCR5)配体人类CC3配体样蛋白1 (CCL3L1)进行果蝇Schneider-2细胞融合蛋白表达,纯化后活性分析。方法克隆人类CCL3L1 cDNA,构建CCL3L1表达载体pMT/BiP/His,获得Schneider-2果蝇细胞表达的His-CCL3L1融合蛋白,同时克隆pCDNA3.1-flag-CCR5表达载体,培养稳定表达flag-CCR5的细胞株,检测表达人趋化因子CCL3L1活性。结果成功构建人趋化因子CCL3L1融合蛋白真核表达载体pMT/BiP/His,表达并纯化出融合蛋白His-CCL3L1。免疫沉淀法和Western印迹法分析发现,纯化的His-CCL3L1蛋白能特异性结合CCR5受体,His-CCL3L1蛋白在浓度1～50 nmol/L存在剂量依赖性,50～100 nmol/L无剂量依赖性。结论果蝇Schneider-2细胞表达的His-CCL3L1蛋白具有与天然CCL3L1相同的生物学活性,为进一步探讨CCL3L1影响HIV-1感染的机制奠定了基础。Objective To express human CC ligand 3-like protein 1（CCL3L1）（ligand of HIV-1 co-receptor, CCR5）fusion protein in Schneider-2 Drosophila cells and evaluate its function after purification. Methods Human CCL3L1 cDNA was cloned, CCL3L1 expression vector pMT/BiP/His was constructed and His-CCL3L1 fusion protein was obtained in Schnelder-2 Drosophila cells, pCDNA3.1- flag-CCR5 expression vector was cloned and a stable flag-CCR5 expression cell line was cultured to detect the activity of human chemokine CCL3L1. Results Eukaryotlc expression vector pMT/BIP/His of human chemokine CCL3L1 fusion protein was constructed and the protein was expressed and purified successfully. Purified His-CCL3L1 protein could specifically bind to CCR5 receptor detected by immunoprecipitation（IP） and Western blot. His-CCL3L] protein was concentration dependent from 1-50 nmol/L and concentration independent from 50-100 nmol/L. Conclusion His-CCL3L1 fusion protein expressed in Schneider-2 cells has similar biological activities with natural CCL3L1 which is the basis of further studies on the function of CCL3L1 in the mechanism of HIV infection.

关 键 词：CCL3L1 HIV-1 获得性免疫缺陷综合征 受体 CCR5 趋化因子类 果蝇属 

分 类 号：R512.91[医药卫生—内科学] R392[医药卫生—临床医学]