海水养殖真鲷弧菌病病原菌外毒素的理化特性

对最小弧菌Vm Pm毒素的分子量、分子结构及氨基酸组分进行了测定 ,同时还研究了温度、酸碱度及胰酶等因子对该毒素生物学活性的影响。研究结果表明 ,该毒素为单一的多肽 ,不具备典型的细菌毒素A、B亚基结构 ,分子量为 30 4 5 6kD ;毒素蛋白含天冬氨酸、苏氨酸、丝氨酸及半胱氨酸等 15种氨基酸 ,其中天冬氨酸的含量最多 ,半胱氨酸的含量最少 ,酸性氨基酸含量为 2 6 6 % ,碱性氨基酸含量为 14 4 % ,疏水性氨基酸含量为 33 4 % ;毒素活性的最适pH为 6— 8,对胰酶有抗性 ;不耐热 ,10 0℃下作用 1min ,其毒性完全消失 ,而在 5 6℃下 ,毒素完全失去活性需要 4 0min。     

绿脓杆菌外毒素A的结构、功能及其重组毒素

绿脓杆菌外毒素A有三个结构功能区.氨基端区(Ⅰ)通过关键位点Lys57结合靶细胞表面受体.中心区(Ⅱ)负责该毒素的跨膜转位功能,Arg276和Arg279是关键位点.在胞吞泡内此毒素于Arg279与Gly280间酶解成28 000和37 000两片段.羧基端区(Ⅲ)所在的37 000片段由其末端氨基酸序列REDLK介导到内质网再转位入胞浆通过Glu553位点结合NAD⁺使延伸因子-2受ADP-核糖基化而抑制细胞蛋白质合成导致细胞死亡.改造的毒素基因同识别蛋白基因融合成的重组毒素有应用前景.     

培养条件对养殖真鲷弧菌病病原菌毒素产生的影响

研究了温度、酸碱度、气体环境、营养成分及培养时间等培养条件对真鲷病原菌最小弧菌Vm-Pm毒素产生的影响。结果表明,温度对Vm-Pm毒素的产生有显著影响,在30℃培养时产生最高;当pH在弱碱性环境（7.2左右）时,Vm-Pm毒素产量最高;振荡通气能极大地促进Vm-Pm毒素的产生,显著提高其产量;Vm-Pm毒素的产量随着培养时间的延长而逐步增加,到36h即可达到最高水平,并维持至72h;改良海水营养培养基与普通海水营养培养基相比,更有利于毒素的产生,毒素产量可提高1倍。     

绿脓杆菌外毒素A的最新研究进展

在免疫缺失、囊肿性纤维化、烧伤烫伤等患者的临床继发感染中,绿脓杆菌是引起感染的主要病原菌之一,其合成的细胞外毒性物质—绿脓杆菌外毒素A(PEA)被认为是引起感染的最关键内在机制。该文对中外学者们在PEA的结构、功能、提取和纯化方法、重组毒素、基因工程疫苗以及应用等方面的研究进行归纳总结,并对研究和应用中存在的问题加以分析,对预防和治疗绿脓菌感染、抗肿瘤、抗移植排斥和治疗自身免疫性疾病等具有重要而深远的意义。     

