鮰爱德华菌外膜微孔蛋白ompN1、ompN2和ompN3基因生物信息学分析及截段基因的原核表达与其抗原性检测     

潘延乐  汪开毓  王均  肖孟玮  李岚敏  陈德芳 《中国兽医学报》2015,35(6):900-908

根据GenBank中鮰爱德华菌(Edwardsiella ictaluri)外膜微孔蛋白N(outer membrane porin protein,OMP)基因序列(NC_012779.2)设计引物,以鮰爱德华菌Ms12基因组DNA为模板,经PCR扩增后克隆至pMD19-T载体,将阳性克隆菌送公司测序。测序结果通过生物信息学软件对ompN1、ompN2和ompN3序列的氨基酸序列相对分子质量、分子式、信号肽、跨膜区、亲/疏水性、B细胞表位进行预测,并构建系统发育树;根据去信号肽的B细胞表位相对集中区域设计特异性引物,经PCR扩增后,将截段序列克隆至pMD19-T载体,鉴定成功后克隆至原核表达载体pET32a(+)并转化至BL21(DE3)/Rosseta(DE3)进行IPTG诱导表达,最后用兔抗鮰爱德华菌血清对重组蛋白OmpN1、OmpN2和OmpN3进行免疫原性检测。结果显示扩增得到ompN1(1 143bp)、ompN2(1 056bp)和ompN3(1 053bp)的序列,获得的基因登录号分别为KJ831564、KJ831565和KJ831566。经生物信息学分析显示,OmpN1、OmpN2和OmpN3蛋白分子式分别为C1940H2993N543O622S7、C1817H2723N497O579S5和C1896H2843N519O563S7,相对分子质量大小分别为44 100、40 900和41 800,均为含有1个信号肽和跨膜区的疏水性蛋白,具有1个革兰阴性细菌OM_channels超家族结构域,是1种嵌入型的膜外蛋白;SDS-PAGE电泳检测发现,诱导表达的融合重组蛋白均以包涵体的形式出现在沉淀中,大小分别约为61 800、58 700和59 100;Western-blot分析表明,重组蛋白OmpN1、OmpN2和OmpN3均能与兔抗鮰爱德华菌全菌血清结合,表明鮰爱德华菌外膜微孔蛋白N具有亚单位疫苗开发的潜力。  相似文献   

鳗鲡爱德华氏病病原菌及一新种   总被引：16，自引：2，他引：16       下载免费PDF全文

王国良  徐兴林  路正 《水产学报》1993,17(3):224-229

本文报道在我国浙江省发现的鳗鲡爱德华氏病病原菌特征。从患病鳗鲡内脏分离到9株菌,用其中的E895205等4株菌进行人工感染,死亡率均为100％,与自然发病症状相似。这些菌株的特征一致。均为G~-短杆菌,单个,周鞭毛,兼性厌氧,发酵葡萄糖产酸产气。氧化酶阴性,接触酶阳性,产H_(?)S。不能利用柠檬酸盐和丙二酸盐作为唯一碳源,鸟氨酸和赖氨酶脱羧酶阳性。属爱德华氏菌属细菌。比较已报道的四个种,认为E895205等菌株为一新种,定名为浙江爱德华氏菌(Edwardsiella zhe jiangensis sp. nov.)。  相似文献   

Intra- and interspecific phenotypic characteristics of fish-pathogenic Edwardsiella ictaluri and E. tarda   总被引：1，自引：0，他引：1  

Victor S Panangala  Craig A Shoemaker  Shawn T McNulty  Covadonga R Arias  & Phillip H Klesius 《Aquaculture Research》2006,37(1):49-60

Intra‐ and interspecific characteristics of fish‐pathogenic Edwardsiella ictaluri, and E. tarda were determined by numerical analysis of gel electrophoresed protein profiles, fatty acid methyl esters (FAMEs) and immunoblotting. The 18 E. ictaluri isolates revealed a high degree of homogeneity (70% similarity or higher) in their protein profiles and 95% similarity in their FAME, while the nine E. tarda isolates revealed 30% similarity in their protein profiles and 95% similarity in their FAME. Immunoblots probed for antigenic epitopes with goat antiserum produced against E. ictaluri and E. tarda, respectively, revealed that E. ictaluri were more homogeneous compared with the E. tarda isolates. Overall, there was a considerable degree of relatedness between the two species. Our findings suggest that phenotypically E. ictaluri represents a clonal bacterial population structure compared with the less monomorphic E. tarda.  相似文献   

