致龋链球菌粘附、增殖相关基因的克隆、原核表达及功能初步研究
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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0n
blot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。
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
last
decade 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 react
with 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 function
We 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,
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0n
blot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。
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
last
decade 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 react
with 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 function
We 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,
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32． Marsh PD, Bradsgaw DJ. Dental plaque as a biofilm. J Indust Microbiol. 1995; 15:169-175
33． Burne RA. Oral streptococcus . products of their environment. J. Dent.Res. 1998; 77:445-449
34． Bowen WH, Schilling K, Giertsen E. et al. Role of a cell surface-associated protein in adherence and dental caries. Infect Immun. 1991; 59:4604-4609
35． Schilling K, Bowen WH. Glucans synthesized in situ in experimental pellicle function as specific binding site for Streptococcus mutans. Infect Immun.1992; 60:284-295
36． Yamashita Y, Bowen WH, Bume RA. et al. Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun. 1993; 61: 3811-3817
37． Burne RA, Chen YY, Wexler DL. et al. Cariogenicity of Streptococcus mutans strain with defects in fructan metabolism assessed in a program-fed specific-pathogen-free rat model. J Dent Res. 1996; 75: 1572-1577
38． Spata G. Rohrer K. Barnarg D. et al. A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo. Infect Immun. 1995;63: 2556-2563
39． Marquis RE. Acid-base physiology, stress responses and effects of weak acids on oral streptococcus. Abstracts of the 5th national symposium of caries research, society of cariology and endodontology, Chinese stomatological association. Peking, 1998．
40． 樊明文主编。龋病学。第一版，贵州：贵州科技出版社，1993：201-208
41． Russell MW, Hajishengallis G, Childers NK. et al. Secretary immunity in defense against careogenic mutans streptococci. Caries Res. 1999; 33:4-7
42． Mestecky J. Common mucosal immune system and current strategies for induction of immune response in external secretions. J Clin Immunol. 1987; 7:165-176
43． Underdown BJ, Schiff JM. Immunoglobulin A: Strategic defense initiative at the mucosal surface. Annu Rev Immunol. 1986; 4:389-417
44． Love RM, Jenkinson HF. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by antigen Ⅰ /Ⅱ family of polypeptides. Infect Immun. 1997; 65:5157-5164
45． Camling E, GahnbergL, Krasse B. et al. IgG antibodies and Streptococcus mutans on erupting human first permanent molars. Arch Oral Biol. 1991; 36:703-708
46． Tenovuo J, Lehtonen O-P, Aaltonen. Caries development in children in relation to the presence of mutans streptococci in dental plaque and serum antibodies against whole cells and protein antigen U Ⅰ / Ⅱ OF Streptococcus mutans. Caries Res. 1990; 24:59-64
47． Legle DW, McGhee JR, Lynch DP. et al. Immunodeficiency disease and dental caries in man. Arch Oral Biol. 1981; 26:905-910
48． McCarthy M. DNA vaccination : A direct line to the immune system. The Lancet. 1996; 348:1232-1233
49． Wolff JA, Malone RW, Williams P. et al. Direct gene transfer into mouse muscle in vivo. Science. 1990; 247:1465-1468
