【作者】 周祖涛；

【导师】 毕丁仁； 李自力；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 博士

【摘要】 鸭疫里氏杆菌（Riemerella anantipestifer,RA）病是由鸭疫里氏杆菌引起的雏鸭的一种急性、接触性败血性传染病,是严重危害我国养鸭业的重要传染病之一。鸭疫里氏杆菌血清型复杂,各血清型之间缺乏有效的交叉免疫保护,这给使用全菌灭活疫苗或弱毒疫苗进行该病的免疫防治带来了诸多困难。临床分离菌株的鉴定和血清定型及鸭群鸭疫里氏杆菌抗体的监测,对了解鸭疫里氏杆菌的流行情况和制定有效的免疫计划以及评价疫苗免疫效果具有重要意义。鸭疫里氏杆菌在入侵宿主后,迅速在感染位点定居、繁殖,经血液扩散到全身各组织器官,引起全身多发性浆膜炎并发展为败血症。而目前对鸭疫里氏杆菌如何在宿主体内生存、繁殖和逃避免疫系统监视并致病的分子机制知之甚少。本研究针对我国流行的血清1型鸭疫里氏杆菌的42kD外膜蛋白OmpA进行了克隆、表达和免疫原性研究,探索外膜蛋白OmpA的免疫保护作用及其作为包被抗原建立用于鸭疫里氏杆菌分子流行病学调查的ELISA诊断方法;在此基础上,利用选择性捕获转录序列技术对鸭疫里氏杆菌在感染鸭肝脏基因差异表达进行了研究,为进一步开展了鸭疫里氏杆菌致病分子机理的研究,阐明雏鸭腹腔广泛性浆液渗出的分子机制奠定基础。本研究取得的结果如下:1.鸭疫里氏杆菌平板染色诊断抗原的制备和应用用本实验室鉴定、保存的血清1型鸭疫里氏杆菌云梦分离株（RA-YM）制备了RA平板染色诊断抗原,该抗原与鸭流感、鸭瘟、鸭肝炎、鸭大肠杆菌和鸭沙门氏菌的等5种病原的阳性血清无交叉反应;染色抗原平板凝集试验与琼脂免疫扩散试验（AGID）对RA疫苗免疫鸭的抗体检出率结果表明,在疫苗免疫后12天,平板凝集法即可检测到RA抗体,检出率为30%,AGID在免疫后31天才能检测到抗体,检出率为10%,而平板凝集法的检出率为80%,平板凝集法抗体检出率明显高于AGID,且染色抗原平板凝集试验比AGID的灵敏度高3个滴度;不同批次的平板染色诊断抗原检测的阳性率基本相当,在凝集程度上有较小的差异,表明不同批次平板染色诊断抗原具有良好的重复性。以RA-YM和纯化的鸭IgG为免疫原免疫鸭和兔,制备了鸭抗1型RA和兔抗鸭IgG的高免血清,平板凝集试验检测鸭抗RA-YM高免血清的效价为1:32,兔抗鸭IgG高免血清的琼扩效价为1:64。以制备的鸭抗血清1型RA血清对分离自湖北省不同地区的23株鸭疫里氏杆菌进行血清型鉴定,结果均为血清1型,表明我省目前流行的RA主要是血清1型。将制备的兔抗鸭IgG高免血清纯化后,经辣根过氧化物酶（HRP）标记,制备出特异性强、免疫学活性高的兔抗鸭IgG-HRP,工作浓度为1:4000,为下一步建立一种用于RA流行病调查的间接ELISA诊断方法奠定了基础。2.鸭疫里氏杆菌OmpA基因的克隆、表达及重组OmpA蛋白免疫保护性试验研究根据RA15型CVL110/89株OmpA基因序列（GeneBank登录号:AF104936）合成特异性引物,PCR扩增了RA-YM株OmpA基因,克隆到pMDl8-T,获得重组质粒pT-OmpA并进行了测序;将OmpA克隆到pGEX-KG,构建了重组原核表达质粒pGEX-OmpA,转化E.coli BL21后经IPTG诱导获得高效表达,经SDS-PAGE分析,表达的重组蛋白的分子量为68kD,表达产物主要以包涵体形式存在,Western-blot证实表达产物具有良好的生物学活性。将重组表达的OmpA蛋白以0.5mg、1.0mg、1.5mg三个免疫剂量分别与弗氏完全佐剂乳化后免疫7日龄樱桃谷肉鸭,17日龄以同样剂量加强免疫,二免后8天攻毒（3×10⁶CFU/只）,在攻毒24h后蛋白免疫组和未免疫空白组均发病,动物试验结果表明重组表达的OmpA不能起到保护作用。3.鸭疫里氏杆菌间接OmpA-ELISA检测方法的建立及初步应用用大肠杆菌表达的重组蛋白OmpA建立了RA抗体OmpA-ELISA检测方法,方阵滴定确定了抗原最佳包被浓度为0.81μg/mL,血清最佳稀释倍数为1:160。通过检测84份鸭疫里氏杆菌病阴性血清,确定该方法的阴阳性临界值为0.22。用该方法检测鸭流感,鸭瘟,鸭肝炎,鸭大肠杆菌和鸭沙门氏菌的标准阳性血清OD₄₅₀值均小于临界值0.22,表明该方法与其它病原抗体无交叉反应,特异性强。重复性试验表明OmpA-ELISA检测方法的批内重复和批间重复的变异系数均小于10%。用建立的OmpA-ELISA检测方法和平板凝集分别对213份送检血清进行检测,结果表明OmpA-ELISA检测方法的敏感性为95.77%,二者的符合率为62.91%。将湖北汉川、荆州和武汉市江夏区、黄陂区等地送检的472份鸭血清采用建立的OmpA-ELISA检测方法进行检测,结果表明RA抗体阳性率为64.6%,说明建立的间接OmpA-ELISA检测方法能够很好的应用于临床RA抗体检测。4.应用选择性捕获转录序列技术（SCOTS）筛选鸭疫里氏杆菌在感染鸭肝脏中差异表达的基因的研究根据RAD-24105株16S rRNA基因序列（GeneBank登录号:AY871819）,合成特异性引物,PCR扩增了RA-YM株16S rRNA基因,克隆到pMD18-T,获得重组质粒pT-16SrDNA并进行测序,结果表明克隆的RA-YM株16S rRNA为1479bp,其GeneBank登录号为FJ031240。比对分析Genbank中已公布的表皮葡萄球菌（GeneBank登录号:NC₀02737）,多杀巴氏杆菌（GeneBank登录号:NC₀02663）、大肠杆菌（GeneBank登录号:NC₀00913）和噬二氧化碳单胞菌（GeneBank登录号:AY661855）23S rRNA基因序列的保守区域,利用Primer premier 5.0设计3对引物,分3段分别扩增了23S rRNA基因,克隆到pMD18-T,获得重组质粒pT-23S2、pT-23S1和pT-23S3并进行测序,拼接结果表明克隆的RA-YM株23S rRNA为2655bp,其GeneBank登录号为FJ031241。对生物素标记的RA-YM基因组DNA和构建好的RA-YM核糖体rDNA质粒（pT-16SrDNA,pT-23S-1rDNA和pT-23S-3rDNA）超声破碎,凝胶电泳结果表明破碎产物大小为100-2000bp。以血清1型RA-YM感染9日龄樱桃谷肉鸭,提取感染鸭肝脏的总RNA和TSB培养基中生长的RA-YM的总RNA,经SuperScript^TMⅢRTase反转录,Klenow酶合成cDNA第二链后,记为体内感染cDNAs（in vivo cDNAs）和RA-YM体外培养cDNAs（in vitro cDNAs）。对in vivo cDNAs和in vitro cDNAs进行3轮SCOTS,得到均一化的RA-YM在体内感染时转录的cDNAs和在TSB培养时转录的cDNAs,通过3轮差异杂交选择性捕获RA-YM在感染鸭肝脏中差异表达的基因,将RA-YM在感染鸭肝脏中差异基因的PCR产物克隆到pMD18-T载体,转化E.coli DH5α感受态细胞,通过PCR和斑点杂交,从文库中筛选出187个差异克隆,经序列测定和比对分析获得了48个RA-YM在感染鸭肝脏中差异表达的基因。Real-time RT-PCR对甘氨酸裂解酶T亚基,尿黑酸1,2-加氧酶,天冬氨酸转氨酶,触酶,磷酸盐转运蛋白,二肽肽酶Ⅳ,肽酶M28,和RA46等8个基因的差异表达情况进行验证,结果表明与生长于TSB培养基RA的基因表达水平相比,这8个基因在RA感染过程中的表达水平上调1.44-4.62倍,t-检验分析表明8个基因的表达水平差异显著。根据基因功能,将48个基因分为六类:合成与代谢,适应性调节,应激,转运系统,蛋白酶和5个未知功能序列。本研究初步揭示了RA在感染鸭肝脏的基因表达情况,获得的差异表达基因对研究RA在宿主体内的存活、增殖及持续感染具有重要的作用。本研究筛选到二肽肽酶Ⅳ（Dipeptidyl peptidaseⅣ,DPPⅣ）可能是RA重要的毒力因子。DPPⅣ是一种丝氨酸蛋白酶,能特异性水解氨基端第二位是脯氨酸的多肽。牙龈卟啉单胞菌的DPPⅣ能够降解结缔组织以及介导牙龈卟啉单胞菌与纤粘蛋白的粘附,缺失dppⅣ基因的牙龈卟啉单胞菌的毒力明显降低。格氏链球菌的DPPⅣ可以酶解P物质,协同胞外精氨酸蛋白酶酶解缓激肽,导致血管通透性的变化及感染的内皮组织平滑肌的收缩。雏鸭感染鸭疫里氏杆菌后,主要病理变化是全身各组织器官的浆膜面出现广泛性的纤维素性渗出,RA感染严重损伤了雏鸭的血管内皮系统,导致血管通透性的增高。因此,二肽肽酶Ⅳ可能是RA关键的毒力因子。下一步研究将克隆、表达和缺失RA的dppⅣ基因,以及利用酵母双杂交系统筛选和鉴定与DPPⅣ互作的宿主蛋白,为进一步研究dppⅣ对RA的毒力以及RA的致病机理奠定基础。更多还原

