变形链球菌葡聚糖结合蛋白C基因特异片段的克隆和在大肠杆菌中的表达

目的：克隆变形链球菌葡聚糖结合蛋白C基因（Glucan-binding protein C gene,gbpC）编码序列的特异片段,并在大肠杆菌中实现原核表达。方法：根据gbpC序列设计并合成一对寡核苷酸引物,PCR扩增位于gbpC编码序列1342bp～1794bp间的一段特异片段,扩增产物经回收、酶切后,定向插入克隆载体puc18中,连接产物转化感受态大肠杆菌DH5a,挑选阳性克隆,鉴定后进行序列测定。将克隆获得的约0．45kb的gbpC基因特异片段经EcoRI／SalI双酶切后,定向插入pGEX-4T-1中,构建pGEX-4T-1／gbpCE原核融合表达载体,转化感受态大肠杆菌DH5α,挑选阳性克隆,酶切及PCR鉴定后,异丙基硫代-β-D-半乳糖苷（Isopropyl-β-D-thiogalactoside,IPTG）诱导表达融合蛋白GST-GbpC^E,SDS-PAGE检测表达产物。结果：测序结果与Sato等报道的序列一致;原核表达后,在SDS-PAGE凝胶上出现一条新生蛋白条带,其相对分子量约43000,与预计大小相符合。结论：成功克隆并表达gbpC特异片段,获得了融合蛋白GST-GbpC^E,为制备GbpC抗体打下了基础。     

变形链球菌乳酸脱氢酶基因及同源区的克隆和序列分析

目的：克隆变形链球菌乳酸脱氢酶基因(ldh)及其两侧同源区基因片段。方法：应用PCR技术扩增乳酸脱氢酶及其两端同源区序列片段，插入克隆载体pMD18-T中，转化大肠杆菌DH5α，Amp^ 抗性挑选阳性克隆，经酶切及PCR鉴定后进行序列测定。结果：PCR扩增产物特异；抗性筛选的4株菌落均为阳性克隆；用DNASIS将序列测定的结果与基因Bank中报道的序列对比分析，同源性为99．1％。结论：根据同源性分析的结果，可确定该克隆片段为变形链球菌乳酸脱氢酶及其两侧同源区序列。     

小鼠釉基质丝氨酸蛋白酶成熟肽编码区cDNA的克隆和序列分析

目的：克隆小鼠釉基质丝氨酸蛋白酶（EMSP）成熟肽编码区的基因。方法：设计引物，以小鼠牙胚总RNA为模板，利用RT-PCR方法，扩增出小鼠EMSP的基因片段，将所得基因片段插入pBS质粒载体。转化到大肠杆菌XL-1-Blue后挑选阳性克隆，提取重组质粒DNA，通过限制性酶切和核苷酸序列分析鉴定阳性克隆。结果：重组质粒pBS-EMSP的酶切图谱和序列分析结果与国外文献报道一致。结论：克隆到小鼠EMSP成熟肽编码区基因。     

牙胚中上游刺激因子部分序列的cDNA克隆

目的：克隆牙胚中上游刺激因子（upstream stimulatory factor,USF)基因编码区特异片段。方法：从生后5d balb/c鼠牙胚中抽提总RNA，用随机引物逆转录合成cDNA，然后 用PCR方法借助特异性引物以cDNA中扩增小鼠USF1基因片段（289bp），电泳检测、回收目的片段，将所得的基因片段连接至Teasy质粒载体，并转化到大肠杆菌DH5α后挑选阳性克隆，扩大培养，提取重组质粒DNA，酶切分析和核苷酸序列分析鉴定阳性克隆。结果：酶切图谱显示质粒重组，克隆成功，序列分析结果与国外报道一致。结论：在牙胚中克隆到USF1基因片段。     

TK基因重组质粒的构建及在ACC-2细胞中的表达

目的：克隆胸腺嘧啶核苷激酶（TK）基因，构建质粒表达载体pIRES—TK，并利用该载体转染ACC-2细胞，建立了稳定表达TK基因的ACC-2细胞克隆。方法：通过PCR从逆转录病毒重组体pLNSX—TK中扩增出TK基因全长序列，将扩增产物定向插入到克隆载体pMD18-T中进行序列测定，测序正确后将其亚克隆到质粒表达载体pIRES中，构建重组表达载体pIRES—TK，用电穿孔法以该质粒转染ACC-2细胞，经G418筛选获得稳定表达TK基因的ACC-2细胞株，提取该细胞株的总RNA，用RT—PCR检测TK基因的表达。结果：PCR扩增出1128 bp大小的片段，测序结果与GeneBank报道的TK序列基本一致；阳性重组质粒pIRES—TK经XhoI和Mull双酶切后获得2692 Bb和1128 bp的片段；RT-PCR从转染细胞的总RNA中扩增出361 bp的预期片段。结论：成功扩增了TK基因的DNA片段；成功构建了质粒表达载体pIRES—TK；建立了稳定表达TK基因的ACC～2细胞株，为TK／GCV自杀基因系统在腺样囊性癌基因治疗中的应用奠定良好的基础。     