50． Tang DC, Devit M, Johnston SA, Genetic immunization is a simple method for eliciting an immune response. Nature. 1992; 356:152-154
51． Jens E, Jasper P, Jack RW. DNA vaccines. Interviol. 1999; 42:117-
52． Madison KM, Bowen WH, Pearson SK, Falany JL. Enhancing the virulence of Streptococcus sobrinus in rats. J Dent Res. 1991; 70:38-43
53． Smith DJ, and Taubman MA. Vaccines against dental caries infection. In Levine MM, Woodrow GC, Kaper JB, and Coban GS, (ed) New generation vaccines, 2nd.ed. Marcel Dekker, Inc., New York.N.Y. 1997, P.914-930．
54． 樊明文，陈平，边专。免疫防龋研究的新进展。中华口腔医学杂志。1999，34：69-72
55． Oishi Y, Onozuka A, Kato H, et al. The effect of amino acid spacers on the antigenicity of dimeric peptide-inducing cross-reacting antibodies to a cell surface protein antigen of streptococcus mutans. Oral Micro Immun. 2001, 16:40-44
56． Shani S. Friedman M. Steinberg D. The anticariogenic effect of Amine Fluorides on Streptococcus sobrinus and glucosyltransferase in biofilms. Caries Research 2000; 34:260-267
57． Katsura H, Tsukiyama RI, Suzuki A, and Kobayashi M. In vitro antimicrobial activities of bakuchio! against oral microorganisms. Antimicrob Agents Chemother. 2001,45:3009-13
58． Shouji N. Takada K. Fukushima K. and Hirasawa M. Anticaries effect of a component from Shiitake (an edible mushroom). Caries Research. 2000; 34:94-100
59． Lutkenhaus J. FtsZ ring in bacterial cytokinesis. Mol.Mcrobiol. 1993; 9:403-410
60． Addinall S.G., Dewer S.J.and Donachie W.D. FtsA is localized to the septum in an
FtsZ-dependent manner. J. Bactreiol. 1996; 178:7167-7172
61． Addinall SG, Cao C and Lutkenhaus J. FtsN, a late recruit to the septum in Escherichia coli. Mol.Microbiol. 1997; 25:303-309
62． Begg KJ, Dewer SJ and Donachie WD. A new Escherichia coli. cell division gene ftsK. J.Bactreiol.1995; 177:6211-6222
63． Boyle DS. Khattar MM and Addinall SG. FtsW is an essential cell-division gene in Escherichia coli. Mol.Microbiol. 1997; 24:1263-1273
64． Rothfiled LI, Justic SS. Bacterial cell division: the cycle of the ring. Cell. 1997; 88:581-584
65． Donachie WD. The cell cycle of Escherichi coli. Annu.Rev.Microbiol. 1997; 47:199-230
66． Wu LJ, and Errington J. Bacillus subtilis SpoⅢE protein required for DNA segregation during asymmetric cell division. Science. 1994; 264:572-575
67． Wu LJ, Lewis PJ, Almansberger R. et al. A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev. 1995; 9:1316-1326
68． Diez A A, Farewell A, Nannmark U, et al. A mutation in the ftsK gene of Escherichi coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J.Bactreiol.1997; 179:5878-5883
69． Yu XC, Weihe EK, Margolin W. Role of the C terminus of ftsK in Escherichi coli chromosome segregation. J. Bactreiol. 1998; 180:6424-6428
70． Draper GC, Mclennan N, Begg K. et al. Only the N-terminal domain of FtsK function in cell division. J. Bactreiol. 1998, 180: 4621-4627
71． Yu XC, Tran AH, Sun Q, et al. Localization of cell division protein FtsK to the Escherichi coli septum and identification of a potential N-terminal. J.Bactreiol. 1998; 180:1296-1304
72． Steiner W, Liu G, Donachie WD, et al. The FtsK cell division protein is required for resolution of chromosome dimers in Escherichi coli. Mol Microbiol. 1999; 31:579-583
73． Boyle DS, Grant D, Draper CC, et al. All major regions of FtsK are required for resolution of chromosome dimers. J.Bactreiol.2000; 182:4124-4127
74． 郭丽宏，刘天佳，曹福贤。变形链球菌表面蛋白真核表达质粒pcDAN3／pacA及 pcDAN3／pacP免疫动物实验研究。牙体牙髓牙周病学杂志。2001；11：287-289
