黄颡鱼溃疡综合征病原菌及主要毒力因子研究
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摘要
黄颡鱼(Pelteobagrus fulvidraco, Yellow catfish)属鲶形目,鲿科,黄颡鱼属。广泛分布于我国的长江、珠江、黑龙江流域及太平洋水系,是我国重要的淡水经济鱼类,也是我国出口创汇的优良鱼种,经济价值超过四大家鱼。在我国,黄颡鱼已人工驯化饲养,年产量已达50万吨左右,产值超过10亿元/年。由于养殖模式的变化和规模不断扩大,新的传染疾病出现了暴发与流行。近年来,重庆市一些集约化鱼场养殖的黄颡鱼出现鳍红肿、出血,肌肉溃疡及败血症的疾病,发病率超过30%,死亡率高达15%,经济损失巨大,严重危及养鱼业的生存与发展。但是,该传染病的病原尚不清楚,故不能有效地进行预防和治疗。确定其病原及致病机理,可为有效预防和治疗黄颡鱼的溃疡综合征提供临床依据,对于黄颡鱼养殖有重要的理论和生产意义。
目前,有关黄颡鱼溃疡综合征的病原尚不清楚,其致病机理也待研究。由于鱼类溃疡综合征的病原细菌有10余种,必须明确黄颡鱼溃疡综合征病原,弄清其发病机理,才能有效控制黄颡鱼溃疡综合征。本研究从患病黄颡鱼分离获得病原,借助细菌生化及分子鉴定方法确定病原种类,对溶血素、气溶素、胞外丝氨酸蛋白、脂肪酶重要毒力因子的基因进行了克隆,并通过大肠杆菌异源表达获得了重组溶血素,为利用重组溶血素蛋白作为抗原,建立检测温和气单胞菌技术及亚单位疫苗研制提供了基础条件。主要研究结果如下：
1.病原分离鉴定与致病性
从黄颡鱼病料的心血获得两株细菌,命名为RC-07-KA株和RC-07-XB株。两株细菌能够在LB、麦康凯、鲜血培养基上生长。生化试验鉴定两株细菌的氧化酶试验、精氨酸试验阳性、O/F发酵,KCN生长试验、七叶苷、水杨苷等试验为阴性与温和气单胞菌(A.sobria)生化特性一致；通过PCR扩增的16S rRNA基因序列与GenBank上公布的25株细菌16S rRNA的基因序列进行同源性分析,结果显示,本次分离菌株与6株不同来源的温和气单胞菌(A.sobria) EU916710、 DQ822702、DQ822759(Lithuania,2008), AY987762(India,2006), DQ133178(Korea,2007), EU069415(China,2007)之间的亲缘关系很近,同源性达到96.4-99.8%。结合两种鉴定方法结果,证明分离的RC-07-KA株和RC-07-XB株为温和气单胞菌(A.sobria)。
RC-07-KA株回归动物试验结果表明,RC-07-KA株对试验的黄颡鱼、鲤鱼、鲫鱼肌肉接种6.15×107CFU/尾,七天内能够导致死亡。鲫鱼病理组织观察表明,肾脏肾小球肿大,肾小球毛细血管内皮细胞肿胀,肾小管上皮细胞肿胀,有的管腔内可见丝状纤维蛋白渗出,肾小管间质中可见炎性白细胞浸润；心脏部分心肌纤维肿胀,横纹不明显；肝脏肝细胞变性坏死,排列紊乱,中央静脉有纤维蛋白渗出,嗜中性白细胞浸润；肠道粘膜部分上皮脱落,肠内有大量中性白细胞和少量的巨噬细胞浸润等病变。表明RC-07-KA株对鲫鱼多器官有明显病理损伤。
RC-07-KA株经LB液体培养基培养,过滤的无菌滤液,在10%鲜血LB琼脂平板上进行溶血活性初步测定,结果显示：无菌滤液的溶血效价达到1×25,表明RC-07-KA株具有较强产生p溶血素特性；无菌滤液经硫酸铵二步盐析法处理得到胞外粗提物。溶血试验结果显示：胞外粗提物有明显的溶血性,表明具有溶血活性的β溶血素可以通过硫酸铵盐析方法制备。
2.RC-07-KA株主要毒力基因的克隆及序列分析
温和气单胞菌的致病力与多种毒力因子相关,包括气溶素、溶血素、蛋白酶、脂肪酶等,为了探讨RC-07-KA株的毒力因子,本试验采用PCR方法,扩增、克隆了该菌株的溶血素、气溶素、脂肪酶和胞外丝氨酸蛋白酶基因,并对其基因序列进行了分析。
2.1气溶素基因
通过PCR扩增得到RC-07-KA株气溶素基因0.7kb片段,克隆到pGEM-T载体,测序结果显示,所得到的基因片段大小为692bp,序列能编码230个氨基酸残基的多肽。Blast分析结果显示：得到的序列与气单胞菌属内菌株的气溶素基因相关,属于气单胞菌属的毒力因子气溶素超家族(aerolysin superfamily),序列含有成孔毒素的保守结构域序列；遗传进化分析结果显示：RC-07-KA株气溶素基因序列与中国、日本和印度等报道菌株A. hydrophila, AEF (HM853019, China,2010), A. sobria, isolate S2-As (AF443392, China,2001), A.sobria isolate S34-As (AF443394, China,2001), A.veronii, S43-Av (AF443395, China,2001), A.veronii bv.sobria (AB109093, Japan,2004), A.veronii, MTCC32497(EF034117, India,2007)的气溶素基因序列亲缘关系较近,为同一分支簇。分析结果同时显示：中国来源的气单胞菌分离株气溶素基因分别属于几个主要的簇。
2.2β溶血素基因
通过序列比对分析结果设计引物,采用PCR方法扩增得到RC-07-KA株p溶血素基因片段,克隆测序结果表明：得到的p溶血素基因片段大小为1467bp,包含一个ORF,编码487个氨基酸残基,编码蛋白分子量约为54KDa。遗传进化分析结果表明,该基因与气单胞菌属中的A. hydrophila菌株Sb(AY611033)、NLEPA-1607(AF410466)、AEF (HM853019), A.sobria菌株357(AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2(X65048)的β溶血素基因亲缘关系较近,同源性大于95%,而与其他菌株的同源性较低。进化树构建结果显示：气单胞菌属的β溶血素基因可分为几个明显分支的基因簇,RC-07-KA株溶血素基因与AY611033(A.hydrophila strain Sb)、EF620533(host=Homo sapiens")、 AY157998(A.sobria strain357)、X65048(A.salmonicida strain:17-2)、AF410466(A.hydrophila strain:NLEP A-1607). HM853019(A.hydrophila strain AEF)为同一簇。
对比分析气单胞菌属内与RC-07-KA株亲缘关系较近及较远的代表菌株p溶血素氨基酸序列的亲水性、抗原性指数及表面可及性,结果显示：气单胞菌属的溶血素具有很强的抗原性,与RC-07-KA株亲缘关系较近菌株β溶血素的抗原性相差较小,揭示p溶血素具有作为候选的蛋白疫苗的潜能。
2.3胞外丝氨酸蛋白酶基因
采用PCR方法扩增得到RC-07-KA株胞外丝氨酸蛋白酶基因得到1.9kb基因片段,克隆测序结果显示：得到的RC-07-KA株胞外丝氨酸蛋白酶基因大小为1875bp,包含一个ORF,编码624个氨基酸残基的多肽。构建系统进化树分析结果显示：RC-07-KA株胞外丝氨酸蛋白酶基因与AF253471(A.sobria strain288)、 AF126213(A.hydrophila, South Korea,1999)和CP002607(A.veronii B565)属于同一遗传衍化分支Ⅱ。序列相似性分析结果显示：RC-07-KA株胞外丝氨酸蛋白酶基因与AF253471、AF126213和CP002607的序列相似性高,分别为93.6%、93.3%和92.5%。这些结果表明我们成功克隆得到RC-07-KA株胞外丝氨酸蛋白酶基因,为进一步深入研究胞外丝氨酸蛋白酶的功能奠定了良好的基础。
2.4脂肪酶基因
采用PCR方法扩增得到RC-07-KA株脂肪酶基因片段,克隆测序显示：得到的脂肪酶基因片段为2445bp,包含一个ORF,编码814个氨基酸残基的多肽,蛋白分子量约为83KDa。构建系统进化树分析结果显示：RC-07-KA株脂肪酶基因与A.sobria strain AS228(AB206038, Japan,2005)、A.sobria strain AS008(AB206037, Japan,2005)、A.sobria strain288(JN019936, Japan,2011)属于同一遗传衍化分支Ⅱ。同源性分析结果显示：RC-07-KA株脂肪酶基因与A.sobria AS228、AS008(AB206038、AB206037, Japan,2005)的序列相似性高,分别为93.6%、93.3%。
3.重组溶血素表达载体构建
将RC-07-KA株溶血素基因片段连接到pET28a(+)载体的EcoR Ⅰ和Xho Ⅰ位点,转化大肠杆菌提取质粒,用EcoR Ⅰ、Xho Ⅰ、Sal Ⅰ进行单、双酶切鉴定,结果表明：正确构建得到重组溶血素表达质粒pET28a-Hly。将pET28a-Hly转化大肠杆菌Rosetta,用IPTG诱导溶血素基因的表达,SDS-PAGE结果显示：重组菌株表达目的蛋白条带位于66.4KDa与44.3KDa之间,结果与预期相符,且重组蛋白多为包涵体,少量为可溶性蛋白。通过His单克隆抗体进行Western-blot鉴定,结果显示在66.4KDa与44.3KDa之间出现印迹条带,表明目的蛋白在大肠杆菌中获得了表达,这为进一步研究重组溶血素的活性奠定了基础。
4.重组溶血素优化表达、纯化及活性检测
为了得到较多的可溶性重组溶血素,对IPTG、诱导温度及诱导表达时间进行了优化,结果显示：IPTG浓度为0.4mmol/L、诱导温度为32℃、诱导表达3-4h可溶目的蛋白的相对含量较高。在优化的条件下表达可溶性溶血素,用Ni2+-Sepharose6Fast Flow凝胶进行目的蛋白的纯化,SDS-PAGE检测结果显示纯化产物在58KDa左右得到单一条带,表明得到纯化的重组溶血素。采用鲫鱼红细胞为靶细胞进行重组溶血素的活性测定,结果显示：重组溶血素在20℃和37℃均具有溶血活性,1.0μg的重组溶血素、反应1h能明显观察到明显的溶血现象,反应3h能使试验红细胞完全溶解,表明重组溶血素具有活性,这为进一步深入研究奠定了基础。
Pelteobagrus fulvidraco, which blongs Siluriformes, Bagridae, Pelteobagrus is in the taxonomy, and widely distributed in China's Yangtze River, Pearl River, Heilongjiang River and the Pacific River system, is one of the important economic freshwater fish of our country, and one of the admirable fingerling export, of which economic value beyond of four domestic fishes. In China, catfish has successfully achieved the domestication of artificial breeding, and the production of artificial breeding catfish reached50million tons, the output value over1billion yuan per year. Because of changes of the breed model and the breeding scale expand ceaselessly. There have been new infectious disease outbreak and epidemic. In recent years, a large number of P. fulvidraco occurred an infectious disease in many Chongqing's intensive fish farms, of which prevalence rate and mortality rate exceed30%and15%, respectively. The disease characterized swelling of fins, bleeding, muscle ulcers and septicemia. The prevalence of disease have caused huge economic losses, and seriously jeopardized the survival and development of fish farming. However, the infectious etiology is unknown; there is no effective methods for prevention and treatment it. Therefore, determination of the etiology and pathogenic mechanism can provide clinical basis for effective prevention and treatment ulcerative disease syndrome of P. fulvidraco. This has important theoretical and practical significance for P. fulvidraco farming.
The etiology, which cause ulcerative disease syndrome of P. fulvidraco, at present, is not clear. Its pathogenic mechanism could be studied. Because of more than10species of pathogenic bacteria can cause fish ulcerative disease syndrome, it is necessary to clear the pathogen of P. fulvidraco ulcerative disease syndrome and understand its pathogenesis for effectively control ulcerative disease syndrome. This study focuses on the isolation and identification of pathogenic bacteria from the prevalence of P. fulvidraco,by using Bacterial biochemistry and molecular identification methods. The virulence associated gene aerolysin, hemolysin, extracellular serine proteases and lipases were cloned and sequenced. Furthermore, the recombinant hemolysin is obtained by heterologous expression in Escherichia coli. This provides a basis for establishment of A. sobria detection technology and subunit vaccine research by use of recombinant hemolysin as antigen. The main results of this research are as follows:
1. Isolation, identification of the pathogen and its pathogenicity
Two isolates of A. sobria, named as strain RC-07-KA and strain RC-07-XB, were isolated from heart blood of P. fulvidraco. Both of them can be cultured on LB medium, MacConkey's agar and fresh blood agar. The biochemical tests results showed that the oidase test and arginine test demonstrate positive, the O/F fermentation, KCN growth test, aescin glycosides test and salicin test demonstrate negative. These results addressed that the biochemical properties consistent with that of A. sobria. The16S rRNA gene fragment was amplified by polymerase chain reaction and the sequence was aligned with the25sequences of bacteria published on GenBank. The results revealed that the isolates of ours showed close phylogenetic relationship with the other6strains of A. sobria, which shared96.4-99.8%homology with each other.
Animal regression experiment results show that A. sobria strain RC-07-KA can kill yellow catfish, carp and crucian by muscle inoculation with6.15×107CFU/fish in7days. Histopathological observation showed that the challenged fish characterized as renal glomerular enlargement, glomerular capillary endothelial cell and renal tubular epithelial cell swelling, the visible filamentous fibrin exudation within some of the lumen, visible inflammatory leukocyte infiltration in renal tubule interstitial; the portion of the cardiac muscle fiber swelling, ambiguous grosgrain; hepatic cellular degeneration and necrosis, derangement, fibrin exudation at central vein, neutrophil infiltration; part of intestinal mucosa epithelial shedding, a large number of neutrophils and a small amount of macrophages infiltrating in enteral. The results demonstrated that A. sobria strain RC-07-KA can cause obvious pathological injury of multiple organ of crucian.
A. sobria strain RC-07-KA was inoculated and cultured in LB medium. The cultures were filtered, and the filtration was employed to detect the hemolytic activity on the LB agar which contained10%fresh blood. The results showed that the cell-free filtration can lysis red blood cells, and the hemolytic titer reached1×25. The results Indicates that the A. sobria strain RC-07-KA posses strong βhemolysin production characteristics. Extracellular crude extracts were obtained by two step salting-out method. The hemolytic test results demonstrated that extracellular crude extracts address hemolytic activity. This suggest that hemolytic activephemolysin can be obtained by ammonium sulfate precipitation method, despite its low yields.
2. Cloning and sequence analysis of main virulence gene of strain RC-07-KA
There are many virulence factors associated with Aeromonas pathogenicity, including aerolysin, hemolysin, extracellular serine proteases and lipases, ect. In order to study the virulence factor of A. sobria strain RC-07-KA, the hemolysin, aerolysin, extracellular serine protease and lipase gene were amplified by polymerase chain reaction. The four gene fragments were cloned and sequenced. The obtained gene sequences were analyzed.
