2型猪链球菌pnp基因缺失株与回补株的构建及其生物学特性的研究

Construction and biological function of pnp gene deletion mutant and complemented strain of Streptococcus suis serotype 2

为研究2型猪链球菌(SS2) pnp基因的生物学功能,本研究通过基因无痕操作技术构建SS2 05ZYH33pnp基因缺失株Δpnp以及回补株CΔpnp,经PCR和测序鉴定结果表明,正确构建了pnp基因无痕缺失株和回补株。将05ZYH33、Δpnp以及CΔpnp分别在不同温度(28℃、37℃、42℃)培养后测定OD_(600nm)值,绘制各菌株的生长曲线;利用革兰氏染色后经镜检及透射电镜观察各菌株的形态结构;通过滴定微孔平板结晶紫法检测各菌株的生物被膜形成能力;采用接触法分析各菌株的溶血特性;于含不同抗生素的THB平板上培养各菌株,分析各菌株的药物敏感性;于氧化条件与酸性条件下培养各菌株,通过统计存活率评价各菌株的氧化耐受与酸耐受能力。结果显示,缺失株的生长速率明显低于野生株和回补株,且在冷应激条件下的生存能力下降;与野生株和回补株相比,缺失株细胞壁肽聚糖层的结构更加松散、溶血能力下降、生物被膜形成能力下降、对不同类型抗生素的敏感性增强、抗氧化与抗酸能力均下降。上述结果表明pnp基因是SS2的一个重要调节因子,参与调节细菌对外界环境变化的适应能力。本研究为科学防控猪链球菌病以及研发新型抗菌药物奠定基础。

To investigate the biological function of pnp gene of Streptococcus suis serotype 2,the pnp gene deletion mutantΔpnp and complementary strain CΔpnp of S.suis serotype 2 strain 05ZYH33 were constructed using markerless genetic manipulation technique,and the growth curve,bacterial morphological structure,biofilm formation ability,hemolysis,antimicrobial susceptibility,oxidative resistance and acid tolerance were detected to evaluate the biological function of the gene.The results showed that theΔpnp mutant and CΔpnp strain were successfully constructed.The OD_(600nm) values of 05ZYH33,Δpnp and CΔpnp were measured after culture at different temperatures,and the growth curves were plotted.The morphological structure of each strain was observed by transmission electron microscopy after Gram staining.The biofilm formation ability of each strain wa detected by titration micropore plate method.The hemolysis characteristics of each strain were analyzed by contact method.The strains were cultured on THB plates containing different antibiotics,and the antimicrobial sensitivity was analyzed.The strain were cultured under oxidative and acidic conditions,and their oxidative tolerance and acid tolerance were evaluated by detecting the survival rate.The growth curve of each strain showed that the growth rate ofΔpnp was significantly lower than that of the wild strain and CΔpnp,and the survival ability ofΔpnp decreased under cold stress.The results of morphological structure and hemolytic test of each strain showed that the cell wall peptidoglycan layer structure of the deletion strainΔpnp was looser and th hemolytic ability decreased.The results of antimicrobial sensitivity test showed that the sensitivity ofΔpnp to many different type of antibiotics was enhanced.The results of oxidative tolerance and acid tolerance test showed that the antioxidant and acid resistance of the deletion strainΔpnp decreased.The above results demonstrated that pnp gene is an important regulator of S.sui serotype 2,which is involved in regulating the adaptability of bacteria to changes in the external environment.This study lays a foundation for scientific prevention and control of swine streptococcosis and development of new antibacterial agents.