鼠疫耶尔森菌caf1基因缺失株体外生物学特性和对小鼠致病性分析

Analysis of biological characteristics and pathogenicity of caf1 deletion mutant strain of Yersina pestis

目的探索caf1基因缺失对鼠疫耶尔森菌体外生物学特性与对小鼠致病性的影响.方法利用CRISPR/Cas12a介导的基因编辑技术构建鼠疫耶尔森菌caf1基因缺失株,并通过PCR技术对caf1基因缺失验证.比较鼠疫耶尔森菌caf1基因缺失株和野生株201在生长速率、液体培养状态、聚集情况等体外生物学特性和对小鼠致病性等方面的差异.结果PCR扩增结果证实,caf1基因缺失株构建成功.体外生物学特征分析表明,与野生株相比,caf1基因缺失株的形态学和生长速率并未发生明显变化,自凝聚率升高.肺递送途径LD50分析表明,caf1的缺失导致鼠疫耶尔森菌气溶胶感染小鼠半数致死剂量显著降低.结论不同途径感染结果表明,caf1基因缺失株相较于野生株,在肺递送、滴鼻、皮下、尾静脉注射4种途径下对BALB/c小鼠的毒力均降低.caf1基因的缺失导致鼠疫耶尔森菌自凝聚率升高,对BALB/c小鼠致病性下降.

To explore the effect of the caf1 gene deletion on biological characteristics and pathogenicity of Y.pestis.A caf1 deletion strain of Y.pestis was constructed using CRISPR/Cas12a based gene editing method,and the deletion of caf1 was confirmed by PCR.The in-vitro biological characteristics(including morphology,growth rate,aggregation rate)and virulence of caf1 deletion mutant strain were determined and compared with those of wild type strain.PCR results showed that caf1 deletion mutant strain was successfully constructed.The morphology and growth rate of the mutant strain did not change significantly compared with those of the wild strain.Compared with the wild strain,the caf1 deletion mutant strain can gather and deposit from the liquid medium more quickly.LD50 analysis showed that the lethal dose of caf1 deletion mutant strain to mice infected via aerosol route reduced significantly.Furthermore,the virulence of caf1 deletion mutant strain to mice was significantly reduced compared with that of wild type strain infected via intratracheal inoculation,nasal drops,subcutaneous and tail vein injections routes.The deletion of caf1 gene enhances the aggregation rate of Y.pestis,but reduce the virulence of Y.pestis to mice.