变形链球菌荧光报告株的构建和鉴定

目的探讨变形链球菌(S.mutans)荧光素酶(luc)基因报告株构建方法,拟为探讨抗菌剂对多菌种生物膜中S.mutans的作用提供研究基础。方法将S.mutans UA159乳酸脱氢酶(ldh)基因及其上游部分约1100000片段克隆至自杀质粒pFW5-luc的多克隆位点,构建重组质粒,并经酶切和测序证实。采用自然转化的方法,实现重组自杀质粒和S.mutans UA159同源序列的单次交换。结果经过聚合酶链反应(PCR)和测序分析,筛选出具有报告活性的S.mutans ldh-luc基因荧光报告株。结论成功构建了S.mutans ldh-luc基因荧光报告株。     

变形链球菌gcp基因突变菌株的构建及鉴定

目的:构建变形链球菌gcp基因突变菌株,以便研究变形链球菌gcp基因功能。方法:厌氧培养变形链球菌UA159,以前期研究中pMD19T-gcp为模板,PCR扩增gcp基因内部序列,连接自杀载体pVA8912,酶切鉴定;将鉴定正确的质粒转化变形链球菌UA159,挑取阳性克隆,提取基因组DNA,用PCR结合酶切鉴定。结果:发现PCR产物及插入片段大小与预期值相符,表明成功构建了重组质粒pVA8912/gcp;经PCR鉴定:gcp基因失活株基因组中gcp基因内部成功插入了重组载体片段。结论:成功构建了变形链球菌gcp基因失活株,为该基因功能的研究奠定了基础。     

含尿素酶基因的重组变链菌JH1140变链素对变链菌UA159的抑制作用

目的:检测重组变形链球菌JH1140的变链素抑制其他变形链球菌(变形链球菌UA159)的效果.方法:以变形链球菌UA159为指示菌,选取野生型变形链球菌JH1]40和重组变形链球菌JH1140(各20例)与变形链球菌UA159共同培养,观察记录抑菌环直径.采用SAS9.1软件包进行方差分析,比较2种细菌的抑菌环直径,分析抑菌能力.以不同浓度变形链球菌UA159为指示菌,取野生型变形链球菌JH1140和重组变形链球菌JH1140(各20例)与变形链球菌UA159共同培养,观察记录抑菌环直径.采用SAS9.1软件包进行方差分析,比较不同浓度指示菌条件下,重组变形链球菌JH1140的抑菌环直径.结果:相同指示菌浓度下,野生型变形链球菌与重组变形链球菌的抑菌环直径之间未见显著差别,P＞0.05.不同浓度指示菌条件下,重组变形链球菌JH1140的抑菌环直径未见显著差别,P＞0.05.结论:重组重组变形链球菌JH1140与野生型变形链球菌JH1140相比,抑菌能力未见改变.重组细菌在表达新基因的同时,其变链素分泌未受影响.     

变形链球菌F-ATP酶基因重组质粒的构建和初步分析

目的：构建用于转化变形链球菌，含有F-ATP酶基因的重组质粒。方法：采用PCR方法，以变形链球菌基因组DNA为模板，扩增F-ATP酶β亚基5′末端序列，将克隆片段与载体pVA891酶切后连接，形成重组质粒，并对变形链球菌的转化作了初步分析。结果：构建的重组质粒经PCR鉴定、酶切鉴定和DNA序列测定，显示插入的目的片段序列正确。结论：变形链球菌目的片段与穿梭载体重组后能有效克隆，为通过同源重组特异突变变形链球菌染色体基因奠定基础。     

变形链球菌recA基因启动子荧光蛋白表达载体的构建

目的 构建由recA基因启动子启动的红色荧光蛋白穿梭表达载体,研究变形链球菌recA基因表达的特点,以期为不同致龋环境中变形链球菌致龋毒力因子基因的表达提供参照.方法 以变形链球菌(UA159)基因组DNA为模板,扩增recA基因启动子,采用双酶切反应连接入载体pDsRed2-N1,构建原核表达载体pRred;通过酶切重组人穿梭载体pDL276,构建红色荧光蛋白表达载体pLRred.结果 经酶切鉴定目的质粒片段插入无误,同时重组载体pLRred转化菌荧光观测结果提示,重组载体pLRred构建成功.结论 本项研究中构建的recA基因启动子红色荧光蛋白同源重组克隆载体正确,可应用于recA基因表达的研究,同时可为研究变形链球菌致龋基因的表达提供内参照.     