2.1Aerolysin gene
0.7kb fragment of aerolysin gene of A. sobria strain RC-07-KA was amplified by use of PCR method and cloned into pGEM-T vector. The sequencing result showed that the gene fragment is size of692bp, which can encode a peptide of230amino acid residues. The results of Blast analysis revealed that the obtained sequence shared similarity with the aerolysin gene of Aeromonas, which belongs to the virulence factors aerolysin superfamily of Aeromonas, and containing conserved domain of pore-forming toxins.
Phylogenetic analysis results showed that A. sobria strain RC-07-KA close to aerolysin gene from China, Japan and India strains(Accession:AB109093, EF034117, HM853019, AF443392, AF443394, AF443395), which located in the same cluster. In the meanwhile, the analysis result display that aerolysin gene of Chinese isolates belong to several main clusters.
2.2β hemolysin gene
A pair of primers were designed and synthesized according to the published hemolysin nucleotide sequence from A. sobira in GenBank. The gene fragment of (3hemolysin was amplified by PCR through the designed primers. Sequencing results of the cloned gene fragment revealed that the obtained β hemolysin gene is in size of1467bp, which can encode a peptide of487amino acid residues, an about54kDa protein. Phylogenetic analysis results showed that β hemolysin gene of A. sobria RC-07-KA close to A. hydrophila strain sb (AY611033), NLEP A-1607(AF410466),AEF (HM853019),A. sobria strain357(AY157998), A. sobria Homo sapiens isolate(EF620533) and A. salmonicida strain17-2(X65048), with shared over95%homology, and lower homology with the other strains. Phylogenetic tree analysis results demonstrated that the β hemolysin gene of Aeromonas can be divided into several distinct gene clusters, the β hemolysin gene of A. sobria strain RC-07-KA located in cluster Ⅱ, with the reference strains of AY611033, EF620533, AY157998, X6504, AF410466and HM853019.
Comparative analysis the hydrophilic, antigenic index and surface accessibility of hemolysin revealed that the hemolysin have strong antigenicity, with a lower antigenic differences between A. sobria strain RC-07-KA and closed strains. This suggests that beta hemolysin may be used as a potential candidate vaccine.
2.3Extracellular serine protease gene
A1.9Kb gene fragment of extracellular serine protease of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results addressed that the extracellular serine protease gene is1875bp, which contains an open reading frame, encoding a peptide of624amino acid residues. Phylogenetic tree analysis results showed that the extracellular serine protease gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain288(AF253471), A. hydrophila, South Korea isolate (AF126213) and A.veronii B565(CP002607).The sequence similarity is93.6%、93.3%and92.5%, respectively. These results suggest that the extracellular serine protease gene of strain RC-07-KA was successfully cloned, an important basics for further studies.
2.4Lipase gene
A lipase gene fragment of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results showed that lipase gene is2445bp of in size, which contains an open reading frame, encoding a peptide of814amino acid residues, an about83kDa protein. Phylogenetic tree analysis results demonstrated that the lipase gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain AS228(AB206038), AS008(AB206037) and288(JN019936). The sequence similarity is93.6%、93.3%with AB206038and AB206037, respectively.
3. construction of recombinant hemolysin expressing vector
The β hemolysin gene was amplified, purified and cloned into the EcoRI and XhoI restriction enzyme digestion site to construction recombinant hemolysin expressing vector pET28a-Hly. The gained recombinant plasmid pET28a-Hly was identified by restriction enzyme digestion. The results demonstrated that pET28a-Hly be successfully constructed. The pET28a-Hly was transformed into E.coli. strain Rosetta for recombinant protein expression. SDS-PAGE analysis results demonstrated that the band of target protein appear between66.4KDa and44.3KDa, in line with expectations. The recombinant protein was confirmed by Western-blot test. The results suggest the target protein have successfully expressed in E.coli, which is an important basics for further studies.
4.Optimal expression, purification and activity analysis of recombinant β hemolysin
In order to get more soluble recombinant hemolysin, the condition of inducible expression were optimized based on IPTG concentration, temperature and the time of induce. The results demonstrated that soluble target protein reached at a relatively high level when IPTG concentration is0.4mmol/L,32℃, induce period3-4hours.
Amount of soluble recombinant β hemolysin were expressed at the optimized conditions, and the recombinant protein were purified by Ni2+-Sepharose6Fast Flow gel affinity chromatography. SDS-PAGE analysis results revealed a single brand between66.4KD and44.3KD, that suggest the recombinant β hemolysin have been purified. The activity of purified recombinant β hemolysin was detected by hemolysis test using crucian carp red blood cells as indicator. The results demonstrated that recombinant hemolysin have hemolysis activity at both20℃and37℃. The obvious can be detected when1.0μg recombinant hemolysin reacts with the indicator cells, and the test red blood cells can be completely dissolved in3hours. The results suggest that the recombinant hemolysin posses hemolysis activity, which is essential for further studies.
目前,有关黄颡鱼溃疡综合征的病原尚不清楚,其致病机理也待研究。由于鱼类溃疡综合征的病原细菌有10余种,必须明确黄颡鱼溃疡综合征病原,弄清其发病机理,才能有效控制黄颡鱼溃疡综合征。本研究从患病黄颡鱼分离获得病原,借助细菌生化及分子鉴定方法确定病原种类,对溶血素、气溶素、胞外丝氨酸蛋白、脂肪酶重要毒力因子的基因进行了克隆,并通过大肠杆菌异源表达获得了重组溶血素,为利用重组溶血素蛋白作为抗原,建立检测温和气单胞菌技术及亚单位疫苗研制提供了基础条件。主要研究结果如下：
1.病原分离鉴定与致病性
从黄颡鱼病料的心血获得两株细菌,命名为RC-07-KA株和RC-07-XB株。两株细菌能够在LB、麦康凯、鲜血培养基上生长。生化试验鉴定两株细菌的氧化酶试验、精氨酸试验阳性、O/F发酵,KCN生长试验、七叶苷、水杨苷等试验为阴性与温和气单胞菌(A.sobria)生化特性一致；通过PCR扩增的16S rRNA基因序列与GenBank上公布的25株细菌16S rRNA的基因序列进行同源性分析,结果显示,本次分离菌株与6株不同来源的温和气单胞菌(A.sobria) EU916710、 DQ822702、DQ822759(Lithuania,2008), AY987762(India,2006), DQ133178(Korea,2007), EU069415(China,2007)之间的亲缘关系很近,同源性达到96.4-99.8%。结合两种鉴定方法结果,证明分离的RC-07-KA株和RC-07-XB株为温和气单胞菌(A.sobria)。
RC-07-KA株回归动物试验结果表明,RC-07-KA株对试验的黄颡鱼、鲤鱼、鲫鱼肌肉接种6.15×107CFU/尾,七天内能够导致死亡。鲫鱼病理组织观察表明,肾脏肾小球肿大,肾小球毛细血管内皮细胞肿胀,肾小管上皮细胞肿胀,有的管腔内可见丝状纤维蛋白渗出,肾小管间质中可见炎性白细胞浸润；心脏部分心肌纤维肿胀,横纹不明显；肝脏肝细胞变性坏死,排列紊乱,中央静脉有纤维蛋白渗出,嗜中性白细胞浸润；肠道粘膜部分上皮脱落,肠内有大量中性白细胞和少量的巨噬细胞浸润等病变。表明RC-07-KA株对鲫鱼多器官有明显病理损伤。
RC-07-KA株经LB液体培养基培养,过滤的无菌滤液,在10%鲜血LB琼脂平板上进行溶血活性初步测定,结果显示：无菌滤液的溶血效价达到1×25,表明RC-07-KA株具有较强产生p溶血素特性；无菌滤液经硫酸铵二步盐析法处理得到胞外粗提物。溶血试验结果显示：胞外粗提物有明显的溶血性,表明具有溶血活性的β溶血素可以通过硫酸铵盐析方法制备。
2.RC-07-KA株主要毒力基因的克隆及序列分析
温和气单胞菌的致病力与多种毒力因子相关,包括气溶素、溶血素、蛋白酶、脂肪酶等,为了探讨RC-07-KA株的毒力因子,本试验采用PCR方法,扩增、克隆了该菌株的溶血素、气溶素、脂肪酶和胞外丝氨酸蛋白酶基因,并对其基因序列进行了分析。
2.1气溶素基因
通过PCR扩增得到RC-07-KA株气溶素基因0.7kb片段,克隆到pGEM-T载体,测序结果显示,所得到的基因片段大小为692bp,序列能编码230个氨基酸残基的多肽。Blast分析结果显示：得到的序列与气单胞菌属内菌株的气溶素基因相关,属于气单胞菌属的毒力因子气溶素超家族(aerolysin superfamily),序列含有成孔毒素的保守结构域序列；遗传进化分析结果显示：RC-07-KA株气溶素基因序列与中国、日本和印度等报道菌株A. hydrophila, AEF (HM853019, China,2010), A. sobria, isolate S2-As (AF443392, China,2001), A.sobria isolate S34-As (AF443394, China,2001), A.veronii, S43-Av (AF443395, China,2001), A.veronii bv.sobria (AB109093, Japan,2004), A.veronii, MTCC32497(EF034117, India,2007)的气溶素基因序列亲缘关系较近,为同一分支簇。分析结果同时显示：中国来源的气单胞菌分离株气溶素基因分别属于几个主要的簇。
2.2β溶血素基因
通过序列比对分析结果设计引物,采用PCR方法扩增得到RC-07-KA株p溶血素基因片段,克隆测序结果表明：得到的p溶血素基因片段大小为1467bp,包含一个ORF,编码487个氨基酸残基,编码蛋白分子量约为54KDa。遗传进化分析结果表明,该基因与气单胞菌属中的A. hydrophila菌株Sb(AY611033)、NLEPA-1607(AF410466)、AEF (HM853019), A.sobria菌株357(AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2(X65048)的β溶血素基因亲缘关系较近,同源性大于95%,而与其他菌株的同源性较低。进化树构建结果显示：气单胞菌属的β溶血素基因可分为几个明显分支的基因簇,RC-07-KA株溶血素基因与AY611033(A.hydrophila strain Sb)、EF620533(host=Homo sapiens")、 AY157998(A.sobria strain357)、X65048(A.salmonicida strain:17-2)、AF410466(A.hydrophila strain:NLEP A-1607). HM853019(A.hydrophila strain AEF)为同一簇。
对比分析气单胞菌属内与RC-07-KA株亲缘关系较近及较远的代表菌株p溶血素氨基酸序列的亲水性、抗原性指数及表面可及性,结果显示：气单胞菌属的溶血素具有很强的抗原性,与RC-07-KA株亲缘关系较近菌株β溶血素的抗原性相差较小,揭示p溶血素具有作为候选的蛋白疫苗的潜能。
2.3胞外丝氨酸蛋白酶基因
采用PCR方法扩增得到RC-07-KA株胞外丝氨酸蛋白酶基因得到1.9kb基因片段,克隆测序结果显示：得到的RC-07-KA株胞外丝氨酸蛋白酶基因大小为1875bp,包含一个ORF,编码624个氨基酸残基的多肽。构建系统进化树分析结果显示：RC-07-KA株胞外丝氨酸蛋白酶基因与AF253471(A.sobria strain288)、 AF126213(A.hydrophila, South Korea,1999)和CP002607(A.veronii B565)属于同一遗传衍化分支Ⅱ。序列相似性分析结果显示：RC-07-KA株胞外丝氨酸蛋白酶基因与AF253471、AF126213和CP002607的序列相似性高,分别为93.6%、93.3%和92.5%。这些结果表明我们成功克隆得到RC-07-KA株胞外丝氨酸蛋白酶基因,为进一步深入研究胞外丝氨酸蛋白酶的功能奠定了良好的基础。
2.4脂肪酶基因
采用PCR方法扩增得到RC-07-KA株脂肪酶基因片段,克隆测序显示：得到的脂肪酶基因片段为2445bp,包含一个ORF,编码814个氨基酸残基的多肽,蛋白分子量约为83KDa。构建系统进化树分析结果显示：RC-07-KA株脂肪酶基因与A.sobria strain AS228(AB206038, Japan,2005)、A.sobria strain AS008(AB206037, Japan,2005)、A.sobria strain288(JN019936, Japan,2011)属于同一遗传衍化分支Ⅱ。同源性分析结果显示：RC-07-KA株脂肪酶基因与A.sobria AS228、AS008(AB206038、AB206037, Japan,2005)的序列相似性高,分别为93.6%、93.3%。
3.重组溶血素表达载体构建
将RC-07-KA株溶血素基因片段连接到pET28a(+)载体的EcoR Ⅰ和Xho Ⅰ位点,转化大肠杆菌提取质粒,用EcoR Ⅰ、Xho Ⅰ、Sal Ⅰ进行单、双酶切鉴定,结果表明：正确构建得到重组溶血素表达质粒pET28a-Hly。将pET28a-Hly转化大肠杆菌Rosetta,用IPTG诱导溶血素基因的表达,SDS-PAGE结果显示：重组菌株表达目的蛋白条带位于66.4KDa与44.3KDa之间,结果与预期相符,且重组蛋白多为包涵体,少量为可溶性蛋白。通过His单克隆抗体进行Western-blot鉴定,结果显示在66.4KDa与44.3KDa之间出现印迹条带,表明目的蛋白在大肠杆菌中获得了表达,这为进一步研究重组溶血素的活性奠定了基础。
4.重组溶血素优化表达、纯化及活性检测
为了得到较多的可溶性重组溶血素,对IPTG、诱导温度及诱导表达时间进行了优化,结果显示：IPTG浓度为0.4mmol/L、诱导温度为32℃、诱导表达3-4h可溶目的蛋白的相对含量较高。在优化的条件下表达可溶性溶血素,用Ni2+-Sepharose6Fast Flow凝胶进行目的蛋白的纯化,SDS-PAGE检测结果显示纯化产物在58KDa左右得到单一条带,表明得到纯化的重组溶血素。采用鲫鱼红细胞为靶细胞进行重组溶血素的活性测定,结果显示：重组溶血素在20℃和37℃均具有溶血活性,1.0μg的重组溶血素、反应1h能明显观察到明显的溶血现象,反应3h能使试验红细胞完全溶解,表明重组溶血素具有活性,这为进一步深入研究奠定了基础。
Pelteobagrus fulvidraco, which blongs Siluriformes, Bagridae, Pelteobagrus is in the taxonomy, and widely distributed in China's Yangtze River, Pearl River, Heilongjiang River and the Pacific River system, is one of the important economic freshwater fish of our country, and one of the admirable fingerling export, of which economic value beyond of four domestic fishes. In China, catfish has successfully achieved the domestication of artificial breeding, and the production of artificial breeding catfish reached50million tons, the output value over1billion yuan per year. Because of changes of the breed model and the breeding scale expand ceaselessly. There have been new infectious disease outbreak and epidemic. In recent years, a large number of P. fulvidraco occurred an infectious disease in many Chongqing's intensive fish farms, of which prevalence rate and mortality rate exceed30%and15%, respectively. The disease characterized swelling of fins, bleeding, muscle ulcers and septicemia. The prevalence of disease have caused huge economic losses, and seriously jeopardized the survival and development of fish farming. However, the infectious etiology is unknown; there is no effective methods for prevention and treatment it. Therefore, determination of the etiology and pathogenic mechanism can provide clinical basis for effective prevention and treatment ulcerative disease syndrome of P. fulvidraco. This has important theoretical and practical significance for P. fulvidraco farming.
