重组LTB蛋白在水弧菌VSP60中的高效表达与纯化  

Expression and purification of recombinant B subunit of Escherichia coli heat-labile enterotoxin in a marine vibrio

作　　者：王静[1] 李琳琳[1] 孔令洪[1] 于军[1] 郑瑾[1] 韩俊宏[1] 来宝长[1] 司履生[1] 王一理[1] 

机构地区：[1]西安交通大学生命科学与技术学院癌症研究所,710061

出　　处：《中华微生物学和免疫学杂志》2003年第6期432-435,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家"8 63"计划 ( 2 0 0 1AA2 15 2 2 1);国家自然科学基金( 3 0 0 70 848)资助项目

摘　　要：目的　优化重组大肠杆菌热不稳定肠毒素B亚单位 (rLTB)工程菌 (VSP6 0 )高效表达的条件。方法　以UVP凝胶照相系统扫描以及改良Lowry′s法检测rLTB表达量并分析各种因素对其影响。利用SephacrylS 10 0凝胶层析纯化rLTB蛋白。结果　工程菌 30℃振荡培养至吸光度 (A6 0 0 )值达0 .2～ 0 .3时加IPTG(终浓度为 0 .5mmol L) ,诱导培养 18～ 2 2h为rLTB蛋白表达的最佳条件。SephacrylS 10 0凝胶柱一步层析后 ,rLTB蛋白纯度可达 98.1%。结论　本研究所用的培养和纯化工艺简单易行 ,可获得高产量、高纯度的rLTB蛋白。Objective To optimize the conditions for expression and purification of Escherichia coli heat-labile enterotoxin B subuit (LTB) in a marine vibrio (VSP60) expression system. Methods The effects of culture temperature、dosage of inducer, growth statement of bacteria and inducing duration on rLTB expression in engineering bacteria VSP60 were observed. Sephacryl S-100 gel filtration chromatography was carried out for purification. Western blot was done for protein identification. Results The optimal conditions for generating LTB protein were as follows: bacteria cultured at 30℃ were induced with IPTG (final concentration 0.5mmol/L ) for 18-22?h when the A 600 value reached 0.2-0.3. After gel filtration with Sephacryl S-100 , the purity of rLTB reached 98.1%, the yield rate was about 52%. Conclusion A set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable.

关 键 词：重组大肠杆菌热不稳定肠毒素B亚单位 高效表达 纯化 水弧菌 

分 类 号：R392[医药卫生—免疫学]