中草药复方对拟态弧菌的体内外抗菌和拮抗外毒素的作用

目的探明拟态弧菌外毒素的致病性,筛选出对拟态弧菌具有良好抗菌和拮抗外毒素作用的中草药复方。方法培养拟态弧菌安徽分离株,采用蛋白分离技术提取其外毒素,测定其对实验草鱼的致病性。以黄连、大黄、金银花、夏枯草、丹皮、地锦草、连翘、茯苓、车前子、黄芪和甘草组成3个复方,采用改良微量稀释法测定不同中草药复方对拟态弧菌的体外抑菌作用。同时使用动物模型测定不同中草药复方拮抗外毒素作用和对拟态弧菌感染草鱼的保护作用。结果4株拟态弧菌产生的外毒素对实验鱼均具有较强的致死性。复方3的体外抑菌作用最强,对4株拟态弧菌的MIC值均为1.96g/L;其次为复方1,其MIC值介于3.92～7.84g/L;复方2的抑菌作用最差,其MIC值介于62.72～125g/L。复方3拮抗外毒素作用最强,其次为复方2,复方1不具有抗毒素作用。复方3对拟态弧菌感染草鱼具有最好的保护作用,保护率为100%;其次为复方2,保护率为23.81%;复方1的保护率最低,仅为19.05%。结论拟态弧菌外毒素在腹水病发生过程中起着重要致病作用。复方3具有良好的抗菌和拮抗外毒素作用,为筛选出的最佳复方,可进一步研制成为治疗腹水病的中草药复方制剂。     

幽门螺杆菌重组vacA-CtxB蛋白的原核表达及免疫学研究

目的原核表达幽门螺杆菌pQE-vctB(含H.pylori细胞空泡毒素毒性片段与霍乱毒素B亚单位的融合基因)在E.coil DH5α中诱导表达,以获得重组蛋白,鉴定其抗原性和免疫原性,为制备防治H.pylori感染的口服疫苗奠定基础。方法 (1)pQE-vctB转化E.coli DH5α,IPTG诱导表达重组蛋白VCTB,Western blot分析抗原性,镍离子柱纯化。(2)重组蛋白VCTB口服免疫小鼠,ELISA检测小鼠血清特异性IgG、小肠冲洗液IgA,以鉴定其免疫原性。结果重组蛋白VCTB经SDS-PAGE分析相对分子量(Mr)约为40kD,亲和层析后可获得纯度为92%以上的蛋白。Western blot分析显示能与VacA抗血清反应。ELISA检测显示,免疫小鼠的血清特异性抗体IgG,肠粘液IgA显著高于VacA对照组(P0.01)。结论 vacA和ctxB融合基因原核表达质粒转化E.coli DH5α表达菌获得了重组蛋白VCTB,表达量较高,纯度较高,有良好的抗原性和免疫原性,口服免疫小鼠可明显提高其免疫效果,产生较高水平的IgA,可用于制备防治H.pylori感染的口服疫苗。     

霍乱毒素B亚基融合蛋白表达系统的构建

霍乱毒素Ｂ亚基（ＣＴＢ）是良好的免疫佐剂和载体蛋白。本研究通过定点突变,在ＣＴＢ基因（ｃｔｘＢ）３′端终止密码前引入了限制性内切酶ＥｃｏＲＩ,构建了质粒ｐＭＣ０５。ｐＭＣ０５中ＣＴＢ与下游ｌａｃＺ′基因阅读框架相同,转化大肠杆菌后能够表达ＣＴＢ与β－半乳糖苷酶α肽的融合蛋白;所表达的融合蛋白能与ＧＭ１结合,说明融合蛋白保持ＣＴＢ的基本高级结构和生物学活性;融合蛋白能与抗－ＣＴＢ抗体结合,说明融合蛋白具有ＣＴＢ的抗原性。以上结果表明：通过将外源抗原决定簇基因融合至ｃｔｘＢ的３′端,在大肠杆菌中表达融合蛋白,构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响,构建了高效表达融合蛋白的载体－宿主系统。     

