副猪嗜血杆菌Wza基因缺失株的构建及生物学特性研究

发布时间：2018-06-03 20:41

本文选题：自然转化 + ΔWza缺失株　；参考：《农业生物技术学报》2015年06期

【摘要】：副猪嗜血杆菌(Haemophilus parasuis,HPs)荚膜多糖目前被认为是其主要的毒力因子之一,其Wza基因在荚膜多糖的合成运输方面起重要作用。本研究通过构建p UC18-LR-Kan同源重组质粒和运用自然转化法,以Kan抗性的TSA平板成功筛选出Wza基因缺失株(ZJ1208-ΔWza)。生物学特性研究表明,ZJ1208-ΔWza缺失株和ZJ1208野毒株的菌落形态无明显差异,荚膜染色镜检表明在体外培养二者都能形成较薄的荚膜。在体外培养的生长速率方面,二者都在约12 h时其OD600达到最大值,其中ZJ1208-ΔWza缺失株OD600为1.246,而ZJ1208野毒株为2.196,高于ZJ1208-ΔWza株0.95;二者活菌数在约8 h多达到最大,ZJ1208野毒株活菌数对数值为9.763 4,ZJ1208-ΔWza缺失株株约为9.322 2,低于ZJ1208野毒株。菌体形态方面,在培养6 h左右,ZJ1208-ΔWza缺失株菌体大多数可形成长链状或长杆状,而野毒株大多数为较松散,短小的单个菌体。与ZJ1208野毒株比较,ZJ1208-ΔWza株有较快的体外沉降速率,对猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)的吞噬作用更加敏感,对小鼠毒力明显减弱;以上结果表明,Wza基因的缺失对HPs ZJ1208株的多项生物学特性产生了影响。本研究为进一步研究Wza基因在HPs荚膜形成中的作用提供基础资料。
[Abstract]:Haemophilus parasuis (HPs) capsule polysaccharide is considered to be one of its main virulence factors, and its Wza gene plays an important role in the synthesis and transport of capsular polysaccharides. In this study, the homologous recombinant plasmid of p UC18-LR-Kan was constructed and the Wza gene deletion strain ZJ1208- 螖 WZA was successfully screened by using Kan resistant TSA plate. There was no significant difference in colony morphology between ZJ1208- 螖 Wza deletion strain and ZJ1208 wild strain, and capsule staining showed that both of them could form a thin capsule in vitro. In the growth rate of culture in vitro, the OD600 of both of them reached the maximum at about 12 h. The OD600 of ZJ1208- 螖 Wza deletion strain was 1.246, while that of ZJ1208 wild strain was 2.196, which was higher than that of ZJ1208- 螖 Wza strain 0.95.The number of live bacteria of ZJ1208- 螖 Wza strain reached the maximum in about 8 hours, and the logarithmic value of live strain of ZJ1208- 螖 Wza deletion strain was 9.322 2, which was lower than that of ZJ1208 strain. In terms of cell morphology, most of ZJ1208- 螖 Wza deletions could form long chain or long rod, while most of wild strains were loose and short. Compared with ZJ1208 wild strain, ZJ1208- 螖 Wza strain had a faster in vitro sedimentation rate, more sensitive to phagocytosis of porcine alveolar macrophage pam, and weakened virulence to mice. These results suggested that the deletion of the Wza gene had an effect on many biological characteristics of HPs ZJ1208 strain. This study provides basic data for further study of the role of Wza gene in the formation of HPs capsule.
【作者单位】：安徽农业大学动物科技学院;浙江省农业科学院畜牧兽医研究所;
【基金】：国家自然科学基金(No.31201934)
【分类号】：S852.61

【参考文献】