【Abstract】 The Riemerella anatipestifer infection is one of the most important diseases to duck industry throughout the world,which usual occurs as an acute,contagious,septic disease in ducks.Infected ducks have fibrinous pericarditis,perihepatitis,meningitis and peritonitis.R.anatipestifer causes increased mortalities,decreased weight gains,and increased condemnations at slaughter and is estimated to cause substantial economic losses to the commercial duck industry each years.R.anatipestifer have complex serotypes,among which lack of effective cross-protection,leading to the difficulty of using inactivated bacterins,live or cell-free filtrates vaccines to immunize ducks against infection.Characterizaton of the field isolates and differentiation of theirs serotype, respectively,and monitoring of antibodies against R.anatipestifer,which play a very important role in understanding epidemic status of R.anatipestifer and formulating immunized procedure and evaluating immunized effect.R.anatipestifer firstly causes local inflammation at the site of entry,then rapidly break through the defensive organizations into blood.After the proliferation in blood,R.anatipestifer located mainly in serous membrane all over the body,cause inflammation in here and result in septicemia. Howerer,at present,little is known about the survive and multiply while avoiding the host immune system within a host organism of R.anatipestifer during infection.Therefore,we explored that development of OmpA-ELISA detecting antibodies against R.anatipestifer and differential expressed genes of R.anatipestifer in infected duck livers by selective capture of transcribed sequence technique.The research contents are summarized as follows:1.Preparation and preliminary of stain plate agglutination disgnostic antigen of R. anatipestiferThe serotype 1 field isolate,R.anatipestifer RA-YM was used to prepare diagosis antigen of stain plate agglutination.The results showed that has a good specificity, sensitivity and repeatability.It has no cross-reaction with five cluck diseases sera,and titer dectected by stain plate agglutination is higher than agar immune diffusion.This work showed stain plate agglutination could be used for antibodies detection and diagnosis for R.anatipestifer,which will play an active role in prevention and control the R. anatipestifer disease.Ducks were immunized with serovar 1,R.anatipestifer RA-YM emusified with Frund’s adjuvant,and sera of duck against RA-YM were acquired after four inoculations,and the titers of plate agglution reach 1:32.Twenty-three R. anatipestifer isolated from Hubei province were identificated by antisera prepared above, the result showed thet are serovar 1.At the same time,purified serum of rabbit anti-duck IgG（AGID:1:64） from rabbit immunized