【作者】 吴涛；

【导师】 陈焕春； 贝为成；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猪链球菌（Streptocuccus suis,S.suis）是当前严重危害养猪业的一种重要病原菌。根据荚膜多糖（capsular polysaccharide,CPS）抗原成分不同,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（Streptococcus suis serotype 2,SS2）是最常见、也是毒力最强的血清型,同时也是一种重要的人兽共患病原菌。猪链球菌2型revS基因是一种反应调控因子（response regulator）,在SS2致病过程中可能起着很重要的作用。研究发现该基因能够在转录水平上对SS2多种毒力因子进行调控,缺失该基因后多种毒力因子表达减弱。鉴于此,本研究构建了猪链球菌2型revS基因缺失菌株和质粒介导的互补菌株,并对缺失菌株和互补菌株的生物学特性进行了研究,进一步研究其所调控毒力因子的表达情况,为深入研究猪链球菌2型的的分子致病机理奠定前期基础。透明质酸酶（hyaluronidase,HYL）由猪链球菌2型Hyt基因编码,该酶为粘多糖分解酶,可水解组织基质中的透明质酸,增加组织的通透性。另外也有研究报道,HYL可以作为一种调控蛋白,通过降低血中的透明质酸酶的浓来诱导血管的增生。在SS2感染过程中,细菌分泌的透明质酸酶是否可以改变宿主细胞通透性,导致链球菌及其分泌的毒力因子更容易进入宿主细胞内而发挥毒性作用有关目前还不清楚。因此,本研究利用酵母双杂交技术,以透明质酸酶作为诱饵蛋白,从小鼠的脑细胞和人的肺细胞cDNA文库中寻找HYL的互作蛋白,旨在为进一步探索HYL在SS2致病中的作用提供理论依据。鉴于以上研究背景,本研究主要开展了以下研究内容:1.猪链球菌2型revS基因缺失菌株和互补菌株的构建与鉴定通过PCR分别扩增了revS基因上下游同源臂p1和p2基因片段、开放阅读框（ORF）和全长序列,利用温度敏感型“自杀性”质粒pSET4s构建了质粒pSET4s-p1-p2,并将该重组质粒电转化进入亲本菌SS2野生菌株SC21中,通过抗生素和温度双重标记筛选,获得revS基因缺失菌株,命名SC211。另外,将revS全长基因定向插入穿梭载体pAT18中,构建了穿梭质粒pAT18-revS,并将该质粒电转入revS基因缺失菌株SC211,通过抗生素筛选,得到质粒介导的revS基因互补菌株,命名为SC212。通过PCR、Southern blotting进一步对基因缺失突变菌株和互补菌株进行了鉴定,证实构建正确。2.猪链球菌2型revS基因缺失菌株和互补菌株的生物学特性研究通过细菌传代培养,对缺失突变菌株SC211和互补菌株SC212的遗传稳定性进行了分析,结果显示缺失突变菌株和互补菌株能够稳定生长。比较了基因缺失突变菌株、互补菌株及野生菌株的生长特性,发现缺失菌株的生长速度明显加快,互补菌株也比野毒菌株的生长快。溶血性试验结果表明,基因缺失后SC211的溶血活性明显降低,而互补菌株SC212的溶血活性虽然有所回复,比基因缺失菌株明显提高,但是没有达到野毒菌株的水平。利用Hep2细胞为模型,研究不同菌株对细胞黏附特性,结果显示,基因缺失后链球菌对Hep2细胞的黏附能力明显下降,只有野毒菌株的29%:互补菌株虽然黏附能力比基因缺失菌株要高,但是没有达到野毒菌株的水平,只有野毒菌株的79%。以CD1小鼠为模型比较了不同菌株的毒力,结果显示基因缺失后链球菌毒力明显下降,基因缺失菌株的LD₅₀（7.78×10⁷）是野毒菌株LD₅₀（7.63×10⁶）的10倍,而互补菌株的毒力有所增高,但是没有达到野毒菌株的水平,其LD₅₀（2.44×10⁷）是野毒菌株的3倍。3.猪链球菌2型revS基因缺失菌株和互补菌株的毒力相关因子的相对定量分析利用定量PCR研究了SS2毒力相关因子体内外表达情况。体外定量结果显示:除了cps和gapdh外,基因缺失菌株其他的毒力相关因子（ef,fops,hyt,sly,sod,mrp,gyrap,和rpob）的表达都要比野毒菌株低,其中ef,fbps,hyl,sly和mrp约下降了50%—80%。而互补菌株在gyrap、rpob和gapdh的表达上和野毒菌株没有明显差别,其他毒力因子（cps,ef,fbps,hyl,sod,sly和mrp）的表达都明显增高,在一定程度上上调了0.5—1.5倍。攻毒后不同时间SS2毒力相关因子的表达水平比较发现:攻毒48h后的组织中链球菌毒力相关因子的表达量要比攻毒24h和72h的高,但是差异不显著。而攻毒后不同组织中的SS2毒力相关因子的表达水平比较发现:攻毒后组织中毒力相关因子的表达量都比体外的高,差异显著（p＜0.01%）。脑组织和肺组织中链球菌毒力相关因子的表达水平比血中的高,但是差异不显著。在体内,不同菌株毒力相关因子的表达水平比较发现:在体内cps的表达野毒菌株和基因缺失菌株之间没有明显差异,但互补菌株的cps表达量是野毒菌株和基因缺失菌株的1.2倍。在基因缺失菌株中,mrp,ef,sly,fbps,sod,rpob,gyra和hyl的表达量分别比野毒菌株降低了41%,37%,29%,39%,33%,46%,36%和49%,而在互补菌株中这些毒力相关因子的表达量比基因缺失菌株增高了1.5—2.5倍,而不同组织中gapdh的表达没有差异。4.猪链球菌2型revS基因缺失菌株和互补菌株的免疫原性的研究免疫保护性实验结果表明:10⁶cfu的基因缺失菌株免疫CD1小鼠后能够对猪链球菌2型野毒菌株的攻击提供90%免疫保护,10⁷cfu的基因缺失菌株免疫CD1小鼠后能够对猪链球菌2型野毒菌株的攻击提供100%免疫保护力;10⁵cfu的互补菌株免疫CD1小鼠后能够对猪链球菌2型野毒菌株的攻击提供90%免疫保护力,10⁶cfu的互补菌株免疫CD1小鼠后能够对猪链球菌2型野毒菌株的攻击提供100%免疫保护力。不同菌株免疫CD1小鼠后ELISA抗体水平测定结果显示:revS基因缺失后,猪链球菌2型的免疫原性并没有太大的变化,基因缺失菌株免疫后所诱导的cps抗体水平与野毒菌株差异不显著,但互补菌株的cps抗体水平明显高于野毒菌株和基因缺失菌株。5.猪链球菌2型HYL蛋白与宿主互作蛋白的筛?通过PCR方法扩增SS2 Hyl全长基因,定向克隆到pSos载体中,构建bait载体（pSos-Hyl）。将pSos-Hyl分别与鼠脑和人肺cDNA文库（pMyr-cDNA）共转化cdc25H酵母细胞,筛选与HYL相互作用的阳性克隆。经过筛选和重复验证,并对所得到的Target载体插入片段进行测序,BLAST分析,从鼠的脑组织cDNA文库中得到2个与HYL相互作用的阳性克隆,两个基因分别与锌指结构基序（widely-interspaced zinc fingermotifs gene of Mus musculus（NT-039649 REGION:18685755..18718324）,WIZF）和血管生长因子抑制因子1（ribonuclease/angiogenin inhibitor 1 gene of Mus musculus（NT-166306 REGION:2098219..2104509）,AI1）基因的同源性为99%和100%;从人的肺组织cDNA文库中得到2个与HYL相互作用的阳性克隆,两个基因分别与γ内收蛋白（Homo sapiens adducin 3（gamma）（NT₀30059.12）,ADD3）和装配架离子转运蛋白（Homo sapiens chromosome 6genomic contig,reference assembly,ion transporter protein（NT₀34880.3）,AITP）基因的同源性为99%和100%。6.猪链球菌2型HYL蛋白与宿主互作蛋白的验证与分析将Hyl克隆到pET-28c载体中,构建重组表达载体（pET28c-Hyl）进行原核表达,同时将筛选得到的WIZF、AI1、ADD3和AITP基因分别进行真核细胞表达后,用免疫共沉淀试验验证了HYL与AI1、ADD3和MTP之间的相互作用,而HYL与WIZF的证实没有发生相互作用。进一步并分析和预测了AI1、ADD3和AITP可能与SS2致病性的关系。血管上皮细胞生长抑制因子（AI1）主要是抑制血管上皮细胞的生长,从而导致血管的通透性的改变,利于细菌的扩散。而ADD3是一种内收蛋白,也是一种细胞膜骨架蛋白,内收蛋白的确切功能尚不明了,但是已知它与细胞膜表面spectrin和肌动蛋白连接部位的定向组装信号传导和跨膜离了转运有关,它的改变可以导致细胞膜的通透性的改变。AITP是一种离子转运蛋白,是细胞内外离子转运的载体,与细胞膜的离子通道和通透性有关。更多还原