75． Oho T, Yu H, Yamashita Y, et al. Binding of salivary Glycoprotein-secretary immunogtobulin A complex to the surface protein antigen of Streptococcus mutans. Infect Immun. 1998; 66:115-121
76． Krieg AM, Yi AK, Matson S, et al. CpG motif in bacterial DNA trigger direct B cell activation. Mature. 1995; 374:546-549
77． Klinman DM, Yi AK, Beaucage SL, et al. CpG motifs present in bacterial DNA rapilidy induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon y. Proc.Natl.Acad. Sci.USA. 1996; 93:2879-2883
78． Cowdery JS, Chace JH, Yi AK, et al. Bacterial DNA induces NK cells to produce IFN-y in vivo and increase the toxicity of lipopolysaccharides. J. Immunol. 1996; 156:4570-4575
79． Bendigs S, Salzer U, Lipford GB, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells. Eur. J. Immunol. 1999; 29:1209-1218
80． Sato Y, Roman M, Tighe J, et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science. 1996; 273:352-354
81． Childers NK, Tong G, Li F, et al. Humans immunized with Streptococcus mutans antigens by mucosal routes. J Dent Res. 2002; 81:48-52
82． Saito M, Otake S, Ohmura M, et al. Protective immunity to Streptococcus induced by nasal vaccination with surface protein antigen and mutan cholera toxin adjuvant. J Infect Dis. 2001; 183:823-825
83． Taubman M, Smith D.J., Holmberg CJ, et al. Coimmunization with complementary glucosyltransferase peptides results in enhanced immunogenicity and protection against dental caries. Infect. Immun. 2000; 68:2698-2703
84． Smith DJ, and Taubman MA. Experimental immunization of rats with a Streptococcus
mutans 59-kilodalton glucan-binding protein protects against dental caries. Infect Immun.1996; 64:3069-3073
85． Oho T, Shimazaki Y, Mitoma M, et al. Bovine milk antibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans. J Nutri Beth. 1999; 129:1836-1841
86． Smith DJ, King WF, Godiska R. Passive transfer of immunogobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect.Immun. 2001; 69:3135-3142
87． 杨锦波。变形链球菌葡糖基转移酶抗原表位基冈真核表达质粒的构建极其免疫动物 实验研究。四川大学博士学位论文。2001。
88． Kawabata S,Terao Y, Fujiwara T, et al. Targeted Salivary Gland Immunization with Plasmid DMA Elicits Specific Salivary Immunoglobulin A and G Antibodies and Serum Immunoglobulin G Antibodies in Mice. Infect Immun. 1999; 67: 5863-5868
89． Ilium L, Jabbal-Gill I, Hinchcliffe M,et al. Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev. 2001 ; 51 :81-96
2． Banas JA, Russell RRB. and Ferretti JJ. Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt Infect Immun. 1990． 58:667-673
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5． Douglas CW1, and Russell RRB. Effect of specific antisera on adherence properties of the oral bacterium streptococcus mutans. Arch.OralBiol. 1982; 27: 1039-1045
6． Smith DJ, and Taubman MA. Vaccines against dental caries infection. P.914-930． In M.M.Levine, G.C.Woodrow, J.B.Kaper, and G.S.Coban,(ed) New generation vaccines, 2nd.ed. Marcel Dekker,Inc., New York, N. Y. 1997．
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16． Aoki H, Shiroza T, Hayakawa M, et al. Cloning of a Streptococcus mutans glucosytransferase gene encoding for insoluble glucan synthesis. Infect Immun. 1986; 53:587-594
17． Shiroza T, Ueda S, Kuramitsu HK. Sequence analysis of the gtfB gene from Streptococcus mutans. J. Bacteriology. 1987; 169;4263-4270
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19． Honda O, Kato C, and Kuramitsu HK. Nucleotide sequence of the Streptococcus mutans gtf D gene encoding the glucosyltransferase-S enzyme. J.Gen.Microbiol. 1990; 136:2099-2105
