【作者】 顾宏伟；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪链球菌（Streptococcus suis）是一种革兰氏阳性菌,有35个血清型（1～34,1/2）,其中猪链球菌2型（SS2）可引起猪的急性败血症及相关人员感染而急性死亡。2005年7月在四川再次暴发,倍受世界关注。目前,对SS2毒力因子的研究仍是零散研究,且处于体外研究阶段,主集中于CPS、MRP、EF、SLY、GDH、GAPDH和FBPs等毒力因子的检测及其重组表达蛋白体外功能等,尚不足以揭示SS2感染宿主时的真实情况。本试验运用IVIAT（in vivo induced antigen technology）通量筛选及鉴定SS2感染相关因子,并对部分基因的特性进行了较为深入的探索,为全面认识SS2感染相关因子在SS2感染过程中的作用铺垫基础。1 SS2基因组表达文库的构建及鉴定根据Sanger研究所提供的SS2基因组序列,DNASTAR软件模拟酶切,依酶切产物大小的分布情况,确定合适的限制性内切酶。提取SS2四川流行株基因组DNA,用限制性内切酶部分酶切,调整酶切时间、温度及酶量等条件,确定最适条件,在此基础上,进行大体积酶切,回收大小合适的片段。同时酶切表达载体系列（pET30abc）,去磷酸化,回收线性化的载体。将基因组酶切片段按适当的比例分别与载体连接,电转化进DH5α,并用载体特异性引物分析片段插入率及其片段大小分布。然后分别提取质粒,将其电转化至高效表达宿主菌BL21（DE3）中,构建出三种基因组表达文库。2 SS2基因组表达文库的免疫筛选、鉴定及功能预测制备并采集猪耐过SS2感染的康复血清,将得到的8份康复血清混合,分步与SS2体外表达的抗原（如全菌、菌裂物及胞外蛋白等）结合去除相应的抗体,同时应用间接ELISA监测每一步的吸附效果。将经上述步骤吸附过的血清对SS2基因组表达文库进行筛选,经4轮筛选及4轮鉴定,共筛选出64个阳性克隆。将阳性克隆送公司测序,分析插入的序列,结果表明,其中60个克隆编码48种蛋白,如代谢及能量代谢、分子合成、胞外蛋白、转运蛋白、调控蛋白等及功能未知蛋白。另参照欧洲株P1/7基因组序列,将上述序列进行基因组定位分析,以了解这些感染相关因子在基因组的分布。3实时荧光定量RT-PCR比较SS2感染相关因子的体内外表达差异根据已公布的SS2欧洲株P1/7株或加拿大株89/1591株的序列,设计13对感染相关因子（trag,ysirk,abc56,sdh,srt,abc15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps）的检测引物,同时以SS2四川分离株ZY05719的gapdh为内参,参照其序列设计引物。体外培养,分别于OD₆₀₀值为0.1,0.2,0.4,0.6和0.8时收集菌体,提取菌体总RNA,反转录cDNA,荧光定量PCR分析SS2体外培养时mRNA表达水平。体内感染,以微型SPF香猪为动物模型,分别采用静脉和肌肉注射的方式感染ZY05719,感染后于不同时间段采血,从菌血中分离SS2,提取总RNA,反转录cDNA,荧光定量PCR分析SS2体内感染培养时mRNA表达水平。在分别分析体外培养和体内感染的基础上,比较体内增殖时mRNA表达差异。结果表明,体外培养时这些感染相关因子在对数生长期时可瞬时转录,但随即mRNA开始降解,这13个基因的mRNA水平均迅速下降;体内感染时,静脉注射途径:orf1616,trag,nlpa,abc56和flps呈持续上升趋势,abc15,srt,ysirk,hp10和ptss呈先上升后下降,cwh,hprk和sdh呈下降。肌肉注射途径:orf1616,trag,hprk,sdh及flps呈上升趋势,abc15,nlpa,srt,ysirk,abc56,cwh,hp10和ptss呈下降趋势。体内外增殖时感染因子mRNA转录水平比较结果表明,各感染相关因子在SS2体内感染时,不同感染途径及不同时间段mRNA转录水平不尽一致,其表达水平相对于体外培养时均有不同程度的上调。4新鉴定的SS2感染相关因子的分布分析参考已公布的SS2欧洲株P1/7株或加拿大株89/1591株的序列,设计13对感染相关因子（trag,ysirk,abc56,sdh,srt,abc15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps）的检测引物,取SS2我国江苏和四川流行株、其它临床分离株和参考株,及猪链球菌1型、1/2型、7型、9型及C群,共39株,分别以其DNA为模板,PCR扩增。结果表明,在SS2菌株中,除SS2-N和无毒株T15全部阴性,其它不同地区不同年份的均有不同程度的分布,2005年四川分离株（含两人源株）全部呈阳性,1998年江苏分离株HA9801除abc15阴性外,其它因子均阳性,但1998年人源株98012全部阳性。其它猪链球菌,C群链球菌均阴性,SS1、SS1/2、SS7和SS9各有其自身分布特点,不尽一致。5 SS2新的感染相关因子自溶素的鉴定与分析根据Sanger研究所公布的SS2型P1/7株的自溶素的基因序列,分2段设计扩增引物以扩出完整orf序列,取SS2我国江苏和四川流行株（HA9801和ZY05719）、人源株（260）、参考株（ATCC43765）和欧洲株（11611）,分别以其DNA为模板,PCR扩增,装入T载体测序,分别拼接得出各自完整序列。结果表明,我国流行株的自溶素orf基因全长为3108bp,软件分析结果显示,该蛋白含有6个重复的“GBS_Bsp-like”域和1个“N-乙酰胞壁酰-L-丙氨酸酰胺酶”域,与SS2欧洲株有较高同源性（99.8%）,但与SS2加拿大株差异较大。另参照已知的N-乙酰胞壁酰-L-丙氨酸酰胺酶三级结构对我国两次流行株等的自溶素进行空间结构预测,结果表明,在该域内,我国株的自溶素与加拿大的在局部结构域上存在差异,与欧洲株的差异较小。在DNASTAR分析所编码蛋白的抗原性的基础上,另设计引物,以四川ZY05719株DNA为模板,PCR扩增具有良好免疫原性的片段基因,并定向克隆至表达载体pET30a（+）中,进行重组表达,SDS-PAGE分析重组蛋白的表达,用康复血清做Western blot,结果表明,所获得重组自溶素与康复血清有着良好反应性。6猪链球菌调控蛋白HprK的鉴定与分子生物学特性分析根据Sanger研究所公布的SS2 P1/7株的hprk的基因序列,设计引物扩增完整orf序列,取SS2我国两次流行株（HA9801和ZY05719）、人源株（260）、参考株（ATCC43765）和欧洲株（11611）,分别以其DNA为模板,PCR扩增,装入T载体测序,分别拼接得出各自完整序列。结果表明,我国两次流行株等的hprk基因全长均为933bp,编码蛋白由310氨基酸组成。比对结果表明,hprk高度保守,各地分离株之间同源性高达98%以上,且与其它细菌的hprk有着较高同源性。另参照已知的HprK三级结构对两次疫情代表株等的HprK进行空间结构预测,结果表明,我国株的HprK与参考蛋白的空间结构非常接近,与欧洲株的差异较小,但加拿大株缺一个β-片层。进化关系分析表明,HprK与其它细菌的HprK有较高的亲缘关系,与链球菌属亲缘最近,各猪链球菌HprK之间虽有差异,但仍在同一个进化分支上。另信号肽分析表明,该蛋白不含信号肽。7猪链球菌一种新的功能未知的表面蛋白的鉴定与分析根据Sanger研究所公布的SS2 P1/7株的第1616编码蛋白的基因序列,分三段设计扩增引物以扩出完整orf序列,取SS2我国两次流行株（HA9801和ZY05719）、人源株（260）、参考株（ATCC43765）和欧洲株（11611）,分别以其DNA为模板,PCR扩增,装入T载体测序,分别拼接得出各自完整序列。结果表明,包括我国两次流行株在内的上述5株的ORF1616均由1121个氨基酸组成,GenBank比对分析表明,上述5株的ORF1616与加拿大株89/1591差异较大,后者的仅由917个氨基酸组成。Sanger数据库比对分析表明,这5株的与P1/7株的有较高的同源性,同源性均在99%以上。进化关系分析表明,ORF1616与猪链球菌功能推测蛋白亲缘关系较近,但与无乳链球菌的关系较远。另信号肽分析表明,该蛋白存在37个氨基酸的信号肽,切割位置在Ala和Asp之间。8 SS2三种感染相关因子突变质粒的构建为进一步研究感染相关因子（cwh,hprk和orf1616）在SS2感染过程中作用。本试验构建SS2三种感染相关因子的突变质粒。参考前期四川流行株ZY05719三个基因全序列结果及欧洲株P1/7的序列,在各基因5’端及其上游区和3’端及其下游区分别设计引物,PCR扩增各基因的上、下游片段,经酶切后插入T载体,经鉴定及测序确认后,分别将这些片段插入链球菌突变质粒pfw5壮观霉素基因的单侧/两侧,酶切鉴定,分别构建出突变质粒:pfw5-cwh,pfwS-Δhprk和pfw5-orf1616,为同源重组突变ZY05719株的上述三种感染相关因子打基础。更多还原