【Abstract】 Streptococcus suis(S.suis) is a common pathogenic microorganism and can cause a variety of clinical diseases in human and swine.Based on the CPS antigens of S.suis,35 serotypes or capsular types have been characterized.S.suis serotype 2(SS2) is most commonly and most virulent of these serotypes.revS gene is the response regulator gene of SS2,belonging to a two component system,which can affect the virulence gene expression of streptococcus suis.For further research the virulence gene expression regulated by revS,revS gene mutant strain and reversible mutant strain were constructed and characteristic analyzed.Hyaluronidase is a kind of protein coding by Hyl gene of SS2,it is mueopolysaccharide lytic enzyme which can hydrolyze the hyaluronic acid of periplast, result in the permeability of the tissue augmentation.In addition some research report HYL can act as a modulin,density of HYL in the blood is descended which can induce Blood vessel accrementition.In the course of SS2 infection,whether the hyaluronidase secreted by SS2 can result in the permeability of the tissue augmentation,ease to the ingress of virulence factors secreted by SS2 into the host and play effect is not clear.So in the research hyaluronidase was used as a bait protein to search its interacted protein from Mouse brain cDNA library and human lung cDNA library using the yeast two hybrid system to explore its function in SS2 infection.So the following researches were explored.1.Construction and verification of SS2 revS gene mutant strain and complementation strainIn the research two DNA fragments flanking the revS gene were amplified by PCR from the genome of S.suis 2 SC21.The fragments of the N-terminus and the C terminus flanking region of the revS gene were digested with BamH I/SalⅠand Sal I/EcoRⅠ, respectively.The revS-flanking PCR products were mixed in equal amounts and inserted directly into BamH I/EcoRⅠdigested and dephosphorylated thermosensitive suicide vector pSET4s,ligated,and transformed into E.coli DH5α.The resulting plasmid, pST4S-revS,was electroporated into S.suis 2 SC21,and the resultant strains were grown at 28℃in the presence of spectinomycin(100μg/mL) selection and subsequently passaged at 37℃in the absence of spectinomycin selection as described previously. Successful deletion of the gene revS was confirmed by PCR and Southern blotting. Southern blotting analysis confirmed the successful deletion of the revS gene in the chromosome of the S.suis 2 revS gene mutant strain named SC211.In addition complementation strain was constructed too.The revS structural gene,including its own promoter,was amplified from chromosomal DNA S.suis 2 SC21 by PCR.The PCR product was cut with Sal I/BamHⅠand ligated into the Sal I/BamHⅠdigested E. coli/Streptococcus shuttle vector pAT18(Trieu-Cuot P et al.1991).The resultant plasmid was termed pAT18-revS.The plasmid pAT18-revS were transformed by electroporation into the revS gene mutant strain with subsequent erythromycin selection. Complementation strain designed SC212.The mutant strain and complementation strain were confirmed by PCR and