医学licD2基因缺陷导致肺炎链菌毒力下降.docVIP

licD2基因缺陷导致肺炎链球菌毒力下降 作者：蓝锴，单幼兰，袁军，孟江萍，张雪梅，尹一兵 【关键词】 缺陷 　　Deletion of licD2 gene leads to reduced virulence of Streptococcus pneumoniae 　　【Abstract】AIM: To assess the role of licD2 gene in the virulence of Streptococcus pneumoniae. METHODS: licD2deficient strain was constructed and identified by PCR. Relative quantitative RTPCR was applied to measure the expression levels of virulent factors and the virulence was studied by mice survival test. RESULTS: The expression of virulent factors in licD2deficient strain was lower than that of widetype strain (Plt;0.05). The transformation capability of licD2deficient decreased obviously, even completely lost. Mice virulence experiments showed that the survival time of widetype strain was less than 1 d, while that of licD2 mutation was more than 2 weeks (Plt;0.001). The elimination speed of licD2 mutation in mice blood was faster than that of the widetype. CONCLUSION: The deficiency of licD2 gene leads to decreased transformation capability and the expression of virulent genes, thus markedly reducing the bacterial virulence. 　　【Keywords】 Streptococcus pneumoniae; virulence; reverse transcriptase polymerase chain reaction 　　【摘要】 目的： licD2是掌控肺炎链球菌生长所需物质胆碱代谢最重要的基因. 本研究探索licD2基因的缺陷是否影响细菌毒力. 方法： 采用插入复制失活的方法缺陷licD2基因，通过相对定量RTPCR分析其毒力因子表达情况，并利用动物体内试验观察licD2基因缺陷菌株毒力改变. 结果： RTPCR显示licD2缺陷菌株毒力因子表达低于野生菌，两组比较P＜0.05；转化试验发现缺陷菌株转化力下降甚至消失；小鼠毒力试验结果野生菌株半数致死时间＜1 d，而缺陷菌株半数致死时间为gt;14 d, Plt;0.001，差异有显著性；缺陷菌在小鼠体内被清除速度也显著快于野生菌. 结论： 结果提示licD2基因的缺陷使自然转化力下降，多种毒力因子表达减弱，细菌毒力降低. 　　【关键词】 链球菌，肺炎；毒力；逆转录聚合酶链反应 　　0引言 　　目前应用最广泛的23价肺炎链球菌（Streptococcus pneumoniae, S.pn）多糖疫苗，虽然效果较好，但是该疫苗不是蛋白类疫苗、免疫原性较弱，对免疫力低下的人群如老年人、幼儿等预防效果较差［1］. 最新研制出以破伤风和白喉类毒素作载体的11价S.pn结合疫苗对婴幼儿有很好的免疫原性，但也仅仅限于2岁以下的幼儿［2］. 因此要彻底解决S.pn感染的防治，仍需深入理解肺炎链球菌致病的分子机制. 胆碱作为S.pn所需的生长因子，在链球菌细胞分裂、转化和自溶等生物学特性中有着不可替代的作用. 链球菌表面的磷脂酰胆碱可直接与宿主细胞表面作用，从而促进黏附和侵袭［3］. 磷脂酰胆碱还是大量内皮和上皮细胞表面血小板活化因子受体（plateletactivating factor receptor, PAFR）的黏附配体，S.pn利用胆碱与此受体结合后对宿主细胞的黏附和侵袭会明显增加［4］. 磷脂酰胆碱还是将胆碱结合蛋白（cholinebinding proteins, CBP