【作者】 耿建新；

【导师】 范红结；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 马链球菌兽疫亚种(Streptococcus equi subsp．zooepidemicus)对集约化养猪业的发展危害严重，同时对养猪业的相关从业人员的健康具有潜在威胁。能引起猪和人的脑膜炎、败血症、关节炎、肺炎、及突发性死亡，是重要的人兽共患病病原。纤连蛋白(fibronectin，Fn)是由α和β链组成的一种二聚体糖蛋白，由多种类型的细胞表达产生，Fn在动物体内以可溶形式存在于血浆及各种体液中，以不溶形式存在于细胞外基质及细胞表面。前者总称血浆Fn，后者总称细胞Fn。纤连蛋白结合蛋白(fibronectin-binding protein，FNZ)是链球菌一种重要的表面蛋白和毒力因子，是链球菌建立感染所依附的重要的表面分子之一，该菌是重要的黏附素。本试验在分子水平上对马链球菌兽疫亚种FNZ蛋白的功能片段进行了研究，为该菌的致病机制和基因工程疫苗的研制进行了有益的探索。根据已经发表的马链球菌兽疫亚种纤连蛋白结合蛋白的基因序列，设计和合成一对引物，并以ATCC35246株的基因组为模板，通过PCR技术，扩增出缺失信号肽的FNZ基因，并定向克隆至PMD19-T载体中。酶切鉴定正确后，对克隆片段进行序列测定，其长度为1895bp。利用DNAstar软件，比较所测序列与不同来源链球菌的纤连蛋白序列的同源性，该序列与已经发表的马链球菌兽疫亚种VTU211菌株FNZ基因的同源性为89．9％，氨基酸同源性为84．4％，其中突变部位较多，而且中间有111个碱基缺失。与马链球菌马亚种的同源性为78％，氨基酸同源性为44．1％，与其他链球菌的同源性较低。根据ATCC35246的FNZ基因序列，设计和合成五对引物，用PCR方法分别扩增出FNZ基因的4-277bp、4-808bp、592-1134bp、895-1615bp区间和全长的基因片段。将扩增片段双酶切后用T4 DNA连接酶定向克隆于融合表达载体pET-32a(+)的相应酶切位点。构建pFNZ(2-91)、pFNZ(2-268)、pFNZ(197-378)、pFNZ(299-538)和pFNZ重组质粒，对阳性插入片段进行酶切测序鉴定，结果表明pFNZ(2-91)、pFNZ(2-268)、pFNZ(197-378)、pFNZ(299-538)和pFNZ的插入序列与ATCC35246FNZ的相应序列完全一致。将重组质粒转化大肠杆菌BL21，经IPTG诱导表达，表达产物大小分别为31、50、40、45、84kD，与预测结果一致。采用配基印迹结合试验确定FNZ上Fn的结合部位，结果表明pFNZ(2-268)、pFNZ(197-378)、pFNZ(299-538)和pFNZ与人的Fn结合。据此推断在FNZ基因的277-808bp、895-1134bp，即197-268aa、299-378aa之间有Fn的线性结合位点。将含有Fn线性结合部位的重组质粒细菌进行表达，SDS-PAGE检测，结果表明表达的蛋白位于上清之中，以His亲和层析柱纯化表达蛋白，将纯化好的蛋白与ISA206佐剂1：1进行乳化后免疫ICR小鼠，融合蛋白免疫方式为pFNZ(2-268)、pFNZ(299-538)和pFNZ(2-268)+pFNZ(299-538)，以0．2ml(1．0×10~8cell／ml)ATCC35246强毒株攻击后，小鼠的存活率分别为20％、30％和30％，证实所表达的蛋白具有一定的保护性，为马链球菌兽疫亚种重要的保护性抗原。更多还原

【Abstract】 Streptococcus equi subsp.zooepidemicus made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry. they are worldwide zoonotic pathogens and lead to many different diseases characterized by meningitis, septicemia, arthritis, endocarditis and may cause sudden dealth in pig or human.Fn is a large dimeric glycoprotein composed of aand (3 polypeptide chains, which is generated by several cells.Fn is present in a soluble form in hematoplasma and erevy body fluids and in a fibrillar form in the extracellular matrix of connective tissue. The former is called by hematoplasma Fn, and the latter is called by cell Fn.Fibronectin-binding protein (FNZ) is an important surface protein and virulenceassociated factor of Streptococcus equi subsp.zooepidemicus, which is to mediate substrate adhesion of eukaryotic cells.It is one of the main adhension of SEZ.In the present study,at the molecular levels,we investigated the virulence factors of FNZ in order to get new insights into the pathogenic mechanism and new prevention methods against Streptococcus equi subsp.zooepidemicus.Based on the sequence of FNZ published on the genbank of Streptococcus equi subsp.zooepidemicus,FNZ gene was amplified from genomic DNA of Streptococcus equi subsp.zooepidemicus ATCC35246 strain by polymerase chain reaction(PCR), then the amplified gene was cloned into pMD-19 vector.The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing.Nucleotide sequencing analysis revealed a 1895bp fragment of FNZ gene of strain ATCC35246,the accession number of this sequence to GenBank is DQ363208.sequence comparison with other published FNZ gene of Streptococci showed that the FNZ gene nucleotide sequence was 89.9% identical to that of Streptococcus equi subsp. zooepidemicus VTU211 strain, and 78 %identical to that of Streptococcus equi subsp. equi, and the FNZ protein sequence was 84.4% identical to that of Streptococcus equi subsp.zooepidemicus VTU211 strain, 44.1 % identical to that of Streptococcus equi subsp. equi. It is low homology with other streptococcus. More mutation exists and 111bp is absent in FNZ gene. Based on the sequence of ATCC35246 FNZ gene, five gene fragments of FNZ gene beween 4-277bp、4-808bp、592-1134bp、895-1615bp and 4-1716bp were amplified from the genomic DNA of SEZ by PCR.Then the amplified fragments were cloned into the plasmid of pET-32a (+),the recombinant plasmids of pFNZ (2-91)、pFNZ (2-268)、pFNZ (197-378)、pFNZ (299-538) and pFNZ were verified by restriction endonuclease analysis and nucleotide sequencing.These recombinant plasmids were trasfomated into its host E.coli strains BL21, recombinant proteins of 31、50、45、25、84kD were highly expressed after induced by IPTG respectively. SDS-PAGE analysis showed that the recombinant proteins had the molecular weight as expected.Western ligand affinity blot assay were used with recombinant fusion protein pFNZ (2-91)、pFNZ(2-268)、pFNZ(197-378)、pFNZ(299-538)and pFNZ,respectively.The results showed that the recombinant fusion protein pFNZ (2-268)、pFNZ (197-378)、pFNZ (299-538) and pFNZ can bind hFn. Therefore, it is speculated that the epitopes are present in FNZ gene 277-808bp and 895-1134bp, the FNZ protein have linear Fn-binding domain between 197-268 and 299-378 amino acid residue.The BL21 was induced by IPTG to over express, which contains the linear Fn-binding domain recombinant plasmids, SDS-PAGE showed that the fusion protein exist in supernatant. The fusion protein was purified by affinity chromatograph column, and emulsionized by ISA206. The ICR mice were immunized with the purified recombinant protein of pFNZ(2-268)、pFNZ(299-538)和pFNZ (2-268)+pFNZ(299-538) respectively, the 20%、30% and 30% of mice were survival after challenge with Streptococcus equi subsp.zooepidemicus strain ATCC35246. It demonstrated that the recombinant protein was a protective antigen and maybe a critical functional fragment of FNZ.更多还原