The etiology, which cause ulcerative disease syndrome of P. fulvidraco, at present, is not clear. Its pathogenic mechanism could be studied. Because of more than10species of pathogenic bacteria can cause fish ulcerative disease syndrome, it is necessary to clear the pathogen of P. fulvidraco ulcerative disease syndrome and understand its pathogenesis for effectively control ulcerative disease syndrome. This study focuses on the isolation and identification of pathogenic bacteria from the prevalence of P. fulvidraco,by using Bacterial biochemistry and molecular identification methods. The virulence associated gene aerolysin, hemolysin, extracellular serine proteases and lipases were cloned and sequenced. Furthermore, the recombinant hemolysin is obtained by heterologous expression in Escherichia coli. This provides a basis for establishment of A. sobria detection technology and subunit vaccine research by use of recombinant hemolysin as antigen. The main results of this research are as follows:
1. Isolation, identification of the pathogen and its pathogenicity
Two isolates of A. sobria, named as strain RC-07-KA and strain RC-07-XB, were isolated from heart blood of P. fulvidraco. Both of them can be cultured on LB medium, MacConkey's agar and fresh blood agar. The biochemical tests results showed that the oidase test and arginine test demonstrate positive, the O/F fermentation, KCN growth test, aescin glycosides test and salicin test demonstrate negative. These results addressed that the biochemical properties consistent with that of A. sobria. The16S rRNA gene fragment was amplified by polymerase chain reaction and the sequence was aligned with the25sequences of bacteria published on GenBank. The results revealed that the isolates of ours showed close phylogenetic relationship with the other6strains of A. sobria, which shared96.4-99.8%homology with each other.
Animal regression experiment results show that A. sobria strain RC-07-KA can kill yellow catfish, carp and crucian by muscle inoculation with6.15×107CFU/fish in7days. Histopathological observation showed that the challenged fish characterized as renal glomerular enlargement, glomerular capillary endothelial cell and renal tubular epithelial cell swelling, the visible filamentous fibrin exudation within some of the lumen, visible inflammatory leukocyte infiltration in renal tubule interstitial; the portion of the cardiac muscle fiber swelling, ambiguous grosgrain; hepatic cellular degeneration and necrosis, derangement, fibrin exudation at central vein, neutrophil infiltration; part of intestinal mucosa epithelial shedding, a large number of neutrophils and a small amount of macrophages infiltrating in enteral. The results demonstrated that A. sobria strain RC-07-KA can cause obvious pathological injury of multiple organ of crucian.
A. sobria strain RC-07-KA was inoculated and cultured in LB medium. The cultures were filtered, and the filtration was employed to detect the hemolytic activity on the LB agar which contained10%fresh blood. The results showed that the cell-free filtration can lysis red blood cells, and the hemolytic titer reached1×25. The results Indicates that the A. sobria strain RC-07-KA posses strong βhemolysin production characteristics. Extracellular crude extracts were obtained by two step salting-out method. The hemolytic test results demonstrated that extracellular crude extracts address hemolytic activity. This suggest that hemolytic activephemolysin can be obtained by ammonium sulfate precipitation method, despite its low yields.
2. Cloning and sequence analysis of main virulence gene of strain RC-07-KA
There are many virulence factors associated with Aeromonas pathogenicity, including aerolysin, hemolysin, extracellular serine proteases and lipases, ect. In order to study the virulence factor of A. sobria strain RC-07-KA, the hemolysin, aerolysin, extracellular serine protease and lipase gene were amplified by polymerase chain reaction. The four gene fragments were cloned and sequenced. The obtained gene sequences were analyzed.
2.1Aerolysin gene
0.7kb fragment of aerolysin gene of A. sobria strain RC-07-KA was amplified by use of PCR method and cloned into pGEM-T vector. The sequencing result showed that the gene fragment is size of692bp, which can encode a peptide of230amino acid residues. The results of Blast analysis revealed that the obtained sequence shared similarity with the aerolysin gene of Aeromonas, which belongs to the virulence factors aerolysin superfamily of Aeromonas, and containing conserved domain of pore-forming toxins.
Phylogenetic analysis results showed that A. sobria strain RC-07-KA close to aerolysin gene from China, Japan and India strains(Accession:AB109093, EF034117, HM853019, AF443392, AF443394, AF443395), which located in the same cluster. In the meanwhile, the analysis result display that aerolysin gene of Chinese isolates belong to several main clusters.
2.2β hemolysin gene
A pair of primers were designed and synthesized according to the published hemolysin nucleotide sequence from A. sobira in GenBank. The gene fragment of (3hemolysin was amplified by PCR through the designed primers. Sequencing results of the cloned gene fragment revealed that the obtained β hemolysin gene is in size of1467bp, which can encode a peptide of487amino acid residues, an about54kDa protein. Phylogenetic analysis results showed that β hemolysin gene of A. sobria RC-07-KA close to A. hydrophila strain sb (AY611033), NLEP A-1607(AF410466),AEF (HM853019),A. sobria strain357(AY157998), A. sobria Homo sapiens isolate(EF620533) and A. salmonicida strain17-2(X65048), with shared over95%homology, and lower homology with the other strains. Phylogenetic tree analysis results demonstrated that the β hemolysin gene of Aeromonas can be divided into several distinct gene clusters, the β hemolysin gene of A. sobria strain RC-07-KA located in cluster Ⅱ, with the reference strains of AY611033, EF620533, AY157998, X6504, AF410466and HM853019.
Comparative analysis the hydrophilic, antigenic index and surface accessibility of hemolysin revealed that the hemolysin have strong antigenicity, with a lower antigenic differences between A. sobria strain RC-07-KA and closed strains. This suggests that beta hemolysin may be used as a potential candidate vaccine.
2.3Extracellular serine protease gene
A1.9Kb gene fragment of extracellular serine protease of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results addressed that the extracellular serine protease gene is1875bp, which contains an open reading frame, encoding a peptide of624amino acid residues. Phylogenetic tree analysis results showed that the extracellular serine protease gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain288(AF253471), A. hydrophila, South Korea isolate (AF126213) and A.veronii B565(CP002607).The sequence similarity is93.6%、93.3%and92.5%, respectively. These results suggest that the extracellular serine protease gene of strain RC-07-KA was successfully cloned, an important basics for further studies.
2.4Lipase gene
A lipase gene fragment of A. sobria strain RC-07-KA was obtained by using PCR method. The fragment was cloned and sequenced. The results showed that lipase gene is2445bp of in size, which contains an open reading frame, encoding a peptide of814amino acid residues, an about83kDa protein. Phylogenetic tree analysis results demonstrated that the lipase gene of A. sobria strain RC-07-KA located in gene cluster Ⅱ, along with A.sobria strain AS228(AB206038), AS008(AB206037) and288(JN019936). The sequence similarity is93.6%、93.3%with AB206038and AB206037, respectively.
3. construction of recombinant hemolysin expressing vector
The β hemolysin gene was amplified, purified and cloned into the EcoRI and XhoI restriction enzyme digestion site to construction recombinant hemolysin expressing vector pET28a-Hly. The gained recombinant plasmid pET28a-Hly was identified by restriction enzyme digestion. The results demonstrated that pET28a-Hly be successfully constructed. The pET28a-Hly was transformed into E.coli. strain Rosetta for recombinant protein expression. SDS-PAGE analysis results demonstrated that the band of target protein appear between66.4KDa and44.3KDa, in line with expectations. The recombinant protein was confirmed by Western-blot test. The results suggest the target protein have successfully expressed in E.coli, which is an important basics for further studies.
4.Optimal expression, purification and activity analysis of recombinant β hemolysin
In order to get more soluble recombinant hemolysin, the condition of inducible expression were optimized based on IPTG concentration, temperature and the time of induce. The results demonstrated that soluble target protein reached at a relatively high level when IPTG concentration is0.4mmol/L,32℃, induce period3-4hours.
Amount of soluble recombinant β hemolysin were expressed at the optimized conditions, and the recombinant protein were purified by Ni2+-Sepharose6Fast Flow gel affinity chromatography. SDS-PAGE analysis results revealed a single brand between66.4KD and44.3KD, that suggest the recombinant β hemolysin have been purified. The activity of purified recombinant β hemolysin was detected by hemolysis test using crucian carp red blood cells as indicator. The results demonstrated that recombinant hemolysin have hemolysis activity at both20℃and37℃. The obvious can be detected when1.0μg recombinant hemolysin reacts with the indicator cells, and the test red blood cells can be completely dissolved in3hours. The results suggest that the recombinant hemolysin posses hemolysis activity, which is essential for further studies.
引文
[1]Ewing W H, R Hugh,and J G Johnson. Studies on the Aeromonas group.U.S.Department of Health and Human Services, Atlanta,1961,GA.
[2]Stanier R Y.A note on the taxonomy of Proteus hydrophilus[J].Bacteriol,46:213-214.
[3]Caselitz F H.How the Aeromonas story started in medical microbiology[J].Med.Microbiol,Lett. 1996,5:46-54.
[4]Von Graevenitz A,and A H Mensch.The genus Aeromonas in human bacteriology:report of 30 cases and review of the literature.N Engl[J]. Med,1968,278:245-249.
[5]Popoff M Y, C Coynault, M Kiredjian,and M Lemelin.Polynucleotide Sequence relatedness among motile Aeromonas species[J].Curr.Microbiol,1981,5:109-114.
[6]Colwell R R,M T Mac Donell,and J De Ley.Proposal to recognize the family Aeromonadaceae fam.nov[J].Syst. Bacteriol,1986,36:473-477.
[7]Seshadri R,S W Joseph,A K Chopra,etc.Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J].Bacteriol,2006,188:8272-8282.
[8]Janda J M,and P D Duffey.Mesophilic aeromonads in human disease;current taxonomy, laboratory identification,and infectious disease spectrum. Rev.Infect.Dis,1988,10:980-997.
[9]Fanning G R,F W Hickman Brenner,J J Farmer Ⅲ, and D J Brenner.DNA relatedness and phenotypic analysis of the genus Aeromonas,abstr,1985,C-116,p.319.Abstr.85th Annu.Meet. Am.Soc.Microbiol.American Society for Microbiology,Washington,DC.
[10]Popoff M Y,C Coynault,M Kiredjian,and M Lemelin.Polynucleotide sequence relatedness among motile Aeromonas species.Curr.Microbiol,1981,5:109-104.
[11]Martin Carnahan A,and S W Joseph.Genus I.Aeromonas Stanier,1943,213AL,2005,p.557-578.
[12]Janda J M,and S L Abbott.16S rRNA gene sequencing for bacterial identification in the diagn-ostic laboratory:pluses,perils,and pitfalls[J].Clin.Microbiol,2007,45:2761-2764.
[13]Christensen H,M Bisgaard,W Frederiksen,R Mutters,P Kuhnert,and J E Olsen.Is characteriza-tion of a single isolate sufficient for validation of a new genus or species? Proposal to modify recommendation 30b of the Bacteriological Code(l990 revision). Int.[J].Syst. Evol. Microbiol, 2001,51:2221-2225.
[14]Stackebrandt E,W Frederiksen,G M Garrity.Report of the adhoc committee for the re-evaluation of the species definition in bacteriology Int.[J]. Syst.Evol.Microbiol,2002,52:1043-1047.
[15]Huang H C,W L Yu,K H Huan,K C Cheng,and Y C Chuang.Aeromonas sobria prostatitis and septic shock in a healthy man with chronic alcoholic consumption.Jpn[J].Infect.Dis,2007,60: 400-401.
[16]Lai C C, C C Shiao,G D Lu,and L W Ding.Aeromonas hydrophila and Aeromonas Sobria bac-teremia:rare pathogens of infection in a burn patient.Burns,2007,33:255-257.
[17]Popoff M,and M Veron.A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group[J].Gen.Microbiol,1976,94:11-25.
[18]J Michael Janda,Sharon L Abbott.The Genus Aeromonas:Taxonomy,Pathogenicity,and Infec-tion.Clincal Microbiology Reviews,2010,23.1.35-73.
[19]Valera L,and C Esteve.Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas[J].Appl.Microbiol,2002,93:77-95.
[20]Minana Galbis D,M Farfan,J G Loren,and M C Fuste.Biochemical identification and numerical taxonomy of Aeromonas spp.Isolated from environmental and clinical samples in Spain[J]. Appl.Microbiol,2002,93:420-430.
[21]Minana Galbis D,M Farfan,M C Fuste, and J G Loren.Aeromonas molluscorum sp.nov,isolated from bivalve mollusks.Int [J].Syst.Evol. Microbiol,2004,54:2073-2078.
[22]Minana Galbis D,M Farfan,M C Fuste,and J G LorenAeromonas bivalvium sp.nov,isolated from bivalve mollusks.Int [J].Syst.Evol.Microbiol,2007,57:582-587.
[23]Pidiyar V,A Kaznowski,N B Narayan,M Patole,and Y S ShoucheAeromonas culicicola sp.nov, from the midgut of Culex quinquefasciatus.Int[J].Syst.Evol Microbiol,2005,2:1723-1728.
[24]Huys G,M Cnockaert,and J Swings.Aeromonas culicicola Pidiyar et al.2002 is a latter subject-tive synonym of Aeromonas veronii Hickman Brenner et al.1987.Syst. Appl.Microbiol,2005, 28:604-609.
[25]Pidiyar V,A Kaznowski,N B Narayan,M Patole,and Y S Shouche.Aeromonas culicicola sp.nov, from the midgut of Culex quinquefasciatus.Int.Int[J].Syst.Evol.Microbiol,2002,52:1723-1728.
[26]Kupfer M,P Kuhnert,B M Korczak,R Peduzzi,and A Demarta.Genetic relationships of Aeromo-nas strains inferred from 16S rRNA, gyrB and rpoB gene sequences.Int. [J].Syst. Evol. Micr-obiol,2006,56:2743-2751.
[27]Soler L,M A Yanez,M R Chacon,M G Aguilera Arreola,V Catalan,M J Figureras, and A J Martinez Murcia.Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.Int[J].Syst.Evol.Microbiol,2004,54:1511-1519.
[28]Dubnau D, I Smith,P Morell,et al.Gene conservation in Bacillus species.I.Conserved genetic and nucleic acid base sequence homologies.Proc Natl Acad Sci USA,1965,54(2):491-498.
[29]Woese C R.Bacterial evolution[J].Microbiol Rev,1987,51(2):221.
[30]Olsen GJ,Woese CR.Ribosomal RNA:a key to phylogeny[J].Faseb,1993,7:113-123.
[31]Rikihisa Y,Kawaham M,Wen B.Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA sequences of H.murs,H.felis and Eperythrozoon suis[J].Clin Micro-biol,1997,35(4):823-829.