变形链球菌耐氟菌株中耐酸相关基因ffh突变的检测

目的检测变形链球菌耐氟菌株中耐酸相关基因ffh是否存在突变。方法体外诱导变形链球菌耐氟菌株,碱裂解法提取细菌基因组,根据GenBank上公布的变形链球菌耐酸相关基因ffh的序列,设计一对引物进行PCR,利用PGEM-TEasy载体进行T-A克隆,构建重组质粒,酶切电泳,并测序鉴定。结果变形链球菌耐氟菌株中耐酸相关基因ffh发生了突变。结论变形链球菌耐氟菌株中耐酸相关基因ffh发生了突变,并可能与其耐酸性增强有关。     

变异链球菌LuxS基因缺陷突变株的构建及其生物膜结构的初步观察研究

目的:通过同源重组法构建变异链球菌LuxS基因缺陷突变菌株,比较其与变异链球菌UA159标准株在生物膜形成上的差异。方法:运用基因同源重组方法将氯霉素抗性基因(Cmr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到PUC载体的多克隆位点中,构建出带氯霉素抗性标志的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用氯霉素抗性筛选出LuxS基因缺陷的突变株,并利用聚合酶链式反应(PCR)和哈氏弧菌发光实验进行检测。以釉质磨片为载体,在扫描电镜下观察上述两菌株含有20 g/L葡萄糖、20 g/L蔗糖的乳酪消化胨酵母(TPY)液体培养基中形成24 h的生物膜。结果:PCR基因扩增结果显示:突变株LuxS基因已被Cmr基因完全替换,成功的构建了变异链球菌LuxS基因突变株,并且突变株不能诱导哈氏弧菌BB170的生物发光。当用葡萄糖作为补充糖源时,变异链球菌UA159标准株和LuxS基因突变株所形成的生物膜表现型未见明显差异;而用蔗糖作为补充糖源时,两菌株所形成的生物膜有明显不同,UA159生物膜相对平滑均质,而LuxS基因突变株生物膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落。结论:成功构建出LuxS基因缺陷的变异链球菌突变株,与变异链球菌UA159标准株其生物膜结构发生改变,提示LuxS会影响变异链球菌生物膜的形成。     

变形链球菌AgⅠ/Ⅱ蛋白编码可变区(Ⅴ+)基因表达质粒的构建

目的 构建变形链球菌（S.mutans血清型f,s.mutnas血清型c)表面蛋白编码Ⅴ 基因重组质粒pCVc,pCVf和重组表达质粒pSRVc,pSRVf。方法 用PCR方法从sr基因中扩增目的基因片段与质粒pCR2.1连接，再将V 区基因片段转移到高效表达质粒pET21a( )。酶切电泳检测，结果 PCR扩增产物为1.16kb的DNA带，测序结果与sr基因序列V 区一致。酶切电泳证实V 区基因片段整合到pET21a( )的适当部位，构建重组表达质粒pSRVc,pSRVf。结论 本试验成功地构建了携带V 基因片段的表达质粒pSRVc,pSRVf。     

变形链球菌耐氟菌株中耐酸相关基因dltC突变的检测及临床意义

目的 检测变形链球菌耐氟菌株中耐酸相关基因dltC是否发生突变,推测该基因对变形链球菌耐氟菌株的影响.方法 体外人工诱导变形链球菌耐氟菌株,根据GenBank发表的变形链球菌dltC基因序列设计引物,对其耐氟菌株基因组进行PCR扩增,对扩增产物用DNA回收试剂盒回收鉴定,对回收产物用PGEM-T载体进行T-A克隆,构建重组质粒并测序.结果 PCR扩增获得与预期结果一致的特异性片段,并发现dltC基因有1个碱基发生突变,突变位点在8310(A→G).提交该序列到GenBank,获得登陆序列号为DQ272518.结论 变形链球菌耐氟菌株耐酸相关基因dltC发生点突变并属于同义突变.     

