clpp基因缺陷对肺炎链球菌毒力影响的研究.doc

clpP基因缺陷对肺炎链球菌毒力影响的研究 张 群，胥文春，尹一兵，朱兴华，李有强，张雪梅(重庆医科大学医学临床检验诊断学 省部共建教育部重点实验室，重庆，400016) 摘 要：目的 探索clpP基因缺陷对肺炎链球菌毒力的影响。方法 用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpP基因，用PCR、western blot鉴定缺陷菌株，通过RT-PCR分析自溶素(major autolysin A，lytA)、表面黏附素A(pneumococcal surface adhesion A，PsaA)、溶血素(pneumolysin，ply)和胆碱结合蛋白A(choline binding protein A, CbpA)的表达，并通过动物实验观察clpP基因缺陷株毒力改变情况。 结果 RT-PCR显示clpP缺陷株的四个毒力因子mRNA表达水平均低于野生菌，两者比较有统计学差异（P0. 05）；小鼠毒力实验表明野生菌株半数致死时间22 h，而缺陷株半数致死时间为8d，两者比较有统计学差异（P0 .0l）；缺陷菌在小鼠体内被清除速度也显著快于野生菌株（P0.05）。结论clpP基因缺陷使肺炎链球菌多种毒力因子表达减弱，毒力降低。 关键词：肺炎球菌；毒力因子；热休克蛋白；clpP基因 中图法分类号：R378 文献标识码：A Effect of clpP gene deletion on virulence of Streptococcus pneumoniae ZHANG Qun, XU Wen-chun, YIN Yi-bing, ZHU Xing-hua, LI You-qiang, ZHANG Xue-mei （Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing University of Medical Science, Chongqing,400016, China） Abstract: Objective To investigate the effect of clpPgene deletion on the virulence of Streptococcus pneumoniae.Methods clpP -deficient strain was constructed by LFH-PCR and identified by PCR and western bloting .Relative quantitative RT-PCR was used to measure the mRNA expression levels of autolysin A, pneumococcal surface adhesion A, pneumolysin and choline binding protein A and study the virulence change by mice virulence experiments.The deficient strain was applied to infect BALB／c mice to determine the virulenceResults clpP gene was replaced completely by erm cassette.The expression of virulent factors in clpP-deficient strain was lower than that of the wide-type(P0.05)，Mice virulence experiments showed the median lethal time of the widetype Was 22 h，while that of mutant was 8d(P0 01). The elimination of clpP mutant in mice blood was faster than that of the wide-type. Conclusion The deficiency of gene decreases the expression of virulent genes，thus finally reduced the bacterial virulence 基金项目：国家自然科学基金 Supported by the National Natural Science Foundation of China(306