LuxS基因报告质粒的构建和在变链菌中表达能力的研究
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摘要
菌斑生物膜是黏附于牙齿表面的微生物团块,是龋病发生的始动因子。生物膜中细菌基因表达和表型明显不同于浮游细菌,突出表现为对抗生素具有较强抵抗力,即具有一定的耐药性,可能与信号传导系统的调节作用有关。
通过运用各种分子生物学技术,对一些信号传导系统在变链菌形成生物膜过程中的作用已有初步了解。变链菌(S.mutans)密度感应系统在生物膜的形成和稳态维持中作用最为关键。其中,AI-2/LuxS可参与调节S.mutans生物膜的形成、糖代谢、耐酸能力以及多种毒力因子的分泌,对S.mutans在口腔这个复杂的环境中生存有着重要意义。S.mutans等口腔定植细菌可通过细菌间的独特联系来利用或干扰AI-2/LuxS介导的群体感应,因此与正常人体内的菌群平衡和菌群失调有着密切的关系。然而,目前对于AI-2的合成、分泌、调节途径,以及在活体生物膜内如何调节相邻细菌的生理活动仍不清楚。因此进一步明确S.mutans中LuxS介导群体感应的作用机制,将为找到降低菌斑生物膜的致病性的方法,摸索出“耐药性”的应对措施提供线索,无疑有着重要的意义。
本研究运用聚合酶链反应(PCR)等分子生物学技术,成功构建了变异链球菌LuxS基因绿色荧光蛋白(GFP)融合报告质粒,并将其转入变异链球菌UA159中,确认该质粒能在变链菌中表达,为进一步的研究打下了基础。主要内容包括:
1.变异链球菌LuxS基因启动子启动gfp表达载体的构建和鉴定
本实验应用PCR方法分别从变异链球菌基因组DNA、载体pEGFP-N1中扩增出LuxS基因启动子(pLuxS)和绿色荧光蛋白基因(gfp)片段,经回收、纯化后,利用分子克隆技术分别克隆入载体pUC19中。通过对重组质粒pUC19-pLuxS-gfp的酶切图谱和DNA序列测定分析证实插入片段序列正确。说明成功构建出转染变链菌所需的重组质粒pUC19-pLuxS-gfp,为下一步功能研究奠定了基础。
2.融合报告菌株表型的初步研究
将构建出的质粒pUC19-pLuxS-gfp转入变链菌UA159中,抗生素筛选阳性单克隆,通过PCR和提质粒进行酶切图谱鉴定,证实转化成功。荧光显微镜观察
将野生型细菌和转化后的细菌在含有0.5﹪蔗糖BHI(含有3﹪的马血清)培养液中培养,然后在荧光显微镜下观察细菌的发光情况。结果显示:转化后的细菌发出不同程度的绿色荧光。
激光扫描共聚焦显微镜(CLSM)观察细菌形态和生物膜的形态
将收集到的牙齿通过慢速切锯机切割成薄片,消毒后置于96孔板中,在96孔板中加入含有0.5﹪蔗糖BHI(含有3﹪的马血清)和菌液培养液中培养24h,然后在CLSM下观察。牙片表面生物膜中形成大量大小不一的散开菌落,菌落之间由细胞间桥互相连接,由于存在蔗糖生物膜状态细菌中绿色荧光强度增加。上述结果表明:我们所构建的重组质粒能在变链菌中表达,为进一步的研究打下了基础。
Plaque biofilm, a well-organized microbial community located on tooth surface, has been recognized as a major factor in the pathogenesis of dental caries. Compared with their planktonic counterparts, the bacteria harbored in biofilms exhibit the different gene expression and phenotype, as well as an increased resistance to antimicrobials, presumably due to the regulation effect exerted by the signal transduction systems.
With various techniques of molecular biology, a preliminary understanding on the role of signal transduction systems in biofilm formation has been achieved.The quorum sensing system of S.mutans has been proven to play a crucial role in the formation of biofilm and maintenance of steady-state. Especially, AI-2/LuxS may be involved in the regulation of S.mutans biofilm formation, glucose metabolism, antiacid capacity, and the production of virulence factors,thus, contributing significantly to the survival of S.mutans in the complex environment. Moreover, AI-2/LuxS mediated quorum sensing can be utilized or interrupted by the interaction of S.mutans and other oral bacteria, directly influencing the balance of oral bacteria flora. However, little has been known about the synthesis and secretion of AI-2, as well as its adjusting means and regulation mechanism on the activity of bacteria adjacent within biofilm. Therefore further investigation on the mechanism of LuxS-mediated induction in S.mutans is of great importance for reducing the cariogenesis of oral biofilm and providing possible solution for the resistance of bacteria to antimicrobials.