[32]Rappe MS,Giovannoni SJ.The uncultured microbial majority[J].Annu Rev.Microbiol,2003,57: 369-94.
[33]Chakravorty S,D Helb,M Burday,N Connell,and D Alland.A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria[J].Microbiologic Methods,2007, 69:330-339.
[34]Ragnhild Wilk,Erko Stackebrandt.Classification of Fish-Pathogenic Vibrios Based on Compar-ative 16S rRNAAnalysis[J]. IJSEM July 1995 vol.45(3):421-428.
[35]Borrell N,S G Acinas,M J Figueras.Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes[J].Clin.Microbiol,1997, 35(7):1671-1674.
[36]Janda J M,and S L Abbott. Evolving concepts regarding the genus Aeromonas:an expanding panorama of species,disease presentation,and unanswered questions.Clin.Infect.Dis,1998,27(2): 332-344.
[37]Mc Mahon M A S,and I G Wilson.The occurrence of enteric pathogens and Aeromonas species in organic vegetables.Int[J].Food Microbiol,2001,70:155-162.
[38]Austin B,and C Adams.Fish pathogens,1996,p.197-243.In B Austin,M Altwegg,P J Gosling,and S Joseph(ed.),The genus Aeromonas.JohnWiley&Sons Ltd,West Sussex,England.
[39]Joseph S W,and A Carnahan.The isolation,identification,and systematics of the motile Aeromo-nas species.Annu.Rev.Fish Dis,1994,4:315-343.
[40]http://www.promedmail.org/pls/otn/f? P=2400:1202:959209208141620::NO::F2400_P1202_CHECK_DISPLAY,F2400_P1202_PUB_MAIL_ID:X,18797.
[41]Michael,Sharon.The Genus Aeromonas:Taxonomy,Pathogenicity,and Infection[J].Clincal Micr-obiology Reviews,Jan,2010,p.35-73.
[42]Leclerc H,L Schwartzbrod,and E Dei Cas.Microbial agents associated with waterborne diseases. Crit.Rev.Microbiol,2002,28:371-409.
[43]Isonhood J H,and M Drake.Aeromonas species in foods[J].Food Prot,2002,65:575-582.
[44]Palumbo S A,F Maxino,A C Williams,R L Buchanan,and D T W Thayer. Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl. Environ. Microbiol,1985,50: 1027-1030.
[45]Borrell N,M J Figueras,and J Guarro.Phenotypic identification of Aeromonas genomespecies from clinical and environmental sources.Can[J].Microbiol,1998,44:103-108.
[46]Neyts K, G Huys, M Uyttendaele,J Swings, and J Debevere.Incidence and identification of mesophilicAeromonas spp.from retail foods.Lett.Appl. Microbio,2000,31:359-363.
[47]Rusin P A,J B Rose,C N Haas,and C P Gerba.Risk assessment of opportunistic bacterial path-ogens in drinking water.Rev.Environ.Contam.Toxicol,1997,152:57-83.
[48]Holmes P,L M Niccolls,and D P Sartory.The ecology of mesophilic Aeromonas in he aquatic environment,1996,p.127-150.In B Austin,M Altwegg, P J Gosling,and S Joseph (ed.),The genus Aeromonas.John Wiley &Sons Ltd,West Sussex,England.
[49]Fiorentini C,E Barbieri,L Falzano.Occurrence,diversity and pathogenicity of mesophilicAero-monas in estuarine waters of the Italian coast of the Adriatic Sea[J].Appl.Microbiol,1998,85: 501-511.
[50]Hazen T C,C B Fliermans,R P Hirsch,and G W Esch. Prevalence and distribution of Aeromonas hydrophila in the United States.Appl.Environ.Microbiol,1978,36:731-38.
[51]Hazen T C,and C B Fliermans.Distribution of Aeromonas hydrophila in Natural and man-made thermal effluents.Appl.Environ.Microbiol,1979,38:166-168.
[52]http://www.who. int/water_sanitation_health/dwq/guidelines/en/index.html.
[53]http://permanent.access.gpo.gov/lps21800/www.epa.gov/safewater/ccl/cclfs.html.
[54]Khardori N,and V Fainstein. Aeromonas and Plesiomonas as etiological agents[J].Annu.Rev. Microbiol,1988,42:395-419.
[55]Kelly K A,J M Koehler,L R and Ashdown.Spectrum of extraintestinal disease due to Aeromon-as species in tropical Queensland,Australia.Clin.Infect.Dis,1993,16:574-579.
[56]Llopis F,I Grau,F Tubau,M Cisnal.Epidemiological and clinical characteristics of bacteraemia caused by Aeromonas spp.as compared with Escherichia coli and Pseudomonas aeruginosa. Scand[J].Infect.Dis,2004,36:335-341.
[57]Bossi Kupfer M,A Genini,R Peduzzi,and A Demarta.Tracheobronchitis caused by Aeromonas veronii biovar sobria after near drowning[J].Med.Microbiol,2007,56:1563-1564.
[58]Centers for Disease Control and Prevention. Aeromonas wound infections associated with outdoor activities-California.Morb.Mortal.Wkly.Rep,1990,39:334-335.
[59]Angel M F,F Zhang,M Jones,J Henderson,and S W Chapman.Necrotizing fasciitis of the upper extremity resulting from a water moccasin bite.South.Med[J].2002,95:1090-1094.
[60]Kunimoto D,R Rennie,D M Citron,and E J C Goldstein.Bacteriology of a bear bite wound to a human:case report[J].Clin.Microbiol,2004,42:3374-3376.
[61]Lamy B,A Kodjo,the colBVH Study Group,and F Laurent.Prospective nationwide study of Aeromonas infections in France[J].Clin.Microbiol,2009,47:1234-1237.
[62]Ceylan E,M Berktas,and Z Agaoglu.The occurrence and antibiotic resistance of motile Aerom-onas in livestock.Trop.Anim.Health Prod,2009,41:199-204.
[63]Moro E M P,R D N Weiss,R S C Friedrich.Aeromonas hydrophila isolated from cases of bovine seminal vesiculitis in south Brazil[J].Vet.Diagn.Invest,1999,11:189-191.
[64]Gosling P J.Aeromonas species in disease of animals,1996,p.175-195.In B Austin,M Altwegg, P J Gosling, and S Joseph (ed.),The genus Aeromonas. John Wiley& Sons Ltd,West Sussex, England.
[65]Thorton S M,S Nolan,and F M Gulland.Bacterial isolates from California sea lions (Zalophus californicus),harbor seals(Phoca vitulina),and northern elephant seals (Mirounga angustirostris) admitted to a rehabilitation center along the central Calif ornia coast[J],1994-1995. Zoo. Wildl. Med,1998,29:171-176.
[66]Josenhans C,S Suerbaun.The role of motility as a virulence factor in bacteria[J].Int Med Micr-obiol,2002,291:605-614.
[67]Berg H C.The rotary motor of bacterial flagella.Annual Rev Biochem,2003,72:19-54.
[68]Rocio Canals,Maria Altarriba, Silvia Vilches,Gavin Horsburgh.Analysis of the Lateral Flagellar Gene System of Aeromonas hydrophila AH-3[J].Bacteriol,2006,188(3):852-862
[69]Sleytr U B.Regular arrays of macromolecules on bacterial cell walls:structure, chemistry, asse-mbly and function[J].Int.Rev.Cytol,1978,53:1-64.
[70]T J Beveridge,L L Graham. surface layers of bacteria. Microbiol Rev.1991,12,55(4):684-705
[71]Boot HJ and PH Pouwels.Expression,secretion and antigenic variation of bacterial S-layer protein[J].Mol Microbiol,1996,21 (6):1117-1123.
[72]H Chart,D H Shaw,E E Ishiguro,and T J Trust.Structural and immune chemical homogeneity of Aeromonas salmonicida lipopolysaccharide[J].Bacteriol,1984,158:16-22.
[73]Sleyt U B, Messner P .Crystalline bacterial cell surface layers[M]. Adv.Microb Physiol,1992, 23:213-275.
[74]B Uwe.Sleytr,Paul Messner,Dietmar Pum,Margit Sara.Crystalline bacterial cell surface layers[J].Mole Micro,1993,10,911-916.
[75]Koval S F,S H Hynes.Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus[J].Bacteriol,1991,173(7):2244-2249.
[76]Blaser M J,P F Smith,J E Repine and K A Joiner.Pathogenesis of Campylobacter fetus infections.Failure of encapsulated Campylobacter Fetus to bind C3b explains serum and phagocytosis resistance[J].Clin.Invest,1988,81 (5):1434-1444.
[77]Rafaela A Garduno and W W Kay.Interaction of the fish pathogen Aeromonas salmonicida with rainbow trout macrophages[J].Infect.Immun,1992,60(11):4612-4620.
[78]Trust T J,W W Kay and E E Ishiguro.Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida[J].Curr.Microbiol,1983,9:315-318.
[79]Sleytr U B, M Sara.Bacterial and archaeal S-layer proteins:structure function relationships and their biotechnological applications[J].Trends Biotechnol,1997,15:20-26.
[80]Sleytr U B,P Messner,D Pum.Immobilised Macromolecules:Application Potentials.1993,26, 212.
[81]Messner P, F M Unger, U B Sleytr.Vaccine development based on Slayer technology.In:Cryst-alline Bacterial Cell Surface,1996.
[82]Laurence Abrami,Marc Fivaz.Sensitivity of Polarized Epithelial Cellstothe Pore-Forming Toxin Aerolysin[J].Infect.Immun,2003,71.2.739-746.
[83]Oliver Knapp,Bradley Stiles,Michel R Popoff.The Aerolysin Like Toxin Family of Cytolytic, Pore-Forming Toxin[J].The Open Toxinology Journal,2010,3,53-68.
[84]Szcesny p,Iacovache.Extending the aerolysin the family:from bacteria to vertebratea.PLoS One 2011,6(6):e20349.
[85]Oliver Knapp,Bradley Stiles.The Aerolysin-Like Toxin Family of Cytolytic,Pore-Forming Toxins[J].The Open Toxinology,2010,3,53-68.
[86]Bischofberger M, M R Gonzalez.Membrane injury by pore forming proteins[J].Curr Opin Cell Biol,2009,21:589-595.
[87]Rosado C J,A M Buckle,R H P Law,et al.A common fold mediates vertebrate defense and bacterial attack[J].Science,2007,317:1548-1551.
[88]Hadders M A,Beringer D X,Gros P.Structure of c8a-MACPF reveals mechanism of membrane attack in complement immune defense[J].Science,2007,317:1552-1554.
[89]Melton J A,Tweten J M.In:Alouf J E,Popoff M R,Eds.The comprehensive sourcebook of bacterial protein toxins.3rded.London:Academic Press,2006,pp.623-630.
[90]Sousa M V,Richardson M,Fontes W,Morhy L.Homology between the seed cytolysin enterolobin and bacterial aerolysins[J].Protein Chem,1994,13:659-667.
[91]Cole AR,Gibert M,Popoff M,Moss DS, Titball RW, Basak AK.Clostridium perfringens epsilon toxin shows structural similarity to the pore-forming toxin aerolysin[J].Nat Struct Mol Biol, 2004,11(8):797-798.
[92]Mancheno J M,Tateno H,Goldstein I J,Martinez Ripoll M,Hermoso J A.Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars[J].Biol Chem, 2005,17251:17259.
[93]Akiba T,Abe Y,Kitada S,et al.Crystal structure of the parasporin 2 Bacillus thuringiensis toxin that recognizes cancer cells[J].Mol Biol,2009,386:121-133.
[94]Caselitz F H and R G Unther.Hemolysin studien mit Aeromonas stammen[J].Zentralbl. Bakteriol.Abt.I.Orig,1960,180:30-38.
[95]Wretlind B,R Mollby and T Wadstrom. Separation of two hemolysins from Aeromonas hydrophila by isoelectric focusing[J].Infect.Immun,1971,4(4):503-505.
[96]Wretlind B,L Hedean,and T Wadstroum.Formation of extracellular haemolysin by Aeromonas hydrophila in relation to protease and staphylolytic enzyme[J].Gen. Microbiol,1973,78(1):57-65.
[97]Bernheimer A W,and L S Avigad.Partial characterization of aerolysin, a lytic exotoxin from Aeromonas hydrophila[J].Infect.Immun,1974,9(6):1016-1021.
[98]Buckley JT,LN Halasa,KD Lund and S Maclntyre.Purification and some properties of the hemolytic toxin aerolysin.Can[J]. Biochem,1981,59(6):430-435.
[99]Howard S P,and J T Buckley.Activation of the hole-forming toxin aerolysin by extracellular processing[J].Bacteriol,1985,163(1):336-340.
[100]Howard S P and J T Buckley.Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila.Mol.Gen.Genet, 1986,204(2):289-295.
[101]Vander Goot F G,Pattus F,Wong K R,Buckley J T.Oligomerization of the channel forming toxin aerolysin precedes insertion into lipid bilayers[J].Biochemistry,1993,32(10):2636-2642.
[102]Vander Goot FG,Lakey J,Pattus F,et al.Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin:identification of the site of proteolytic activation[J].Bioch-emistry,1992,31 (36):8566-8570.
[103]Yeongjin Hong,Kazuhito Ohishi.Requirement of N-gly-can on GPI-anchored proteins for efficie-nt binding of aerolysin but not Clostridium septicum α-Toxin[J].The Embo Journal,2002,21, 5047-5056.
[104]Fukushima K L,Kehara Y,Kanai M.A β-N-Acetylglucosaminyl Phosphate Diester Residue Is Attached to the Glycosylphosphatidylinositol Anchor of Human Placental Alkaline Phosphatase [J].Biological Chemistry Vol,2003,278:36296-36303.
[105]Parker M W,Feil S C.Pore-forming protein toxins:from structure to function[J].Prog Biophys Mol Biol,2005,88(1):91-142.
[106]Krause K H, M Fivaz, A Monod.Aerolysin Induces G-protein Activation and Ca2+ Release from Intracellular Stores in Human Granulocytes[J].Biological Chemistry 1998,273,29,18122-18129.
[107]Nelson K L,Brodsky R A,Bucky J T.Channels formed by subnanomola reconcentrations of the toxin aerolysin trigger apoptosis of T lymphomas[J].Cell Microbiol,Jul,1999,1(1):69-74.
[108]Brodsky R A,Mukhina G L,Nelson K L,Lawrence T S, Jones R J,Buckley J T.Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin [J].Blood,1999,93(5):1749-1756.
[109]Rita Brenden,J M Janda.Detection,quantitation and stability of the β haemolysin of Aeromonas spp[J].Med Microbiol,1987,24(3):247-251.
[110]Tsutomu Aaao,Yoshio Kinshita.Purification and Some Properties of Aeromonas hydrophila Hemolysin[J].infection and immunity,1984,46(l):122-127
[111]Hirono I,Aoki T.Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila[J].Micro Pathog.1991,11(3):189-197.
