【作者】 程方俊；

【导师】 黄建国；

【作者基本信息】 西南大学 ， 农业资源利用， 2012， 博士

【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。更多还原

【Abstract】 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 pathogenicityTwo 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-KAThere 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 gene0.7kb 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 geneA 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 geneA1.9Kb 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 geneA 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 hemolysinIn 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.更多还原