铜绿假单胞菌外毒素衍生物PE38KDEL的原核表达载体构建及其鉴定  

Construction and expression of a prokaryotic expression vector encoding truncated Pseudomonas exotoxin A(PE38KDEL)

作　　者：张琴[1] 李明远[1] 张林[2] 蒋忠华[1] 祁志荣[1] 李虹[1] 

机构地区：[1]四川大学基础医学与法医学院微生物学教研室 [2]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《免疫学杂志》2007年第3期291-294,共4页Immunological Journal

基　　金：国家自然科学基金(30470613);教育部博士点专项基金(20040610053)资助

摘　　要：目的构建铜绿假单胞菌外毒素衍生物(PE38KDEL)的原核表达载体并对其表达的蛋白进行鉴定。方法采用PCR方法扩增本实验所需要的PE38KDEL基因片段,再通过酶切及连接反应构建原核表达载体pGEX-4T-1-PE38KDEL,重组载体经过限制性内切酶酶切、PCR扩增鉴定及DNA序列测定证实插入片段正确后,转化感受态大肠杆菌BL21,经IPTG诱导表达,表达产物经SDS-PAGE电泳后及蛋白免疫印迹法分别测定其大小和特异性。结果经鉴定证实原核表达载体pGEX-4T-1-PE38KDEL构建成功,且在大肠杆菌BL21中获得了PE38KDEL与GST的融合表达,且表达蛋白产物的分子质量大小与预期值一致,并可被PE的特异性抗体所识别。结论PE38KDEL在大肠杆菌中获得了高效的融合表达,为下一步研究其功能奠定了基础。Objective To construct a prokaryotic expression vector encoding a tnmcated form （PE38KDEL） of Pseudomonas exotoxin A, and express the vector in E coli BL21. Methods PE38KDEL gene was cloned by polymerase chain reaction （PCR）, and then inserted into the prokaryotic expression plasmid pGEX-4T-1. The recombinant vector confirmed by PCR, restriction endonucleases digestion, and DNA sequence analysis was transformed into E. coli BL21. The expression of the protein was induced by IPTG. Specificity and relative molecular weight of the expression product were detected by SDS-PAGE and Westem-bloting, respectively. Results The prokaryotic expression vector pGEX-4T-I-PE38KDEL was constructed successfuUy. The E. coli BL21 transformed with recombinant plasmid pGEX-4T-I-PE38KDEL had expressed GST-PE38KDEL recombinant protein. The expression product could react with the specific antibody, and the relative molecular weight of the expression product was identical to the expected value. Conclusion PE38KDEL protein is expressed stably in E. coli BL21, which will provide a basis for the study on function of the protein.

关 键 词：铜绿假单胞菌外毒素 PE38KDEL 原核表达 

分 类 号：R378[医药卫生—病原生物学]