【作者】 苏凌云；

【导师】 吴补领；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2002， 博士

【摘要】 龋病是三大非传染性疾病之一，发病率高，危害大。变形链球菌族(MS)是目前公认的致龋菌，其中与人类龋病发生关系密切的是变形链球菌和茸毛链球菌。MS在牙面的粘附、增殖是其致龋的基础。目前防龋研究主要围绕链球菌在牙面的粘附、聚集及增殖几个环节进行，介导MS在牙面粘附、聚集的毒力因子主要有表面蛋白抗原Ⅰ/Ⅱ(AgⅠ/Ⅱ)、葡糖基转移酶(GTFs)及葡聚糖结合蛋白(Gbps)。AgⅠ/Ⅱ、GTFs及GbpB在免疫防龋中具有明显的保护性防龋作用，GbpA在氨基酸序列上与GTFs的葡聚糖结合区(GLU)高度同源，但其在防龋免疫中的作用还无报道，基因疫苗是上世纪90年代发展起来的新型疫苗，对变形链球菌表面蛋白T、B细胞表位基因防龋疫苗及葡糖基转移酶抗原表位基因防龋疫苗研究表明，它们均可产生保护性免疫反应。细菌增值方面的防龋研究主要集中在抗菌制剂抑制细菌生长。对茸毛链球菌增殖过程中细胞分裂调控基因的研究及其在细菌致龋过程中的作用还未见报道，本课题通过克隆变形链球菌gbpA的葡聚糖结合区GBD片段基因，在大肠杆菌表达并纯化，同时构建GBD真核表达质粒，用重组蛋白及真核表达质粒免疫大鼠观察其在防龋中的作用，另外，我们通过“genome walking system”克隆茸毛链球菌细胞分裂蛋白ftsK全长，并在大肠杆菌中表达，观察过表达的茸毛链球菌细胞分裂蛋白相关基因ftsK对大肠杆菌生长的影响。 本研究分为两个部分： 第四军医大学博士学位论文第一部分：变形链球菌gbpA的葡聚糖结合区GBD多肽和基因疫苗的制备及 免疫防龋研究 主要实验内容包括： 1．应用 PCR技术扩增出 S．ffistsflS gbpA的 GBD基因片段，定向插入 PUC载体，构建 PUC－GBD质粒，挑取鉴定后的含 PUC－GBD质粒的菌液送测序，测序结果与文献报道一致。 2．将测序的GBD定向插入原核表达载体pPROEXW HTb，构建原核融合表达质粒 pPROEX””HTbGBD，转化至 Eec ＊ DHS a，IPTG诱导表达融合蛋白GBD，通过金属螫合亲和层析｛N尸－N仪介质）纯化GBD蛋白，做丑0nblot免疫印迹杂交证实所纯化的融合蛋白与S．mutans gbpA具有抗原－抗体免疫反应。 3．构建真核表达质粒pcDNA3．IGBD，将其瞬时转染至哺乳动物细胞COS7，pCDNA3二－GBD在COS7细胞的胞浆呈阳性表达。 4．用纯化的GBD及pCDNA3．IGBD基因疫苗免疫大鼠，血清中IgG及唾液中 IgG明显升高，Western blot免疫印迹杂交血清中的 IgG与融合蛋白具有特异性免疫反应，大鼠高糖饮食及口腔接种S．mutans Ingbritt，免疫组的龋患率明显低于对照组，说明GBD多肽及基因疫苗具有免疫防龋作用。 第二部分：茸毛链球菌新基因细胞分裂蛋白ftSK的克隆及功能初步研究 在实验中我们筛选出S．s加bus的一段基因，geneb舢同源性分析，其为一新基因的一个片段，同源性比较，与大肠杆菌的细胞分裂蛋白tSK及枯草芽抱杆菌的SpollJE同源，故将其命名为茸毛链球菌细胞分裂蛋白tSK，本实验拟克隆此新基因的全长，并对其功能做初步研究，主要实验包括： l．“genome walking system”的建立，根据“genome walking”原理，设计接头bdaptor）和引物，酶切细菌基因组 DNA及 adaptor，将 adaptor与基因组DNA连接，连接产物作为PCR的模板。 二．通过PCR扩增出S．sobrinus细胞分裂蛋白tsK的未知区域，将PCR产l〕尸厂aftment of opel、atlve Dentistry and Endodontlcs －3－ 第四军医大学博士学位论文物送测序，将所测序列连接，共长 318 8 hp，包括细胞分裂蛋白伦K门 00 hp）全长。 3．将新基因的核酸序列及氨基酸序列与gengbank进行同源性分析，并用蛋白质分析软件对新基因的氨基酸序列进行分析，结果显示，所克隆的基因的中部有 ZI 00碱基组成的的开放读框（Ony），编码 699个氨基酸，推测其分子量为 77KD。用 GOR4软件对 S．sobrinus tsK的二级结构预测，显示其主要山。螺旋和无j见则卷曲组成，另有小部分伸展线，但无p折叠，在其N末端有一跨膜区。对氨基酸序列进行保守区分析，氨基酸335－530 序列具有ftSK／SpothE保守序列，同源仕分析，除N末端226个氨基酸外，其余氨基酸序列与大肠杆菌的C末端2＊序列同源，217七35氨基酸与枯草杆菌SpOLllE的C末端同源，N末端165氨基酸与SpolllE的N末端同源，但与大肠杆菌的N木端完全不同。 3．构建Ssobrinus新基因细胞分裂蛋白tsK的原核表达质粒pPROEX””HTc／tsK，诱导其在大肠杆菌DHS a表达，观察S．sobrinus的细胞分裂蛋白 tsK过表达对大肠杆菌生长的影响，结果表明，S．sobrinus的细胞分裂蛋白tsK在大肠杆菌过表达可抑制大肠杆菌的生长。更多还原