[112]Michael E Reith,Rama K Singh,Bruce Curtis,Jessica M Boyd.The genome of Aeromonas salmonicida subsp.salmonicida A449:insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9:427
[113]Epple H J, Mankertz J R,et al.Aeromonas hydrophila Beta Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells(HT-29/B6).Infect Immun,2004,72 (8):4848-4858.
[114]Hage C M,Chahine,G Del Giudice,P H Lambert,and J C Pechere.Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independ-ent antigens[J].Infect Immun,April,1992,60(4):1415-1421.
[115]Siritat T,Intuseth J,Chanphone J,et al.Characterisation of Aeromonas hydrophila extracellar prodcts with reference to toxicity,virulence, protein profiles and antigencity[J].Asian Fisheris Science,1999,12:371-379.
[116]Christopher Y F W,Michael W H and Robert L P F.Inactivation of two haemolytic toxin genes in Aeromonas hydrophla attenuates virulence in a suckling mouse model[J]. Microbiology, 1998,144(2):291-298.
[117]Moral C H,Castillo E F D.Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophicaroA mutant as a live attenuated vaccine[J].Infect and Immun,1998,66(5):1813-1821.
[118]T C Hsu,W D Waltman, and E B Shots.Correlation of extracellular enzymatic activity and biochemical characteristics with regard to virulence of Aeromonas hydrophila.Dev.Biol.Stand. 1981.49:101-111.
[119]K Y.R M WStevenson,.Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish[J].Infect.Immun,1988,56(10):2639-2644.
[120]K Y Leung, R M W Stevenson.Characteristics and distribution of extracellular proteases from Aeromonas hyrophila[J].Microbiology,1988,134:151-160.
[121]Kanai K,Wakabayashi H.Purification and some proterties of protease from Aeromonas hydrophila[J].Bulletin of the Japanese Society of Scientific Fisheries,1984,50(8):1367-1374.
[122]Nieto T P,Ellis.Characterization of extracellular metallo and serine proteases of Aeromonas hydrophila strain B51[J] Journal of General Microbiology,1986,132(7):1975-1979.
[123]Chabot D J,Thuner L.Protease of the Aeromonas hydrophila complex:identification, characte- rization and relation to virulence in channel catfish,lctaulrus pantatus(Rafinesque)[J].Journal of Fish Disease,1991,14(2):171-183.
[124]Alberto cason.Javier yugueros,Alandro Temprano.A Major secreted elastase is essential for pathogenicity of Aeromonas hydrophila[J].Infection and Immunity,2000,68(6):3233-3241.
[125]Chang TM,Liu C C,Chang M C.Cloning and sequence analysis of the gene(eprA1)encoding an extracellular protease from Aeromonas hydrophila[J].Gene,1997,199:225-229.
[126]Rivero O,Anguita J,Paniagua C.et al.Molecular cloning and characterization of an extracellu-lar protease gene from Aeromonas hydrophila[J].Journal of Bacteriology,1990,172:3905-3908.
[127]Kanai K,Wakabayshi H.Purification and some proterties of protease from Aeromonas hydrophila[J].Bulletin of the Japanese Society of Scientific Fisheries,1984,50(8):1367-1374.
[128]Thune R L,Grahanm T E.Extracellular protease from Aeromonas hydrophila:Partial purifica-tion and effects on age-0 shannel catfish[J].Transaction of the American Fisheries Society, 1982,111:749-754.
[129]Allan B J, Stevenson R M W.Extracellular virulence factors of Aeromonas hydrophila in fish infections[J].Canadian Journal Microbiology,1981,27(10):1114-1122.
[130]Harwood J.The versatility of lipases for industrialuses.Trends Biochem.Sci.1989.14:125-126.
[131]Hilton S, J T Buckley.Studies on the reaction mechanism of a microbial lipase acyltransferase using chemical modification and site-directed mutagenesis[J].Biol.Chem,1991,256:997-1000.
[132]Hilton S,W D Me Cubbin,C M Kay,and J T Buckley.Purification and spectral study of a microbial fatty acyltransferase:activation by limited proteolysis.Biochemistry,1990,29(38): 9072-9078.
[133]Hedstrm S A,and P Nilsson.Lipolytic activity of Staphylococcus aureus strains from cases of human chronic osteomyelitis and other infections[J].Acta Pathol.Microbiol.Scand.Sect.1975, 83(3):285-292.
[134]Rollof J,J H Braconier and P Nilsson Ehle.Interference of Staphylococcus aureus lipase with human granulocyte function[J].Eur.Clin.Microbiol.Infect.Dis,1988,7(4):505-510.
[135]Eftimiadi C,E Buzzi,M Tonetti,P Buffa,D Buffa,M T J van Steenbergen,J Graaff, and G A Botta. Short-chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide[J].Infect,1987,14(l):43-53.
[136]Hewley H,and G Gordon.The effects of long chain free fatty acids on human neutrophil function and structure[J].Lab.Invest,1976,34(2):216-222.
[137]武寒雪,祝建波等,细菌脂肪酶基因的生物信息学分析[J].江苏农业学报,2009,25(4)：942-944.
[138]Chakraborty T,M A Montenegro,S C Sanyal.Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotoxic enterotoxin[J].Infect. Immun,1984,46(2):435-441.
[139]S P Howard,S Macintyre,J T Buckley.The genus Aeromonas,1996,267-286.In B Austin,M Altwegg,P J Gosling,and S Joseph (ed.),Toxin.John Wiley and Sons, Singapore.
[140]Leung K Y,and R M Stevenson.Tn5-induced protease-deficient strains of Aeromonas hydrop-hila with reduced virulence for fish[J].Infect.Immun,1988,56(10):2639-2644.
[141]Nieto T P,Y Santos,L A Rodriguez, A E Ellis.An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish[J].Microb.Pathog,1991,11:101-110.
[142]Cornelis G R,and F Van Gijesem.Assembly and function of type III secretory systems[J]. Annu.Rev.Microbiol,2000,54:735-774.
[143]Grizzle J M,Kiry U Y.Histopathology of gill,liver and pancreas and serum enzyme levels of channel catfish infected with Aeromonas hydrophila complex[J].Journal of Aquatic Animal Health,1993,5(1):36-50.
[144]Cruze Silvap,Grazina Freitas S.Parasites and parasitoses of the European eel(Anguilla japonica L)in Poaugal[J].Bull Soc Franc Parasitol,1990,8(S1):287.
[145]马国文,温海深,刘振歧,等.鲤鱼竖鳞病病原和病理研究[J].水利渔业,1999,19(3)：37-40.
[146]Hanninen M L,Samalmis S,Mattila L,et al.Association of Aeromonas spp.with travellers diarrhoea in Finland[J] Journal of Medical Microbiology,1995,42(l):26-32.
[147]Angka S L,Lam T J,Siny M.Some virulence characteristics of Aeromonas hydrophila in walking catfish(Clarias gariepinus)[J].Aquaculture,1995,130(2/3):103-112.
[148]Baloda S B,Krovacek K,et al.Detection of aerolysin gene in Aeromonas strains isolated from drinking water,fish and foods by polymerase chain reaction[J].Comparative Immunology Microbiology and Infection Disease,1995,18(1):17-26.
[149]Vadiveu I J.Puthuheary S D,Phpps M,et al.Possible virulence factors involved in bacteraemia caused by Aeromonas hydrophila[J]. Journal of Medical Microbiology,1995,42(3):171-174.
[150]Thomas S R,TrustR T J.Tyrosine phosphorylation of the tetragonal paracrystalline array of Aeromonas hydrophila:Molecular cloning and high level expression of the S-layer protein gene[J].Journal of Molecular Biology,1995,245(5):568-581.
[151]陈怀青,陆承平,陈琼,等.用点酶法检测鱼类致病性嗜水气单胞菌HEC毒素[J].动物检疫,1993,10(4)：7-9.
[152]EurellT E,Lewis D H,Grumblel L C.Comparison of selected diagnostic tests for detection of motile aeromonas septicemia in fish[J].American Journal of Veterinary Research,1978,39(8): 1384-1386.
[153]李焕荣,陈怀青,陆承平,等.嗜水气单胞菌胞外蛋白酶的检测[J].水生生物学报,1997,21(1)：97-100.
[154]Gruber H J,WilmsonH U,et al.Partial purification of the rat erythroeyte receptor for the chanel forming toxin aerolysin and reconstitution into planar lipid bilayers[J].Molecular Microbiology, 1994,14(5):1093-1101.
[155]Pollard D R,Johnson W M,Lior H.Detection of the aerolysin Gene in Aeromonas hydrophila by PCR[J].Clinical Microbiology,1990,28(11):2477-2481.
[156]R Y C Kong, S K Y Lee, T W F Law.Rapid detection of six types of bacterial pathogens inmaine waters by multiplex PCR[J].Water Raserch,2002,36:2802-2812.
[157]Alberto Casson,Juan Angutta,,et al. Identification of Aeromonas hydrophila hybridization group 1 by PCR assays[J].Applied and Environmental Microbio logy,1996,62(4):1167-1170.
[158]Micheal W H.ChristopherY F.Distribution of two hemolytic toxin genes in clinical and environm-ental isolates of Aeromonas spp:correlation with virulence in a suckling mouse model[J].FEMS Microbiology Letters,1999,174(1):131-136.
[159]Gonzlez O,Serano C J,Santos J A.Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case[J].Journal of App lied Microbiol ogy,2002,93(3):414-419.
[160]朱大玲,李爱华,汪建国,等.嗜水气单胞菌毒力与毒力基因分布的相关[J].中山大学学报：自然科学版,2006,45(1)：82-85.
[161]宋铁英,包晓东,郑伟文.中华鳖爆发性传染病研究--Ⅳ不同生化类型嗜水气单胞菌aer基因的PCR比较[J].江西农业大学学报：自然科学版,2002,24(2)：164-167.
[162]吴淑勤.鳗鲡混合感染嗜水气单胞菌和迟缓爱德华氏菌的快速诊断[J].鱼类病害研究,1993,3：40-42.
[163]高汉娇.水库暴发性鱼病及其防治技术研究2：直接荧光抗体法对白鲢病原菌的鉴定[J].水利渔业,1995,2：11-13.
[164]陈昌福.用直接荧光抗体和细菌培养法对中华鳖体内的嗜水气单胞菌的检测[J].华中农业大学学报,1998,17(3)：264-266.
[165]李卫军.应用间接荧光抗体法检测鱼类嗜水气单胞菌的试验研究[J].中国动物检疫,1998,15(4)：5-6.
[166]钱冬,陈月英.应用酶联免疫吸附实验检测暴发病病原-嗜水气单胞菌的研究[J].水产养殖,1993,4：14-17.
[167]陈怀青,陆承平,陈琼,等.用点酶法检测鱼类致病性嗜水气单胞菌HEC毒素[J].动物检疫,1993,10(4)：7-9.
[168]沈素芳,储卫华,陆承平.鱼类致病性气单胞菌诊断试剂盒的研制[J].中国兽医学报,2000,20(5)：462-464.
[169]刘颖.应用DOT-ELISA技术快速检测鱼类细菌性疾病病原的研究[J].内陆水产,1998,1：6-7.
[170]Anderson D P.Fish immunology[M].T F H public company limited,1974,296.
[171]Ward P D.The Development of Bacterial Vaccines for Fish[M].ROBERTS R J.Microbial Disease of Fish.Ed.London:Academic Press,1982:47-58.
[172]Amend D F,Johnson K A.Evidence for lack of antigenic competition among various combinat-ions of Vibrio anguillarum, Yersinia ruckeri,Aerolmonas salmonicida and Renibacterium salmoninarum bacterins when administered to salmonid fishes[J]. Journal of Fish Disease,1984, 7(4):293-299.
[173]Thune R L,PlumbJ A.Efect of delivery method and antigen preparation on the production of antibodies against Aeromonas hydrophila in channel catfish[J].Prog Fish Cult,1982,44:53-54.
[174]Ruangpan L,Kitao,Yoshida T. Protective efficacy of Aeromonas hydrophila vaccines in nileti-laph[J]. Veterinary Immunology Immunopathology,1986,12:345-350.
[175]Loghoth Oesis P N.Immune response of rainbow trout(Oncorhynchus mykiss,Walbaum) to Aer-omonas hydrophila[J].Fish and Shellfish Immunology,1994,4:239-254.
[176]Shieh H S.Correlation between cell virulence and protease toxicity of Aeromona salmonicida isolates[J].Microbiology letter,1985,28:69-72.
[177]Ford L A,Thune R L.Immunization of channel catfish with a crude, acid extracted preparation of motile aeromonad S-layer protein[J]. Biome dical Letters,1992,47:355-362.
[178]Adams A,ThompsonH K D.Fish Vaccination Development. FAO Manual for the production and quality control of veterinary vaccine for use in developing countries[J].FAO Animal Produ-ction and Health Series,1997(35).
[179]Leszer.GUZ,A licja Kozinskal.Antibiotic susceptibility of aeromonas hyrophilad and A.sobria isolated from carp(cyprinuscarpiol)[J].bullvet inst pulawy,2004,48,391-395.
[180]T Wahil, S E Burr.Aeromonas sobria,a causative agent of disease in farmed perch, Perca fluviatilis L[J].Fish Dis.2005,28(3):141-150.
[181]ya li,Hu Cai.Identification and Pathogenicity of Aeromonas sobria on Tail rot Disease in Juvenile Tilapia Oreochromis niloticus [J].Current Microbiology 62,623-627.
[182]McGarey DJ,Milanesi L,Foley DP,Reyes B Jr.The role of motile aeromonads in the fish disease, ulcerative disease syndrome (UDS).Experientia.1991,May,15,47(5):441-4.
[183]F.奥斯伯,R.布伦特,R.E金斯顿,等.精编分子生物学实验指南[M].北京：科学出社,2001：39.
[184]冯东岳.2009年我国大宗淡水养殖鱼病害调查及分析[J].水产学杂志,2010,23(4)：60-65.
[185]田雨,王旭,贾光风,等.铁岭市主要养殖区鱼病调查与分析[J].河北渔业,2011,5：33-37.
[186]郭闯,朱国强,王永坤,等.14株患病水生动物气单胞菌属细菌的分离鉴定与最佳治疗药物筛选试验[J].水产科学,2003,22(4)：15-17.
[187]韩英,刘敏,姜艳平.鲤鱼出血病病原的研究和药敏试验[J].东北农业大学学报,2005,36(5)：593-597.
[188]陈爱平,江育林,钱冬,等.淡水鱼细菌性败血症[J].中国水产,2011,3：54-55.
[189]徐海圣,舒妙安.黄鳝温和气单胞菌病病原的分离鉴定及免疫应答[J].中国兽医学报,2003,23(3)：240-245.
[190]胡琳琳,房文红,梁思成,等.金鱼温和气单胞菌的分离鉴定及药敏试验[J].上海水产大学学报,2008,17(3)：285-290,
[191]叶雪平,顾金华,杨广智.牛蛙温和气单胞菌病病原及防治[J].上海水产大学报,2000,9(1)：78-80.
[192]樊海平,吴斌,曾占壮,等.日本鳗鲡体表溃疡病病原菌的分离鉴定及单克隆抗体制备[J].中国水产科学,2009,16(2)：295-302.