Antigenic relationships between the glucosyltransferase activities of Streptococcus mutans subspecies mutans.

Antibodies, directed against the partially-purified glucosyltransferase-A (synthesizing primarily insoluble glucans) and purified glucosyltransferase-B (producing predominantly soluble glucans) of Streptococcus mutans GS5 (serotype c), were used to assess the antigenic relatedness of the corresponding enzyme activities of strains MT703 (serotype e) and OMZ-175 (serotype f). Both anti-GTF-A and anti-GTF-B inhibited the corresponding activities from strains GS5, MT703 and OMZ-175 equally well. Furthermore, the cell-associated activities and sucrose-dependent adherence capabilities of all three organisms were strongly inhibited in a similar way by anti-GTF-A. The adherence-inhibiting activity of anti-GTF-A was demonstrated to be directed against a heat-sensitive component of the cell surface, probably the cell-associated GTF activity. The results are discussed in terms of the previously proposed close genetic relationship between Strep. mutans serotype c, e and f organisms.     

质粒介导变形链球菌转化的研究

本研究建立了变形链球菌ＭＴ８１４８转化系统。在本组转化培养条件下，变形链球菌ＭＴ８１４８在对数生长早期出现感受态但维持时间很短，供体ＤＮＡ最适浓度为２５μｇ／ｍｌ，超螺旋闭环质粒与线性质粒均可转化变链ＭＴ８１４８。与变链ＧＳ５相比，变链ＭＴ８１４８的转化率约低１００倍     

Transmission of mutans streptococci.

It has been shown that early establishment of mutans streptococci in the mouth of infants increases risk of caries. Extensive studies on the timing of infection, and on sources and routes of transmission of the organism have been performed. Results of many studies suggest that the source of mutans streptococci is the mother, especially her saliva. Methods used to attempt to verify this hypothesis have developed considerably in recent years. Recent research and work in progress using methods based on DNA analysis have increased and will extend knowledge about transmission. This review summarizes the results obtained by different methods of transmission of mutans streptococci. The final goal of studies is to accumulate adequate information for prevention strategies of mutans streptococcal infection.     

Intraindividual variations in counts of mutans streptococci measured by "Strip mutans" method

Abstract— Intraindividual variations in the mutans streptococci measured by Strip mutans, in a 1-day and a 5-day period, as well as the effect of toothbrushing, were investigated. 30 subjects participated, and their levels of salivary mutans were estimated by a scale containing four steps. With the exception of three comparisons, time intervals of different lengths did not cause a deviation of more than one step. Comparisons made before and after toothbrushing yielded the same score in 22 cases, whereas two subjects showed a discrepancy of two steps. Clearly, in a short-term perspective, variations can certainly be found, but more pronounced discrepancies are rare.     

Intraindividual variations in counts of mutans streptococci measured by "Strip mutans" method

Intraindividual variations in the mutans streptococci measured by Strip mutans, in a 1-day and a 5-day period, as well as the effect of toothbrushing, were investigated. 30 subjects participated, and their levels of salivary mutans were estimated by a scale containing four steps. With the exception of three comparisons, time intervals of different lengths did not cause a deviation of more than one step. Comparisons made before and after toothbrushing yielded the same score in 22 cases, whereas two subjects showed a discrepancy of two steps. Clearly, in a short-term perspective, variations can certainly be found, but more pronounced discrepancies are rare.