Polymerase Chain Reaction Analysis of the Clonality of Porphyromonas Gingivalis and Collagenase Gene

目的：本研究旨在分析牙龈卟啉菌基因型和其重要毒力因子胶原酶基因（collagenase gene,PrtC）的遗传异质性，了解细菌遗传变异与其对牙周致病性的相关性。方法：采用随机引物聚合酶链反应（AP－PCR）的方法，对从24例牙周炎患者口腔中分离的79株牙龈卟啉菌和参考菌株ATCC33277进行DNA指纹分析。并随机选择24株临床菌株，进一步检测是否存在特异性的胶原酶基因片段。对照菌株为伴放线放线标菌Y4和中间普氏菌ATCC25611，通过酶切鉴定和DNA序列分析，了解PrtC基因遗传多样性的变化。结果：80株牙龈卟啉菌共获得7种基因型（I－Ⅶ），以Ⅶ所占比例最高（25．8％）。24株临床菌株和ATCC33277中均扩增出特异性的胶原酶基因片段（548bp）。将4株细菌的序列分析结果与国外的报道相比较，发现一些基因序列存在差异，其中有6个核苷酸碱基缺失。结论：临床分离的牙龈卟啉菌中可检测到多种基因型，存在明显的个体差异，这些菌株具有合成胶原酶的能力，不同菌株胶原酶基因之间存在遗传异质性的变化。     

牙龈卟啉单胞菌prtH基因水解活性区域的表达

目的 克隆、表达牙龈卟啉单胞菌（P．gingivalis）prtH蛋白水解酶活性结构域（prtHN），制备多克隆抗体，检测其特异性。方法 采用Generunner软件分析prtH蛋白水解酶活性结构域prtHN片断，PCR扩增prtHN片段并进行DNA序列测定；构建原核表达质粒pQE30-prtHN。E．coliJM109表达prtHN重组蛋白．镍亲和柱纯化，SDS—PAGE分析鉴定；以兔抗P．g菌体抗体和抗膜泡抗体进行Western blot分析重组蛋白免疫原性。制备兔抗prtHN多克隆抗体，不同菌种牙周致病菌Western blot检测抗体特异性。结果 克隆重组基因测序结果与GenBank中P．gingivalis RgpA蛋白酶的基因序列U15282一致。SDS—PAGE显示，IPTG诱导的E．eoliJM109（pQF30-prtHN）以包涵体形式表达14KDa的重组蛋白。Western blot检测证实其具有免疫原性。所制备的兔抗prtHN多克隆抗体具有特异性。结论 成功克隆，原核表达的prtHN蛋白具有免疫原性。prtHN蛋白的多克隆抗体具有菌种反应特异性。     

细菌array-CGH技术用于比较牙龈卟啉单胞菌不同菌株基因组差异的研究

目的:构建牙龈卟啉单胞菌(简称P.gingivalis)全基因组规模的微阵列比较基因组杂交(简称array-CGH)芯片平台,分析不同菌株间基因组的差异,为后续检测临床分离株的毒力基因,进一步阐述牙周炎发病机制提供依据。方法:综合已经全基因组测序的12株P. gingivalis菌的序列信息,设计探针序列,定制芯片。提取P. gingivalis高毒力株W83和低毒力株ATCC 33277的基因组DNA,利用array-CGH检测其全基因组的差异DNA片段,并运用PCR验证结果的准确性。结果:Array-CGH结果显示两组菌株间存在几个连续片段的拷贝数不同。P.gingivalis W83的部分特异片段参与编码毒力致病因子。在P.gingivalis ATCC 33277的特有片段中,部分编码蛋白可增强细菌表明粘附力,使其具有高粘附力。从中挑选12个基因进行PCR差异性验证,证实与芯片结果一致。结论:用array-CGH芯片的方法来分析全基因组拷贝数的变化具有分辨率高,能精确定位异常片段的特性。本文成功构建了array-CGH技术检测P.gingivalis不同毒力株基因差异的平台,为后续比较临床分离株的差异DNA片段,筛查毒力基因,深入阐述牙周炎的发病机制奠定基础。     

牙龈卟啉单胞菌不同毒力株基因差异的比较研究

目的比较牙龈卟啉单胞菌（Porphyromonas gingivalis，Pgingivalis）高毒力株W83与低毒力株标准参考菌ATCC 33277之间的差异基因。方法采用抑制消减杂交技术（SHH）对比牙龈卟啉单胞菌高毒力株W83与低毒力株标准参考菌ATCC 33277的基因差异。以高毒力株W83为被检菌，低毒力株ATCC 33277为参考菌，将提取的基因组DNA用内切酶Rsa Ⅰ酶切，连接特殊设计的接头进行两次消减杂交和PCR扩增，得到消减混合物，与TA克隆载体连接，转化到JM109中，建立消减文库，经PCR筛选鉴定阳性克隆，进而对部分片段进行测序和同源分析。结果经SSH筛选鉴定得到36个片段大小为88～372bp的阳性克隆基因片段。结论从全基因角度研究牙龈卟啉单胞菌高毒力株W83与标准株ATCC 33277之间的分子遗传差异，为牙龈卟啉单胞菌致病基因的筛选及今后牙周病预防、诊治的靶标提供依据。     

