伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

伴放线放线杆菌血清型分析

目的 PCR法检测伴放线放线杆菌（Actinobacillus actinomy＇etemcomitans，Aa）临床分离菌株血清型，分析其与flp-1基因型的关系。方法用血清型特异性引物，通过普通PCR和多重PCR的方法对60株Aa临床分离菌株的血清型进行鉴定，并分析其与flp-1基因型的关系。结果 60株Aa临床分离菌株中血清型c型63，33％，e型23．33％，b型6.67％，a，f型各占3．33％，未检测到d型菌株；在24名被检测者中，15名检测到c型An菌株，3名检测到b型菌株，各有2名分别检测到a、e、f型菌株。fip-1基因型Ⅰ型菌株的血清型均为a型，40株Ⅱ型菌株中38株为c型，Ⅳ型菌株均为b型，11株Ⅴ型菌株中9株为e型，Ⅵ型菌株均为e型。结论 Aa血清型分布以c型为主，fip-1基因型与菌株血清型存在一定对应关系。     

Cloning and sequencing of part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4

We have cloned and sequenced the 5.2 kb Eco RI fragment that contained part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4. The order of the ribosomal protein genes was identical to that of the S10 operon of Haemophilus influenzae and Escherichia coli . The deduced amino acid sequences of ribosomal proteins in this operon displayed significant homologies (65.3%-100%) to those of H. influenzae, E. coli, Yersinia enterocolitica and Yersinia pseudotuberculosis . Phylogenetic trees obtained for these ribosomal proteins were similar to that obtained for 16S rRNA.     

Simultaneous isolation of Actinobacillus actinomycetemcomitans from subgingival and extracrevicular locations of the mouth

Abstract In the present study, a total of 619 subgingival and extracrevicular samples from 66 early-onset periodontitis, 42 adult periodontitis/gingivitis and 36 treated Actinobacillus actinomycetemcomitans-associated periodontitis patients were selectively cultivated for presence of A. actinomycetemcomitans. The organism was recovered from 68% cases with early-onset periodontitis, 24% cases with adult periodontitis/gingivitis and 50% of treated patients. Associations between recovery from pooled subgingival plaque and samples from extracrevicular locations as well as between different extracrevicular samples, were not heterogeneous with regard to different groups with the exception for cheek/saliva comparisons (odds ratios: early-onset periodontitis 825; adult periodontitis 8.1; treated patients 117; 0.05<p<0.1). For associations between recovery of A. actinomycetemcomitans from pooled subgingival plaque/extracrevicular samples, Mantel-Haenszel's odds ratios of between 12.2 and 21.6 were calculated (p<0.0001). The organism was isolated from 17 cheek mucosa samples of 18 patients identified as still harboring the organism after therapy. Present results point to the considerable value of cheek mucosa samples especially in treated patients to diagnose persistent A. actinomycetemcomitans colonization of the oral cavity.     

伴放线放线杆菌粘附特性研究

目的分析伴放线放线杆菌的粘附特性及菌毛结构基因tip-1的遗传多样性对菌株粘附活动的影响。方法检测不同孵育条件下5种tip-1基因型临床分离菌株和光滑型菌株的粘附活动。结果临床分离菌株的粘附量随菌液浓度,孵育时间的增加而增加。tip-1基因型Ⅱ型菌株的粘附量高于其它4型菌株,光滑型菌株的粘附量低于临床分离菌株。生理温度下菌株粘附数高,低温下明显降低。厌氧条件和有氧条件下的粘附量无显著性差异。结论伴放线放线杆菌临床分离菌株的粘附存在时间和菌量依赖性,并要求一定新陈代谢活性,粘附效率在氧浓度改变时没有明显变化。伴放线放线杆菌表型影响菌株的粘附作用。不同tip-1基因型菌株粘附能力存在差异,Ⅱ型菌株粘附能力最强。     

In vitro antimicrobial susceptibility of different serotypes of Actinobacillus actinomycetemcomitans

In vitro susceptibility of Actinobacillus actinomycetemcomitans ( A.a .) serotypes to selected antimicrobial agents was investigated by the agar dilution method on supplemented Mueller-Hinton test medium. Eighty-three A.a . strains, 80 recent isolates from 40 periodontally healthy or diseased subjects, and three type strains were included in the study. Serotype a represented 20, serotype b 32, serotype c 17, and serotype e 7 and nontypable 4 of the tested strains. The most effective drugs against all A.a . serotypes in vitro were cefaclor, cefuroxime, tetracycline hydrochloride, doxycycline, trimethoprim-sulfamethoxazole (cotrimoxazole), and ciprofloxacin, which inhibited 100% of the strains at 4.0 μg/ ml, 4.0 μg/ml, 1.0 μg/ml, 2.0 μg/ml, 0.06 μg/ml, and 0.015 μg/ml, respectively. Serotypes a and e were more susceptible to cafaclor and cefuroxime than were serotypes b and c; 100% of the first two groups were inhibited at 2.0 μg/ml and 1.0 μg/ml. Ampicillin inhibited 92% of the tested strains at 1.0 μg/ml. Serotype b was always susceptible to ampicillin. Metronidazole exhibited the best activity against serotype a strains. The lowest minimal inhibitory concentration values for benzylpenicillin, ampicillin, erythromycin, doxycycline, and metronidazole were encountered among serotype b isolates. The results of the present study indicate minor differences in the in vitro antimicrobial susceptibility patterns of different A.a . serotypes, except to metronidazole. Also, the new oral cephalosporins and cotrimoxazole, rare antimicrobial agents in periodontology, showed promising efficacy against all A.a . strains.     

