F3重组毒素的克隆表达及纯化  

The Cloning, Expression and Purification of F3 Recombinant Toxin

作　　者：朱海兵[1] 黄轶[1] 曾昭淳[1] 

机构地区：[1]重庆医科大学生物化学及分子生物学教研室,重庆400016

出　　处：《科技导报》2009年第12期19-22,共4页Science & Technology Review

基　　金：国家自然科学基金项目(C030404)

摘　　要：以pEKH-F3质粒为模板,PCR扩增F3片段,将F3插入克隆质粒PQE80L,转化大肠杆菌,筛选,获得含有F3的PQE80L重组载体的克隆。提取绿脓杆菌菌株染色体DNA为模板,PCR扩增绿脓杆菌外毒素A催化区,PE40。然后将PQE80-F3和PE40重组,得到表达PQE80L-F3-PE40载体。转化至大肠杆菌DH5a,BL21,表达融合蛋白F3-PE40。结果显示,大肠杆菌表达了融合蛋白F3-PE40,表达的融合蛋白量大概占菌体总体蛋白量的20%。通过质粒提取、PCR扩增构建F3-PE40表达载体转化大肠杆菌,成功地表达了融合蛋白F3-PE40,为大规模表达、纯化F3-PE40的进一步功能奠定了基础。F3 is inserted into PQE80L carrier, after being amplified by PCR, with the plasmid pEKH-F3 as the template, to obtain PQE80L-F3 recombinated plasmid. With extracted pseudomonas aeruginosa strain chromosome DNA as the template, PE40, the pseudomonas aeruginosa exotoxin A catalysis area, is amplified by PCR. Then PQE80-F3 is recombinated with PE40, to obtain PQE8OL-F3-PE40 carrier. The DH5a and BI221 are obtained by transformation. The results show that F3-PE40 fusion protein is expressed and the quantity of expressed fusion protein accounts for about 20% of the total bacterial proteins. Through extraction of plasmid, PCR amplification, and F3-PE40 construction, E. coli expression vector is obtained by transformation to successfully express the fusion protein F3-PE40, which lays a foundation for large-scale expression and purification of F3-PE40 for further functionality.

关 键 词：融合基因 克隆 表达 绿脓杆菌外毒素A催化区 

分 类 号：R73-3[医药卫生—肿瘤]