This study successfully constructed S.mutans LuxS gene green fluorescent protein (gfp) fusion report plasmid through polymerase chain reaction (PCR) technique, and transferred this plasmid into S.mutans to verify it’s expression. 1. Construction of a vector expressing LuxS gene promoter and gfp LuxS promoter and gfp region was amplified by PCR from S.mutans DNA and pEGFP-N1 vector, respectively, then cloned into the pUC19 vector following routine procedures by using molecular cloning technique.
Identification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector pUC19-pLuxS-gfp. The successful construction of the recombinant plasmid pUC19-pLuxS-gfp will be further studied in this field.
2. The phenotype detection of the fusion reporter strain
Transfer the prokaryotic expression recombinant plasmid pUC19-pLuxS-gfp into S.mutansUA159, using selection with 30μg ampicillin ml-1and isolating the transformants.
Fluorescence microscope observation
Cultures of wild-type S. mutans strains and the reporter strain were grown in the brain heart infusion(BHI) which contain 3 % Horse serum and supplemented with 0.5% sucrose.
The cell cultures were analyzed directly by using phase-contrast fluorescent microscopy. Fluorescence was readily detected in the plasmid-containing strain but not in the wild-type.
CLSM of GFP-expressing S. mutans cells and biofilm morphology The collection teeth were cut into thin slice with the slow cut saw, and then placed in 96-well plates after disinfection, The 96-well plates contained the brain heart infusion(BHI) which supplemented 3 % Horse serum 0.5 % sucrose.After 24h incubation the reporter strain cells formed several large amorphous microcolonies and many small microcolonies were interspersed across the biofilm, The micrographs of single sections indicated an increase of fluorescence in the biofilm as a function of sucrose. These results demonstrated that: the recombinant establisthed can expressing in S.mutans, laying a foundation for further study.
通过运用各种分子生物学技术,对一些信号传导系统在变链菌形成生物膜过程中的作用已有初步了解。变链菌(S.mutans)密度感应系统在生物膜的形成和稳态维持中作用最为关键。其中,AI-2/LuxS可参与调节S.mutans生物膜的形成、糖代谢、耐酸能力以及多种毒力因子的分泌,对S.mutans在口腔这个复杂的环境中生存有着重要意义。S.mutans等口腔定植细菌可通过细菌间的独特联系来利用或干扰AI-2/LuxS介导的群体感应,因此与正常人体内的菌群平衡和菌群失调有着密切的关系。然而,目前对于AI-2的合成、分泌、调节途径,以及在活体生物膜内如何调节相邻细菌的生理活动仍不清楚。因此进一步明确S.mutans中LuxS介导群体感应的作用机制,将为找到降低菌斑生物膜的致病性的方法,摸索出“耐药性”的应对措施提供线索,无疑有着重要的意义。
本研究运用聚合酶链反应(PCR)等分子生物学技术,成功构建了变异链球菌LuxS基因绿色荧光蛋白(GFP)融合报告质粒,并将其转入变异链球菌UA159中,确认该质粒能在变链菌中表达,为进一步的研究打下了基础。主要内容包括:
1.变异链球菌LuxS基因启动子启动gfp表达载体的构建和鉴定
本实验应用PCR方法分别从变异链球菌基因组DNA、载体pEGFP-N1中扩增出LuxS基因启动子(pLuxS)和绿色荧光蛋白基因(gfp)片段,经回收、纯化后,利用分子克隆技术分别克隆入载体pUC19中。通过对重组质粒pUC19-pLuxS-gfp的酶切图谱和DNA序列测定分析证实插入片段序列正确。说明成功构建出转染变链菌所需的重组质粒pUC19-pLuxS-gfp,为下一步功能研究奠定了基础。
2.融合报告菌株表型的初步研究
将构建出的质粒pUC19-pLuxS-gfp转入变链菌UA159中,抗生素筛选阳性单克隆,通过PCR和提质粒进行酶切图谱鉴定,证实转化成功。荧光显微镜观察
将野生型细菌和转化后的细菌在含有0.5﹪蔗糖BHI(含有3﹪的马血清)培养液中培养,然后在荧光显微镜下观察细菌的发光情况。结果显示:转化后的细菌发出不同程度的绿色荧光。
激光扫描共聚焦显微镜(CLSM)观察细菌形态和生物膜的形态
将收集到的牙齿通过慢速切锯机切割成薄片,消毒后置于96孔板中,在96孔板中加入含有0.5﹪蔗糖BHI(含有3﹪的马血清)和菌液培养液中培养24h,然后在CLSM下观察。牙片表面生物膜中形成大量大小不一的散开菌落,菌落之间由细胞间桥互相连接,由于存在蔗糖生物膜状态细菌中绿色荧光强度增加。上述结果表明:我们所构建的重组质粒能在变链菌中表达,为进一步的研究打下了基础。
Plaque biofilm, a well-organized microbial community located on tooth surface, has been recognized as a major factor in the pathogenesis of dental caries. Compared with their planktonic counterparts, the bacteria harbored in biofilms exhibit the different gene expression and phenotype, as well as an increased resistance to antimicrobials, presumably due to the regulation effect exerted by the signal transduction systems.
