【作者】 毛颖；

【导师】 范红结；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 马链球菌兽疫亚种(Streptococcus equi ssp. zooepidemicus)属于兰氏分群的C群链球菌,是我国猪链球菌病的主要病原。主要引起猪的脑膜炎、败血症、关节炎及突发性死亡,现有灭活疫苗和弱毒活疫苗的免疫效果存在不足；所以,发掘新的免疫原是研制新型亚单位疫苗的当务之急。本试验分别提取马链球菌兽疫亚种ATCC35246株的胞外蛋白(extracellular proteins, EP).胞膜相关蛋白(membrane-associated proteins, MAP)和胞壁相关蛋白(cell wall-associated proteins, CWP),采用免疫蛋白组学的方法,以微型定远猪的ATCC35246康复血清作为一抗,辣根过氧化物酶标记的葡萄球菌蛋白A(HRP-SPA)为二抗,鉴定具有免疫原性的蛋白。将重复胶上与之对应的蛋白点进行飞行时间质谱(MALDI-TOF-MS)或串联飞行时间质谱(MALDI-TOF／TOF-MS),并进行肽指纹图谱分析。最后,一共有8个胞外蛋白、11个膜相关蛋白和13个胞壁相关蛋白被成功鉴定出来。BLAST结果(www.sanger.ac.uk)显示所有成功鉴定的蛋白对应核酸序列与马链球菌兽疫亚种具有很高的同源性(≥61%),除AE6(UDP-氮乙酰葡萄糖胺焦磷酸化酶)、AE7(UDP葡萄糖焦磷酸化酶)外的所有鉴定蛋白在马链球菌兽疫亚种中均为第一次报道。为了检测这些蛋白对应基因片段在猪源链球菌不同中国分离株中的分布状况,从而进一步确定疫苗候选抗原靶分子提供依据,我们选择了同源性大于等于60%的蛋白,然后根据Sanger中心公布的SEZ序列设计并合成引物、进行PCR检测。比较弱毒株ST171和强毒株ATCC35246的结果,我们发现AE4(3-磷酸甘油醛脱氢酶)、AM1(黄素单核苷酸腺苷酰转移酶)、AM8(DNA导向RNA聚合酶α亚单位)和AC11(丙酮酸激酶)这四对引物仅在强毒株中被检测到,这预示这四个蛋白与ATCC35246的毒力因子有关.更让我们感兴趣的是,CY株的PCR检测结果与ATCC35246的相一致。因此,我们有理由相信,这株有自主知识产权的菌株在以后的试验中能逐步替代ATCC35246,成为毒力研究的标准模式强毒株。更多还原

【Abstract】 Streptococcus equi ssp. zooepidemicus (SEZ) is classified in Lancefield’s group C, and it is the main pathogen causing meningitis, septicemia, arthritis and sudden death in swine. Subunit vaccine is preferred in our vaccine development strategies due to its high efficiency and safety, while the killed whole-cell vaccines and the living vaccines both have severe defects. The development of an effective vaccine is vital to circumvent the widespread economic losses in the event of a pandemic. Therefore, the identification of novel immunogens is key to which may become potential vaccine candidates.In our research, the extracellular proteins (EP), membrane-associated proteins (MAP) and cell wall-associated proteins (CWP) of ATCC35246 strain were extracted separately. An immunoproteomic approach combining two-dimensional electrophoresis (2-DE), Western blot and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) or tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been used to rapidly survey the all proteins of ATCC35246, to recognize new immunogens and direct vaccine selecting research. At last, there are 8 EPs (AE1, AE4-10),11 MAPs (AM1-8, AM10,12,13),13 CWPs (AC1-13) being successfully identified. The BLAST (www.sanger.ac.uk) showed all of these proteins shared high identities with SEZ (≥61%), and none of these proteins had been reported before, except for AE6 and AE7.The PCR detection was performed to detect the presence of genes encoding the identified proteins in 5 swine origin group C streptococci (Chinese isolates). We only chose the proteins with high identities (over 61%) to be our objectives, and the primers were designed and synthesized, according to the sequence of SEZ in Sanger database. This procedure would provide additional information for vaccine candidates.Comparing avirulent ST171 and virulent ATCC35246, the products of AE4, AM1, AM8 and AC11 were only detected in virulent strain ATCC35246, and they could be the candidates of subunit vaccine. Interestingly the virulent strain CY shared the same results with ATCC35246, so we believe CY will replace ATCC35246 as a new standard strain in future, for it has independent intellectual properties.更多还原