构建变形链球菌psm基因同源重组质粒的实验  

Construction of Streptococcus mutans psm gene homologous recombinant plasmids

作　　者：段劲[1] 陈伟[1] 刘筱娣[2] 郭丽宏[2] 

机构地区：[1]湖州师范学院,浙江省湖州市313000 [2]北京大学口腔医学院,北京市100081

出　　处：《中国组织工程研究与临床康复》2008年第20期3918-3921,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：北京市自然科学基金(7042035)“c血清型变形链球菌新基因的克隆及功能研究”~~

摘　　要：背景:为了深入研究磷酸蔗糖变位酶基因psm与变形链球菌S.mutans致龋性之间的关系,需要利用同源重组原理构建psm基因功能丧失菌株,因此构建一个符合同源重组条件的合格质粒载体就成为实验工作的重要前提。目的:构建变形链球菌S.mutans UA159psm的两种同源重组质粒。设计、时间及地点:于2005-12/2006-07在北京大学医学部病原微生物实验室完成的单一样本观察实验。材料:实验所用口腔链球菌均来自四川大学华西口腔医学院微生物实验室。方法:首先应用Southern Blot对psm基因的保守性进行分析,再将S.mutans UA159psm基因内部序列分别克隆至自杀质粒pSF151和pVA981上的多克隆位点,构建重组质粒。主要观察指标:psm基因的保守性及重组质粒pSF151及pVA981的构建结果。结果:psm基因在实验中所列8种S.mutans中保守存在;经过聚合酶链反应和酶切分析,两个重组质粒所插入片断无误。重组质粒pSF151(空质粒大小为3.5kbp)和pVA981(空质粒大小为7.1kbp)构建成功。结论:两种可以用于转化S.mutans UA159的重组质粒的构建可用于今后S.mutans UA159psm基因功能的研究,且重组质粒大小是否影响转化效率需后续观察。BACKGROUND： To further study the correlation between phospho-sugar mutase gene （psm） and Streptococcus mutans （S.mutans）, a psm-knocked strain was established based on homologous recombination principle. A homologous recombinant plamsid vector should be firstly constructed. OBJECTIVE： To construct two kinds of homologous recombinant plasmids of Streptococcus mutans strain UA 159 psm gene. DESIGN, TIME AND SETTING： Single sample observation was performed at Laboratory of Pathogenic Microorganism, Peking University Medical Department from December 2005 to July 2006. MATERIALS： Oral Streptococcus was provided by Microorganism Laboratory of West China College of Stomatology, Sichuan University. METHODS： Southern Blot was used to analyze the distribution ofpsm gene in oral streptococci. Two DNA sequences in the psm gene were selected and cloned respectively into multiple cloning sites of suicide plasmid pSF151 and pVA981 to construct the recombinant plasmids. MAIN OUTCOME MEASURES： Distribution ofpsm gene and construction of pSF151 and pVA981 recombinant plasmids. RESULTS： The psm gene was conserved in 8 strains of S. mutans in this test. By PCR analysis and enzyme digesting, it was confirmed that two kinds of S. mutans psm gene homologous recombinant plasmids were successfully constructed, recombinant plasmid pSF151 （empty plasmid was 3.5 kbp） and pVA981 （empty plasmid was 7.1 kbp）. CONCLUSION： Two psm gene homologous recombinant plasmids are successfully constructed and can be used in future research of construction of psm-negative mutans of S. mutans strain UA 159. Whether the size of recombinant plasmids can influence transfection rate needs further observation.

关 键 词：链球菌 变异 基因 质粒 重组 遗传 

分 类 号：R378.12[医药卫生—病原生物学]