牛源A型多杀性巴氏杆菌hyaD基因缺失株的构建及其在小鼠上的交叉保护性分析

hyaD为A型多杀性巴氏杆菌荚膜多糖合成相关基因，为探讨该基因对多杀性巴氏杆菌毒力及其免疫保护特性的影响，本研究利用同源重组方法，构建了牛源A型多杀性巴氏杆菌CQ2株（PmCQ2）的hyaD基因缺失株（ΔhyaD）。结果发现，与野生株相比，ΔhyaD的荚膜产生量及其感染后在脏器中的细菌定殖量均显著下降，其毒力显著降低。细胞试验发现，ΔhyaD更易黏附于巨噬细胞，被吞噬数量显著多于野生株，致使巨噬细胞相关炎性因子表达显著上调。hyaD基因的缺失，可调控与荚膜合成、LPS合成转运、铁转运等相关的基因表达显著下调，促使相关保护性抗原基因表达显著上调。以制备的PmCQ2株和ΔhyaD株灭活苗免疫小鼠（加强免疫1次），免疫后第21天分别采用同源和异源多杀性巴氏杆菌攻毒，ΔhyaD株免疫小鼠肺组织感染后24 h无明显或轻微病理损伤，对牛源A型、B型和F型多杀性巴氏杆菌的免疫保护率分别为100%、100%和80%，对兔源、猪源和禽源A型多杀性巴氏杆菌的免疫保护率分别为90%、100%、100%；而野生株PmCQ2除对牛源A型多杀性巴氏杆菌的保护率在80%以上外，对牛源B型和F型及兔、猪、禽源A型多杀性巴氏杆菌均无明显交叉保护作用。研究结果表明，hyaD基因可通过调控荚膜产生及毒力相关因子表达影响菌株毒力；hyaD基因缺失可调控相关交叉保护性抗原表达，赋予菌株交叉免疫保护特性。该研究为多杀性巴氏杆菌通用型疫苗的研发提供了参考。

Construction of Bovine Pasteurella multocida Type A hyaD Mutant and Its Cross-protection Analysis in Mice

hyaD is a gene involved in capsular polysaccharide synthesis of Pasteurella multocida type A. To investigate the effect of hyaD gene on P. multocida virulence and immune protection characteristics, an hyaD mutant (ΔhyaD) derived from bovine P. multocida type A strain CQ2 (PmCQ2) was constructed by homologous recombination. The results showed that, compared to the wild type strain, the capsule production and virulence of ΔhyaD were significantly reduced, and the bacterial loads in the mice organs infected with ΔhyaD were also decreased. The mutant strains were easier to adhere to mice peritoneal macrophage and were easily been swallowed, leading to the up-regulation of the expression of related inflammatory factors in macrophage. The hyaD gene deletion significantly down-regulated the expression of genes involved in capsule synthesis, LPS synthesis and transport, and iron transport. While significantly up-regulated the expression of protective antigen genes. The inactivated bacterin of wild type PmCQ2 and ΔhyaD strains were prepared by conventional methods, and the mice were immunized with both bacterin, respectively (one booster following the primary immunization). On the day 21 after first immunization, the immunized mice were challenged with homologous and heterologous serotypes P. multocida, the lungs of mice immunized with ΔhyaD had none or slight histopathological lesion at 24 h after challenged. Immunization of mice with ΔhyaD conferred significant protection against challenge from bovine P. multocida type A, type B and type F strains, as well as rabbit, porcine and avian P. multocida type A strains, with protection rate at 100%, 100%, 80%, 90%, 100%, and 100%, respectively; While immunization with similar doses of killed wild-type was failed to confer protections against heterologous and homologous serotypes excepting bovine P. multocida type A strains challenge, with protection rate above 80%. Taken together, the results show that the hyaD gene can affect the virulence of P. multocida by regulating capsule production and expression of virulence-related factors, and the hyaD gene deletion endows mutant cross-protection characteristics by up-regulating the expression of cross-protective antigens, and the mutant can be used as a candidate for the development of P. multocida vaccine. This study lays the foundation for the development of a universal vaccine against P. multocida infection.