with purified duck IgG was labeled with horseradish peroxidase（HRP）.The conjugate is characteristic of high activity of immunity and its work concentration reached 1:4000.This laid a foundation for the development of indirect enzyme-linked immunosorbent assay（ELISA） for R. anatipestifer.2.Cloning,expression of the OmpA gene of Riemerella anatipestifer and protection test of rOmpA in ducksThe OmpA gene of RA-YM was amplified by PCR technique and cloned into pMD 18-T,and recombinant plasmid of pT-OmpA was sequenced by Sanger’s sequencing techqique.Then,the OmpA from pT-OmpA was inserted into the pGEX-KG vector,to yield the recombinant plasmid pGEX-OmpA.After induced by IPTG,a high expression of fusion proteins was obtained.SDS-PAGE analysis showed that the fusion proteins was 68 kD in size.rOmpA existed mainly in form of inclusion bodies and was specific to antisera against R.anatipestifer by western blot analysis.The rOmpA was used to immunize ducks,the result showed rOmpA couldn’t protect vaccinated ducks from challenge with RA-YM.3.Development and preliminary of OmpA-ELISA diagnostic methodThe OmpA-ELISA was developed using expressed rOmpA in E.coli.The suitable concentration of coated antigen,0.81μg/mL,and the serum dilutions,1:160,were selected by checkerboard.The cut-off value determined by testing 84 negative sera was 0.22.In the analysis of indirect ELISA cross specificity,it revealed a negative reaction with positive sera of duck influenza,duck hepatitis,duck plague,s.anatum,E.coli(O₇₈),serum of duck uninfected with R.anatipestifer and a positive reaction with standard positive serum of or R.anatipestifer.In order to know its reliability,a repetitive experiment was conducted in different ELISA in the same time and in the different time that result in less than 10%variable coefficient of absoprotion.To evaluate the specificity and sensitivity of the OmpA-ELISA,213 field sera samples were collected from chickens.Using flat plate agglutination test as the reference methods,the sensitivity of the OmpA-ELISA was 95.77%and coincidence 62.91%.Then a total of 472 duck sera samples were axamined that were sent from several field farms such as Hubei e.t.The results above indicated the assay to be a good method with strong specificity,high sensitivity and excellent coincidence and it could be used to fastly,accurately detect the antibodies against R. anatipestifer in the field sera samples.4.Identification of Riemerella Anatipestifer Genes Differentially Expressed in Infected Duck Livers by Selective Capture of Transcribed Sequences TechniqueThe published sequences for R.anatipestifer 16S rDNA（AY871819） were used to design primers for the amplification of 16S rDNA of R.anatipestifer RA-YM.Because the 23S rDNA sequence of R.anatipestifer is not available,the 23S rDNA sequences of S. pyogenes（NC₀02737）,P.multocida（NC₀02663）,E.coli（NC₀00913） and C. canimorsus strain 24231（AY661855） were aligned to identify the conservative regions for designing three sets of primers for the amplification of 23S rDNA of R.anatipestifer RA-YM.The primers were designed by using Primer premier 5.0.The amplified DNA fragments were then ligated into pMD 18-T vector and confirmed by sequencing analysis. The confirmed plasmids（pMD-16rRNA,pMD-23S1rRNA,and