20． Giffard PM, and Jacques NA. Definition of a fundamental repeating unit in streptococcal glucosytranaferase glucan-binding regions and related sequence. J. Dent. Res. 1994; 73:1133-1141
21． Gilmore KS, Russell RRB, and Ferretti. Analysis of the Streptococcus downei gtfS gene, which specifies a glucosytransferase that synthesizes soluble glucan. Infect Immun. 1990; 58:2452-2458
22． Mooser G, Wong C. Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan syntheses) from Streptococcus sobrinus. Infect Immun. 1988; 56:880-884
23． Kobayashi S, Koga K, Hayashida O. et al. Glucan-binding domain of a glucosyltransferase from Streptococcus sobrinus: isolation of a 55-kilodalton peptide from a trypsin digest of glucosyltransferase prebound to insoluble glucan. Infect Immun. 1989; 57:2210-2213
24． Abo H, Matsumura T, Kodama T.et al. Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synmetase). J Bacterial. 1991; 173:989-996
25． Lis M, Shiroza T, and Kuramitsu H. Role of the C-terminal direct repeating units of the Streptococcus mutans glucoytransferase-S in glucan binding. Appli Envio. Micro. 1995; 61:2040-2042
26． Kingston KB, Allen DM, Jacques NA. Role of the C-terminal YG repeats of the primer-dependent streptococcal glucosyltransferase, GtfJ, in binding to dextran and mutan. Microbio. 2002; 148:549-58
27． 卢圣栋，主编。现代分子生物学实验技术。第一版。北京：高等教育出版社，1993
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29． Lee SS. Intravesical gene therapy: in vivo gene transfer using recombinant vaccinia virus vector. Cancer Res. 1994; 54:3325-3330
30． Mellon P, Parker V, Gluzman Y, et al. Identification of DNA sequences required for transcription of the human alpha 1-globin gene in a new SV40 host-vector system. Cell. 1981; 27:279-88．
31． Sabbinoi S, Negrini M, Rimessi P, et al. ABK virus episomal vector for constitutive high expression of exogenous cDNA in human cell. Arch Viro. 1995; 140:335-339
32． Marsh PD, Bradsgaw DJ. Dental plaque as a biofilm. J Indust Microbiol. 1995; 15:169-175
33． Burne RA. Oral streptococcus . products of their environment. J. Dent.Res. 1998; 77:445-449
34． Bowen WH, Schilling K, Giertsen E. et al. Role of a cell surface-associated protein in adherence and dental caries. Infect Immun. 1991; 59:4604-4609
35． Schilling K, Bowen WH. Glucans synthesized in situ in experimental pellicle function as specific binding site for Streptococcus mutans. Infect Immun.1992; 60:284-295
36． Yamashita Y, Bowen WH, Bume RA. et al. Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun. 1993; 61: 3811-3817
37． Burne RA, Chen YY, Wexler DL. et al. Cariogenicity of Streptococcus mutans strain with defects in fructan metabolism assessed in a program-fed specific-pathogen-free rat model. J Dent Res. 1996; 75: 1572-1577
38． Spata G. Rohrer K. Barnarg D. et al. A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo. Infect Immun. 1995;63: 2556-2563
39． Marquis RE. Acid-base physiology, stress responses and effects of weak acids on oral streptococcus. Abstracts of the 5th national symposium of caries research, society of cariology and endodontology, Chinese stomatological association. Peking, 1998．
40． 樊明文主编。龋病学。第一版，贵州：贵州科技出版社，1993：201-208
41． Russell MW, Hajishengallis G, Childers NK. et al. Secretary immunity in defense against careogenic mutans streptococci. Caries Res. 1999; 33:4-7
42． Mestecky J. Common mucosal immune system and current strategies for induction of immune response in external secretions. J Clin Immunol. 1987; 7:165-176