[193]王永坤,李志健,朱国强,等.温和气单胞菌和嗜水气单胞菌致病力的比较研究[J].鱼类病害研究,1997,19(2)：18-24.
[194]蔡完其,孙佩芳.罗非鱼温和气单胞菌病的病原研究和药敏试验[J].中国水产科学,2002,9(3)：243-246.
[195]陈翠珍,张晓君,房海.草鱼肠炎的病原菌检验与分析[J].中国兽医科技,1999,29(1)：5-7.
[196]陈怀青,陆承平.家养鲤科鱼爆发性传染病的病原研究[J].南京农业大学学报,1991,14(4)：87-91.
[197]耿毅,汪开毓,吴麟,杜宗君.齐口裂腹鱼败血症的病原分离与鉴定[J].水利渔业.2004,24卷第4期,71-73.
[198]韩继卫,罗文,郑大恒.黄颡鱼温和气单胞菌的分离鉴定与药敏试验绍兴文理学院学报.2011第31卷第9期,19-21.
[199]丁磊,吴康,蔡春芳,等.嗜水气单胞HEC毒素对鲤鱼内皮细胞的毒性[J].中国水产科学,2002,9(2)：117-130.
[200]Tunlid,et al Purification and characterizition of an Extracellular serine protease from the nematode-trapping fungus Arthrobotrysoligo-spore[J].microbiology,1994,140:1687-16952.2.
[201]储卫华,陆承平.嗜水气单胞菌J21株丝氨酸蛋白酶基因克隆与序列分析[J].水产学报,2004,28(1)：84-88.
[202]沈锦玉,李新华,潘晓艺等.温和气单胞菌TL97528株丝氨酸蛋白酶基因片段克隆与序列分析[J].上海海洋大学2010,19.(2)151-156.
[203]张翠娟,于宙亮,田莉瑛,等.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[J].生物工程学报,25(2)：251-256.
[204]潘晓艺,郝贵杰,姚嘉赞,等.嗜水气单胞菌TPS-30株丝氨酸蛋白酶基因与溶血素基因在大肠杆菌中的融合表达.水生生物学报,2010,34(3)：591-597.
[205]赵秀敏,高勤学,陈鸿军,等.嗜水气单胞菌溶血素A表达、免疫原性及活性检测.中国动物传染病学 报,2011,19(1)：45-50.
[206]武斌,于宙亮,于珊,等.人源温和气单胞菌溶血素基因的克隆与表达.河北师范大学学报(自然科学版),2008,32(2)：239-242.
[207]Donovan R S,Robinson C W, Glick B R. Review:optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J].Ind Microbiol. 1996,16(3):145-54.
[208]Harcum SW,Bentley WE.Response dynamics of 26-,34-,39-,54-,and 80-kDa proteases in induced cultures of recombinant Escherichia coli[J].Biotechnol Bioeng,1993,42(6):675-85.
[209]Pack P,Kujau M,Schroeckh V,et al.Improved bivalent miniantibodies, with identical avidity as whole antibodies,produced by high cell density fermentation of Escherichia coli[J]. Biotechnology (N Y),1993,11(11):1271-1277.
[210]Winograd E, Pnlido M A,Wasserman M. Production of DNA-recombinant peptides by tac-inducible vectors using micromolar concentrations of IPTG [J].Biotechniques[J],1993,14: 886-887.
[211]Auger E A,Bennett G N.Temperature optimization of in vivo expression from the E.coli trp and trp:lac promoters[J].Biotechnol Lett 1987,8:157-162.
[212]Kosinski M J,Rinas U, Bailey J E.Isopropyl-13-D-thiogalacto-pyranoside influences the meta-bolism of Escherichia coll[J]. Appl Environ Microbiol,1992,36:782-784.
[213]Surek B,Wilhelm M,Hillen W.Optimizing the promoter and ribosome binding sequence for expression of human single chain mokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response[J].Appl Microbiol Biotech-nol.1991,34:488-494.
[214]Chesshyre J A,Hipkiss A R.Low temperatures stabilize interferon alpha-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli[J].Appl Microbiol Biotechnol,1989,31: 158-162.
[215]Chalmers J J,Kim E,Telford J N,et al.Effects of temperature on Escherichia coli overprodu-cing/3-lactamase or human epidermal growth factor[J]. Appl Environ Microbiol.1990,56: 104-111.
[216]Bentley W E,Mirjalili N,Anderson D C,et al. Plasmid encoded protein:the principal factor of metabolic burden associated with recombinant bacteria[J].Biotechnol Bioeng.1990,35: 668-681.
[217]Bentley W E,Davis R H,Kompala D S.Dynamics of CAT expression in E.coli.Biotechnol Bioeng[J].1991,38:749-760.
[2]Stanier R Y.A note on the taxonomy of Proteus hydrophilus[J].Bacteriol,46:213-214.
[3]Caselitz F H.How the Aeromonas story started in medical microbiology[J].Med.Microbiol,Lett. 1996,5:46-54.
[4]Von Graevenitz A,and A H Mensch.The genus Aeromonas in human bacteriology:report of 30 cases and review of the literature.N Engl[J]. Med,1968,278:245-249.
[5]Popoff M Y, C Coynault, M Kiredjian,and M Lemelin.Polynucleotide Sequence relatedness among motile Aeromonas species[J].Curr.Microbiol,1981,5:109-114.
[6]Colwell R R,M T Mac Donell,and J De Ley.Proposal to recognize the family Aeromonadaceae fam.nov[J].Syst. Bacteriol,1986,36:473-477.
[7]Seshadri R,S W Joseph,A K Chopra,etc.Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J].Bacteriol,2006,188:8272-8282.
[8]Janda J M,and P D Duffey.Mesophilic aeromonads in human disease;current taxonomy, laboratory identification,and infectious disease spectrum. Rev.Infect.Dis,1988,10:980-997.
[9]Fanning G R,F W Hickman Brenner,J J Farmer Ⅲ, and D J Brenner.DNA relatedness and phenotypic analysis of the genus Aeromonas,abstr,1985,C-116,p.319.Abstr.85th Annu.Meet. Am.Soc.Microbiol.American Society for Microbiology,Washington,DC.
[10]Popoff M Y,C Coynault,M Kiredjian,and M Lemelin.Polynucleotide sequence relatedness among motile Aeromonas species.Curr.Microbiol,1981,5:109-104.
[11]Martin Carnahan A,and S W Joseph.Genus I.Aeromonas Stanier,1943,213AL,2005,p.557-578.
[12]Janda J M,and S L Abbott.16S rRNA gene sequencing for bacterial identification in the diagn-ostic laboratory:pluses,perils,and pitfalls[J].Clin.Microbiol,2007,45:2761-2764.
[13]Christensen H,M Bisgaard,W Frederiksen,R Mutters,P Kuhnert,and J E Olsen.Is characteriza-tion of a single isolate sufficient for validation of a new genus or species? Proposal to modify recommendation 30b of the Bacteriological Code(l990 revision). Int.[J].Syst. Evol. Microbiol, 2001,51:2221-2225.
[14]Stackebrandt E,W Frederiksen,G M Garrity.Report of the adhoc committee for the re-evaluation of the species definition in bacteriology Int.[J]. Syst.Evol.Microbiol,2002,52:1043-1047.
[15]Huang H C,W L Yu,K H Huan,K C Cheng,and Y C Chuang.Aeromonas sobria prostatitis and septic shock in a healthy man with chronic alcoholic consumption.Jpn[J].Infect.Dis,2007,60: 400-401.
[16]Lai C C, C C Shiao,G D Lu,and L W Ding.Aeromonas hydrophila and Aeromonas Sobria bac-teremia:rare pathogens of infection in a burn patient.Burns,2007,33:255-257.
[17]Popoff M,and M Veron.A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group[J].Gen.Microbiol,1976,94:11-25.
[18]J Michael Janda,Sharon L Abbott.The Genus Aeromonas:Taxonomy,Pathogenicity,and Infec-tion.Clincal Microbiology Reviews,2010,23.1.35-73.
[19]Valera L,and C Esteve.Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas[J].Appl.Microbiol,2002,93:77-95.
[20]Minana Galbis D,M Farfan,J G Loren,and M C Fuste.Biochemical identification and numerical taxonomy of Aeromonas spp.Isolated from environmental and clinical samples in Spain[J]. Appl.Microbiol,2002,93:420-430.
[21]Minana Galbis D,M Farfan,M C Fuste, and J G Loren.Aeromonas molluscorum sp.nov,isolated from bivalve mollusks.Int [J].Syst.Evol. Microbiol,2004,54:2073-2078.
[22]Minana Galbis D,M Farfan,M C Fuste,and J G LorenAeromonas bivalvium sp.nov,isolated from bivalve mollusks.Int [J].Syst.Evol.Microbiol,2007,57:582-587.
[23]Pidiyar V,A Kaznowski,N B Narayan,M Patole,and Y S ShoucheAeromonas culicicola sp.nov, from the midgut of Culex quinquefasciatus.Int[J].Syst.Evol Microbiol,2005,2:1723-1728.
[24]Huys G,M Cnockaert,and J Swings.Aeromonas culicicola Pidiyar et al.2002 is a latter subject-tive synonym of Aeromonas veronii Hickman Brenner et al.1987.Syst. Appl.Microbiol,2005, 28:604-609.
[25]Pidiyar V,A Kaznowski,N B Narayan,M Patole,and Y S Shouche.Aeromonas culicicola sp.nov, from the midgut of Culex quinquefasciatus.Int.Int[J].Syst.Evol.Microbiol,2002,52:1723-1728.
[26]Kupfer M,P Kuhnert,B M Korczak,R Peduzzi,and A Demarta.Genetic relationships of Aeromo-nas strains inferred from 16S rRNA, gyrB and rpoB gene sequences.Int. [J].Syst. Evol. Micr-obiol,2006,56:2743-2751.
[27]Soler L,M A Yanez,M R Chacon,M G Aguilera Arreola,V Catalan,M J Figureras, and A J Martinez Murcia.Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.Int[J].Syst.Evol.Microbiol,2004,54:1511-1519.
[28]Dubnau D, I Smith,P Morell,et al.Gene conservation in Bacillus species.I.Conserved genetic and nucleic acid base sequence homologies.Proc Natl Acad Sci USA,1965,54(2):491-498.
[29]Woese C R.Bacterial evolution[J].Microbiol Rev,1987,51(2):221.
[30]Olsen GJ,Woese CR.Ribosomal RNA:a key to phylogeny[J].Faseb,1993,7:113-123.
[31]Rikihisa Y,Kawaham M,Wen B.Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA sequences of H.murs,H.felis and Eperythrozoon suis[J].Clin Micro-biol,1997,35(4):823-829.
[32]Rappe MS,Giovannoni SJ.The uncultured microbial majority[J].Annu Rev.Microbiol,2003,57: 369-94.
[33]Chakravorty S,D Helb,M Burday,N Connell,and D Alland.A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria[J].Microbiologic Methods,2007, 69:330-339.
[34]Ragnhild Wilk,Erko Stackebrandt.Classification of Fish-Pathogenic Vibrios Based on Compar-ative 16S rRNAAnalysis[J]. IJSEM July 1995 vol.45(3):421-428.
[35]Borrell N,S G Acinas,M J Figueras.Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes[J].Clin.Microbiol,1997, 35(7):1671-1674.
[36]Janda J M,and S L Abbott. Evolving concepts regarding the genus Aeromonas:an expanding panorama of species,disease presentation,and unanswered questions.Clin.Infect.Dis,1998,27(2): 332-344.
[37]Mc Mahon M A S,and I G Wilson.The occurrence of enteric pathogens and Aeromonas species in organic vegetables.Int[J].Food Microbiol,2001,70:155-162.
[38]Austin B,and C Adams.Fish pathogens,1996,p.197-243.In B Austin,M Altwegg,P J Gosling,and S Joseph(ed.),The genus Aeromonas.JohnWiley&Sons Ltd,West Sussex,England.
[39]Joseph S W,and A Carnahan.The isolation,identification,and systematics of the motile Aeromo-nas species.Annu.Rev.Fish Dis,1994,4:315-343.
[40]http://www.promedmail.org/pls/otn/f? P=2400:1202:959209208141620::NO::F2400_P1202_CHECK_DISPLAY,F2400_P1202_PUB_MAIL_ID:X,18797.
[41]Michael,Sharon.The Genus Aeromonas:Taxonomy,Pathogenicity,and Infection[J].Clincal Micr-obiology Reviews,Jan,2010,p.35-73.
[42]Leclerc H,L Schwartzbrod,and E Dei Cas.Microbial agents associated with waterborne diseases. Crit.Rev.Microbiol,2002,28:371-409.
[43]Isonhood J H,and M Drake.Aeromonas species in foods[J].Food Prot,2002,65:575-582.
[44]Palumbo S A,F Maxino,A C Williams,R L Buchanan,and D T W Thayer. Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl. Environ. Microbiol,1985,50: 1027-1030.
[45]Borrell N,M J Figueras,and J Guarro.Phenotypic identification of Aeromonas genomespecies from clinical and environmental sources.Can[J].Microbiol,1998,44:103-108.
[46]Neyts K, G Huys, M Uyttendaele,J Swings, and J Debevere.Incidence and identification of mesophilicAeromonas spp.from retail foods.Lett.Appl. Microbio,2000,31:359-363.
[47]Rusin P A,J B Rose,C N Haas,and C P Gerba.Risk assessment of opportunistic bacterial path-ogens in drinking water.Rev.Environ.Contam.Toxicol,1997,152:57-83.
[48]Holmes P,L M Niccolls,and D P Sartory.The ecology of mesophilic Aeromonas in he aquatic environment,1996,p.127-150.In B Austin,M Altwegg, P J Gosling,and S Joseph (ed.),The genus Aeromonas.John Wiley &Sons Ltd,West Sussex,England.
[49]Fiorentini C,E Barbieri,L Falzano.Occurrence,diversity and pathogenicity of mesophilicAero-monas in estuarine waters of the Italian coast of the Adriatic Sea[J].Appl.Microbiol,1998,85: 501-511.
[50]Hazen T C,C B Fliermans,R P Hirsch,and G W Esch. Prevalence and distribution of Aeromonas hydrophila in the United States.Appl.Environ.Microbiol,1978,36:731-38.
[51]Hazen T C,and C B Fliermans.Distribution of Aeromonas hydrophila in Natural and man-made thermal effluents.Appl.Environ.Microbiol,1979,38:166-168.
[52]http://www.who. int/water_sanitation_health/dwq/guidelines/en/index.html.
[53]http://permanent.access.gpo.gov/lps21800/www.epa.gov/safewater/ccl/cclfs.html.
[54]Khardori N,and V Fainstein. Aeromonas and Plesiomonas as etiological agents[J].Annu.Rev. Microbiol,1988,42:395-419.
[55]Kelly K A,J M Koehler,L R and Ashdown.Spectrum of extraintestinal disease due to Aeromon-as species in tropical Queensland,Australia.Clin.Infect.Dis,1993,16:574-579.
[56]Llopis F,I Grau,F Tubau,M Cisnal.Epidemiological and clinical characteristics of bacteraemia caused by Aeromonas spp.as compared with Escherichia coli and Pseudomonas aeruginosa. Scand[J].Infect.Dis,2004,36:335-341.
