布鲁氏菌S2 WboA基因缺失株的构建及免疫效果  

Construction of a WboA-Deficient Brucella suis S2 Strain and Its Immune Effect

作　　者：丁家波[1] 程君生[1] 牟巍[1] 毛开荣[1] 张尔利[1] 蒋玉文[1] 

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《中国农业科学》2008年第8期2448-2453,共6页Scientia Agricultura Sinica

基　　金：国家"863"项目(2006AA10A203)

摘　　要：【目的】WboA基因编码光滑型布鲁氏菌脂多糖(LPS)O-侧链合成必须的糖基转移酶,该基因的缺失或破坏会影响布鲁氏菌光滑型表型的形成。本研究拟构建WboA基因缺失的重组粗糙型布鲁氏菌,以使布鲁氏菌弱毒疫苗与野毒株布鲁氏菌感染相区分。【方法】以猪源光滑型布鲁氏菌S2株为研究对象,通过同源重组将氯霉素抗性基因(Cmr)完全替代S2株的WboA基因,获得重组粗糙型布鲁氏菌rS2-WboA株。【结果】rS2-WboA株在适宜的培养基(TSA)上传50代后仍保持对氯霉素的抗性。用1×107 CFU rS2-WboA免疫Balb/c小鼠和豚鼠,1个月后均能通过平板凝集试验检测到粗糙型抗体。1×1011 CFU rS2-WboA菌攻毒Balb/c小鼠后,不会引起死亡,并且能抵抗200 CFU强毒M28的攻击。小鼠试验显示了rS2-WboA与原始株S2相似的保护性和更高的安全性,其保护性也略高于另一重组株rS2-WbkC。【结论】WboA可作为构建重组粗糙型布鲁氏菌疫苗株缺失的靶基因。[Objective] WboA gene encodes a glycosyl transferase, an enzyme essential for the synthesis of O antigen. Disruption of the WboA gene in smooth B. abortus and B. melitensis results in rough, attenuated mutants which fail to produce the O polysaccharide （O antigen）. In this study, whether the wboA gene disruption is responsible for the rough phenotype of B. suis 2 strain （S2） was explored. [ Method] Chloramphenicol resistance gene （Cm^r） was cloned into the genomic DNA of S2 by homologous recombination with knocking out the WboA gene, and the recombinant rS2-WboA was obtained. [Result] rS2-WboA was conversed into rough-phenotype from smooth-phenotype. The recombinant kept the ability to chloramphenicol resistance after 50 passages in tryptic soy agar （TSA） free of chloramphenicol. 1×10^7 CFU rS2-WboA could induce specific rough antiserum in mice or guinea pigs, which could be detected by aggregation tests. No mice were died after inoculation of 1×10^11 CFU rS2-WboA. Afterwards, the rS2-WboA immunized mice could resist the challenge of 200 CFU virulent M28. Mice tests showed rS2-WboA offered similar protection to S2 strain, but more safe than S2. Its protective effect was a little better than the other recombinant strain rS2-WbkC. In view of these results, rS2-WboA is a promising candidate for vaccine strain. [Conclution] WboA is a prospective candidate as a gene deletion target for brucella recombination.

关 键 词：布鲁氏菌 WboA基因 免疫性 

分 类 号：S852.5[农业科学—基础兽医学]