布鲁氏菌M5-90疫苗株磷酸葡萄糖变位酶基因(pgm)突变株的构建及免疫评价  

Construction and Immune Evaluation on Mutation of phosphoglucomutase gene (pgm) Attenuates of Brucella Melitensis Vaccine M5-90

作　　者：李天森[1] 张辉[1] 张艳[1] 孟茹[1] 张豫[1] 孙志华[1] 蒋攀文[1] 王震[1] 陈瑞花[1] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,石河子832000

出　　处：《农业生物技术学报》2013年第4期448-455,共8页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2010CB30203);国家自然科学基金项目(No.31001046/30960288);高层次人才科研启动资金(No.RCZX200914)

摘　　要：为获得可以区分自然感染和疫苗免疫且毒力较弱的布鲁氏菌候选疫苗株,本实验研究了磷酸葡萄糖变位酶基因(pgm)对布鲁氏菌(Brucella melitensis)M5-90疫苗株毒力的影响,并对其进行了免疫评价。本实验构建了重组质粒pGEM-7zf-Δpgm,电转化布鲁氏菌M5-90感受态细胞,筛选获得布鲁氏菌疫苗株M5-90的pgm基因缺失株(Δpgm),并对获得的M5-90 Δpgm株进行遗传稳定性、免疫原性检测。结果显示,Δpgm能稳定传15代;Δpgm组的抗体含量较亲本株M5-90组差异不显著;Δpgm在诱导机体产生IL-2的能力要强于M5-90,产生INF-γ的能力低于M5-90;该基因缺失株采用虎红平板凝集试验和试管凝集试验不发生凝集反应;Western blot证实,Δpgm不能诱导小鼠产生抗pgm蛋白的抗体。本研究构建的Δpgm具有很好的遗传稳定性和免疫原性,为研制布鲁氏菌基因缺失疫苗提供了实验依据。In order to obtain a Brucella vaccine candidate which can distinguish between natural infection and attenuated vaccine, this paper studied the phosphoglucomutase gene （pgm） influence on Brucella M5-90 vaccine strain virulence and immune evaluation. The recombinant plasmid pGEM-Tzf-Apgm was constructed, and which was electroporated into Brucella melitensis M5-90 competent cells, screening for Brucella vaccine strain M5-90 pgm gene deletion mutant strains （△pgm） and to detect the M5-90 △pgm strain genetic stability, and immunogenicity. The test results showed that the reverse mutation did not occur within 15 passages; the △pgrn antibody content and the parent strain M5-90 group had no significant difference; △pgm had the ability to produce the IL-2 was stronger than that of M5-90, the ability to produce INF-γ was lower than the M5-90; the rose bengal plate agglutination test and tube agglutination test of this gene deletion strains was not agglutinated; the results of Western blot confirmed that the △pgm could not induce the mice to produce anti- pgm protein antibody. The △pgm constructed in this study, had good genetic stability and immunogenicity, provided basic data for the next step to more in-depth develop Brucella gene-deleted vaccine.

关 键 词：布鲁氏菌 磷酸葡萄糖变位酶基因(pgm) 同源重组 免疫原性 

分 类 号：S852.614[农业科学—基础兽医学]