支气管败血波氏杆菌皮肤坏死毒素的重组表达及其生物学特性

皮肤坏死毒素(DNT)是支气管败血波氏杆菌的主要致病因子之一.通过PCR分段扩增和克隆获得了全长4 356 bp的dnt基因,并利用pET-28a/BL21系统对其进行了融合表达.Western blotting检测结果表明,表达产物具有良好的免疫学活性.使用His-band purification kit纯化试剂盒纯化后,得到纯度为93.2％的融合蛋白His6-DNT.在乳鼠皮肤坏死试验中,表达产物His6-DNT和天然DNT均能导致乳鼠皮肤产生坏死性病变.在乳鼠皮肤坏死阻断试验中,兔抗His6-DNT血清能中和天然DNT使其失去对乳鼠的皮肤坏死毒性.试验结果表明,重组蛋白具有天然DNT的生物学毒性和免疫原性,所产生的抗体具有中和活性.文中所获得的重组DNT蛋白具有良好的生物学活性,为DNT的结构和功能研究奠定基础.     

猪源支气管败血波氏杆菌百日咳杆菌黏附素基因的原核表达及其抗体检测ELISA方法

[目的]以猪源支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)百日咳杆菌黏附素(PRN)基因的原核表达产物为抗原建立检测PRN抗体的间接ELISA方法.[方法和结果]利用谷胱甘肽-S-转移酶(GST)表达系统对PRN基因在大肠杆菌中进行融合表达.SDS-PAGE和Western blot检测证实该基因获得高效表达,产物易于纯化且具有良好的免疫学活性.通过凝血酶酶切GST-PRN并回收,获得不含GST载体蛋白的PRN蛋白片段.以PRN蛋白片段为抗原建立检测天然PRN抗体的间接ELISA方法.该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性,其敏感性比乳胶凝集试验提高4～128倍,能检测到人工感染14 d后的仔猪血清抗体IgG,对临床送检的1,229份猪血清的检测阳性率为32.7%.ELISA方法对阳性猪场的监测结果预示了保育期仔猪的合群导致猪群大量感染支气管败血波氏杆菌.[结论]该方法具有特异性强、敏感性高、重复性好的特点,可用于猪群PRN抗体水平监测和猪波氏菌病流行病学调查.     

兔支气管败血波氏杆菌PCR检测方法的建立及应用

目的建立一种简便、快速、敏感、特异的适用于支气管败血波氏杆菌的PCR检测方法。方法根据兔支气管败血波氏杆菌（Bordetella bronchiseptica）的fim2基因序列设计了一对特异性引物,进行PCR扩增、特异性和敏感性试验,并将其应用于临床样品的检测。结果利用该PCR方法扩增出425bp的目的基因片段,该产物序列与GeneBank上公布的基因序列同源性为100％。特异性试验表明,该方法对大肠埃希氏菌、多杀性巴氏杆菌、魏氏梭菌和金黄色葡萄球菌均无交叉性反应;并且最小可检出菌液浓度为3．6CPU。用该PCR方法检测了从江苏、山东等地采集的146份兔鼻拭子,结果检出支气管败血波氏杆菌阳性92例,阳性率为63．01％。结论建立了快速检测支气管败血波氏杆菌的PCR方法。     

兔支气管败血波氏杆菌单克隆抗体的制备及鉴定

目的建立能稳定分泌抗兔支气管败血波氏杆菌（Bb）的单克隆抗体杂交瘤细胞株,为今后进一步建立该菌的免疫检测技术奠定基础。方法以Bb分离株BLJ05的灭活菌液为免疫原,腹腔免疫BALB/c小鼠,采用常规杂交瘤技术制备Bb单克隆抗体（McAb）,用间接ELISA、Western-blot等方法对McAb特性进行鉴定。结果获得两株能稳定分泌抗Bb单克隆抗体的杂交瘤细胞株,分别命名为A7D5和D6B2,其小鼠腹水抗体效价分别为1∶409600和1∶102400;且不与兔大肠杆菌、多杀性巴氏杆菌、产气荚膜梭菌等兔的常见病原菌反应,特异性强。两株单抗亲和力实验表明A7D5亲和力略高于D6B2。ELISA相加试验表明它们针对相同的抗原表位。结论成功建立了两株能稳定分泌抗兔支气管败血波氏杆菌单克隆抗体的杂交瘤细胞株,效价高、特异性强,为今后建立该菌的免疫检测技术建立奠定了基础。     

支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测

本研究以小鼠为实验动物模型,研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用.本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP,利用SDS-PAGE比较了2株之间差异,采用Western-blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分,采用电洗脱方法获的分子量为68 kD的P13-OMP有效保护抗原成分(OMP68),然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原.抗体动态变化:将清洁级小鼠70只随机分成7组,10只/组,用油乳剂P13的全茵和不同剂量的P13-OMP和OMP68免疫抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体水平并分析抗体动态变化;主动免疫保护试验:将清洁级小鼠80只随机分成8组,10只/组,分别采用制备的油乳剂P13-OMP(OMP25 μg/只)、OMP68(25 μg/只)和P13菌体免疫抗原在免疫10 d后,分别用100 LD50的P11和P13腹腔攻毒,然后统计保护率并分析免疫原所提供的免疫保护力.免疫P13-OMP和OMP68油乳剂抗原后,7 d时,抗体水平开始逐渐上升,42 d时,抗体水平分别达到215.3和214.8,然后逐渐下降,70 d时,抗体水平分别达到29.1和210.2.主动免疫时,P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护,OMP68免疫组分别均得到9/10和10/10的保护,而P13全菌免疫组分别得到4/10和6/10保护,对照组全部死亡.P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用,为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.     