Actinobacillus actinomycetemcomitans recovery from extracrevicular locations of the mouth

Associations between recovery of Actinobacillus actinomycetemcomitans from samples of subgingival plaque, and samples of buccal mucosa, tongue and unstimulated saliva were studied in 107 subjects. Ten subjects had gingivitis, 18 localized juvenile periodontitis, 45 rapidly progressive periodontitis and 32 adult periodontitis. Two children suffered from prepubertal periodontitis. Heterogeneity tests for associations in different study populations yielded nonsignificant results. Mantel-Haenszel's common odds ratios were 52.9, 37.2 and 19.8 for respective associations between pooled subgingival samples, and cheek, saliva and tongue samples. Significant McNemar's chi-square of 5.88, 11.25 and 16.96 for respective associations pointed to secondary occurrence of A. actinomycetemcomitans in extra-crevicular samples. Multiple linear regression yielded a significant influence of the number of deep periodontal pockets of 7 mm or more and a negative influence of the diagnosis "adult periodontitis" on the log-transformed number of colony-forming units of A. actinomycetemcomitans in samples from cheek mucosa in patients infected with the organism. Extracrevicular occurrence of A. actinomycetemcomitans seems to reflect total subgingival numbers of the organism. Especially sampling check mucosa appears to be a promising tool in the diagnosis of a periodontal infection with A. actinomycetemcomitans .     

Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism

The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a.     

The production of 6-deoxy-L-talan from Actinobacillus actinomycetemcomitans via bacterial coupling in vitro

A method of producing 6-deoxy-L-talan, the serotype c-specific polysaccharide antigen (SPA) from Actinobacillus actinomycetemcomitans,was established using a whole-cell reaction with two recombinant Escherichia coli strains. The production of serotype c-SPA was investigated using the dot blot assay with anti-A. actinomycetemcomitans NCTC 9710 serum after an 18-h reaction, starting with a solution containing the recombinant E. coli cells, alpha-d-glucose-1-phosphate, and dTTP. Moreover, examination of the time course for 6-deoxy-L-talan production proved that this system ran satisfactorily. This paper is the first report of a convenient method to readily produce the exopolysaccharide from A. actinomycetemcomitans in vitro.     

Occurrence of Actinobacillus actinomycetemcomitans in patients wearing orthodontic appliances

Abstract The aim of the present study was to assess: (1) the occurrence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque from young patients undergoing orthodontic treatment with fixed appliances; (2) a possible relationship between the presence of Aa and the clinical conditions; (3) a relation between the duration of orthodontic treatment and the microbiological and clinical parameters; (4) whether differences exist when taking into consideration the different type of appliances, i.e., bands or brackets. 34 subjects aged between 12 and 20 years participated in the study. Of these, 20 subjects had worn orthodontic appliances (test group), while the remaining 14 subjects served as matched control (control group). 4 to 8 sites in each patient were available for clinical and microbiological examination. Clinical parameters consisted of presence/absence of plaque and gingival bleeding index (GBT), Microbiological sampling was performed in the same sites as in the clinical examination. A statistically significant difference was present when comparing %s of GB1 positive scores between teeth from the test group (57.5%) and teeth from the control group (25%). Plaque was present in 53% of test sites and 37% of control sites, but this difference was not statistically significant. Aa was detected, from at least one site in 85% of test subjects and in 15% of the control subjects (p<0.001). Among the subjects, 41% harboured Aa at a concentration between 0.1% and 1.0%, whereas another 40% yielded Aa at a concentration greater than 1.0%. Finally, a positive correlation was noted between the % of sites positive for Aa and the % of sites displaying a positive GB1 score (r=0.41; p<0.005). No relation was found between the duration of orthodontic treatment and the microbiological or clinical parameters; neither were statistically significant differences found when we compared results from sites wearing bauds or brackets. In conclusion, the present study showed that young subjects wearing orthodontic appliances harbour Aa with a remarkable frequency of detection, although plaque levels do not significantly differ from those of a matched control group.