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【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6h;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6～8h后涂至含10%的蔗糖及NAD的TSA培养基,培养24h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。
【Objective】 To construct the attenuated serotype of Actinobacillus pleuropneumoniae serotype 10 and lay the foundation for the study of attenuated live attenuated A. pleuropneumoniae vaccine. 【Method】 The construction and screening of mutant strains were carried out by bacterial conjugation and SacB negative screening, and the mutants were identified by PCR, Western blot and recombinant loci. First, the pEICALDH recombinant transfer plasmid containing P36 gene of Mycoplasma pneumoniae was constructed and transformed into donor E. coli X7213. The positive clones transformed were mixed with wild-type APP serum type 10 parental bacteria for 6h. Resistant and niacinamide adenine dinucleotide (NAD) TSA culture medium, positive clones were picked and inoculated into non-resistant NAD-containing TSB liquid medium, cultured 6 ~ 8h after coating with 10% Of sucrose and NAD TSA medium, cultured 24h picked sucrose-resistant clones, to obtain the desired mutant. 【Result】 The results of virulence test in mice showed that the virulence of mutant strain was significantly lower than that of the parent strain. The growth characteristics analysis showed that there was no significant difference in proliferation between the mutant strain and the parent strain. Meanwhile, Strain can induce mice to produce a better immune response, proof of apxIC gene deletion does not affect the APP’s immunocompetence. 【Conclusion】 The 10 strains of Actinobacillus pleuropneumoniae serotype 10 containing Mycoplasma hyopneumoniae P36 gene were successfully constructed and the mutants obtained were expected to become the attenuated porcine infectious bronchitis vaccine strain.