百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌     

Effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation in calvaria of neonatal rats

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on bone formation were investigated using a purified toxin preparation. Single injection of 4.3 ng of the toxin into the subcutaneous tissue overlying the calvariae of neonatal rats necrotized periosteum of parietal bone and degenerated osteoblasts within two days. Nine days after the injection, the lesion of the bone tissue became severe; the bone matrix became thin and fragmented. These observations indicate that dermonecrotizing toxin without other factors produced by the organisms impairs bone formation.     

Long-term survival of Bordetella bronchiseptica in lakewater and in buffered saline without added nutrients

Abstract Bordetella bronchiseptica grew from small inocula, and retained viability for at least 24 weeks, in unsupplemented lakewater or phosphate-buffered saline. From washed inocula of around 10³ colony-forming units/ml, there was growth at both 10°C and 37°C to give 10⁶–10⁷ colony-forming units/ml. At 10°C, these counts were maintained with little diminution up to week 24 when observations ceased. In the tests at 37°C, two of three strains tested showed similar retention of viability. These results suggest that B. bronchiseptica may exist as hitherto unsuspected reservoirs of infection in freshwater habitats.     

In vivo colonization profile study of Bordetella bronchiseptica in the nasal cavity

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.     

Role played by the response regulator Ris in Bordetella bronchiseptica resistance to macrophage killing

Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired. This suggested that ris-dependent products are required for the intracellular survival of bacteria. In this study we demonstrate that ris-regulated products play a role in B. bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.     

家兔支气管败血波氏杆菌重组百日咳杆菌粘附素（PRN）蛋白的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）PRN蛋白,并以此建立检测Bb抗体的间接ELISA方法。方法参照GenBank公布的猪源支气管败血波氏杆菌prn基因序列（AY376325）设计了一对特异性引物,PCR扩增出相应的核苷酸片段。将PCR扩增产物连接至原核表达载体pGEX-4T-1中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白PRN作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数等,建立检测支气管败血波氏杆菌抗体的间接ELISA方法。结果成功克隆了prn全基因序列,并在E.coliBL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白PRN具有良好的抗原性。应用重组蛋白PRN为抗原建立了检测Bb血清抗体的间接ELISA诊断方法。试验确定重组蛋白PRN抗原的包被浓度为500ng/mL,最适血清稀释度为1∶40。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础。     

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.     

家兔支气管败血波氏杆菌重组蛋白DNT1的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）DNT蛋白,并以此建立检测Bb抗体的间接ELISA方法。方法参照GenBank公布的猪源支气管败血波氏杆菌dnt基因序列（AB020025）针对其N-端设计了一对特异性引物,PCR扩增出相应的核苷酸片段。将PCR扩增产物连接至原核表达载体pET-28a（＋）载体中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白DNT1作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数,建立检测支气管败血波氏杆菌重组蛋白DNT1抗体的ELISA方法。结果成功克隆了dntN-端的基因序列,并在E.coli BL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白DNT1具有良好的抗原性。应用重组蛋白DNT1为抗原建立了检测Bb血清抗体的间接ELISA诊断方法。试验确定重组蛋白DNT1抗原的包被浓度为6.25μg/mL,最适血清稀释度为1∶100。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了一种比较实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础。     

Bordetella pertussis respiratory infection in C57B1/6 and Balb/c mice: Pathophysiology and immune responses

A virulent clone of Bordetella pertussis, injected intranasally into C57B1/6 or Balb/c mice, induced a respiratory tract infection that mimicked the infectious process of whooping cough. The density of the inoculum influenced the kinetics of in vivo bacterial growth, as well as the associated leucocytosis, which were of equivalent intensity in both strains of mice. Convalescing mice became resistant to re-infection, but not to the effects of the leucocytosis-promoting factor of the pertussis toxin. Prominent immune response, associated with acquired resistance, was a delayed type hypersensitivity (DTH) reaction, the intensity of which depended upon the genotype of the mice when the eliciting antigen contained the pertussis toxin in a biologically active form. Intranasal infection of congenic mice may represent an improved quantitative test for reproducible measurement of virulence and immunogenicity of B. pertussis.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica

The gene coding for a thermostable alpha-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulfurogenes carrying the alpha-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.