RsbV基因缺失对单核细胞增生李斯特菌毒力的影响

Effects of the deletion of RsbV gene on virulence of Listeria monocytogenes

摘要:

摘要:【目的】探讨RsbV基因缺失对单核细胞增生李斯特菌(LM)毒力的影响。【方法】运用基因重叠延伸PCR(SOE-PCR)技术扩增出LM-XS5野毒株的RsbV基因缺失片段，然后用同源重组方法构建RsbV基因缺失株;通过肝脾细菌计数、LD50的测定和毒力基因转录水平的检测(qRT-PCR)，研究LM野毒株和缺失株在毒力上的差异。【结果】RsbV基因缺失株LD50是野毒株的104倍(P＜0.01);缺失株在小白鼠肝和脾内的载菌量均明显减少(P＜0.05);实时荧光定量PCR检测结果发现，缺失株4个毒力因子的表达水平均显著低于野毒株(P＜0.05);缺失株免疫小白鼠后对野毒株的攻毒具有良好的免疫保护作用。【结论】RsbV对LM的4个毒力基因inlA、LLO、PlcA和PrfA的表达具有调控作用;RsbV基因缺失株毒力明显减弱，但仍保留了较强的免疫原性。

Abstract:

Abstract:［Objective］To study the effect of RsbV gene deletion on the virulence of Listeria monocytogenes.［Methods］The fragment with RsbV deletion was generated by gene overlap extension PCR (SOE-PCR) and the mutant with RsbV deletion was obtained by homologous recombination based on the wild strain LM-XS5．The differences in virulence between the two strains were determined by LD50，bacterial counts in liver and spleen，and qRT-PCR experiments．［Results］LD50 of the RsbV gene-deleted strain was 104higher than that of the wild strain．The numbers of gene-deleted strain in the mouse's liver and spleen were significantly fewer than that of the wild strain (P＜0.05)．Results of qRT-PCR show that four virulence factors' expression levels of the RsbV gene-deleted strain were significantly lower than that of the wild strain (P＜0.05). The RsbV gene-deleted strain induced a strong immune response in mice against the wild strain．［Conclusion］RsbV regulates the expression of four virulence gene (inlA，LLO，PlcA and PrfA) of Listeria monocytogenes; The virulence of the RsbV genedeleted strain is significantly reduced，but it still has good immunogenicity．