Southern blotting.2.Bionomics research of the mutant strain and complementation strainThe results of the growth curve of the mutant strain、complementation strain and wide type strain show that the mutant strain and complementation strain grow faster than that of wide type strain.The hemolytic activity of wild-type strain was three times higher than that of mutant strain.The hemolytic activity of complementation strain was restored, and was about two times higher than mutant strain SC211.but it did not reach the level of wild-type strain.Reduction of 79%in the adherence to the Hep2 cell of mutant strain compared with wild-type.The adherence of complementation strain was about three times that of mutant strain,but only 81%of the wild-type strain.LD50 values were 7.63×10~6 CFU per mouse for wild-type strain,7.78×10~7 CFU per mouse for mutant strain,and 2.44×10~7 CFU per mouse for complementation strain.Compared with parent strain, mutant strain was attenuated at least ten fold.Complementation strain was restored three fold than the mutant strain.3.Immunogenic research of the mutant strain and complementation strainA vaccine dose of 10~7 CFU mutant strain and 10~6 CFU complementation strain was protective against an extremely high challenge dose of 4×10~7 CFU.These results demonstrated that the Streptococcus suis-2 revS gene mutant was highly attenuated in the mouse model,and 100%protective against homologous bacterial challenge at 10~7 CFU vaccine-challenge doses.The complementation strain was attenuated in the mouse model, and was 100%protective against homologous bacterial challenge at 10~6 CFU vaccine-challenge doses.The ELISA antibody level of mutant strain has no difference with wide type strain,but the ELISA antibody level of the complementation is higher than that of mutant strain and wide type strain.4.Relative quantitative PCR assay of virulence associated gene of SS2The characteristic of SC211 and SC212 were researched further.The virulence of different strains were determined by animal infection,the virulence is decreased clearly when the revS gene knocked out,the virulence is reverted when the revS gene reverted, but which can not get to the level of the wide type.Many virulence associated genes, including mrp,ef,sly,fbps,sod,rpob,gyra and hyl,are shown to be effeeted by revS gene, the expression level decreased obviously when revS gene knocked out,but the cps and gapdh is not effected by revS,the virulence gene expression of the reversible mutant were exceeded the wide type.In this study,the differential expression of the key S.suis serotype 2 virulence-associated genes of different strains was further investigated in bacteria harvested from the blood,lungs and brains of mice 24,48,and 72h post intraperitoneal infection with different strain S.suis.Bacterial RNA was extracted and gene expression was monitored using real-time reverse transcriptase-PCR at various time points.In all organs the expression level of wide type strain is higher than mutant strain at all time,the reversible mutant strain were higher than the other two strains,the virulence gene expression were similar to that observed in vitro,while the expression level in vivo were higher than their in vitro levels.But at different time the difference of same virulence gene expression between in vivo and in vitro is changed.With the time of infection delay the difference of same virulence gene expression