【作者】 杨俊兴；

【导师】 陈焕春； 金梅林；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪链球菌(Streptocuccus suis,S.suis)是一种重要的人畜共患病原菌。根据荚膜多糖(capsular polysaccharide，CPS)抗原成分不同，可以将猪链球菌分为35个血清型(1.34型和1／2型)。其中，猪链球菌2型(Streptocuccussuistype 2，SS2)是最常见、也是毒力最强的血清型。猪链球菌是一种条件致病性病原菌，成年猪感染后一般不表现任何临床症状。但是，受到应激因素的刺激，往往会加剧发病率和死亡率。血清学调查(sero-surveillance)或抗体水平监测(antibody monitoring)有助于了解猪群中链球菌感染状况，为控制猪链球菌病提供科学的依据．目前，有一些方法可以用于SS2抗体检测，如ELISA和琼脂扩散试验等，但这些方法由于费时或成本高等因素的制约，只能用于实验室诊断，不适合用于大量样品的检测。因此，急需研制一种快速检测SS2抗体的方法，对我国SS2血清流行病学进行调查。本试验应用胶体金标记技术(immunogold labeling technique)，根据免疫层析原理，用胶体金标记葡萄球菌蛋白A(SPA)作为探针，用纯化的SS2 CPS和健康猪IgG分别作为检测线试剂和对照线试剂，研制了一种SS2抗体快速检测试纸条。该试纸条具有检测快速和使用方便等优点，可以适用于临床样品的大量检测，为SS2血清抗体检测提供了有效的工具。用该试纸条检测16份SS2感染康复猪血清抗体和24份SS2高免猪血清，试纸条检测为100％阳性，与ELISA法检测结果完全一致；检测68份其它细菌高免血清或健康猪血清，结果除一份链球菌1型(SS1)高免血清呈弱阳性外，其余均为阴性。用试纸条检测20份阳性高免血清抗体滴度，结果表明试纸条的敏感性和ELISA相当。用试纸条和ELISA检测486份临床血清样品，结果表明试纸条的特异性(specificity)为94.6％，敏感性(sensitivity)为91.7％，两种方法检测结果的符合度很高(kappa=0.863)。由此表明，试纸条法可以代替ELISA在临床检测中的应用。深入了解猪链球菌的致病机理，可以为链球菌病的预防和治疗提供科学的依据。病原菌与宿主细胞之间的相互作用，在致病过程中发挥着重要的作用。研究病原与宿主细胞相互作用，可以促进对致病机理的探索。并可以为药物筛选、疫苗设计提供理论依据，以便制定科学的疫病控制措施。纤连蛋白结合蛋白(FBPS)由链球菌Fbps基因编码，可以与人的纤维结合蛋白和纤维蛋白素原结合。在SS2感染过程中，FBPS与细菌在特定器官中的定殖有密切关系，推测FBPS与SS2的致病性有关。因此，本试验用CytoTrap酵母双杂交技术，研究FBPS与宿主细胞蛋白质相互作用，旨在探索FBPS在SS2致病性中的作用。用PCR方法扩增SS2 Fbps全长基因，定向克隆到pSos载体多克隆位点中，构建bait载体(pSos-Fbps)。并将Fbps克隆到pET-28c载体中，构建重组表达载体。将pSos-Fbps和鼠cDNA文库(pMyr-cDNA)共转化cdc25H酵母细胞，筛选与FBPS相互作用的克隆。经过筛选和重复验证，得到2个与FBPS相互作用的阳性克隆，并对Target载体插入片段进行测序，经BLAST分析，两个基因分别与凋亡抑制因子5(apoptosis inhibitor 5，API 5)基因和锌指结构FYVE结构域27(zinc finger FYVE domain containing 27，ZFYVE 27)基因的同源性为99％和100％。将重组原核表达载体pET-28c-Fbps转化BL21大肠杆菌细胞，进行FBPS蛋白质表达。分别用PCR扩增API 5和ZFYVE 27全长基因，定向克隆到pcDNA3.1/myc-His载体中，构建真核表达载体(pcDNA3.1/myc-His-API 5和pcDNA3.1/myc-His-ZFYVE 27)，并分别转染293T细胞进行表达。将细胞裂解后，用兔抗Myc抗体共沉淀FBPS和ZFYVE 27或FBPS和API 5复合物，再用SS2高免血清进行Western-blotting分析。用免疫共沉淀试验分别证了FBPS与API 5和ZFYVE 27之间的相互作用。更多还原

【Abstract】 Streptococcus suis (S. suis) is a common pathogenic microorganism and can cause avariety of clinical diseases in human and swine. Based on the CPS antigens of S. suis, 35serotypes or capsular types have been characterized. S. suis serotype 2 (SS2) is mostcommonly associated with severe disease and is the serotype most frequently isolatedfrom pigs.S. suis is an opportunistic bacterium of pigs. Crowding, poor ventilation, suddenweather changes, mixing, relocation, immunization and the presence of concurrentinfections could predispose pigs to SS2 infection and morbidity. Thereforesero-surveillance or antibody monitoring may be useful in determining the infection statusand can play an important role in the control Streptococcosis in pigs.In this study, an immunochromatographic strip (ICS) was developed for thedetection of antibody against SS2. Colloidal gold particles labeled with staphylococcalprotein A (SPA) were used as the. detector reagent. The capsular polysaccharide (CPS) ofSS2 and affinity purified IgG from a healthy naive pig were immobilized on test andcontrol regions of a nitrocellulose membrane, respectively. An Enzyme linkedimmunosorbent assay (ELISA) for SS2 antibody detection based on purified CPS wasused as a control method.The ICS was used to (i) detect anti-CPS antibody in 16 sera taken from 4 SS2infected pigs, 24 sera from pigs hyper-immunized with SS2 and 68 sera from pigsinoculated or infected with bacteria other than SS2; (ⅱ) determine anti-CPS antibodytiters of 20 positive sera for comparison with ELISA; (ⅲ) detect anti-CPS antibody in 486clinical sera taken from diseased pigs also for comparison with ELISA.For 16 sera from pigs that had survived challenge with SS2, both ICS and ELISAgave all samples positive results. For the 24 hyper-immune sera raised against SS2, therewas also a 100% correlation between ELISA and ICS results. All of the 68 sera againstnon-SS2 bacteria, except one antisera against S. suis type 1 determined as weak positiveby both ICS and ELISA, were negative in both ELISA and ICS tests. Antibody titers of 20hyper-immune sera raised against SS2 were tested by ICS and