[57]Bossi Kupfer M,A Genini,R Peduzzi,and A Demarta.Tracheobronchitis caused by Aeromonas veronii biovar sobria after near drowning[J].Med.Microbiol,2007,56:1563-1564.
[58]Centers for Disease Control and Prevention. Aeromonas wound infections associated with outdoor activities-California.Morb.Mortal.Wkly.Rep,1990,39:334-335.
[59]Angel M F,F Zhang,M Jones,J Henderson,and S W Chapman.Necrotizing fasciitis of the upper extremity resulting from a water moccasin bite.South.Med[J].2002,95:1090-1094.
[60]Kunimoto D,R Rennie,D M Citron,and E J C Goldstein.Bacteriology of a bear bite wound to a human:case report[J].Clin.Microbiol,2004,42:3374-3376.
[61]Lamy B,A Kodjo,the colBVH Study Group,and F Laurent.Prospective nationwide study of Aeromonas infections in France[J].Clin.Microbiol,2009,47:1234-1237.
[62]Ceylan E,M Berktas,and Z Agaoglu.The occurrence and antibiotic resistance of motile Aerom-onas in livestock.Trop.Anim.Health Prod,2009,41:199-204.
[63]Moro E M P,R D N Weiss,R S C Friedrich.Aeromonas hydrophila isolated from cases of bovine seminal vesiculitis in south Brazil[J].Vet.Diagn.Invest,1999,11:189-191.
[64]Gosling P J.Aeromonas species in disease of animals,1996,p.175-195.In B Austin,M Altwegg, P J Gosling, and S Joseph (ed.),The genus Aeromonas. John Wiley& Sons Ltd,West Sussex, England.
[65]Thorton S M,S Nolan,and F M Gulland.Bacterial isolates from California sea lions (Zalophus californicus),harbor seals(Phoca vitulina),and northern elephant seals (Mirounga angustirostris) admitted to a rehabilitation center along the central Calif ornia coast[J],1994-1995. Zoo. Wildl. Med,1998,29:171-176.
[66]Josenhans C,S Suerbaun.The role of motility as a virulence factor in bacteria[J].Int Med Micr-obiol,2002,291:605-614.
[67]Berg H C.The rotary motor of bacterial flagella.Annual Rev Biochem,2003,72:19-54.
[68]Rocio Canals,Maria Altarriba, Silvia Vilches,Gavin Horsburgh.Analysis of the Lateral Flagellar Gene System of Aeromonas hydrophila AH-3[J].Bacteriol,2006,188(3):852-862
[69]Sleytr U B.Regular arrays of macromolecules on bacterial cell walls:structure, chemistry, asse-mbly and function[J].Int.Rev.Cytol,1978,53:1-64.
[70]T J Beveridge,L L Graham. surface layers of bacteria. Microbiol Rev.1991,12,55(4):684-705
[71]Boot HJ and PH Pouwels.Expression,secretion and antigenic variation of bacterial S-layer protein[J].Mol Microbiol,1996,21 (6):1117-1123.
[72]H Chart,D H Shaw,E E Ishiguro,and T J Trust.Structural and immune chemical homogeneity of Aeromonas salmonicida lipopolysaccharide[J].Bacteriol,1984,158:16-22.
[73]Sleyt U B, Messner P .Crystalline bacterial cell surface layers[M]. Adv.Microb Physiol,1992, 23:213-275.
[74]B Uwe.Sleytr,Paul Messner,Dietmar Pum,Margit Sara.Crystalline bacterial cell surface layers[J].Mole Micro,1993,10,911-916.
[75]Koval S F,S H Hynes.Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus[J].Bacteriol,1991,173(7):2244-2249.
[76]Blaser M J,P F Smith,J E Repine and K A Joiner.Pathogenesis of Campylobacter fetus infections.Failure of encapsulated Campylobacter Fetus to bind C3b explains serum and phagocytosis resistance[J].Clin.Invest,1988,81 (5):1434-1444.
[77]Rafaela A Garduno and W W Kay.Interaction of the fish pathogen Aeromonas salmonicida with rainbow trout macrophages[J].Infect.Immun,1992,60(11):4612-4620.
[78]Trust T J,W W Kay and E E Ishiguro.Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida[J].Curr.Microbiol,1983,9:315-318.
[79]Sleytr U B, M Sara.Bacterial and archaeal S-layer proteins:structure function relationships and their biotechnological applications[J].Trends Biotechnol,1997,15:20-26.
[80]Sleytr U B,P Messner,D Pum.Immobilised Macromolecules:Application Potentials.1993,26, 212.
[81]Messner P, F M Unger, U B Sleytr.Vaccine development based on Slayer technology.In:Cryst-alline Bacterial Cell Surface,1996.
[82]Laurence Abrami,Marc Fivaz.Sensitivity of Polarized Epithelial Cellstothe Pore-Forming Toxin Aerolysin[J].Infect.Immun,2003,71.2.739-746.
[83]Oliver Knapp,Bradley Stiles,Michel R Popoff.The Aerolysin Like Toxin Family of Cytolytic, Pore-Forming Toxin[J].The Open Toxinology Journal,2010,3,53-68.
[84]Szcesny p,Iacovache.Extending the aerolysin the family:from bacteria to vertebratea.PLoS One 2011,6(6):e20349.
[85]Oliver Knapp,Bradley Stiles.The Aerolysin-Like Toxin Family of Cytolytic,Pore-Forming Toxins[J].The Open Toxinology,2010,3,53-68.
[86]Bischofberger M, M R Gonzalez.Membrane injury by pore forming proteins[J].Curr Opin Cell Biol,2009,21:589-595.
[87]Rosado C J,A M Buckle,R H P Law,et al.A common fold mediates vertebrate defense and bacterial attack[J].Science,2007,317:1548-1551.
[88]Hadders M A,Beringer D X,Gros P.Structure of c8a-MACPF reveals mechanism of membrane attack in complement immune defense[J].Science,2007,317:1552-1554.
[89]Melton J A,Tweten J M.In:Alouf J E,Popoff M R,Eds.The comprehensive sourcebook of bacterial protein toxins.3rded.London:Academic Press,2006,pp.623-630.
[90]Sousa M V,Richardson M,Fontes W,Morhy L.Homology between the seed cytolysin enterolobin and bacterial aerolysins[J].Protein Chem,1994,13:659-667.
[91]Cole AR,Gibert M,Popoff M,Moss DS, Titball RW, Basak AK.Clostridium perfringens epsilon toxin shows structural similarity to the pore-forming toxin aerolysin[J].Nat Struct Mol Biol, 2004,11(8):797-798.
[92]Mancheno J M,Tateno H,Goldstein I J,Martinez Ripoll M,Hermoso J A.Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars[J].Biol Chem, 2005,17251:17259.
[93]Akiba T,Abe Y,Kitada S,et al.Crystal structure of the parasporin 2 Bacillus thuringiensis toxin that recognizes cancer cells[J].Mol Biol,2009,386:121-133.
[94]Caselitz F H and R G Unther.Hemolysin studien mit Aeromonas stammen[J].Zentralbl. Bakteriol.Abt.I.Orig,1960,180:30-38.
[95]Wretlind B,R Mollby and T Wadstrom. Separation of two hemolysins from Aeromonas hydrophila by isoelectric focusing[J].Infect.Immun,1971,4(4):503-505.
[96]Wretlind B,L Hedean,and T Wadstroum.Formation of extracellular haemolysin by Aeromonas hydrophila in relation to protease and staphylolytic enzyme[J].Gen. Microbiol,1973,78(1):57-65.
[97]Bernheimer A W,and L S Avigad.Partial characterization of aerolysin, a lytic exotoxin from Aeromonas hydrophila[J].Infect.Immun,1974,9(6):1016-1021.
[98]Buckley JT,LN Halasa,KD Lund and S Maclntyre.Purification and some properties of the hemolytic toxin aerolysin.Can[J]. Biochem,1981,59(6):430-435.
[99]Howard S P,and J T Buckley.Activation of the hole-forming toxin aerolysin by extracellular processing[J].Bacteriol,1985,163(1):336-340.
[100]Howard S P and J T Buckley.Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila.Mol.Gen.Genet, 1986,204(2):289-295.
[101]Vander Goot F G,Pattus F,Wong K R,Buckley J T.Oligomerization of the channel forming toxin aerolysin precedes insertion into lipid bilayers[J].Biochemistry,1993,32(10):2636-2642.
[102]Vander Goot FG,Lakey J,Pattus F,et al.Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin:identification of the site of proteolytic activation[J].Bioch-emistry,1992,31 (36):8566-8570.
[103]Yeongjin Hong,Kazuhito Ohishi.Requirement of N-gly-can on GPI-anchored proteins for efficie-nt binding of aerolysin but not Clostridium septicum α-Toxin[J].The Embo Journal,2002,21, 5047-5056.
[104]Fukushima K L,Kehara Y,Kanai M.A β-N-Acetylglucosaminyl Phosphate Diester Residue Is Attached to the Glycosylphosphatidylinositol Anchor of Human Placental Alkaline Phosphatase [J].Biological Chemistry Vol,2003,278:36296-36303.
[105]Parker M W,Feil S C.Pore-forming protein toxins:from structure to function[J].Prog Biophys Mol Biol,2005,88(1):91-142.
[106]Krause K H, M Fivaz, A Monod.Aerolysin Induces G-protein Activation and Ca2+ Release from Intracellular Stores in Human Granulocytes[J].Biological Chemistry 1998,273,29,18122-18129.
[107]Nelson K L,Brodsky R A,Bucky J T.Channels formed by subnanomola reconcentrations of the toxin aerolysin trigger apoptosis of T lymphomas[J].Cell Microbiol,Jul,1999,1(1):69-74.
[108]Brodsky R A,Mukhina G L,Nelson K L,Lawrence T S, Jones R J,Buckley J T.Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin [J].Blood,1999,93(5):1749-1756.
[109]Rita Brenden,J M Janda.Detection,quantitation and stability of the β haemolysin of Aeromonas spp[J].Med Microbiol,1987,24(3):247-251.
[110]Tsutomu Aaao,Yoshio Kinshita.Purification and Some Properties of Aeromonas hydrophila Hemolysin[J].infection and immunity,1984,46(l):122-127
[111]Hirono I,Aoki T.Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila[J].Micro Pathog.1991,11(3):189-197.
[112]Michael E Reith,Rama K Singh,Bruce Curtis,Jessica M Boyd.The genome of Aeromonas salmonicida subsp.salmonicida A449:insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9:427
[113]Epple H J, Mankertz J R,et al.Aeromonas hydrophila Beta Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells(HT-29/B6).Infect Immun,2004,72 (8):4848-4858.
[114]Hage C M,Chahine,G Del Giudice,P H Lambert,and J C Pechere.Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independ-ent antigens[J].Infect Immun,April,1992,60(4):1415-1421.
[115]Siritat T,Intuseth J,Chanphone J,et al.Characterisation of Aeromonas hydrophila extracellar prodcts with reference to toxicity,virulence, protein profiles and antigencity[J].Asian Fisheris Science,1999,12:371-379.
[116]Christopher Y F W,Michael W H and Robert L P F.Inactivation of two haemolytic toxin genes in Aeromonas hydrophla attenuates virulence in a suckling mouse model[J]. Microbiology, 1998,144(2):291-298.
[117]Moral C H,Castillo E F D.Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophicaroA mutant as a live attenuated vaccine[J].Infect and Immun,1998,66(5):1813-1821.
[118]T C Hsu,W D Waltman, and E B Shots.Correlation of extracellular enzymatic activity and biochemical characteristics with regard to virulence of Aeromonas hydrophila.Dev.Biol.Stand. 1981.49:101-111.
[119]K Y.R M WStevenson,.Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish[J].Infect.Immun,1988,56(10):2639-2644.
[120]K Y Leung, R M W Stevenson.Characteristics and distribution of extracellular proteases from Aeromonas hyrophila[J].Microbiology,1988,134:151-160.
[121]Kanai K,Wakabayashi H.Purification and some proterties of protease from Aeromonas hydrophila[J].Bulletin of the Japanese Society of Scientific Fisheries,1984,50(8):1367-1374.
[122]Nieto T P,Ellis.Characterization of extracellular metallo and serine proteases of Aeromonas hydrophila strain B51[J] Journal of General Microbiology,1986,132(7):1975-1979.
[123]Chabot D J,Thuner L.Protease of the Aeromonas hydrophila complex:identification, characte- rization and relation to virulence in channel catfish,lctaulrus pantatus(Rafinesque)[J].Journal of Fish Disease,1991,14(2):171-183.
[124]Alberto cason.Javier yugueros,Alandro Temprano.A Major secreted elastase is essential for pathogenicity of Aeromonas hydrophila[J].Infection and Immunity,2000,68(6):3233-3241.
[125]Chang TM,Liu C C,Chang M C.Cloning and sequence analysis of the gene(eprA1)encoding an extracellular protease from Aeromonas hydrophila[J].Gene,1997,199:225-229.
[126]Rivero O,Anguita J,Paniagua C.et al.Molecular cloning and characterization of an extracellu-lar protease gene from Aeromonas hydrophila[J].Journal of Bacteriology,1990,172:3905-3908.
[127]Kanai K,Wakabayshi H.Purification and some proterties of protease from Aeromonas hydrophila[J].Bulletin of the Japanese Society of Scientific Fisheries,1984,50(8):1367-1374.
[128]Thune R L,Grahanm T E.Extracellular protease from Aeromonas hydrophila:Partial purifica-tion and effects on age-0 shannel catfish[J].Transaction of the American Fisheries Society, 1982,111:749-754.
[129]Allan B J, Stevenson R M W.Extracellular virulence factors of Aeromonas hydrophila in fish infections[J].Canadian Journal Microbiology,1981,27(10):1114-1122.
[130]Harwood J.The versatility of lipases for industrialuses.Trends Biochem.Sci.1989.14:125-126.
[131]Hilton S, J T Buckley.Studies on the reaction mechanism of a microbial lipase acyltransferase using chemical modification and site-directed mutagenesis[J].Biol.Chem,1991,256:997-1000.
[132]Hilton S,W D Me Cubbin,C M Kay,and J T Buckley.Purification and spectral study of a microbial fatty acyltransferase:activation by limited proteolysis.Biochemistry,1990,29(38): 9072-9078.
[133]Hedstrm S A,and P Nilsson.Lipolytic activity of Staphylococcus aureus strains from cases of human chronic osteomyelitis and other infections[J].Acta Pathol.Microbiol.Scand.Sect.1975, 83(3):285-292.
[134]Rollof J,J H Braconier and P Nilsson Ehle.Interference of Staphylococcus aureus lipase with human granulocyte function[J].Eur.Clin.Microbiol.Infect.Dis,1988,7(4):505-510.
[135]Eftimiadi C,E Buzzi,M Tonetti,P Buffa,D Buffa,M T J van Steenbergen,J Graaff, and G A Botta. Short-chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide[J].Infect,1987,14(l):43-53.
