牛布鲁氏菌A19突变株的构建及在BALB/c鼠中的免疫保护评估  

Construction of Brucella abortus A19-Δ VirB12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB / c mice

作　　者：易新萍[1] 叶锋[1] 姚刚[2] 谷文喜[1] 马晓菁[1] 吴冬玲 钟旗[1] 

机构地区：[1]新疆畜牧科学院兽医研究所,乌鲁木齐830000 [2]新疆农业大学动物医学学院,乌鲁木齐830052 [3]新疆天康畜牧生物技术股份有限公司,乌鲁木齐830032

出　　处：《微生物学报》2013年第11期1213-1220,共8页Acta Microbiologica Sinica

基　　金：自治区高新技术研究发展计划(200911109);国家科技支撑项目(2012BAD13B03);2012年新疆维吾尔自治区博士后资助;农业部公益性行业专项(201103008)~~

摘　　要：【目的】构建牛布鲁氏菌A19-ΔVirB12突变株并免疫BALB/c鼠,初步评估了其免疫保护效果。【方法】应用PCR方法扩增A19疫苗株VirB12基因的上下游同源臂序列,构建重组质粒pBK-CMV-SacBVirB12,将该质粒电击转化至布鲁氏菌A19感受态细胞中,筛选得到布鲁氏菌疫苗株A19的VirB12基因缺失株。以A19疫苗株为参照,应用A19-ΔVirB12疫苗接种BALB/c小鼠,免疫45d后布鲁氏菌2308强毒株攻毒,攻毒15d后取BALB/c鼠的脾脏进行克脾指数测定和病理组织学检测。Western-blotting鉴定VirB12蛋白的免疫反应性。【结果】构建了牛布鲁氏菌A19-ΔVirB12突变株,小鼠免疫攻毒后15d,A19-ΔVirB12免疫组和A19免疫组的克脾指数与对照组之间有显著性差异(P<0.05)。A19免疫组与A19-ΔVirB12免疫组之间克脾指数差异不显著(P>0.05)。Western blotting实验表明VirB12蛋白具有免疫反应性。【结论】牛布鲁氏菌A19-ΔVirB12突变株与亲本株A19免疫保护性无明显差异,通过血清学方法可区分疫苗免疫与野生型牛种布鲁氏菌(Brucella abortus)感染动物,具备作为标记疫苗的潜力。[ Objective] A19-ΔVirB12 deletion mutant of Brucella abortus was constructed by using homologous recombination technology. BALB/c mice were vaccinated intraperitoneally with the mutant to evaluate protective efficacy against Brucella abortus 2308 challenge. [ Method] A SaeB gene was amplified by PCR from pIB279 plasmid. The sequences upstream and downstream of the VirB12 gene were amplified by PCR from Brucella abortus A19. These three PCR products were subsequently inserted into pBK-CMV vector, namely pBK-CMV-SaeB-VirB12. This construct was transformed into Brucella abortus A19. The A19-Δ VirB12 mutants were obtained by Kanrand 5% sucrose selection. Six- week-old female BALB/c mice were distributed into three treatment groups, ineludingA19-Δ VirB12 group, A19 group and PBS control group. BALB/c mice were vaccinated intraperitoneally at a dose of 5.0 ×10^4CFU. At the 45-dayspost- immunization, all of mice were challenged with 2308 strain. Fifteen days after thechallenge, the levels of infection were expressed as means of the log10 CFU/spleen values. The histological changes were assessed among the groups. [ Results] Compared with PBS control group, the A19-Δ VirB12 deleted mutant had astatistically significant protection against 2308 challengesimilar to A19 strain. Western blotting showed that A19-A VirB12 mutant did not express VirB12 protein. [ Conclusion] The A19-ΔVirB12 deleted mutantelicits a strong protective immunity, and may beeomea promising vaccine candidate.

关 键 词：牛布鲁氏菌 A19-ΔVirB12突变株 同源重组 免疫保护 

分 类 号：S852.61[农业科学—基础兽医学]