小鼠釉丛蛋白成熟肽编码区cDNA克隆

克隆小鼠釉丛蛋白成熟肽编码区基因。方法：设计引物，以小鼠牙胚ｃＤＮＡ文库为模板，利用ＰＣＲ方法，扩增出小鼠釉丛蛋白成熟区的基因片段，将所得基因片段插入ｐＢＳ质粒载体，转化到大肠杆菌ＸＬ－１－Ｂｌｕｅ后挑选阳性克隆，提取重组质粒ＤＮＡ，通过限制性酶切和部分核苷酸序列分析鉴定性克隆。结果：重组质粒ｐＢＳ－ｔｕｆｔｅｌｉｎ的酶切图谱和序列分析结果与国外文献报道一致。结论：克隆到小鼠釉丛蛋白成熟编码区基因     

小鼠牙本质磷蛋白cDNA的克隆和部分序列分析

目的：克隆小鼠牙本质磷蛋白（ＤＰＰ）ｃＤＮＡ。方法：用异硫氰酸胍一步法从小鼠牙胚组织中抽提总ＲＮＡ，用ｏｌｉｇｏ（ｄｔ） 作引物逆转录合成牙胚ｃＤＮＡ，然后利用ＰＣＲ 方法，从ｃＤＮＡ 中扩增出小鼠ＤＰＰｃＤＮＡ基因片段（ 约１ ．６ｋｂ），将所得基因片段插入ｐＢｌｕｅｓｃｒｉｐｔ 质粒载体，转化到大肠杆菌ＸＬ１ － Ｂｌｕｅ 后挑选阳性克隆，提取重组质粒ＤＮＡ，通过酶切分析和核苷酸序列分析鉴定阳性克隆。结果：酶切图谱和部分序列分析结果与国外文献报道一致。结论：克隆到小鼠ＤＰＰｃＤＮＡ 基因片段。     

小鼠牙本质涎磷蛋白基因启动子的克隆与序列分析

目的 克隆小鼠牙本质涎磷蛋白(dentin sialophosphoprotein，DSPP)基因启动子片段。方法 培养MDPC一23细胞，从培养的细胞中提取基因组DNA，利用设计的上下游引物，进行PCR反应。将扩增得到的DSPP基因启动子片段克隆到pMD18-T栽体，酶切鉴定后，进一步进行DNA序列测定。结果 酶切结果表明成功构建重组质粒，序列分析结果与国外报道一致。结论 成功克隆获得小鼠DSPP基因的启动子片段。     

伴放线放线杆菌cdtC基因的克隆及其原核表达载体pET15b-cdtC的构建

目的：本实验设计克隆Aa CdtC亚基蛋白的编码基因cdtC并构建其原核表达载体pET 15b-cdtC,为研究CdtC亚基对CDT全毒素功能的分子机制奠定基础。方法：厌氧培养Aa ATCC 29523,提取Aa基因组DNA,PCR反应扩增cdtC基因片段。将此片段克隆入pMD 19-T Vector,经限制性内切酶BamHI和Xho I酶切得到粘性末端cdtC片段,克隆入原核表达载体pET 15b中。结果：产物经PCR初步鉴定后进行DNA序列分析,测序结果与Genbank公布序列一致性达100%。结论：本实验成功克隆了cdtC基因,并成功构建了其原核表达载体pET15b-cdtC。为深入研究CdtC亚基以及CDT全毒素分子作用机制奠定基础。     

Identification of a glucan-binding protein C gene homologue in Streptococcus macacae

BACKGROUND/AIMS: The past few decades have seen the isolation of certain glucosyltransferases and a number of proteins from mutans streptococci. Some of these proteins have been shown to possess glucan-binding capabilities which confer an important virulence property on mutans streptococci for the role of these bacteria play in dental caries. Among these proteins is glucan-binding protein C, which is encoded by the gbpC gene, and which we have identified as being involved in the dextran-dependent aggregation of Streptococcus mutans. However, gbpC homologues have yet to be identified in other mutans streptococci. METHODS: We carried out polymerase chain reaction amplification of Streptococcus macacae using primers that were designed based on conserved sequences of S. mutans gbpC and identified a gbpC gene homologue. The gene of that homologue was then characterized. RESULTS: Nucleotide sequencing of the S. macacae gbpC homologue revealed a 1854 bp open reading frame encoding a protein with an N-terminal signal peptide. The molecular mass of the processed protein was calculated to be 67 kDa. We also found an LPxTG motif, the consensus sequence for gram-positive cocci cell wall-anchored surface proteins, which was followed by a characteristic sequence at the carboxal terminal region of the putative protein. This suggests that the S. macacae GbpC homologue protein was tethered to the cell wall. CONCLUSION: Based on these results, together with the demonstrated glucan-binding ability of the S. macacae GbpC homologue protein, we suggest that S. macacae cells are capable of binding dextran via the GbpC homologue protein, which is similar to the S. mutans GbpC protein. In addition, Southern hybridization analysis using the S. macacae gbpC homologue as a probe showed a distribution of gbpC homologues throughout the mutans streptococci.     