【Abstract】 Streptococcus suis is an important swine pathogen that causes many pathological conditions,such as arthritis,endocarditis,meningitis,pneumonia,and septicemia.It is also an important zoonotic agent for humans in contact with colonized,otherwise healthy pigs or their by-products,causing meningitis and endocarditis.Thirty-five serotypes（types 1 to 34,and 1/2） based on capsular antigens are currently known.Type 2 is considered the most virulent and prevalent type in diseased pigs.The mechanisms involved in the pathogenesis and virulence of S.suis are not completely understood.Attempts to control the disease are still hampered by the lack of sufficient knowledge about the pathogenesis of the disease and the lack of effective vaccines and sensitive diagnostic methods.In this study,In vivo-induced antigen technology（IVIAT） has been employed to study SS2 infection-related factors.1 Construction of Streptococcus suis serotype 2 genomic expression librariesReferred to published European SS2 strain P1/7 genomic DNA sequence,software DNASTAR was used to select proper restriction enzyme for construct SS2 genomic expression library.Vector DNA was respectively digested with BamHI,gel-purified by using a gel extraction kit,and treated with alkaline phosphatase.Genomic DNA from strain ZY05719（isolate from Sichuan SS2 outbreak） was partially digested with Sau3AI,and fractionated on a 0.7%agarose gel.DNA fragments ranging from 0.1 to 5.0 kbp（insert DNA） were recovered and cloned into the three BamH I-digested and dephosphorylated vectors,then each library was electroporated into competent E.coli DH5αand BL21（DE3） to create three genomic expression libraries,pET30 vectors specific primers were used to detect the of efficiency of insert ratio.2 Selection and identification of infection-associated factors from Streptococcus suis serotype 2 expression libraries with swine convalescent seraSera from swine convalescing from SS2 challenge were collected,adsorbed against in vitro-grown SS2 strain ZY05719（such as bacterial whole cells,cell lysates and extracellular proteins）,then used to screen SS2 genomic expression libraries,after rigorous selection,which output 64 positive clones.Sequencing analysis revealed 60 clones encoded 48 proteins,which had homology to proteins involved in various aspects of cell surface structure,molecule synthesis/degradation,energy metabolism,regulation,transport systems, and unknown functions.Align these sequences with P 1/7 whole genomic sequence to learn about the distribution of infection-related factors in genome.Alignment result some of them showed high similarity with 89/1589 but not in P1/7,or vice versa,about 44 insert DNA sequence can be located on P1/7 genome.3 Development of a real-time fluorescent quantitative polymerase chain reaction for assessment of differential expression of newly identified