Over-expression, purification and immune responses to Aeromonas hydrophila AL09-73 flagellar proteins

Aeromonas hydrophila is ubiquitous in aquatic environments worldwide and causes many diseases in fish as well as human. Recent outbreaks of aeromonad diseases in channel catfish prompted us to investigate catfish immune responses during infection of A. hydrophila. In this communication, we report to amplify, over-express, purify and characterize 19 A. hydrophila flagellar proteins. All recombinant proteins were confirmed by nucleotide sequencing of expression plasmids, SDS-PAGE analysis and His tag Western blot of induced proteins. Our preliminary result also showed that the purified recombinant FlgK protein reacted strongly to sera from experimentally infected catfish, suggesting that this protein has potential for a novel target for vaccine development. It is also anticipated that these recombinant proteins will provide us with very useful tools to investigate host immune response to this microorganism.     

Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

嗜水气单胞菌BYKAH2008AC株外膜蛋白和溶血素双基因的融合表达及免疫原性分析

嗜水气单胞菌(Aeromonas hydrophila)的外膜蛋白基因(OmpTS)和溶血素基因(Hly)是其重要的保护性抗原。根据Genbank已发表的两个基因的序列设计引物,扩增其去除信号肽部分的基因片段,再将二者通过柔性片段融合,定向插入到质粒pET32a中构建重组融合表达载体,并转化到大肠杆菌BL21(DE3),经IPTG诱导获得与预期大小108 kD相吻合的融合蛋白条带。用纯化的重组蛋白免疫新西兰大白兔制备兔抗血清。ELISA和Western Blot检测该抗体效价为1∶4000以上,且该重组蛋白与抗OmpTS兔血清和抗Hly兔血清都呈阳性反应,表明该融合蛋白保留了外膜蛋白和溶血素的免疫原性,可作为基因工程亚单位疫苗的候选成分。     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.     

嗜水气单胞菌溶血素基因的克隆表达及其类毒素的免疫原性分析

根据嗜水气单胞菌溶血素保护性抗原基因序列(GenBank Accession No. AF539467)设计一对引物, 以嗜水气单胞菌河北分离株基因组为模板, 经PCR扩增得到hly基因。序列分析表明, 该基因产物大小为1485 bp, 经测序与GenBank报道的多个嗜水气单胞菌hly基因序列一致性高于99%。将得到的hly基因定向克隆到原核表达载体pET-28a中构建原核重组质粒pET-28a- hly, 转化大肠杆菌 BL21(DE3)中, 得到重组菌株BL21(DE3)(pET-28a-hly), 经IPTG诱导后, SDS-PAGE分析可见一条56 kD的特异条带。Western blotting分析结果显示表达的Hly蛋白能与抗体发生特异性结合,说明其具有较好的免疫原性。将表达的溶血素蛋白制成类毒素疫苗免疫小鼠后, 具有较高的保护效力, 表明该类毒素疫苗有望作为预防由嗜水气单胞菌引起疾病的基因工程类毒素疫苗。     

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.     

草鱼胶原凝集素基因的克隆及其功能分析

从草鱼Ctenopharyngodon idella肝肾cDNA文库中克隆得到胶原凝集素基因。草鱼胶原凝集素全长cDNA为1128bp,其中5′非编码区229bp,3′非翻译区104bp,最大开放阅读框为795bp,编码264个氨基酸。系统进化分析表明草鱼胶原凝集素与斑马鱼的亲缘关系最近。根据草鱼胶原凝集素序列特征,克隆了包含糖基识别域(CRD)的cDNA,并进行原核表达、纯化获得其重组蛋白PCRD。进行PCRD与6种细菌的凝集和糖抑制实验,结果表明半乳糖、葡萄糖、甘露糖和麦芽糖4种糖都会使PCRD与嗜水气单胞菌的凝集明显下降甚至极大地干扰凝集;麦芽糖使金黄色葡萄球菌的凝集明显下降,而肽聚糖和甘露糖会使凝集受到抑制;此外,PCRD的凝集反应不依赖Ca2+。     

Complete Genome Sequence of Aeromonas hydrophila Phage CC2

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.     

Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

幽门螺杆菌AlpA基因中四种黏附素基因保守区的克隆、表达、纯化及鉴定

幽门螺杆菌（Helicobacter pylori,Hp）感染是慢性活动性胃炎和消化性溃疡的主要病因,与胃腺癌、胃黏膜相关淋巴样组织（MALT）淋巴瘤的发生亦密切相关.鉴于Hp已证实的四种粘附素保守区（AB）是外膜蛋白（OMP）和膜孔素(porin)样成分,而外膜蛋白和膜孔素样成分是优秀的疫苗候选抗原.用PCR技术扩增AB基因,将其定向插入pET-22b（+）载体,在BL21（DE3）大肠杆菌中表达.测序显示AB基因长588 bp,编码195个氨基酸.SDS-聚丙烯酰胺凝胶电泳和凝胶扫描分析,AB基因表达的蛋白质分子质量约为22.5 ku,其重组蛋白质表达量占菌体总蛋白质的29%,表达产物经亲和层析纯化后蛋白质纯度达96%.经免疫印迹证实该重组蛋白可以被AlpA免疫兔血清所识别.AB蛋白的获得为进一步研究Hp黏附素保守区的分子黏附机制和免疫防治作用提供了基础.     

草鱼β-防御素1的趋化活性及免疫佐剂效应研究

为了探究草鱼防御素的免疫调节作用, 研究通过Clustal Omega多序列比对和I-TASSER二级结构预测, 发现草鱼β-防御素1(Ctenopharyngodon idella β-defensin 1, CiBD1)是一类具有两亲性的富含半胱氨酸的小分子阳离子肽, 其结构在硬骨鱼类中高度保守; 通过构建pET-32a-CiBD1原核表达载体, IPTG诱导表达后经过Ni2+亲和层析法纯化和肠激酶酶切获得CiBD1重组蛋白, 通过CFU平板法验证了CiBD1的抑菌活性, 当蛋白浓度达到200 μg/mL时, 对革兰氏阴性菌(G+)及革兰氏阳性菌(G–)都具有显著的抑制活性; 趋化实验表明CiBD1在50 ng/mL时对草鱼原代白细胞趋化活性最佳; 通过个体实验探究了CiBD1重组蛋白对嗜水气单胞菌灭活疫苗的免疫佐剂效应, 发现添加 CiBD1佐剂组死亡率显著低于单独疫苗组, 且该组IgM和MHC Ⅱ转录水平及血清中补体3(C3)和溶菌酶含量都显著高于对照组。研究表明CiBD1重组蛋白在细菌抗感染免疫中发挥着重要作用, 在水产养殖中具有一定的应用前景。     

Expression and purification of Arisaema heterophyllum agglutinin in Escherichia coli

Recombinant Arisaema heterophyllum agglutinin (AHA) was expressed in Escherichia coli as N-terminal His-tagged fusions. After induction with isopropylthio-beta-D-galactoside, the recombinant AHA was purified by metal-affinity chromatography. The purified AHA protein was incorporated into artificial diet at 0.1% (w/v) concentration in insect bioassay trial and the result showed that artificial diet containing AHA could significantly inhibit the growth of the third-instar nymphs of peach potato aphid (Myzus persicae). This study suggested that AHA could be an effective candidate for the control of peach potato aphid, one of the most serious sap-sucking insect pests causing significant yield loss of crops.     

Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein

To develop a vaccine, which can simultaneously prevent the diseases caused by various pathogenic bacteria in fish, we try to find a conserved outer membrane protein (OMP) antigen from different bacterial pathogens. In this study, an OMP fragment of 747 bp (named as Omp-G), which was highly conserved in seven Aeromonas OMP sequences from the NCBI database, was amplified by PCR from one Aeromonas sobria strain (B10) and two Aeromonas hydrophila strains (B27 and B33) with the designed specific primers. The sequence was cloned into pGEX-2T (6 × His-tag) vector, expressed in Escherichia coli system, and then the recombinant protein (named as rOmp-G) was purified with nickel chelating affinity chromatography. The purified rOmp-G showed a good immunogenicity in rabbits and well-conserved characteristics in these three pathogens by enzyme-linked immunosorbed assay. Furthermore, the rOmp-G also showed good immunogenicity in eels (Anguilla anguilla) for eliciting significantly increased specific antibodies (P < 0.01), and providing higher protection efficiencies (P < 0.05) after the pathogens challenge. The values of the relative percent survival in eels were 70% and 50% for two A. hydrophila strain challenge, and 75% for A. sobria strain challenge. This is the first report of a potential vaccination in eels that simultaneously provide protectiveness against different Aeromonas pathogens with a conserved partial OMP.     

嗜水气单胞菌J-1株弹性蛋白酶的表达、纯化及特性分析

摘要：【目的】表达、纯化嗜水气单胞菌J-1株弹性蛋白酶,并对弹性蛋白酶的性质进行分析。【方法】以pET-32a为表达载体将弹性蛋白酶基因ahyB转化至大肠杆菌BL21菌株中进行诱导表达,表达重组酶用His TaqNi2+亲和层析柱纯化并用6 mol/L盐酸胍进行复性;利用硫酸铵分级沉淀、阴离子交换层析和分子筛层析对嗜水气单胞菌培养上清液中的弹性蛋白酶进行纯化。将【结果】从嗜水气单胞菌培养上清液中获得的弹性蛋白酶原酶的最适pH 为8.5,而表达重组酶为 10.0;对热的稳定性,原酶高于表达酶。两种形式酶的性     

多鳞白甲鱼X型凝集素(OmITLN)基因克隆及在抗菌免疫中的功能研究

为探究秦巴山溪半洞穴鱼类——多鳞白甲鱼的先天性免疫因子X型凝集素的免疫功能,研究从多鳞白甲鱼(Onychostoma macrolepis)肝脏转录组数据库筛选出一种Intelectin基因片段,通过克隆鉴定获得Intelectin基因cDNA序列全长,命名为OmITLN;采用qRT-PCR分析OmITLN基因在健康多鳞白甲鱼8个组织和嗜水气单胞菌感染后两个免疫组织的表达情况;构建OmITLN基因的原核表达载体并在大肠杆菌BL21中诱导表达,获得Om ITLN重组蛋白,采用ELISA法以及荧光标记法检测Om ITLN对病原菌的凝集效果及糖结合情况。结果显示, OmITLN是一种X型凝集素基因,其cDNA全长960 bp,编码315个氨基酸。Om ITLN蛋白含有1个N端的纤维蛋白原相关结构域(Fibrinogen-Related Domain, FReD)和1个C端Intelectin特异性区域,不具有信号肽和跨膜区。系统进化树分析发现, Om ITLN与鲫、团头鲂、鳙、草鱼等鲤科鱼类的Intelectin聚为一支,亲缘关系最近。OmITLN基因在所检测组织均有表达,且在脾脏和肝脏中的...     

Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila.

Aeromonas hydrophila is a significantly important pathogen causing major diseases in humans and fresh water fish. The outer membrane proteins (OMP) which are strong immunogens have been reported to act as adhesins aiding in the attachment of enteropathogenic bacteria. It is of interest to investigate the role of OMP in pathogenesis and their potential as vaccine candidates. In our laboratory, we cloned the gene encoding channel protein LamB porin of A. hydrophila. DNA sequence analysis revealed a full length gene of 1345 bp having a high level of homology with the lamB gene of different bacteria. Open reading frame of A. hydrophila lamB consists of a signal peptide of 25 amino acids, two protein translation start sites ATG present at the 31st and 37th base pairs, a translation termination codon, TAA at 1333rd base pair.     

Electroporation-mediated transformation of Aeromonas hydrophila

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4x10(2) CFU/microg DNA at 12.5 kV/cm and 1000 Omega, resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the first report of transformation of bacteria A. hydrophila.