pMD-23S3rRNA） and biotinylated R.anatipestifer RA-YM genomic DNA were sonicated microprobe.The fragment of sample were 100-2000bp after soniacation.The sequence data of R. anatipestifer RA-YM rDNA are accessible with GenBank accession numbers:FJ031240 and FJ031241.The ducks（n=7,9-days old） were experimental infected with R.anatipestifer RA-YM and 24 hours later,the liver samples from these dusks were collected and immediately put into liquid nitrogen.Total RNA from R.anatipestifer RA-YM infected livers or from TSB controls was isolated,and double cDNAs were synthesized,which were named by in vivo and in vitro cDNAs,respectively.After three rounds of normalization,the cDNAs from infected liver-grown R.anatipestifer were enriched and cloned into pMD18-T vectors to generate R.anatipestifer RA-YM-infected liver-specific cDNA libraries. Individual SCOTS clones obtained from liver-specific cDNA libraries were verified by PCR with SCOTS01 primers,and the PCR products of these SCOTS clones were spotted on positively charged membranes in duplicate subjected to dot blot analysis.The positive clones obtained from the dot blot analysis were identified and then sequenced by Sanger’s sequencing techqique.BLASTn was performed to identify sequences of genes,and Swiss-Prot database was performed to identify sequences of translated products,there were up to 48 genes that were differentially expressed in the duck livers after R. anatipestifer infection compared to the control cultures.After that,eight genes（gcvT, hmgA,aspC,cat,ptfp,dppⅣ,m28,and RA46） were random chosen and verified by real-time RT-PCR analysis.All were upregulated by R.anatipestifer in infected duck livers,with changes ranging from 1.44- to 4.62- fold compared to in vitro cultures. Totally 48 genes were identified by SCOTS analysis,which can be divided into five functional groups:metabolism,regulatory,stress,transporter and proteinase.In our study, five genes of R.anatipestifer RA-YM encode different proteinase were identified,which have the highly homology with other bacteria.DPP IV is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal ends of polypeptide chains.It is indicated that DPPIV is a important virulence factor of P.gingivalis by contributing to the degradation of connective tissues,and also mediates the adhesion of P.gingivalis to fibronectin.The proteolysis of substance P by Sg-xPDPP was observed,and the concerted action of an extracellular Arg aminopeptidase and Sg-xPDPP produces a truncated form of bradykinin.The combined effect of these modifications may result in local changes in vascular permeability and smooth muscle contraction at the infected endothelium.The fibrnous exudate is the prominent pathological characterization of R.anatipestifer infection,so the DPPⅣmay serve as a critical virulence factor of this organism for pathogenicity.Inactivation of the DPPⅣgene in R.anatipestifer RA-YM using genetic tools should make it possible to determine the contribution of this protease to bacterial growth and pathogenicity.In the future,studies will focus on the isolation,expression and knockout of this gene or more of the in vivo expressed genes in order to evaluate their relative contributions to R.anatipestifer RA-YM virulence and survival at the site of infection.更多还原