[136]Hewley H,and G Gordon.The effects of long chain free fatty acids on human neutrophil function and structure[J].Lab.Invest,1976,34(2):216-222.
[137]武寒雪,祝建波等,细菌脂肪酶基因的生物信息学分析[J].江苏农业学报,2009,25(4)：942-944.
[138]Chakraborty T,M A Montenegro,S C Sanyal.Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotoxic enterotoxin[J].Infect. Immun,1984,46(2):435-441.
[139]S P Howard,S Macintyre,J T Buckley.The genus Aeromonas,1996,267-286.In B Austin,M Altwegg,P J Gosling,and S Joseph (ed.),Toxin.John Wiley and Sons, Singapore.
[140]Leung K Y,and R M Stevenson.Tn5-induced protease-deficient strains of Aeromonas hydrop-hila with reduced virulence for fish[J].Infect.Immun,1988,56(10):2639-2644.
[141]Nieto T P,Y Santos,L A Rodriguez, A E Ellis.An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish[J].Microb.Pathog,1991,11:101-110.
[142]Cornelis G R,and F Van Gijesem.Assembly and function of type III secretory systems[J]. Annu.Rev.Microbiol,2000,54:735-774.
[143]Grizzle J M,Kiry U Y.Histopathology of gill,liver and pancreas and serum enzyme levels of channel catfish infected with Aeromonas hydrophila complex[J].Journal of Aquatic Animal Health,1993,5(1):36-50.
[144]Cruze Silvap,Grazina Freitas S.Parasites and parasitoses of the European eel(Anguilla japonica L)in Poaugal[J].Bull Soc Franc Parasitol,1990,8(S1):287.
[145]马国文,温海深,刘振歧,等.鲤鱼竖鳞病病原和病理研究[J].水利渔业,1999,19(3)：37-40.
[146]Hanninen M L,Samalmis S,Mattila L,et al.Association of Aeromonas spp.with travellers diarrhoea in Finland[J] Journal of Medical Microbiology,1995,42(l):26-32.
[147]Angka S L,Lam T J,Siny M.Some virulence characteristics of Aeromonas hydrophila in walking catfish(Clarias gariepinus)[J].Aquaculture,1995,130(2/3):103-112.
[148]Baloda S B,Krovacek K,et al.Detection of aerolysin gene in Aeromonas strains isolated from drinking water,fish and foods by polymerase chain reaction[J].Comparative Immunology Microbiology and Infection Disease,1995,18(1):17-26.
[149]Vadiveu I J.Puthuheary S D,Phpps M,et al.Possible virulence factors involved in bacteraemia caused by Aeromonas hydrophila[J]. Journal of Medical Microbiology,1995,42(3):171-174.
[150]Thomas S R,TrustR T J.Tyrosine phosphorylation of the tetragonal paracrystalline array of Aeromonas hydrophila:Molecular cloning and high level expression of the S-layer protein gene[J].Journal of Molecular Biology,1995,245(5):568-581.
[151]陈怀青,陆承平,陈琼,等.用点酶法检测鱼类致病性嗜水气单胞菌HEC毒素[J].动物检疫,1993,10(4)：7-9.
[152]EurellT E,Lewis D H,Grumblel L C.Comparison of selected diagnostic tests for detection of motile aeromonas septicemia in fish[J].American Journal of Veterinary Research,1978,39(8): 1384-1386.
[153]李焕荣,陈怀青,陆承平,等.嗜水气单胞菌胞外蛋白酶的检测[J].水生生物学报,1997,21(1)：97-100.
[154]Gruber H J,WilmsonH U,et al.Partial purification of the rat erythroeyte receptor for the chanel forming toxin aerolysin and reconstitution into planar lipid bilayers[J].Molecular Microbiology, 1994,14(5):1093-1101.
[155]Pollard D R,Johnson W M,Lior H.Detection of the aerolysin Gene in Aeromonas hydrophila by PCR[J].Clinical Microbiology,1990,28(11):2477-2481.
[156]R Y C Kong, S K Y Lee, T W F Law.Rapid detection of six types of bacterial pathogens inmaine waters by multiplex PCR[J].Water Raserch,2002,36:2802-2812.
[157]Alberto Casson,Juan Angutta,,et al. Identification of Aeromonas hydrophila hybridization group 1 by PCR assays[J].Applied and Environmental Microbio logy,1996,62(4):1167-1170.
[158]Micheal W H.ChristopherY F.Distribution of two hemolytic toxin genes in clinical and environm-ental isolates of Aeromonas spp:correlation with virulence in a suckling mouse model[J].FEMS Microbiology Letters,1999,174(1):131-136.
[159]Gonzlez O,Serano C J,Santos J A.Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case[J].Journal of App lied Microbiol ogy,2002,93(3):414-419.
[160]朱大玲,李爱华,汪建国,等.嗜水气单胞菌毒力与毒力基因分布的相关[J].中山大学学报：自然科学版,2006,45(1)：82-85.
[161]宋铁英,包晓东,郑伟文.中华鳖爆发性传染病研究--Ⅳ不同生化类型嗜水气单胞菌aer基因的PCR比较[J].江西农业大学学报：自然科学版,2002,24(2)：164-167.
[162]吴淑勤.鳗鲡混合感染嗜水气单胞菌和迟缓爱德华氏菌的快速诊断[J].鱼类病害研究,1993,3：40-42.
[163]高汉娇.水库暴发性鱼病及其防治技术研究2：直接荧光抗体法对白鲢病原菌的鉴定[J].水利渔业,1995,2：11-13.
[164]陈昌福.用直接荧光抗体和细菌培养法对中华鳖体内的嗜水气单胞菌的检测[J].华中农业大学学报,1998,17(3)：264-266.
[165]李卫军.应用间接荧光抗体法检测鱼类嗜水气单胞菌的试验研究[J].中国动物检疫,1998,15(4)：5-6.
[166]钱冬,陈月英.应用酶联免疫吸附实验检测暴发病病原-嗜水气单胞菌的研究[J].水产养殖,1993,4：14-17.
[167]陈怀青,陆承平,陈琼,等.用点酶法检测鱼类致病性嗜水气单胞菌HEC毒素[J].动物检疫,1993,10(4)：7-9.
[168]沈素芳,储卫华,陆承平.鱼类致病性气单胞菌诊断试剂盒的研制[J].中国兽医学报,2000,20(5)：462-464.
[169]刘颖.应用DOT-ELISA技术快速检测鱼类细菌性疾病病原的研究[J].内陆水产,1998,1：6-7.
[170]Anderson D P.Fish immunology[M].T F H public company limited,1974,296.
[171]Ward P D.The Development of Bacterial Vaccines for Fish[M].ROBERTS R J.Microbial Disease of Fish.Ed.London:Academic Press,1982:47-58.
[172]Amend D F,Johnson K A.Evidence for lack of antigenic competition among various combinat-ions of Vibrio anguillarum, Yersinia ruckeri,Aerolmonas salmonicida and Renibacterium salmoninarum bacterins when administered to salmonid fishes[J]. Journal of Fish Disease,1984, 7(4):293-299.
[173]Thune R L,PlumbJ A.Efect of delivery method and antigen preparation on the production of antibodies against Aeromonas hydrophila in channel catfish[J].Prog Fish Cult,1982,44:53-54.
[174]Ruangpan L,Kitao,Yoshida T. Protective efficacy of Aeromonas hydrophila vaccines in nileti-laph[J]. Veterinary Immunology Immunopathology,1986,12:345-350.
[175]Loghoth Oesis P N.Immune response of rainbow trout(Oncorhynchus mykiss,Walbaum) to Aer-omonas hydrophila[J].Fish and Shellfish Immunology,1994,4:239-254.
[176]Shieh H S.Correlation between cell virulence and protease toxicity of Aeromona salmonicida isolates[J].Microbiology letter,1985,28:69-72.
[177]Ford L A,Thune R L.Immunization of channel catfish with a crude, acid extracted preparation of motile aeromonad S-layer protein[J]. Biome dical Letters,1992,47:355-362.
[178]Adams A,ThompsonH K D.Fish Vaccination Development. FAO Manual for the production and quality control of veterinary vaccine for use in developing countries[J].FAO Animal Produ-ction and Health Series,1997(35).
[179]Leszer.GUZ,A licja Kozinskal.Antibiotic susceptibility of aeromonas hyrophilad and A.sobria isolated from carp(cyprinuscarpiol)[J].bullvet inst pulawy,2004,48,391-395.
[180]T Wahil, S E Burr.Aeromonas sobria,a causative agent of disease in farmed perch, Perca fluviatilis L[J].Fish Dis.2005,28(3):141-150.
[181]ya li,Hu Cai.Identification and Pathogenicity of Aeromonas sobria on Tail rot Disease in Juvenile Tilapia Oreochromis niloticus [J].Current Microbiology 62,623-627.
[182]McGarey DJ,Milanesi L,Foley DP,Reyes B Jr.The role of motile aeromonads in the fish disease, ulcerative disease syndrome (UDS).Experientia.1991,May,15,47(5):441-4.
[183]F.奥斯伯,R.布伦特,R.E金斯顿,等.精编分子生物学实验指南[M].北京：科学出社,2001：39.
[184]冯东岳.2009年我国大宗淡水养殖鱼病害调查及分析[J].水产学杂志,2010,23(4)：60-65.
[185]田雨,王旭,贾光风,等.铁岭市主要养殖区鱼病调查与分析[J].河北渔业,2011,5：33-37.
[186]郭闯,朱国强,王永坤,等.14株患病水生动物气单胞菌属细菌的分离鉴定与最佳治疗药物筛选试验[J].水产科学,2003,22(4)：15-17.
[187]韩英,刘敏,姜艳平.鲤鱼出血病病原的研究和药敏试验[J].东北农业大学学报,2005,36(5)：593-597.
[188]陈爱平,江育林,钱冬,等.淡水鱼细菌性败血症[J].中国水产,2011,3：54-55.
[189]徐海圣,舒妙安.黄鳝温和气单胞菌病病原的分离鉴定及免疫应答[J].中国兽医学报,2003,23(3)：240-245.
[190]胡琳琳,房文红,梁思成,等.金鱼温和气单胞菌的分离鉴定及药敏试验[J].上海水产大学学报,2008,17(3)：285-290,
[191]叶雪平,顾金华,杨广智.牛蛙温和气单胞菌病病原及防治[J].上海水产大学报,2000,9(1)：78-80.
[192]樊海平,吴斌,曾占壮,等.日本鳗鲡体表溃疡病病原菌的分离鉴定及单克隆抗体制备[J].中国水产科学,2009,16(2)：295-302.
[193]王永坤,李志健,朱国强,等.温和气单胞菌和嗜水气单胞菌致病力的比较研究[J].鱼类病害研究,1997,19(2)：18-24.
[194]蔡完其,孙佩芳.罗非鱼温和气单胞菌病的病原研究和药敏试验[J].中国水产科学,2002,9(3)：243-246.
[195]陈翠珍,张晓君,房海.草鱼肠炎的病原菌检验与分析[J].中国兽医科技,1999,29(1)：5-7.
[196]陈怀青,陆承平.家养鲤科鱼爆发性传染病的病原研究[J].南京农业大学学报,1991,14(4)：87-91.
[197]耿毅,汪开毓,吴麟,杜宗君.齐口裂腹鱼败血症的病原分离与鉴定[J].水利渔业.2004,24卷第4期,71-73.
[198]韩继卫,罗文,郑大恒.黄颡鱼温和气单胞菌的分离鉴定与药敏试验绍兴文理学院学报.2011第31卷第9期,19-21.
[199]丁磊,吴康,蔡春芳,等.嗜水气单胞HEC毒素对鲤鱼内皮细胞的毒性[J].中国水产科学,2002,9(2)：117-130.
[200]Tunlid,et al Purification and characterizition of an Extracellular serine protease from the nematode-trapping fungus Arthrobotrysoligo-spore[J].microbiology,1994,140:1687-16952.2.
[201]储卫华,陆承平.嗜水气单胞菌J21株丝氨酸蛋白酶基因克隆与序列分析[J].水产学报,2004,28(1)：84-88.
[202]沈锦玉,李新华,潘晓艺等.温和气单胞菌TL97528株丝氨酸蛋白酶基因片段克隆与序列分析[J].上海海洋大学2010,19.(2)151-156.
[203]张翠娟,于宙亮,田莉瑛,等.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[J].生物工程学报,25(2)：251-256.
[204]潘晓艺,郝贵杰,姚嘉赞,等.嗜水气单胞菌TPS-30株丝氨酸蛋白酶基因与溶血素基因在大肠杆菌中的融合表达.水生生物学报,2010,34(3)：591-597.
[205]赵秀敏,高勤学,陈鸿军,等.嗜水气单胞菌溶血素A表达、免疫原性及活性检测.中国动物传染病学 报,2011,19(1)：45-50.
[206]武斌,于宙亮,于珊,等.人源温和气单胞菌溶血素基因的克隆与表达.河北师范大学学报(自然科学版),2008,32(2)：239-242.
[207]Donovan R S,Robinson C W, Glick B R. Review:optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J].Ind Microbiol. 1996,16(3):145-54.
[208]Harcum SW,Bentley WE.Response dynamics of 26-,34-,39-,54-,and 80-kDa proteases in induced cultures of recombinant Escherichia coli[J].Biotechnol Bioeng,1993,42(6):675-85.
[209]Pack P,Kujau M,Schroeckh V,et al.Improved bivalent miniantibodies, with identical avidity as whole antibodies,produced by high cell density fermentation of Escherichia coli[J]. Biotechnology (N Y),1993,11(11):1271-1277.
[210]Winograd E, Pnlido M A,Wasserman M. Production of DNA-recombinant peptides by tac-inducible vectors using micromolar concentrations of IPTG [J].Biotechniques[J],1993,14: 886-887.
[211]Auger E A,Bennett G N.Temperature optimization of in vivo expression from the E.coli trp and trp:lac promoters[J].Biotechnol Lett 1987,8:157-162.
[212]Kosinski M J,Rinas U, Bailey J E.Isopropyl-13-D-thiogalacto-pyranoside influences the meta-bolism of Escherichia coll[J]. Appl Environ Microbiol,1992,36:782-784.
[213]Surek B,Wilhelm M,Hillen W.Optimizing the promoter and ribosome binding sequence for expression of human single chain mokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response[J].Appl Microbiol Biotech-nol.1991,34:488-494.
[214]Chesshyre J A,Hipkiss A R.Low temperatures stabilize interferon alpha-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli[J].Appl Microbiol Biotechnol,1989,31: 158-162.
[215]Chalmers J J,Kim E,Telford J N,et al.Effects of temperature on Escherichia coli overprodu-cing/3-lactamase or human epidermal growth factor[J]. Appl Environ Microbiol.1990,56: 104-111.
[216]Bentley W E,Mirjalili N,Anderson D C,et al. Plasmid encoded protein:the principal factor of metabolic burden associated with recombinant bacteria[J].Biotechnol Bioeng.1990,35: 668-681.
[217]Bentley W E,Davis R H,Kompala D S.Dynamics of CAT expression in E.coli.Biotechnol Bioeng[J].1991,38:749-760.