牙本质磷蛋白基因突变和序列多态性分析

目的：检测牙本质磷蛋白基因在牙本质发育不全Ⅱ型患者中是否存在突变，并对其序列多态性进行分析。方法：采用PCR方法扩增牙本质磷蛋白基因编码区，通过DNA直接测序方法对其核苷酸序列进行分析。结果：在牙本质磷蛋白基因序列中未发现与疾病相关的特异性改变，但检测到该基因的核苷酸序列在不同个体间具有多态性，存在部分核苷酸的缺失以及广泛的单核苷酸多态现象。结论：牙本质磷蛋白基因在牙本质发育不全Ⅱ型患者中不存在突变，但牙本质磷蛋白基因序列具有多态性。     

远缘链球菌ftsK基因敲除菌株的构建

目的:构建远缘链球菌细胞分裂蛋白相关基因ftsK敲除菌株。方法:首先根据已知的远缘链球菌的ftsK基因序列,设计PCR引物,扩增并克隆了ftsK内部结构基因,亚克隆于自杀质粒后,通过同源重组替换野生型ftsK基因。结果:经过western杂交及southern杂交鉴定,证实重组质粒已插入细菌的基因组中。结论:成功构建远缘链球菌ftsK基因敲除菌株,为研究ftsK在致龋过程中细菌的扩增所起的作用奠定了基础。     

牙龈卟啉单胞菌clpP基因缺失株和回补株的构建和鉴定

目的 构建并鉴定牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）W83的丝氨酸蛋白酶(caseinolytic protease,Clp)基因缺失株和回补株,为探索clpP基因在P.g致病过程中的作用和机制奠定基础。方法 设计clpP基因的引物对,PCR扩增其上下游同源片段,克隆入质粒pUC18中,插入红霉素抗性基因ermB作为筛选标记,构建重组质粒,电转化入P.g W83构建缺失株;PCR扩增clpP基因片段与重组质粒pUC18-ΔclpP-ermB连接,电转化入P.g W83缺失株中构建回补株。采用PCR和酶切电泳鉴定序列的正确性,利用抗性培养基筛选高表达菌株。结果 clpP基因克隆成功并顺利导入质粒pUC18中,P.g W83的clpP基因缺失株和回补株构建成功,并能在体外稳定传代。结论 本研究运用同源重组技术构建了P.g W83的clpP基因缺失株,并建立了以pUC18质粒为载体的clpP基因回补方法,为进一步研究clpP基因在P.g致病过程中的作用打下了基础。     

采用PCR方法鉴别伴放线放线杆菌的6种血清型

目的：探索采用PCR的方法对伴放线放线杆菌的不同菌株进行血清型分类。方法：根据伴放线放线杆菌不同血清型特异性多糖抗原基因序列设计6对不同的寡核苷酸引物，用这6对引物分别对所选择伴放线放线杆菌6种不同的血清型菌株各3株，共18株，其中参考菌株6株，系ATCC29523（血清型a），ATCC43718（血清型b），ATCC33384（血清型c），IDH781（血清型d），IDH1705（血清型e）以及CU1000（血清型f），其余12个菌株均为临床分离株的DNA进行PCR扩增分析。结果：每一对引物均针对相应的血清型产生特异性单一条带PCR产物，产物大小分别为428bp（a），298bp（b），559bp（c），690bp（d），211bp（e），232bp（f）。全部18个菌株均能够被准确识别，无交叉反应。结论：PCR方法可以快速准确地鉴别伴放线放线杆菌目前已知的全部6种血清型。     

放线共生放线杆菌白细胞毒素水平检测

目的　检测放线共生放线杆菌 (Actinobacillusactinomycetemcomitans,Aa)临床分离菌株白细胞毒素水平 ,区分高毒株与低毒株。方法　应用聚合酶链反应 (polymerasechainreaction ,PCR)检测白细胞毒素操纵子启动子区域基因序列的差异。检测临床菌株 6 8株 ,其中b型 17株 ,c型 42株 ,a型9株。阳性对照高毒株为JP2 ,低毒株为ATCC43717等 5株Aa国际参考菌株 ,阴性对照为 12株异种菌国际参考菌株。结果　 6 8株临床分离菌株扩增片段均为 10 2 2bp ,JP2扩增片段为 492bp ,ATCC43717等 5株扩增片段均为 10 2 2bp ,12株异种菌参考菌株无扩增片段出现。结论　 6 8株临床分离菌株全部为低毒株     