口服霍乱活疫苗的重组体

霍乱弧菌(Vibrio cholerae)的病原株由于切除其编码霍乱肠毒的A_1亚基的DNA顺序而被减毒。     

纯化重组霍乱毒素B亚单位与天然霍乱毒素B亚单位性质的对比研究

霍乱毒素Ｂ亚单位（ＢＳ）已用于新型口服霍乱疫苗、佐剂及蛋白质载体,但成本高,来源困难．用重组霍乱毒素Ｂ亚单位（ｒＢＳ）代替ＢＳ可克服上述缺点．ｒＢＳ用于上述目的前必须证实其在物理、化学及免疫学性质方面与天然同类产品的一致性．用亲和层析法从各批次大罐发酵所获工程菌Ｅ．ｃｏｌｉＭＭ２（ｐＭＭ－ＣＴＢ）培养物上清中制备得到了小批量ｒＢＳ纯品,在同等条件下与ＢＳ（Ｓｉｇ－ｍａ公司产品）进行理化、免疫学性质的对比研究,证实二者在ＳＤＳ－ＰＡＧＥ中电泳带位置一致、分子量相同,纯度达９９％;在反相ＨＰＬＣ中出峰行为一致,纯度达１００％;在半干式聚焦电泳分析中电泳带分布相同,等电点为７．９１．ｒＢＳＮ端起的２０个氨基酸序列为ＴＰＱＮＩＴＤＬＣＡＥＹＨＮＴＱＩＨＴＬ,与克隆基因来源株的毒素Ｂ亚单位同一段序列完全一致．氨基酸组成分析证实ｒＢＳ与ＢＳ相近．在免疫学性质分析中,ｒＢＳ与ＢＳ在免疫双扩散试验中与抗ＣＴ均出一条沉淀线且相互吻合;在免疫电泳试验中二者与抗ＣＴ在相应位置上产生一条沉淀弧;二者均能与神经节苷脂ＧＭ１结合且这种结合均可通过二者与抗ＣＴ的预保温处理而被阻断．对比研究结果揭示ｒＢＳ与ＢＳ性质完全一致,可代替ＢＳ用于     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.     

Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains

Abstract An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT). The culture filtrates prepared in the M4 medium caused significantly ( P < 0.05) more fluid accumulation than that in syncase medium. Crude toxin, prepared in the M4 medium with V. cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 μg) prepared in the syncase medium. The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media. These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization. All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.     

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3′-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Secretory and GM1 receptor binding role of N-terminal region of LTB in Vibrio cholerae

Heat labile enterotoxin from enterotoxigenic Escherichia coli is similar to cholera toxin (CT) and is a leading cause of diarrhea in developing countries. It consists of an enzymatically active A subunit (LTA) and a carrier pentameric B subunit (LTB). In the current study, we evaluated the importance of the N-terminal region of LTB by mutation analysis. Deletion of the glutamine (ΔQ3) residue and a substitution mutation E7G in the α1 helix region led to defects in LTB protein secretion. Deletion of the proline residue (ΔP2) caused a decrease in α helicity. The ΔP2 mutant affected GM₁ ganglioside receptor binding activity without affecting LTB pentamer formation. Upon refolding/reassembly, the ΔP2 mutant showed defective biological activity. The single substitution mutation (E7D) strengthened the helix, imparting structural stability and thereby improved the GM₁ ganglioside receptor binding activity. Our results demonstrate the important role of N-terminal α1 helix in maintaining the structural stability and the integrity of GM₁ ganglioside receptor binding activity.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Distribution of the zot (zonula occludens toxin) gene among strains of Vibrio cholerae 01 and non-01

Abstract The distribution of the zot gene that encodes the zonula occludens toxin, a newly described toxin of Vibrio cholerae , among clinical, environmental and food isolates of V. cholerae 01 and non-01 was investigated. Both the zot gene and the ctx gene that encode cholera toxin were found in 247 of 257 clinical strains and 62 of 415 environmental or food isolates of V. cholerae 01. The zot gene, but not the ctx gene was found in 37 strains (one clinical strain and 36 environmental or food isolates). In addition, two of 31 clinical strains and six of 98 environmental or food isolates of V. cholerae non-01 possessed both the zot gene and the ctx gene. These results demonstrated the predominantly concurrent occurrence of the zot gene and ctx genes among strains of V. cholerae 01 which suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea produced by V. cholerae 01.     

Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.     

Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.