鮰爱德华菌单克隆抗体的制备及其在黄颡鱼“红头病”研究中的应用   总被引：2，自引：0，他引：2  

李强  黄华  张显昱  叶仕根  李华 《大连海洋大学学报》2014,(4):323-328

以黄颡鱼Pelteobagrus fulvidraco “红头病”病原---鮰爱德华菌 Edwardsiella ictaluri A86为抗原,利用杂交瘤技术制备了鮰爱德华菌黄颡鱼分离株的菌体单抗5D11、1B8、1G7、3G8、5A9、3H8,其效价分别为1：1280、1：1280、1：2560、1：320、1：160和1：80。结果表明：6株单抗与11株鮰爱德华菌黄颡鱼分离株均可以结合,与迟缓爱德华菌、气单胞菌、大肠杆菌、链球菌、弧菌等10株参考菌株均无交叉反应,单抗5D11和1B8可与鮰爱德华菌参考菌株结合。应用免疫荧光和免疫组织化学法检测了鮰爱德华菌对黄颡鱼的侵染规律。结果表明：黄颡鱼经人工注射鮰爱德华菌A86感染6 h后,在其肝、肾、脾、心、肠和胃中检测到了阳性细菌,其中脾脏中最多,心脏中最少;感染24 h后,脑中检测到阳性细菌;30 h后,鳃中显示有阳性信号;54 h后,在眼的外边缘检测到少量阳性细菌。研究结果可为进一步研究鮰爱德华菌在黄颡鱼体内的致病机理提供技术手段及基础,对揭示黄颡鱼“红头病”的发病机制具有重要意义。  相似文献   

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)     

Seong Bin Park  Kyoung Kwon  In Seok Cha  Ho Bin Jang  Seong Won Nho  Fernand F. Fagutao  Young Kyu Kim  Jong Earn Yu  Tae Sung Jung 《Journal of veterinary science (Suw?n-si, Korea)》2014,15(1):163-166

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.  相似文献   

A novel 'skinny pot-belly' disease in Asian seabass fry, Lates calcarifer (Bloch)     

Gibson-Kueh S  Crumlish M  Ferguson HW 《Journal of fish diseases》2004,27(12):731-735

  相似文献   

水产动物6种主要病原菌与抗血清的免疫交叉反应   总被引：14，自引：0，他引：14       下载免费PDF全文

战文斌  齐继光  刘洪明  周丽  邢婧 《中国水产科学》2004,11(1):14-19

采用ELISA、试管凝集、Western-blot等方法,分析了鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(V. harveyi)、溶藻胶弧菌(V. alginolyticus)、副溶血弧菌(V. paraheamolyticus)、迟钝爱德华氏菌(Edwardsiella tarda)和荧光假单胞菌(Psedomonas fluorescens)等水产养殖中主要病原细菌与抗血清之间的免疫交叉反应.结果表明,弧菌属细菌之间的交叉反应程度比较大,而与其他两属的细菌之间存在的交叉反应程度小,或不存在交叉反应;Western-blot分析结果显示,哈维氏弧菌、溶藻胶弧菌和副溶血弧菌抗血清分别与其他3种弧菌在分子量为135.6 kD和121.5 kD;95.6 kD,48.4 kD,39.2 kD和34.9 kD;55.1 kD的蛋白带处存在交叉反应,而这些分子量的蛋白带与其他两属的抗血清均不发生反应.  相似文献   

Blood parameters in Nile tilapia (Oreochromis niloticus L.) spontaneously infected with Edwardsiella tarda   总被引：6，自引：0，他引：6  

A Ca&#;lan Karasu Benli  & Hijran Yavuzcan Yildiz 《Aquaculture Research》2004,35(14):1388-1390

  相似文献   

A multi-epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia     

Vimbai Irene Machimbirike  Natapol Pornputtapong  Saengchan Senapin  Eakapol Wangkahart  Prapansak Srisapoome  Pongsak Khunrae  Triwit Rattanarojpong 《Journal of fish diseases》2022,45(1):1-18

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E. ictaluri immunogenic proteins previously reported. The 49.32 kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E. ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E. ictaluri infections.  相似文献   

地图鱼迟钝爱德华氏菌病病原菌的鉴定及毒力基因的检测   总被引：3，自引：0，他引：3  

赵飞  邹为民  谭爱萍  罗理  姜兰  陆小萏 《大连水产学院学报》2007,22(6):403-408

从患病地图鱼Astronotus ocellatus肝脏组织中分离到菌株AO1,经人工感染试验证实其为致病菌。通过ATB Expression半自动细菌鉴定仪和常规生理生化特性的分析,初步鉴定该菌株为迟钝爱德华氏菌Ed-wardsiella tarda。测定菌株AO1的16S rRNA基因序列并进行分析,结果显示,该菌株与迟钝爱德华氏菌的同源性最高,达99.9%。因此,可鉴定该菌株为迟钝爱德华氏菌。根据迟钝爱德华氏菌的毒力基因菌毛亚基(fimA)基因序列设计引物,进行PCR扩增,得到383 bp序列,该序列与迟钝爱德华氏菌的fimA基因序列相似性达99.0%,进一步说明菌株AO1具有fimA基因,有一定的致病力。药物敏感试验结果表明,菌株AO1对阿米卡星、氧氟沙星、庆大霉素、新生霉素、氨苄西林、氨曲南、卡那霉素、头孢咪啉、诺氟沙星、环丙沙星、呋喃妥因、妥布霉素、氨苄青霉素13种抗生素较为敏感。  相似文献