人趋化因子CCL3L1融合蛋白表达和活性分析  

Human CC ligand 3-like protein 1(CCL3L1)fusion protein expression and function assay

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

机构地区：[1]首都医科大学附属北京佑安医院,100069

出　　处：《中华微生物学和免疫学杂志》2007年第8期688-692,共5页Chinese Journal of Microbiology and Immunology

基　　金：北京市科委科技计划重大项目(D0906003040591)

摘　　要：目的人趋化因子CCL3L1进行融合蛋白原核表达和真核表达,纯化后活性分析。方法克隆人类CCL3L1 cDNA,构建两种CCL3L1表达载体,获得两个CCL3L1融合蛋白,一个在BL21大肠杆菌表达的GST-CCL3L1融合蛋白,另一个在S2果蝇细胞表达的His-CCL3L1融合蛋白。同时克隆了pcDNA3.1-flag-CCR5表达载体,培养了稳定表达flag-CCR5的细胞株,进行人趋化因子CCL3L1活性分析。结果成功构建人趋化因子CCL3L1融合蛋白原核表达载体pGEX-4T和真核表达载体pMT/ BiP/V5-His,免疫沉淀法检测和Western blot法分析His-CCL3L1蛋白在浓度1nmol/L到50 nmol/L存在剂量依赖性,浓度50 nmol/L到100 nmol/L没有剂量依赖性。纯化的His-CCL3L1蛋白能特异性结合CCR5受体。结论成功表达了融合蛋白GST-CCL3L1和His-CCL3L1,果蝇细胞表达的His-CCL3L1蛋白具有与天然CCL3L1相同的生物学活性,为进一步制备CCL3L1单克隆和多克隆抗体及研究CCL3L1影响HIV-1感染的机制提供基础资料。Objective To express the fusion protein glutathione S-transferase （GST） and human CC ligand 3-like protein 1 in BL21 E. coli, His-CCL3L1 （C-terminal His tag） fusion protein in S2 Drosophila cells, and to purify these proteins and analyze their bioactivities. Melhods Human CCL3L1 cDNA was cloned. Two expression vectors, pGEX-4T-CCL3L1 and pMT/BiP/V5-His-CCL3L1 were constructed. GST-CCL3L1 was expressed in BL21 E. coli and His-CCL3L1 was expressed in S2 Drosophila cells, pcDNA3.1-flag-CCR5 expression vector was cloned and a stable flag-CCR5 expression cell line was cultured. The binding capability between His-CCL3L1 and CCR5 receptor was detected with immunoprecipitation. Results pGEX-4X-CCL3L1 and pMT/BiP/V5-His- CCL3L1 vectors were constructed successfully. His-CCL3L1 showed dose dependence from 1 nmol/L to 50 nmol/L, but not from 50 nmol/L to 100 nmol/L. The results suggested that purified His-CCL3L1 protein can specifically bind to CCR5 receptor. Conclusion GST-CCL3L1 and His-CCL3L1 fusion protein were obtained successfully. His-CCL3L1 expressed in S2 Drosophila cells had the same bioactivity as that of the natural CCL3L1, which could provide the data to produce the monoantibody and polyantibody against CCL3L1, as well as to study the effects of CCL3L1 to HIV-1 ilffection.

关 键 词：CCL3L1 HIV AIDS CCR5 趋化因子 

分 类 号：R392[医药卫生—免疫学]