鸡白痢沙门菌ssaV基因缺失株的构建及其生物学特性分析

Construction and Biological Characterization of ssaV Gene Mutant of Salmonella pullorum

为探究ssaV基因对鸡白痢沙门菌致病性的影响,利用同源重组技术,以pKD3质粒为模板,设计同源臂,扩增外源线性DNA片段,将该片段电转化入含有pKD46的鸡白痢沙门菌C79-13,丢失pKD46后,获得重组菌C79-13ΔssaV∶∶Cm,将pCP20导入该重组菌,通过筛选获得ssaV基因缺失株C79-13ΔssaV,同时构建其回补株C79-13ΔssaV(pBR322-ssaV),并对突变株C79-13ΔssaV的生长特性和生化特性、遗传稳定性及毒力等基本生物学特征进行分析。PCR扩增和测序结果显示,成功构建了缺失株C79-13ΔssaV,野生株C79-13、缺失株C79-13ΔssaV和回补株C79-13ΔssaV(pBR322-ssaV)呈现出一致的生长特性和生化特性,突变株C79-13ΔssaV的遗传稳定性良好,其在雏鸡体内的定殖能力降低,且口服攻毒后,缺失株C79-13ΔssaV对雏鸡半数致死量(LD50)至少是野生株(C79-13)的100倍。以上结果表明,ssaV基因与鸡白痢沙门菌的致病性相关,其缺失会明显降低鸡白痢沙门菌的毒力。

The aim of this study was to investigate the effect of ssaV gene on the pathogenicity of Salmonella pullorum. According to the homologous recombination technique,the target DNA with the homologous arms was amplified in the plasmid pKD3. Next the DNA fragment was transformed into Salmonella pullorum C79-13 which already had the plasmid pKD46,after loss of pKD46,the plasmid pCP20 was transformed into the recombinant strain C79-13ΔssaV∶∶Cm to delete the chloramphenicol resistance gene,then the mutant C79-13ΔssaV was confirmed through screening,and its complement strain C79-13ΔssaV(pBR322-ssaV)was also constructed. The results of basic biological characteristics showed that the growth characteristics and biochemical characteristics of C79-13ΔssaV did not change compared to those of C79-13,and the mutant C79-13ΔssaV was stable after lacking of ssaV,the loss of ssaV reduced the colonial ability of Salmonella pullorum in chickens,the LD50 of C79-13ΔssaV was at least 100 times that of C79-13 for chickens after oral challenge. This study demonstrated that loss of ssaV gene could decrease significantly the virulence of Salmonella pullorum,which laid a foundation for further research on the function of ssaV and development of an attenuated vaccine against Salmonella pullorum infection.