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

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.

Construction and expression of a prokaryotic expression vector encoding truncated Pseudomonas exotoxin A (PF38KDEL)

Objective To construct a prokaryotic expression vector encoding a truncated form(PE38KDEL) of Pseudomonas exotoxin A,and express the vector in E coli BL21.MethodsPE38KDEL 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 Western-bloting,respectively.ResultsThe prokaryotic expression vector pGEX-4T-I-PE38KDEL was constructed successfully.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.ConclusionPE38KDEL protein is expressed stably in E.coli BL21,which will provide a basis for the study on function of the protein.