重组铜绿假单胞菌外毒素A的原核表达和多克隆抗体制备及免疫保护功能分析  

Prokaryotic expression, polyclonal antibody preparation and immunoprotection potential of exotoxin A from recombinant Pseudomonas aeruginosa

作　　者：刘祥[1] 

机构地区：[1]陕西理工学院生物科学与工程学院,陕西汉中723001

出　　处：《湖南农业大学学报（自然科学版）》2016年第1期33-38,共6页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家大学生创新创业训练计划项目(201510720556);陕西省大学生创新创业训练计划项目(1949);陕西理工学院大学生创新创业训练计划项目(UIRP15006)

摘　　要：为探索铜绿假单胞菌ETA蛋白的免疫保护功能,采用分子克隆方法获得铜绿假单胞菌ETA表达菌株;利用切胶纯化获得ETA蛋白并免疫小鼠,制备ETA蛋白多克隆抗体;用ELISA方法,检测出ETA抗血清滴度达1∶8 000;蛋白质印迹分析结果表明,抗血清具有很好的特异性;小鼠免疫保护试验结果表明,ETA对铜绿假单胞菌感染的保护率达50%,显著高于对照组的免疫保护率。用DNAman软件对ETA序列同源性进行分析,发现革兰氏阴性菌的同源性高于革兰氏阳性菌,铜绿假单胞菌种间的同源性高于其他种类细菌的同源性;用MEGA软件对ETA的进化分析结果表明,不同血清型铜绿假单胞菌的亲缘关系高于其他细菌,据此可推测ETA免疫动物产生的特异性抗体可能为不同种类铜绿假单胞菌的感染提供交叉免疫保护。To explore the immunoprotection function of Pseudomonas aeruginosa（P. aeruginosa） exotoxin A（ETA） protein, its strain was obtained by approach of molecular clone; ETA was purified by the way of gel slices, and immunized mice was employed to prepare the polyclonal antibody. Antibody titer was 1 ∶ 8 000 detected by enzyme-linked immuno sorbent assay（ELISA）, and results of western blotting proved that the antiserum had a good specificity; mice specific immune was activated by ETA, and immune protection ratio for mice against P. aeruginosa infection was significant higher than the control group, reached to 50%. Results from ETA homology analysis showed that the homology of gram-negative bacteria was higher than gram-positive bacteria, and P. aeruginosa homology was higher than that of other species analyzed from software of DNAman. Results of phylogenetic analysis showed that the genetic relationship among various P. aeruginosa were higher than that of other bacteria. The specific antibodies produced by ETA immune animals might provide cross protection from the infection of different kinds of P. aeruginosa.

关 键 词：铜绿假单胞菌 外毒素A 原核表达 多克隆抗体 酶联免疫法 免疫保护 

分 类 号：Q789[生物学—分子生物学]