【作者】 徐仰龙；

【导师】 杨德琴；

【作者基本信息】 遵义医学院 ， 口腔临床医学， 2009， 硕士

【摘要】 致龋菌通过多个毒力因子在龋病发生过程中发挥重要作用,其中耐酸是致龋菌最重要的生物学特征和龋病发生的前提。只有耐受生物膜的酸性环境,致龋菌才能生存并持续代谢和产酸,导致牙体硬组织脱矿与再矿化失衡而引起龋病发生。质子移位膜ATP酶（membrane-bound proton-translocating ATPase,FoF₁ATPase,F-ATPase）是致龋菌最重要的耐酸毒力因子,通过将胞内氢离子外运维持胞内相对稳定的中性代谢环境,同时使细胞周围生物膜环境中酸性物质不断堆积,pH持续下降直至临界pH。牙菌斑是生物膜的一种,是病原菌致龋的原位环境,研究表明致龋菌毒力因子在浮游状态与生物膜中的表达有很大差异性,在生物膜中观察F-ATPase的表达能更准确反应致龋菌耐酸因子在龋病发生过程中的作用机理,为深入研究龋病的病因学及预防提供理论基础。目的:本课题以主要致龋菌变异链球菌（Streptococcus mutans,S.mutans,变链菌）为代表,构建变链菌（UA159）F-ATPase操纵元启动子启动的绿色荧光蛋白穿梭表达载体,为原位环境中研究变链菌致龋过程中的耐酸力调控提供物质条件和基础,同时为有效监控其表达,我们构建以管家基因重组结合蛋白A（recombination protein A,RecA）操纵元启动子启动的红色荧光蛋白穿梭载体作为内参照,对F-ATPase的表达强度进行定量。方法:一:构建F-ATPase操纵元启动子启动的绿色荧光蛋白穿梭表达载体以变链菌（UA159）基因序列设计并合成F-ATPase操纵元启动子引物,PCR扩增并插入到pMD18-T载体测序,确认目的片段后经该载体将F-ATPase操纵元启动子插入到绿色荧光蛋白表达载体（pEGFP-N1）,构建原核荧光蛋白表达载体（pFgfp）;从中分离pFgfp中目的片段（F-ATPase操纵元启动子启动的绿色荧光蛋白表达载体）,连接至穿梭载体（pDL276）多克隆位点,构建肠杆菌-链球菌荧光蛋白表达载体。二:构建管家基因RecA操纵元启动子启动的红色荧光蛋白穿梭表达载体于变链菌UA159中扩增获得RecA启动子并测序验证后定向重组至红色荧光蛋白载体pDSred2-N1中,构建荧光蛋白表达载体（pRred）;分离pRred中目的基因片段定向插入pDL276多克隆位点,构建穿梭表达载体pLRred。三:重组载体转化变链菌验证利用基因转化试剂盒将重组表达载体转化变链菌并初步验证其表达。结果:（1）pMD18-F中的目的片段测序结果与变链菌基因组进行对比,证实为目的片段—F-ATPase操纵元启动子序列;荧光显微镜对转化菌落观测及菌落PCR鉴定原核表达载体pFgfp正确;重组载体（pLFgfp）转化菌落具有绿色荧光,提取菌落中质粒双酶切（EcoRⅠ/XbaⅠ）后电泳分析,目的片段大小相一致,表明pLFgfp构建成功。（2）对插入T载体的片段测序,与UA159全基因组对比,证实为RecA操纵元启动子序列;插入RecA启动子的pDsRed2-N1重组载体pRred,转化大肠杆菌后菌落见红色荧光,对荧光菌落行PCR鉴定;连接入RecA启动子启动的荧光蛋白编码片段的重组质粒pLRred转化到DH5α中,观测其成功表达,荧光菌落质粒抽提、酶切、电泳均证实插入片段与目的片段一致,表明pLRred构建成功。（3）转化有穿梭表达载体的变链菌能在含有kan+的BHI培养基上生长,证实菌体荧光,表明重组载体可于变链菌内表达。结论:本实验成功构建由变链菌F-ATPase启动子及内参照RecA启动子启动的荧光蛋白穿梭表达载体,通过转化鉴定,两种穿梭表达载体中的启动子均能正确启动报告基因荧光蛋白在变链菌中的表达,并随变链菌的扩增而扩增。本课题为进一步在生物膜中原位研究致龋菌耐酸因子的表达和调控提供物质条件和基础。更多还原

【Abstract】 Objective: F-ATPase（membrane-bound proton-translocating ATPase, F₀F₁ATPase）, an important aciduric factor of caries pathogen, is essential prerequisite for cariogenesis. As a major component of acid-adaptive response mechanism of cariogenic bacteria, F-ATPase makes bacteria keep activity even in low pH biofilms environment through maintaining internal pH values by pumping protons out of cells. Meanwhile, with more and more protons excretion and accumulation, the pH value in dental plaque reaches 5.4 or 5.5 and results in dental caries. Therefore, F-ATPase plays a key role in dental caries. It is well known that both phenotype characteristics and gene expression of cariogenic pathogens in biofilms are different from in planktonic status. Also it is undoubted that only in the dental pique biofilms can the pathogens performance their cariogenic function. In order to illustrate the F-ATPase’s role of the cariogenic pathogens in dental plaque, we constructed a green fluorescent protein expression vector driven by promoter of F-ATPase operon of Streptococcus mutans（S.mutans）, and a red fluorescent protein shuttle vector promoted by RecA operon’s promoter as a control.Methods:1. Construction of the green fluorescent protein expression vector driven by F-ATP operon promoter:The promoter sequence of F-ATPase operon amplified from PCR was inserted into a plasmid express vector pEGFPN1 to construct a Prokaryotic expression vector pFgfp. Then the promoter gene of F-ATPase operon and a green fluorescent protein gene were ligated together into a shuttle vector pDL276 named as pLFgfp.2. Construction of the red fluorescent protein expression vector driven by RecA operon promoter: pRred, a recombinant vector containing RecA operon promoter which was separated from T vector, was constructed and transformed into DH5α. After that the red fluorescent protein gene promoted by RecA operon promoter, separated from pRred, was inserted into a shuttle vector pDL276, named as pLRred.3. Transformed into host cell-UA159:These above two shuttle vectors were transformed into S. mutans and assayed with fluorescence microscopy.Results:1. pLFgfp, the recombinant shuttle vector containing a green fluorescent protein promoted by F-ATPase operon promoter, expressed green fluorescent protein in DH5a cells.2. pLRred, containing RecA operon promoter and red fluorescent protein gene, was successfully constructed and -expressed red fluorescent protein in cells.3. Both of the two vectors respectively containing F-ATPase and RecA promoters were separately transformed into the host cells S.mutans and express the fluorescent proteins.Conclusion: The recombinant vectors pLFgfp and pLRred were successfully constructed, which provide an important foundation for further study on relationship between acid-adaptive response of S. mutans F-ATPase and cariogenesis under different pH condition in biofilms.更多还原