马耳它布氏杆菌bp26基因缺失株的构建及鉴定

Development of a mutant Brucella Melitensis as vaccine in mice

为了建立马耳他布氏杆菌M5-90株弱毒疫苗株与野生株的鉴别诊断,本研究以bp26基因作为重组靶位点,以M5-90为亲本,利用bp26基因ORF外侧序列作为同源序列,将卡那霉素抗性基因(Kanr)整合到细菌基因组中,双交叉重组阳性菌株,经SDS-PAGE和western blot试验表明迁移率约为29ku的BP26蛋白在亲本菌中表达并可被鼠抗BP26高免血清识别,而突变株M5-90-26反应结果为阴性,表明BP26蛋白在突变疫苗株M5-90-26中未表达。M5-90-26具备从血清学角度区分M5-90-26免疫与野生型布氏杆菌感染,对布氏杆菌病防制、监测及净化具有重要的意义。

Brucella melitensis swain M5-90 is an attenuated vaccine strain with high protective rate and safety. The Mutant bp26 gene of Brucella melitensis named M5-90-26 was constructed by replacing the bp26 gene of attenuated M5-90 with kanamycin resistence gene. Double across recombinant mutant was selected by adding kanamyein ampicillin in culture and confirmed by PCR. SDS-PAGE and western blot showed that an 29 ku protein was expressed in the parent M5-90, which reacted with BP26 antiserum of the hyper-immunized mice, but not with the serum against mutant M5-90-26. Therefore the mutant strain would allow differentiation animals vaccinated with M5-90-26 from those infected with field strains by detetcing the anti-bp26 antibodies using rBP26 as antigen. Altogether these results indicate that Brucella melitensis M5-90-26 might be a promising vaccine against brucellosis.