布鲁氏菌S2WboA基因缺失株的构建及免疫效果-生物通.DOC

布鲁氏菌S2 WboA基因缺失株的构建及免疫效果 丁家波，程君生，牟 巍，毛开荣，张尔利，蒋玉文 （中国兽医药品监察所，北京 100081） 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。 关键词：布鲁氏菌；WboA基因；免疫性 Construction of a WboA-Deficient Brucella suis S2 Strain and Its Immune Effect DING Jia-bo, CHENG Jun-sheng, MU Wei, MAO Kai-rong, ZHANG Er-li, JIANG Yu-wen (China Institute of Veterinary Drug Control, Beijing 100081) Abstract:【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（Cmr）was cloned into the genomic DNA of S2 by homologous recombination with knocking out the WboA gene, and the recombinant rS2-WboA obtained. 【Result】 rS2-WboA was conversed into rough-phenotype from smooth-phenotype. The recombinant kep the ability to chloramphenicol resistance after 50 passages in tryptic soy agar (TSA) free of chloramphenicol. 1×107 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 1×1011 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