43． Underdown BJ, Schiff JM. Immunoglobulin A: Strategic defense initiative at the mucosal surface. Annu Rev Immunol. 1986; 4:389-417
44． Love RM, Jenkinson HF. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by antigen Ⅰ /Ⅱ family of polypeptides. Infect Immun. 1997; 65:5157-5164
45． Camling E, GahnbergL, Krasse B. et al. IgG antibodies and Streptococcus mutans on erupting human first permanent molars. Arch Oral Biol. 1991; 36:703-708
46． Tenovuo J, Lehtonen O-P, Aaltonen. Caries development in children in relation to the presence of mutans streptococci in dental plaque and serum antibodies against whole cells and protein antigen U Ⅰ / Ⅱ OF Streptococcus mutans. Caries Res. 1990; 24:59-64
47． Legle DW, McGhee JR, Lynch DP. et al. Immunodeficiency disease and dental caries in man. Arch Oral Biol. 1981; 26:905-910
48． McCarthy M. DNA vaccination : A direct line to the immune system. The Lancet. 1996; 348:1232-1233
49． Wolff JA, Malone RW, Williams P. et al. Direct gene transfer into mouse muscle in vivo. Science. 1990; 247:1465-1468
50． Tang DC, Devit M, Johnston SA, Genetic immunization is a simple method for eliciting an immune response. Nature. 1992; 356:152-154
51． Jens E, Jasper P, Jack RW. DNA vaccines. Interviol. 1999; 42:117-
52． Madison KM, Bowen WH, Pearson SK, Falany JL. Enhancing the virulence of Streptococcus sobrinus in rats. J Dent Res. 1991; 70:38-43
53． Smith DJ, and Taubman MA. Vaccines against dental caries infection. In Levine MM, Woodrow GC, Kaper JB, and Coban GS, (ed) New generation vaccines, 2nd.ed. Marcel Dekker, Inc., New York.N.Y. 1997, P.914-930．
54． 樊明文，陈平，边专。免疫防龋研究的新进展。中华口腔医学杂志。1999，34：69-72
55． Oishi Y, Onozuka A, Kato H, et al. The effect of amino acid spacers on the antigenicity of dimeric peptide-inducing cross-reacting antibodies to a cell surface protein antigen of streptococcus mutans. Oral Micro Immun. 2001, 16:40-44
56． Shani S. Friedman M. Steinberg D. The anticariogenic effect of Amine Fluorides on Streptococcus sobrinus and glucosyltransferase in biofilms. Caries Research 2000; 34:260-267
57． Katsura H, Tsukiyama RI, Suzuki A, and Kobayashi M. In vitro antimicrobial activities of bakuchio! against oral microorganisms. Antimicrob Agents Chemother. 2001,45:3009-13
58． Shouji N. Takada K. Fukushima K. and Hirasawa M. Anticaries effect of a component from Shiitake (an edible mushroom). Caries Research. 2000; 34:94-100
59． Lutkenhaus J. FtsZ ring in bacterial cytokinesis. Mol.Mcrobiol. 1993; 9:403-410
60． Addinall S.G., Dewer S.J.and Donachie W.D. FtsA is localized to the septum in an
FtsZ-dependent manner. J. Bactreiol. 1996; 178:7167-7172
61． Addinall SG, Cao C and Lutkenhaus J. FtsN, a late recruit to the septum in Escherichia coli. Mol.Microbiol. 1997; 25:303-309
62． Begg KJ, Dewer SJ and Donachie WD. A new Escherichia coli. cell division gene ftsK. J.Bactreiol.1995; 177:6211-6222
63． Boyle DS. Khattar MM and Addinall SG. FtsW is an essential cell-division gene in Escherichia coli. Mol.Microbiol. 1997; 24:1263-1273
64． Rothfiled LI, Justic SS. Bacterial cell division: the cycle of the ring. Cell. 1997; 88:581-584
65． Donachie WD. The cell cycle of Escherichi coli. Annu.Rev.Microbiol. 1997; 47:199-230
66． Wu LJ, and Errington J. Bacillus subtilis SpoⅢE protein required for DNA segregation during asymmetric cell division. Science. 1994; 264:572-575
67． Wu LJ, Lewis PJ, Almansberger R. et al. A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev. 1995; 9:1316-1326