With various techniques of molecular biology, a preliminary understanding on the role of signal transduction systems in biofilm formation has been achieved.The quorum sensing system of S.mutans has been proven to play a crucial role in the formation of biofilm and maintenance of steady-state. Especially, AI-2/LuxS may be involved in the regulation of S.mutans biofilm formation, glucose metabolism, antiacid capacity, and the production of virulence factors,thus, contributing significantly to the survival of S.mutans in the complex environment. Moreover, AI-2/LuxS mediated quorum sensing can be utilized or interrupted by the interaction of S.mutans and other oral bacteria, directly influencing the balance of oral bacteria flora. However, little has been known about the synthesis and secretion of AI-2, as well as its adjusting means and regulation mechanism on the activity of bacteria adjacent within biofilm. Therefore further investigation on the mechanism of LuxS-mediated induction in S.mutans is of great importance for reducing the cariogenesis of oral biofilm and providing possible solution for the resistance of bacteria to antimicrobials.
This study successfully constructed S.mutans LuxS gene green fluorescent protein (gfp) fusion report plasmid through polymerase chain reaction (PCR) technique, and transferred this plasmid into S.mutans to verify it’s expression. 1. Construction of a vector expressing LuxS gene promoter and gfp LuxS promoter and gfp region was amplified by PCR from S.mutans DNA and pEGFP-N1 vector, respectively, then cloned into the pUC19 vector following routine procedures by using molecular cloning technique.
Identification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector pUC19-pLuxS-gfp. The successful construction of the recombinant plasmid pUC19-pLuxS-gfp will be further studied in this field.
2. The phenotype detection of the fusion reporter strain
Transfer the prokaryotic expression recombinant plasmid pUC19-pLuxS-gfp into S.mutansUA159, using selection with 30μg ampicillin ml-1and isolating the transformants.
Fluorescence microscope observation
Cultures of wild-type S. mutans strains and the reporter strain were grown in the brain heart infusion(BHI) which contain 3 % Horse serum and supplemented with 0.5% sucrose.
The cell cultures were analyzed directly by using phase-contrast fluorescent microscopy. Fluorescence was readily detected in the plasmid-containing strain but not in the wild-type.
CLSM of GFP-expressing S. mutans cells and biofilm morphology The collection teeth were cut into thin slice with the slow cut saw, and then placed in 96-well plates after disinfection, The 96-well plates contained the brain heart infusion(BHI) which supplemented 3 % Horse serum 0.5 % sucrose.After 24h incubation the reporter strain cells formed several large amorphous microcolonies and many small microcolonies were interspersed across the biofilm, The micrographs of single sections indicated an increase of fluorescence in the biofilm as a function of sucrose. These results demonstrated that: the recombinant establisthed can expressing in S.mutans, laying a foundation for further study.
引文
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[2] Costerton JW. Introduction to biofilm. Int JAntimierob Agents, 1999,11:217-221
[3] Wilson M, Patel H, Fletscher J. Susceptibility of biofilms of Streptococcus sanguis to chlorhexidine gluconate and cetylpyridinium chloride. Oral Micmbiol Immunol,1996,11(3):188-192
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