铜绿假单胞菌多表位核酸疫苗的构建及其生物学作用的研究  

Construction and biological effect of Pseudomonas aeruginosa multiepitope vaccine

作　　者：陈建国[1] 苏兆亮[2] 柳迎昭[1] 王胜军[2] 彭素芳[2] 李雅贞[2] 邵启祥[2] 许化溪[2] 

机构地区：[1]江苏大学附属人民医院检验科,江苏镇江212002 [2]江苏大学检验医学研究所,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2008年第6期484-486,490,共4页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省社会发展基金资助项目(BS2007041);镇江市社会发展基金资助项目(SH2008042);江苏大学临床医学基金资助项目(JLY20080049)

摘　　要：目的:构建铜绿假单胞菌(Pseudomonas aeruginosa,PA)多表位-MyD88基因疫苗并对其免疫原性进行研究。方法:将PA外膜蛋白F(OprF)的三个中和性B细胞表位及一个外源通用T细胞表位串联,采用重叠PCR方法合成DNA,并在此序列前插入组织型纤溶酶原(tPA)的信号肽基因,将此获得的片段与MyD88基因分别克隆入pIRES载体构建重组质粒pIRES-tPA-OprF:MyD88,并将该质粒免疫Balb/c小鼠,采用ELISA法测定特异性抗体水平。气管内接种PA,进行肺组织匀浆PA细菌计数。结果:成功构建真核表达质粒pIRES-tPA-OprF:MyD88,用该质粒免疫小鼠,能刺激机体产生高度特异性抗体,气管接种PA后,免疫组小鼠肺匀浆细菌计数明显低于其他对照组。结论:成功构建了重组有MyD88基因的PA表位核酸疫苗,接种小鼠后可诱导特异性免疫应答,产生较好的抗铜绿假单胞菌感染的免疫保护作用。Objective： To construct the Pseudomonas aeruginosa multiepitope-MyD88 gene vaccine and to study its immunogenicity in Balb/c mices. Methods： The epigene was designed and synthesized, containing three B cell epitopes of OprF and one foreign ＇promiscuous＇T cell epitope by overlap extension PCR, and tPA signal encoding sequence was amplified by PCR method and then was inserted to the 5＇terminus of epigene to construct tPA-OprF DNA fragment. The DNA fragment and MyD88 gene were cloned into expression vector pIRES to construct recombinant plasmid pIRES-tPA-OprF：MyD88. Balb/c mice were inoculated with pIRES-tPA-OprF：MyD88 plasmid, and the serum antibody titer was measured by ELISA. The number of viable bacteria in lung homogenate was examined after intratracheal challenge with Pseudomonas aeruginosa. Results： The recombinant plasmid pIRES-tPA-OprF： MyD88 was successfully constructed. Mice were immunized with this epigene vaccine, and the result showed that the higher level of antigen spe- cific antibody was detected, and resulted in a fewer number of bacteria in the lung as compared with control plasmid treatment. Conclusion： The construction of the Pseudomonas aeruginosa muhiepitope-MyD88 gene vaccine was proved to be successful and it could elicit dramatic immune response, and then can protect mice against Pseudomonas aeruginosa infection when it was applied as the epitope vaccine in experimental animal.

关 键 词：铜绿假单胞菌 外膜蛋白F MYD88 表位疫苗 免疫原性 

分 类 号：R378.99[医药卫生—病原生物学]