68． Diez A A, Farewell A, Nannmark U, et al. A mutation in the ftsK gene of Escherichi coli affects cell-cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J.Bactreiol.1997; 179:5878-5883
69． Yu XC, Weihe EK, Margolin W. Role of the C terminus of ftsK in Escherichi coli chromosome segregation. J. Bactreiol. 1998; 180:6424-6428
70． Draper GC, Mclennan N, Begg K. et al. Only the N-terminal domain of FtsK function in cell division. J. Bactreiol. 1998, 180: 4621-4627
71． Yu XC, Tran AH, Sun Q, et al. Localization of cell division protein FtsK to the Escherichi coli septum and identification of a potential N-terminal. J.Bactreiol. 1998; 180:1296-1304
72． Steiner W, Liu G, Donachie WD, et al. The FtsK cell division protein is required for resolution of chromosome dimers in Escherichi coli. Mol Microbiol. 1999; 31:579-583
73． Boyle DS, Grant D, Draper CC, et al. All major regions of FtsK are required for resolution of chromosome dimers. J.Bactreiol.2000; 182:4124-4127
74． 郭丽宏，刘天佳，曹福贤。变形链球菌表面蛋白真核表达质粒pcDAN3／pacA及 pcDAN3／pacP免疫动物实验研究。牙体牙髓牙周病学杂志。2001；11：287-289
75． Oho T, Yu H, Yamashita Y, et al. Binding of salivary Glycoprotein-secretary immunogtobulin A complex to the surface protein antigen of Streptococcus mutans. Infect Immun. 1998; 66:115-121
76． Krieg AM, Yi AK, Matson S, et al. CpG motif in bacterial DNA trigger direct B cell activation. Mature. 1995; 374:546-549
77． Klinman DM, Yi AK, Beaucage SL, et al. CpG motifs present in bacterial DNA rapilidy induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon y. Proc.Natl.Acad. Sci.USA. 1996; 93:2879-2883
78． Cowdery JS, Chace JH, Yi AK, et al. Bacterial DNA induces NK cells to produce IFN-y in vivo and increase the toxicity of lipopolysaccharides. J. Immunol. 1996; 156:4570-4575
79． Bendigs S, Salzer U, Lipford GB, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells. Eur. J. Immunol. 1999; 29:1209-1218
80． Sato Y, Roman M, Tighe J, et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science. 1996; 273:352-354
81． Childers NK, Tong G, Li F, et al. Humans immunized with Streptococcus mutans antigens by mucosal routes. J Dent Res. 2002; 81:48-52
82． Saito M, Otake S, Ohmura M, et al. Protective immunity to Streptococcus induced by nasal vaccination with surface protein antigen and mutan cholera toxin adjuvant. J Infect Dis. 2001; 183:823-825
83． Taubman M, Smith D.J., Holmberg CJ, et al. Coimmunization with complementary glucosyltransferase peptides results in enhanced immunogenicity and protection against dental caries. Infect. Immun. 2000; 68:2698-2703
84． Smith DJ, and Taubman MA. Experimental immunization of rats with a Streptococcus
mutans 59-kilodalton glucan-binding protein protects against dental caries. Infect Immun.1996; 64:3069-3073
85． Oho T, Shimazaki Y, Mitoma M, et al. Bovine milk antibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans. J Nutri Beth. 1999; 129:1836-1841
86． Smith DJ, King WF, Godiska R. Passive transfer of immunogobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect.Immun. 2001; 69:3135-3142
87． 杨锦波。变形链球菌葡糖基转移酶抗原表位基冈真核表达质粒的构建极其免疫动物 实验研究。四川大学博士学位论文。2001。
88． Kawabata S,Terao Y, Fujiwara T, et al. Targeted Salivary Gland Immunization with Plasmid DMA Elicits Specific Salivary Immunoglobulin A and G Antibodies and Serum Immunoglobulin G Antibodies in Mice. Infect Immun. 1999; 67: 5863-5868
89． Ilium L, Jabbal-Gill I, Hinchcliffe M,et al. Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev. 2001 ; 51 :81-96