多杀性巴氏杆菌H蛋白介导牦牛出血性败血症的致病作用及机制

Pasteurellamultocida,（P.multocida）is the aetiologicalbacteria of hemorrhagic septicaemia in yak of Qinghai-Tibet Plateau, however, its virulence factors and pathogenesis are poorly understood.The outer membrane protein H (OmpH) is the most abundant protein in the outer membrane of P. multocida and also has been identified as a multifunctional crucial protein involved in pathogenicityand invasiveness ..Previously, we showed that the recombinant OmpH protein from the virulent strain P0910 isolated from yak could produce 90% of the protection in mice, which suggested that the OmpH is an important protective antigen in P. multocida. However, whether the OmpH is involved in the initial adhesion to cell and invasion into host tissues has not been studied yet. .To study the precise function of the OmpH in P. multocida, the suicide vector pEX-ompH containing counter-selection gene sacB will be constructed and the deletion mutant strain and the complementary strain will be constructed by homologous recombination and counter-selection in this study. Moreover, the difference of serum resistance，antiphagocytic activity，adhesion and invasion to bronchial mucosal epithelial cells of yak，immune protection to mice among parents strain deletion mutant strain and the complementary strain will be compare in vitro and in vivo. Meanwhile，its difference of pathogenicity will also be evaluated in yak. This study will develop the research of gene-deleted vaccine , and provide a new way in preventing, monitoring and cleaning hemorrhagic septicemia of yak in Tibetan-plateau.

多杀性巴氏杆菌（P.multocida）是青藏高原牦牛出血性败血症的病原菌。但其毒力因子和致病机制尚不清楚。H蛋白（OmpH）是P.multocida外膜蛋白中的重要组分，对其致病力和侵袭力至关重要。本研究前期从牦牛源强毒株P0910中克隆并表达了OmpH，发现重组OmpH对小鼠可产生90%的保护力，是一种重要的免疫保护性蛋白，而OmpH是否参与P.multocida对细胞的粘附及对宿主的致病过程等则有待于进一步研究。. 本研究拟构建含有负筛选基因sacB的自杀载体pEX-ompH，利用同源重组和负筛选技术获得无抗性基因标记的P0910菌株ΔompH缺失株和互补株，通过体内外试验比较亲本株、缺失株及互补株在血清抵抗性、抗吞噬作用、对牦牛支气管粘膜上皮细胞的黏附和入侵、对小鼠的免疫保护力以及对牦牛的致病性的不同，以期为阐释P.multocida的致病机理，研制基因缺失疫苗提供理论依据。

本研究以多杀性巴氏杆菌（Pasteurella multocida ,Pm）地方血清型分离株P0910为亲本株，根据GenBank（登录号：NC017027）中PmHN06设计ompH基因上下游引物，利用同源重组技术，成功构建了P0910ompH基因缺失株（ΔompH）及互补株（P0910C），为多杀性巴氏杆菌致病机制的研究提供了技术平台。. 通过体内外试验对Pm P0910亲本株、ΔompH缺失株及互补株的遗传稳定性、生长速率、血清抵抗性、抗吞噬作用、对牦牛的致病性以及对牦牛支气管黏膜上皮细胞的黏附能力等生物学特性进行了研究，结果表明OmpH调节了Pm的生长，具有抗小鼠腹腔巨噬细胞的吞噬作用，且Pm依靠其菌体表面的H蛋白能够逃避血清中补体介导的杀菌作用；人工侵染牦牛试验结果表明胸腺可能是Pm 侵染宿主的靶器官；首次建立了牦牛支气管黏膜上皮细胞黏附模型，结果表明P0910亲本株对牦牛支气管黏膜上皮细胞的黏附能力远强于牦牛成纤维细胞。以上结果初步证实了ompH基因是Pm的关键毒力基因之一，H蛋白在Pm侵入宿主并对宿主细胞进行粘附的过程中起着重要作用。. 应用TMT技术对P0910亲本株和ΔompH缺失株进行比较蛋白质组学研究，通过质谱鉴定，成功鉴定出 773种差异蛋白，其中上调差异蛋白439种，下调差异蛋白334种，按照GO注释和KEGG代谢通路分析，发现这些差异蛋白以参与生物过程为主，它们显著富集到糖类和脂质代谢相关信号通路；筛选出了ABC转运蛋白、转录终止/抗终止蛋白Rpoe、热休克蛋白Dna J 这3个显著差异表达蛋白，它们在维持细菌正常的营养吸收及代谢产物的排出、ATP水解、细菌侵染以及抗逆境中至关重要，进一步解释了ΔompH缺失株新生物学表型产生的原因，提示Pm与宿主的互作可能与青藏高原牦牛的低氧适应机制有关，初步阐释了多杀性巴氏杆菌OmpH介导牦牛出血性败血症的致病作用机制，为未来制备ompH基因缺失疫苗奠定了重要的理论基础。