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【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 investigate the effect of deletion of RsbV gene on the virulence of Listeria monocytogenes (LM). 【Methods】 The deletion fragment of RsbV gene of LM-XS5 was amplified by SOE-PCR. Then the deletion of RsbV gene was constructed by homologous recombination. And virulence gene transcription level (qRT-PCR) to study the virulence differences of LM wild-type and deletion strains. 【Results】 The results showed that the LD50 of RsbV gene was 104 times of that of wild type (P <0.01), and the number of bacterium in the liver and spleen of mice was significantly decreased (P <0.05). The result of real-time fluorescence quantitative PCR , And the expression levels of four virulence factors of the deleted strains were significantly lower than those of the wild strains (P <0.05). After the mice were immunized with the deleted strains, they had good immune protection against the challenge of the wild strains. 【Conclusion】 RsbV regulates the expression of 4 virulence genes inlA, LLO, PlcA and PrfA in LM. The virulence of RsbV gene deletion strain is obviously weakened, but still retains strong immunogenicity.