ELISA. The quantifyingtest result indicated that the sensitivity of the strip test is close to or slightly less than thatof ELISA.Of the 486 clinical sera from sick pigs, 228 were antibody positive and 258 werenegative by ELISA, whereas 223 were positive and 263 were negative by ICS. Additionally 209 were positive in both assays and 244 were negative in both tests. Thus,the specificity and sensitivity of ICS, compared to ELISA, was 96.4% and 91.7%,respectively. There was an excellent agreement (kappa=0.863) between ICS and ELISA.Although some assays, e.g. the indirect ELISA and agar gel precipitation tests, havebeen developed for antibody detection for S. suis, these methods are not suitable for useoutside of the research laboratory. In contrast, the ICS is easy to use and rapid. In thiscontext, the ICS can be used as a tool for seroepidemiological survey of pigs fromdifferent areas in China. This would in turn facilitate the formulation of effective controlmeasures.To understand the pathogenesis of S. suis infection contributes to develop strategiesfor prevention or therapy. The interactions between pathogenic microorganism and hostcells may play an important role in its pathogenicity. In this study, the CytoTraptwo-hybrid syste was used to investigate the protein-protein interaction between FBPSand the host cells.FBPS, which can bind to human FN and FGN, is a protein coded by Fbps gene of S.suis. And Fbps gene is present in all known serotypes of S. suis except serotype 32 andserotype 34. A considerable number of FN and FGN binding proteins of various bacterialspecies have been described. Most of these proteins have been shown to be involved inadhesion to epithelial and/or endothelial cells. FBPS play a role in the SS2 colonization ofspecific organs.The full length Fbps gene was amplified from chromosomal DNA of SS2 strainSCZY05 by PCR. Fbps gene was unidirectional cloned into pSos after the amplified PCRproduct and pSos vector were digested with BamHⅠand NotⅠ, yielding the bait plasmidpSos-Fbps. The recombinant expression plasmid pET-28c-Fbps was constructed by thesame way.The library was screened by co-transformation of the pSos-Fbps bait construct into atemperature-sensitive cdc25H yeast strain that can not grow at 37℃. If the bait proteinphysically interacts with the target protein, the hSos protein is recruited to the membraneactivating the Ras signaling pathway and allowing the cdc25H yeast strain to grow at 37℃. The clones growing at 37℃on SD/galactose (-UL) plates but not on SD/glucose(-UL) plates were selected as "putative positive" clones and were analyzed further.The Target plasmid DNA was isolated from pMyr eDNA putative positive clones andwas transformed into E. coli cells for plasmid amplification. Colonies contains eDNAfragment insertion identified by restriction digest analysis. The positive Target plasmid was sequenced on a 3730 Sequencing system. The sequencing results were sent to NCBIfor BLAST searches on line. In this study, we got two positive target plasmid. One ofthese genes is identical to the apoptosis inhibitor 5 (API 5) gene. The other showedhomology to genes encoding zinc finger FYVE domain containing 27 (ZFYVE 27).The protein-protein interaction between FBPS and API 5 or ZFYVE 27 wasconfirmed by ProFound Co-immunoprecipation assay. Recombinant plasmid pET-28c-Fbps was transformed into E. coli BL21 cells for protein expression. The full length ofZFYVE 27 and API 5, obtained by PCR from the target vectors were subcloned intopcDNA3.1/myc-His, yielding pcDNA3.1/myc-His-ZFYVE 27 and pcDNA3.1/myc-His-API 5. The recombinant expression plasmids were transfected into 293T cells for proteinexpression. Anti-mye probe antibody was used to immunopreeipitate the expressedprotein (API 5 or ZFYVE 27) from the whole cell lysate and supernatant of BL21transformed with pET-28c-Fbps culture. The complex was then resolved on a 10% SDSpolyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane.Pig anti-sera against SS2 were used for western-blotting.更多还原