慢性牙周炎患者龈下菌斑中不同基因型牙龈卟啉单胞菌的检测

摘要 目的:建立临床标本中牙龈卟啉单胞菌(P.g)的PCR检测方法,探讨慢性牙周炎患者不同牙位的龈下菌斑中P.g基因型的差异。方法:采用培养法分离鉴定慢性牙周炎患者不同牙位龈下菌斑中P.g,同时采用PCR检测 P.g16SrDNA、prtC和fimA基因。部分扩增产物测定了核苷酸序列。结果:在66例患者的127个龈下菌斑标本中, P.g16SrDNA、prtC和fimA多重引物扩增的阳性率为9814%;PCR阳性率显著高于培养法P.g的检出率(P< 0101)。3010%的患者(18/60)同时感染了不同基因型的P.g菌株。P.g16SrDNA、prtC和fimA扩增片段的核苷酸序列同源性在98162%~100%之间。结论:本文所建立的P.g的PCR检测方法具有较高的敏感性和特异性,适用于P.g的快速临床诊断。同一患者可被不同感染来源的多株P.g同时感染。     

Polymerase chain reaction for the identification of Porphyromonas gingivalis collagenase genes

Porphyromonas gingivalis has been shown to exhibit genetic diversity possibly resulting in variation of virulence. In the present study a potential virulence factor was targeted for the detection of P. gingivalis. A 548 bp fragment of the collagenase gene ( prtC ) from Porphyromonas gingivalis ATCC 33277 was amplified by polymerase chain reaction (PCR) using oligonucleotides derived from the middle portion of prtC. From 16 of 21 clinical P. gingivalis strains, a PCR product of similar size to the prtC could be obtained. These 16 P. gingivalis strains were confirmed as positive for prtC using DNA hybridization with a digoxigenin-labeled prtC PCR product as a probe. In 12 of the 16 prtC positive strains, the restriction analysis of the PCR products revealed fragment patterns identical to the known sequence. In the other 4 prtC positive strains, 4 distinct patterns were found. Of these strains, nucleotide sequence analysis of a 400 bp PCR product stretch revealed 79.1%, 83.0%, 84.8 and 89.5% homology with the known nucleotide sequence for this specific region. Sequence analysis of the PCR products from the ATCC 33277 strain demonstrated 93.7% homology. The limit of detection for the PCR was about 100 organisms. None of the other 48 tested strains of 16 bacterial species derived from oral and extraoral infections yielded a PCR product. The PCR was also used for the detection of prtC sequences in dental plaque. Our data indicate that not all P. gingivalis strains have prtC. Nucleotide heterogeneity exists among P. gingivalis with prtC. Using a potential virulence factor for the detection of putative periodontal pathogens such as P. gingivalis may be valuable for the epidemiology of infection and clinical diagnosis of periodontal diseases.     

Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique

Background/aims:  Porphyromonas gingivalis , a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P .  gingivalis .
Methods:  The subtractive hybridization technique was employed to identify the genes specific to P .  gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P .  gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains.
Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.
Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P .  gingivalis strains.     

Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections

Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease. The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections. Type strains for P. gingivalis (ATCC 33277), P. endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls. When PCR primers specific for the 16S ribosomal RNA gene of P. gingivalis or P. endodontalis were used, 16 of the strains were identified as P. gingivalis, and five strains were identified as P. endodontalis. The presence of the prtC gene for collagenase was detected using PCR. Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene. Type strain ATCC 33277 and all 16 clinical isolates of P. gingivalis produced the collagenase gene amplicon. Neither type strain ATCC 35406 nor the five strains from clinical isolates of P. endodontalis produced the collagenase gene amplicon. These results indicate that P. gingivalis from endodontic infections possesses the prtC gene. P. endodontalis does not seem to exhibit prtC. The virulence of P. gingivalis may be related to its production of collagenase.     

Characterizing the specific coaggregation between Actinobacillus actinomycetemcomitans serotype c strains and Porphyromonas gingivalis ATCC 33277