【Abstract】 Dental caries is one of the three chief non-contagious diseases which threaten the human health. Mutans streptococci (MS) are the primary cariogenic agents, and Streptococcus mutans and Streptococcus sobrinus are believed to have the closest relationship with human caries. Adhesion and proliferation of MS are the basis of cariogensis. At present, the study of anticaries mainly focuses on a series of events involving the adherence, accumulation and proliferation of MS on tooth surfaces. Researchers have done many studies on cloning, functions and anticaries immunization of the genes relative to adherence and accumulation. These genes contain surface protein antigen I /II (Ag I / II) , glucosytransferase (GTFs) and glucan binding protein (Gbps). Ag I /II, GTFs and GbpB can protect against dental caries. GbpA of S. mutans contains a C-terminal GBD homologous to the GBDs of the GTFs, but the function of anticaries immunization of GbpA has not been reported. DNA vaccines are new methods that were developed in the lastdecade of the 20th century. The animal immunization experiments on DNA vaccines of T cell and B cell epitope in Streptococcus mutans surface protein antigen and GTF have shown that these DNA vaccines can induce protective immune response. The studies on the proliferation of bacteria mainly focused on anticaries agents which could inhibit bacterial growth. But there has not been any report about the study of the control genes in the cell division of S. sobrinus and their functions in cariogenic process. In this study, the GBD of glucan binding protein of 5. mutans was cloned,found expressed in E. coll and was purifed. The fusion protein was used to immunize rats. At the same time, the rats were infected by S. mutans. The antiserum was collected and the caries score was evaluated. In addition, we used the "genome walking system" to clone the complete sequence of putative cell division protein ftsK of S. sobrinus, then expressed it in E. coli and observed the impact of the overexpression of the putative cell division protein ftsK of S. sobrinus on the growth of E. coli.Our study consists of two parts:Part One: Preparation of GBD polypeptide and DNA vaccine of glucan binding protein of Streptococcus mutans and study on anticarious immunization.The following are the main experiments conducted:1. The GBD of gbpA of S. mutans was cloned by PCR and then was inserted into vector PUC19 to constructed plasmid PUC19-GBD. The identified plasmid was sequenced. The sequenced result was consistent with other reports.2. The sequenced fragment was subcloned into prokayotic gene expression vector pPROEX?HTb in a certain direction and the resulted plasmid pPROEX?HTb-GBD was used to transform competent E. coli DH5a. The fusion protein was induced by IPTG. The target protein was purified by Ni2+-NTA affinity chromatography. Western blot showed the purified fusion protein could cross reactwith gbpA antiserum.3. The plasmid pcDNA3.1-GBD was constructed and was introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein was detected by immunochemistry technique in cell plasma of COS-7 cells.4. The rats were immunized respectively with GBD fusion protein and pcDNA3.1-GBD DNA vaccines. Immunized animals demonstrated significantly higher serum IgG and salivary IgA antibody levels to GBD than non-immunized animals. Serum IgG could react with GBD fusion protein in Western blot. Immunization with GBD fusion protein and pcDNA3.1-GBD DNA vaccines resulted in significantly reduced dental caries after infection with S. mutans Ingbritt.Part Two: Cloning cell division protein ftsK of Streptococcus sobrinus. and study on its basic functionWe cloned a fragment from S. sobrinus genome DNA. Homology analysis by genebank demonstrated that it is a fragment of a new gene. The new gene has homology with cell division protein ftsK of E. coli. and SpoIIIE gene of B. subtilis. So it has been named as cell division protein ftsK of S. sobrinus. In this study,更多还原