变形链球菌葡聚糖结合蛋白研究进展

葡聚糖结合蛋白是变形链球菌致龋的重要毒力因子。与变形链球菌的粘附、聚集、细胞壁生理功能、酶活性和形成生物膜并维持其结构稳定起重要作用。该文就变形链球菌4种葡聚糖结合蛋白研究现状,葡聚糖结合蛋白与生物膜、防龋疫苗的关系及当前存在的问题作一综述。     

变形链球菌葡聚糖结合蛋白B的纯化

目的：从变形链球菌S.mutans Ingbritt(serotype c)培养液上清中纯化葡聚糖结合蛋白B。方法：Sephadx G-200凝胶（α-1,6键葡聚糖）亲和层析和Q－SephroseFF离子交换层析。结果：纯化出约10μg具有葡聚糖结合特性的GbpB蛋白。结论：通过Sephadx G-200凝胶（α-1,6键葡聚糖）亲和层析和Q－Sephrose FF离子交换层析纯化出变链菌葡聚糖结合蛋白B。     

Inhibition of catalytic and glucan-binding activities of a streptococcal GTF forming insoluble glucans

The ability of a range of potential inhibitors to affect the catalytic activity or binding of dextran by a glucosyltransferase (GTF-I) that synthesises insoluble alpha1,3-linked glucan was tested. Acarbose, deoxynojirimycin, N-dodecyldeoxynojirimycin and Tris, which are thought to interfere with the active site of the enzyme of GTF and related glycosidases, inhibited glucan synthesis but not glucan binding. Tris was found to act as a competitive inhibitor of GTF-I. The effectiveness of the active site inhibitors was not altered by immobilisation of GTF-I on saliva-coated hydroxyapatite. In contrast, three amine hydrofluorides were markedly less effective against immobilised GTF than soluble GTF. The pH of the reaction mixture was found to have a strong influence on inhibition by acarbose, Tris and amine hydrofluorides, a finding that is of direct relevance to use of inhibitors in vivo.     

Biological functions of glucan-binding protein B of Streptococcus mutans

INTRODUCTION: Streptococcus mutans has been implicated as a major causative agent of dental caries in humans. Bacterial components associated with the adhesion phase of S. mutans include glucosyltransferases, protein antigen C and proteins that bind glucan. At least four glucan-binding proteins (Gbp) have been identified; GbpA, GbpB, GbpC and GbpD. METHODS: In our previous study, the contributions of GbpA and GbpC to the virulence of S. mutans were investigated; however, the biological function of GbpB and its role in the virulence of S. mutans remain to be elucidated. Using a GbpB-deficient mutant strain (BD1), we demonstrated in the present study that GbpB has a role in the biology of S. mutans. RESULTS: The growth rate of BD1 was lower than that of other strains, while it was also shown to be less susceptible to phagocytosis and to form longer chains than the parental strain MT8148. In addition, electron microscope observations of the cell surfaces of BD1 showed that the cell-wall layers were obscure. CONCLUSION: These results suggest that GbpB may have an important role in cell-wall construction and be involved in cell separation and cell maintenance.     

Comparison of glucan-binding proteins in cariogenicity of Streptococcus mutans

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. Bacterial components associated with the adhesion phase of S. mutans include cell-associated and cell-free glucosyltransferases (GTFs), as well as protein antigen c and proteins that bind glucan. At least four types of S. mutans glucan-binding protein (Gbp) have been identified; GbpA, GbpB, GbpC and GbpD. In the present study, GbpA-, GbpB- and GbpC-deficient mutants (AD1, BD1 and CD1, respectively) were constructed, and their cariogenic properties were evaluated by comparing them to those of their parent strain MT8148. All of the Gbp mutants showed lower levels of dextran binding, while the sucrose-dependent adhesion levels of AD1 and CD1 were lower than in the parental strain. The expression of each GTF was detected in the Gbp mutants, however, they had lower levels of cell-free-GTF activity than the parental strain. On the other hand, in acid tolerance assays, BD1 was the most sensitive among all of the tested strains. These results suggest that GbpA and GbpC in S. mutans have strong relationships with cariogenicity, while GbpB may have another biological function.     

