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。
[ 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 LDso, bacterial counts in liver and spleen, and qRT-PCR experiments. [Results] LDso 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, PIcA and PrfA） of Listeria monocytogenes ; The virulence of the RsbV genedeleted strain is significantly reduced, but it still has good immunogenicity.
出处
《微生物学报》
CAS
CSCD
北大核心
2013年第1期59-65,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金(30960274)
家畜疫病病原生物学国家重点实验室开放基金(KEYLAB200908)
石河子大学高层次人才专项(RCZX200801)~~