A visual coaggregation study showed specific interspecies coaggregation between an Actinobacillus actinomycetemcomitans serotype c strain and Porphyromonas gingivalis strains ATCC 33277 and 381. We mutagenized A. actinomycetemcomitans SUNYaB 67 (serotype c) with transposon IS903phikan and isolated three transposon insertion mutants that had a reduced ability to aggregate with P. gingivalis ATCC 33277. The three transposon insertions in the mutant strains mapped to the genes at ORF12, ORF13 and ORF16 of the gene cluster responsible for producing serotype c-specific polysaccharide antigen (SPA). Western blot analysis with serotype c-specific antibody showed that these strains did not produce the high-molecular-mass smear of SPA. Furthermore, two SPA-deficient mutants and an SPA-producing mutant were constructed. The two SPA-deficient mutants were deficient for ORF12 and ORF14, which are necessary for the synthesis of serotype c-SPA, and the SPA-producing mutant was deficient for ORF17, which is not related to SPA synthesis. The ORF12- and ORF14-deficient mutants showed reduced ability to aggregate with P. gingivalis ATCC 33277, while the ORF17-deficient mutant aggregated with ATCC 33277 to the same extent as wild-type SUNYaB 67. Our findings suggest that serotype c-SPA of A. actinomycetemcomitans mediates coaggregation with P. gingivalis ATCC 33277.     

牙龈卟啉单胞菌肽酰精氨酸脱亚氨酶的克隆与表达

目的:构建牙龈卟啉单胞菌外膜蛋白肽酰精氨酸脱亚氨酶(PAD)克隆表达重组子,转化于大肠杆菌BL21中,并在最适宜条件下诱导表达。方法:以牙龈卟啉单胞菌ATCC33277全基因组DNA为模板,利用PCR技术获得目的基因PAD,将扩增得到的PAD基因定向插入线性克隆载体PMD18-T Vector中,得到克隆重组子PMD18-T-PAD。经PCR和双酶切鉴定正确的克隆重组子PMD18-T-PAD与表达载体PET-28a经Xhol和Ncol双酶切后,在一定连接体系下,连接构建表达质粒PET-28a-PAD。鉴定正确的原核重组表达质粒PET-28a-PAD,转化大肠杆菌BL21感受态细胞,在不同浓度异丙基硫代-β-D-半乳糖苷(IPTG)及时间诱导下表达融合蛋白。以抗His Tag单克隆抗体为一抗,Western免疫印迹鉴定。结果:DNA测序结果表明,PAD与NCBI核酸数据库中收录的PAD序列同源性达100%;37℃,IPTG浓度为0.5mmol/L,250r/min振摇培养6h的诱导条件下,PAD可高效表达。结论:本实验成功构建了PAD的克隆表达重组子,并在大肠杆菌中表达了PAD蛋白,为进一步研究PAD的免疫学性能及相应的抗体制备奠定了基础。     

prtH in Tannerella forsythensis is not associated with periodontitis

BACKGROUND: It has been suggested that prtH in Tannerella forsythensis encodes for a cystein proteinase that is associated with its pathogenic potential and can discriminate between periodontal health and disease. The aim of this investigation was to further establish this potentially important observation. METHODS: A group of 33 consecutive adult patients with periodontitis (mean age: 47.6 +/- 10.1 years) harboring T. forsythensis was selected to investigate the presence of prtH by polymerase chain reaction (PCR). The T. forsythensis strains were isolated by anaerobic culture techniques. To investigate the association of this gene with periodontitis, a group of 14 age-matched subjects (mean age: 56.4 +/- 6.9 years) without any signs of periodontal disease (probing depths <3 mm and no radiographic attachment loss) was tested for comparison. Pure isolates and crude subgingival plaque samples were used as a template for the PCR. RESULTS: In the group of 33 T. forsythensis-positive patients, we found two T. forsythensis isolates to be prtH negative. Despite repeated analyses, testing of the whole subgingival plaque samples revealed only 17 of 33 samples to be prtH positive. The T. forsythensis isolates from the 14 periodontally healthy subjects were all prtH positive. The odds ratio of the presence of prtH in T. forsythensis in periodontitis patients versus healthy controls is 1.06 (P >0.05). CONCLUSIONS: On the basis of our data, we conclude that the presence of prtH in T. forsythensis is not discriminative for patients with T. forsythensis-associated periodontitis compared to healthy carriers of T. forsythensis. In addition, the use of whole subgingival plaque samples to test for the prevalence of prtH in bacteria appeared unreliable. Culture of the microorganism is an important condition to receive a sufficient amount of template DNA to detect the specific locus of the genome.     

牙龈卟啉单胞菌PG1055基因在临床菌株中的表达

目的应用基因芯片技术检测PG1055基因在不同人群的牙龈卟啉单胞菌(P.gingivalis)中分布,探讨这些基因与牙周临床指数之间的关系。方法取龈下菌斑进行细菌分离培养,以临床采集样本提取的DNA为探针,以抑制消减杂交技术获得P.gingivalisW83的特异基因片段PG1055为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG1055基因在牙周病患者及健康人群的牙龈卟啉单胞菌中的分布。结果PG1055基因在牙周病患者及健康人群中的检出率有统计学差异,并且与牙周临床指数相关。结论PG1055基因与P.gingivalis的致病性有关。