Contribution of glucan-binding protein C of Streptococcus mutans to bacteremia occurrence

Our previous analysis of major cell surface proteins of Streptococcus mutans isolated from the blood of a patient with bacteremia showed variations of glucan-binding protein C (GbpC) expression. In the present study, we analyzed the contribution of GbpC of S. mutans to bacteremia occurrence. A GbpC-defective mutant strain (C1) was significantly less susceptible to phagocytosis by human polymorphonuclear leukocytes than its parent strain (MT8148) (P < 0.001). When 21 rats were injected with C1 or streptomycin-resistant MT8148R into the jugular vein, strain C1 was recovered from blood in larger numbers and for a longer duration than MT8148R. Further, infection with C1 resulted in significant increases in serum sialic acid (SSA) concentrations, and splenomegaly, as well as body weight reduction. We also evaluated GbpC expression in 20 clinical oral isolates by immunoblotting with anti-GbpC serum, and found that expression intensity was positively correlated to phagocytosis rate (P < 0.05). These results suggest that S. mutans GbpC may be associated with systemic virulence, since a weak expression of GbpC causes the organisms to be refractory to phagocytosis, resulting in a longer survival of the bacterium in the bloodstream.     

变形链球菌葡聚糖结合蛋白D基因的分子克隆及其原核表达和初步纯化

目的:克隆变形链球菌葡聚糖结合蛋白D(gbpD)基因,在大肠杆菌中表达并对重组蛋白进行初步纯化,为进一步的蛋白功能研究奠定基础。方法:体外培养变形链球菌UA159菌株并以其基因组为模板,对gbpD基因编码区进行PCR扩增,连接pMD-18T载体并测序。随后将该片段克隆入原核表达载体pPROEX HTb中,构建表达质粒pPROEX/gbpD,转化大肠杆菌DH5α并用IPTG诱导表达。表达产物经镍-次氮基三乙酸(Ni-NTA)柱进行亲和层析纯化。结果:成功克隆变形链球菌gbpD基因并在大肠杆菌中得到可溶性表达,经Ni-NTA柱纯化后获得纯度大于95%、相对分子量(Mr)为76×103的变链菌GbpD蛋白。结论:获得Mr为76×103的变形链球菌GbpD蛋白。对进一步确定GbpD的葡聚糖结合能力,探讨其在变链菌致龋过程中的作用,以及在龋病预防领域的应用前景都具有重要的意义。     

Inactivation of srtA gene of Streptococcus mutans inhibits dextran-dependent aggregation by glucan-binding protein C

A sortase-deficient mutant of Streptococcus mutans was prepared by insertional inactivation of a sortase gene (srtA). The srtA mutant was defective in cell wall-anchoring of two surface proteins 200 and 75 kDa in size. A previous study has shown that the 200 kDa protein is a surface protein antigen PAc and that the sortase catalyzes cell wall-anchoring of PAc in S. mutans. In this study another surface protein 75 kDa in size was examined by immunologic and physiologic methods. Western blot analysis with a specific antiserum showed that the 75 kDa protein was a surface protein, glucan-binding protein C. The protein was overexpressed under a stress condition including a sublethal concentration of tetracycline. The srtA mutant cells also lost the ability of dextran-dependent aggregation. These results suggest that the S. mutans sortase mediates cell wall-anchoring of the glucan-binding protein C and dextran-dependent aggregation of this organism.     

Binding of glucan-binding protein C to GTFD-synthesized soluble glucan in sucrose-dependent adhesion of Streptococcus mutans

Streptococcus mutans produces glucan-binding proteins (Gbp proteins) which promote the adhesion of the organism to teeth. Three Gbp proteins, GbpA protein, GbpB protein, and GbpC protein have been identified; however, the mechanism of adhesion between glucans and bacterial cell surfaces is unknown. We used glucosyltransferase (GTF)- and/or Gbp-deficient mutants to examine the role of GbpC protein in the sucrose-dependent cellular adhesion of S. mutans to glass surfaces. The wild-type strain MT8148 and a GbpA-deficient mutant strain displayed increased sucrose-dependent adhesion following the addition of rGTFD. However, a GbpC-deficient mutant strain demonstrated no changes in the level of sucrose-dependent adhesion in spite of the addition of rGTFD. Further, the binding of rGbpC protein to the glucan synthesized by rGTFD was significantly higher than that to the glucan synthesized by either rGTFB or rGTFC. These results suggest that GbpC protein may play an important role in sucrose-dependent adhesion by binding to the soluble glucan synthesized by GTFD.