胸膜肺炎放线杆菌血清10型apxIC-/p36+弱毒株的构建与鉴定

摘要：【目的】构建血清10型胸膜肺炎放线杆菌弱毒菌株，为胸膜肺炎放线杆菌减毒活疫苗研究奠定基础。【方法】通过细菌接合转移和SacB负向筛选标记完成突变株的构建与筛选，用PCR、Western blot、重组位点序列对突变株进行鉴定分析。首先构建含肺炎支原体p36基因的pEICALDH重组转移质粒，并转化供体大肠杆菌( E. coliX7213)，将转化的阳性克隆子与野生型APP血清10型亲本菌混合培养6 h；然后涂至含氯霉素抗性和烟酰胺腺嘌呤二核苷酸（NAD）的TSA培养基培养，挑取阳性克隆，接种至无抗性的含NAD的TSB液体培养基，培养6～8 h后涂至含10%的蔗糖及NAD的TSA培养基，培养24 h后挑取蔗糖抗性的克隆，即得到目的突变株。【结果】小鼠毒力试验结果表明突变株比亲本株的毒力显著降低；生长特性分析结果显示突变株与亲本株的增殖能力无显著差异；同时免疫试验结果表明突变株与安全剂量的亲本株均可诱导小鼠产生较好的免疫反应，证明apxIC基因缺失并不影响APP的免疫活性。【结论】成功构建了含猪肺炎支原体p36基因的胸膜肺炎放线杆菌血清10型突变株，所获得的突变株有望成为猪传染性胸膜肺炎弱毒疫苗株。

Construction and characterization of an attenuate strain apxIC-/p36+ of Actinobacillus pleuropneumoniae serovar 10

Abstract: Objective] To construct an attenuate Actinobacillus pleuropneumoniae serovar 10 strain apxIC-/p36+ for new vaccine development. Methods] The mutant was constructed by transconjugation and counter-selection and then verified by PCR, western blot and sequence analysis. A transconjugation plasmid pEICALDH was constructed and transformed into donor strain Escherichia coli X7213. After mixing the donor cells with A. pleuropneumoniae acceptor cells, we cultivated the mixture for 6 hours and plated on solid medium containing chloromycetin. Then the CmR positive clones were picked and inoculated into liquid medium without any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies(SucBR) were selected and considered as mutant. Results] Compared with parental strain, the mutant have the same growth rate in vitro and reduced virulence in mice; additionally, the animal experiment indicated that the mutant strain can successfully induce as good immune response as the parental strain, despite of deletion of apxIC gene. Conclusion] In conclusion, we successfully constructed the attenuate strain apxIC-/p36+ of Actinobacillus pleuropneumoniae serovar 10, and this mutation system will facilitate development of live attenuated vaccines.