infection-related factors between in vitro and in vivo conditionVirulence factors and the pathogenesis of SS2 infection are not well understood.In this study,13 out of 48 newly identified infection-related factors（trag,ysirk,abc56,sdh,srt, abc15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps） were selected to analyze their differential expression between in vitro and in vivo condition,gapdh was used reference gene.In vitro, ZY05719 was harvested at OD₆₀₀ of 0.1,0.2,0.4,0.6 and 0.8 respectively.In vivo, specific-pathogen free piglets（SPF Bama minipigs） were used as experimental model to leam about mRNA expression difference at in vivo condition.SPF Bama minipigs were challenged with ZY05719 intravenous（i.v.） or intramuscular（i.m.） injection,then bacterial cells were recovered from blood at 12h,24h and 36h（i.v.） and 24h and 48h（i.m.）.RNA was isolated and used as template for assess their differential expression by quantitative real-time PCR between in vitro and in vivo.During i.v.approach orf1616,trag,nlpa,abc56 and flps upregulated continuously;abc15,ysirk,hp10 and ptss upregulated before 24h then went down;cwh,hprk,srt and sdh went down from 12h.During i.m.approach, orf1616,trag,hprk,sdh and flps climbed up from 24h to 48h,and abc15,nlpa,srt, ysirk,abc56,cwh,hp10 and ptss went down form 24h to 48h.Results mentioned above suggesting that these genes may play a role during SS2 invasive course.4 Distribution of newly identified SS2 infection-related factors in Streptococcus suis strainsStreptococcus suis serotype 2 is an important pathogen for pig and human.About 48 infection-related factors had been selected by IVIAT in previous study.In this study,13 factors（trag,ysirk,abc56,sdh,srt,abe15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps） were selected to analyze their distribution in Streptococcus suis strains from different sources, regions and times.According to published SS2 genomic sequence of European strain P1/7 or Canadian strain 89/1591,primers for these 13 genes have been designed and used to detect their distribution.Genomic DNA of SS2 outbreak represent strains（Jiangsu isolate HA9801 and Sichuan isolate ZY05719）,reference strain ATCC43765,SS2 isolates from different sources,and other serotypes or group（SS1,SS1/2,SS7,SS9 and C group） were used as template for polymerase chain reaction（PCR）.Results showed that these 13 factors widely distributed in most SS2 strains,but SS2-N and non-virulent strain T15 didn’t have. In addition,these factors could be detected in other serotypes but each serotype had different distribution,and negative in Lancefield group C.5 Identification and characterization of a novel infection-related factor（cell wall hydrolase/autolysin） of Streptococcus suis serotype 2Cell wall autolysins have been implicated in various biological functions,including cell separation,cell wall turnover,restructuring of cell walls,and bacterial autolysis.In this study,the complete autolysin genes of HA9801,ZY05719,ATCC43765,260 and 11611 were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software.Results showed that open reading frame（orf） length of SS2 autolysin is 3108bp,they were consisted of 6 repeated domain "GBS_Bsp-like" and 1 domain "N-acetylmuramoyl-L-alanine amidase",while 89/1591 only has 5 repeated domain "GBS_Bsp-like".Alignment results showed that autolysins of strain HA9801 