between reversible mutant strain and wide type strain or mutant strain is contracted.In vivo the cps expression was changed obviously.At all the time postinfection,all genes examined were higher than their in vitro levels in the blood,lungs and brains,revS gene can affect the virulence associated gene expression and contribute to S.suis pathogenesis.5.Screen of the interaction protein of HYL of SS2The full length Hyl gene was amplified from chromosomal DNA of SS2 strain SC21 by PCR,Hly gene was directional cloned into pSos vector after the amplified PCR product and pSos vector were digested with BamHⅠand SalⅠ,yielding the bait plasmid pSos-Hyl.The recombinant expression plasmid pET-28c-Hyl was constructed by the same way at the same time.The library was screened by co-transformation of the pSos-Hyl bait constructed into a temperature- sensitive cdc25H yeast strain that can not grow at 37℃. If the bait protein physically interacts with the target protein,the hSos protein is recruited to the membrane activating 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-cDNA putative positive clones and was transformed into E.coli cells for plasmid amplification.Colonies contains cDNA fragment insertion identified by restriction digest analysis.The positive Target plasmid was sequenced on a 3730 Sequencing system.The sequencing results were sent to NCBI for BLAST searches on line.In this study two positive target plasmid received from the Mouse brain cDNA library.One of these genes is identical to the widely-interspaced zinc finger motifs gene of Mus museulus(WIZF).The other showed homology to ribonuclease/angiogenin inhibitor 1 gene of Mus musculus(AI1).At the same time two positive target plasmid received from the human lung cDNA library.One of these genes is identical to Homo sapiens adducin 3 gene(ADD3).The other showed homology to assembly ion transporter protein gene of homosapiens(AITP).6.Confirmation of the protein-protein interaction between HYL and targets receivedThe protein-protein interaction between HYL and targets received was confirmed by ProFound Co-immunoprecipation assay.Recombinant plasmid pET-28c-Hyl was transformed into E.coli BL21 cells for protein expression.The coding sequence of WIZF、AI1,ADD3 and AITP obtained by PCR from the target vectors respectively,then they were subcloned into pcDNA3.1/myc-His,yielding four recombinant expression plasmids.The recombinant expression plasmids were transfected into 293T cells for protein expression.Anti-myc probe antibody was used to immunoprecipitate the expressed protein(WIZF、AI1、ADD3 or AITP) from the whole cell lysate and supernatant of BL21 transformed with pET-28c-Hyl culture.The complex was then resolved on a 10%SDS polyacrylamide gel.The separated proteins were transferred to a nitrocellulose membrane.Pig anti-sera against SS2 were used for western-blotting.But the interaction between HYL and WIZF were not confirmed by this way.The relation between the function of AI1、ADD3 or AITP and the pathopoiesis was analyzed.AI1 can inhibit the growth of blood vessel cellula epithelialis,which result in the permeability of the blood vessel changed ease to the diffuse of SS2.ADD3 is a kind of adducing,cell membrane skeleton protein.AITP is ion transporter protein of the cell membrane.These two protein belongs to the ion channel of the cell membrane,which is associated with the permeability of the cell membrane.更多还原