血小板活化因子乙酰水解酶融合蛋白的表达、纯化及鉴定  

Expression,purification and identification of recombinant platelet-activating factor acetylhydrolase fusion protein

作　　者：邹颖[1] 毕新岭[1] 顾军[1] 缪明永[2] 王梁华[2] 时多[2] 

机构地区：[1]第二军医大学长海医院皮肤科,上海200433 [2]第二军医大学基础部生物化学与分子生物学教研室,上海200433

出　　处：《第二军医大学学报》2007年第5期504-507,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30271197).~~

摘　　要：目的:获得血小板活化因子乙酰水解酶(PAF-AH)全长编码基因并在GST原核表达系统中表达。方法:通过RT- PCR方法,从正常人外周血单个核细胞(PBMC)中扩增出PAF-AH全长编码基因,将其克隆至大肠杆菌表达载体pGEX-4T- 3,转化大肠杆菌BL21并诱导表达,采用GST蛋白纯化系统进行纯化。所得纯化蛋白以SDS-PAGE及Western印迹方法鉴定,并进行酶活性测定。结果:获得了PAF-AH基因,序列分析与GenBank数据库中序列一致。SDS-PAGE及Westem印迹结果显示,诱导表达出可溶性的融合蛋白,其相对分子质量与预期相符。纯化蛋白经Western印迹检测能够被特异性抗体所识别,具有免疫学活性;酶活性检测证实其具有特异的水解PAF的活性。结论:本实验在大肠杆菌表达系统中高效表达了有活性的PAF-AH蛋白。Objective：To obtain the full length of platelet activating factor acetylhydrolase （PAF-AH） gene and express it in prokaryocytes. Methods： The full length PAF-AH gene was amplified from peripheral blood mononuclear cells（PBMCs） by RT PCR and then cloned into the expression vector pGEX-4T-3. The PAF-AH GST was expressed in E. coli BL21 and purified with GST purifying system. The products was identified by SDS-PAGE and Western blot, and their enzymatic activities were detected. Results： Sequence analysis indicated that the sequence of the obtained PAF AH gene was identical with that in the GenBank. SDS-PAGE and Western blot confirmed the successful expression and purification of PAF-AH, with the molecular weight as expected. Western blotting showed that the purified protein could be recognized by specific antibodies and had immune activities. Enzymatic activity detection confirmed that the protein could specifically hydrolyzing PAF. Conclusion： Recombinant PAF-AH protein with biological activity has been efficiently expressed in E. coli in the present study.

关 键 词：血小板活化因子 乙酰水解酶 基因表达 纯化 

分 类 号：Q786[生物学—分子生物学]