and ZY05719 showed high homologue to that of European strain P1/7（99.8%）,but differ from Canada strain 89/1589.Based on published N-acetylmuramoyl-L-alanine amidase tertiary structure, we forecast N-acetylmuramoyl-L-alanine amidase tertiary structure of these five strains and Canadian strain 89/1591 and European strain P1/7.Tertiary structures of these five strain were similar with each other,and showed high homology with that of P1/7,but differ from that of 89/1591 at partial domains.Partial gene of autolysin was cloned and inserted into expression vector pET30a（+）,and induced by IPTG to express recombinant autolysin. SDS-PAGE and western blot were used to analyze its antigenicity.6 Identification and characterization of a novel infection-related factor HprK/P of Streptococcus suis serotype 2P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria,but also plays a role in virulence development of certain pathogens.In this study,the complete hprk genes of HA9801,ZY05719,ATCC43765,11611（isolated from pig） and 260（isolated from human） were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software.Results showed that open reading frame（orf） length of SS2 HprK/P is 933bp,and HprK/P molecular weight is about 35kDa.Alignment results showed that HprK/P is highly conserved at Genbank database,SS2 HprK/P demonstrated high similarity with HprK/P of almost all bacteria.Based on published HprK/P tertiary structure,we forecast HprK/P tertiary structure of these five strains and Canadian strain 89/1591 and European strain P1/7.Results showed that tertiary structures of these five strain were similar with each other,and showed high homology with that of P 1/7,but differ from that of 89/1591 at partial domains.In addition,signal peptide of SS2 HprK/P were analyzed by software,result showed that it’s a mature protein,has no signal peptide.7 Identification and characterization of a novel function-unknown infection-related factor ORF1616 of Streptococcus suis serotype 2ORF1616 is a putative cell surface protein,but its function unknown,may play a role during SS2 infection.In this study,the complete orf1616 genes of HA9801,ZY05719, ATCC43765,11611（isolated from pig） and 260（isolated from human） were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software. Results showed that open reading frame（orf） length of SS2 orf1616 is 3366bp,ORF1616 is consisted of 1121 amino acids（Aa） and molecular weight is about 122kDa.ORF1616 sequence information is limited at Genbank database,Alignment results showed high homologue to that of European strain P1/7（99.8%）,but differ from Canada strain 89/1589, the latter only have 917Aas.In addition,signal peptide of SS20RF1616 were analyzed by software,result showed that it harbor 37Aa signal peptide,was cleaved between Ala and Asp.8 Construction of mutation plasmid of three infection-realted factors（cwh,hprk and orf1616）Three genes of cwh,hprk and orf1616 has been studied in previous study.In this study, to construct the homologous recombination plasmids of cwh,hprk and orf1616 of Streptococcus suis serotype 2 so as to provide the basis for further study of their function during infection course.5’-upstream and 3’-upstream of cwh,hprk and orf1616 and their flanking fragments were amplified and cloned into streptococcal integration vector pfw5,to construct pfw5-cwh,pfw5-Ahprk and pfw5-orf1616.更多还原