【作者】 李健；

【导师】 陈焕春； 周锐；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 产毒素多杀性巴氏杆菌（Toxigenic Pasteurella multocida，T⁺Pm）是猪进行性萎缩性鼻炎（Swine progressive atrophic rhinitis，PAR）的主要病原菌之一。它可以产生一种分子量为146kDa的慢性皮肤坏死性毒素——多杀性巴氏杆菌毒素（Pasteurella multocida toxin，PMT）。该毒素由toxA基因编码，是产毒素多杀性巴氏杆菌重要的毒力因子和保护性抗原。为了分析和鉴定toxA基因中的毒力结构域和免疫原性结构域，本研究主要开展了以下几方面的工作：1．toxA基因及其截短片断的克隆与表达：以本实验室分离保存的产毒素多杀性巴氏杆菌HN-13株为研究对象。首先通过GenBank上已经公布的各种不同株的多杀性巴氏杆菌的基因序列，进行序列分析，找出其中比较保守的部位，分别设计了3对引物，以高保真DNA聚合酶用PCR方法分3段扩增了toxA基因，连接酶连接，得到全长的toxA基因，共3858bp。对获得的全长toxA基因进行基因测序，与GenBank上公布的登陆号为Z28388序列的核苷酸100％同源。然后根据相关文献的报道，对全长toxA基因进行分段截短或连接，共获得9个大小不同的片段，并构建了9个原核表达载体。将各个表达载体转化大肠杆菌BL21（DE₃）感受态细胞，其中5个获得了表达，对重组蛋白进行了纯化和Western-blot分析，结果表明：5种重组蛋白均能与HN-13株多克隆抗体发生特异性反应。2．重组蛋白的免疫原性分析：将5种重组蛋白分别用弗氏佐剂乳化，分组免疫昆明小鼠，2周后加强免疫一次，免疫前后用PMT-ELISA检测免疫组和对照组的抗体水平。二免后14天，以1个LD50的HN-13株进行攻击，统计各组小鼠在攻毒后24h和48h的存活率；取各组小鼠的肝脏和肺组织，进行组织病理学分析。结果表明，重组蛋白的免疫保护性从C端截短片段到N端截短片段依次减弱，全长片段有良好的免疫保护力。3．重组蛋白的细胞毒性分析：以小鼠成纤维细胞（NIH-3T3细胞）为材料，实验评估了5种重组蛋白的细胞毒性，观察细胞的死亡数量和病变状况。结果表明，各重组蛋白的毒性较天然毒素有所降低，毒力从C端截短片段到N端截短片段依次减弱。本课题对产毒素多杀性巴氏杆菌toxA基因的功能结构域进行了研究。分段PCR扩增并连接得到了toxA基因的全基因和缺失部分核苷酸的截短片段，分别研究了全基因与截短片段的免疫保护性和毒力，并开展了动物试验和细胞毒力试验。试验表明，截短后的重组蛋白毒力明显下降，具有较好的免疫保护力，为今后进一步研究toxA基因和新型疫苗的开发提供了理论依据。更多还原

【Abstract】 Toxigenie Pasteurella multocida （T⁺Pm） is a major pathogen of progressive atrophicrhinitis （PAR）. T⁺Pm secretes P. multocida toxin （PMT）, which is a dermonecrotic toxinabout 146 kDa, and PMT was encoded by toxA gene. PMT is one of the most importantvirulence factors and protective antigens of Toxigenic Pasteurella multocida. In order toanalyses and accredits the structural domains of virulence and immunoprotection on toxAgene, The projects are as follows:1. cloning and expressing of toxA gene and its truncated segments: T⁺Pm, which wasisolated and preserved by our lab, named HN-13. First, according to the toxA gene whichwas published in GenBank, compare their gene sequence, and find the conservation one.Three pairs of palmers were designed to clone three fragments of toxA gene with thePyrobest^TM DNA polymerase. Connect with ligase, and procured the whole toxA gene.The whole toxA gene is 3858bp. The compared result shows that the length of the toxAgene was same to the toxA gene, which was published in GenBank Z25388. Andaccording to the literature, Absenced the whole toxA gene, procured nine fragments, andgot the nine prokaryote recombinant expressive vectors. Prokaryote recombinantexpressive vectors were transformed into E.coli BL21（DE3） competent cells and theninduced to express. Cleansing five of them, procured inclusion body. Purifiedrecombination protein. And the result of Western-blot shows that the five recombinationprotein can specific reaction with polyclonal antibody of HN-13.2. Immunogenicity analysis of recombination protein: using the Freund’s adjuvant,emulsifying five of the recombination proten, immunize KUN-Ming mice of each group,immunize the KUN-Ming mice again after two weeks. Using the PMT-ELISA todetermine the level of the antibody of each group before and after immunize. Fourteendays after second time immunize, Inject of toxigenic Pasteurella multocida（HN-13） toeach group, observe the survival percentage of each group at 24h and 48h. Take liver andlung of each group, analysis in histopathology. This work shows that theimmunoprotection of recombination protein from C- fragment to N- fragment isattenuated, and the full length fragment have good immunoprotection.3. Cytotoxicity analysis of recombination protein: using the collagenoblast ofmice（NIH-3T3 cell）, appraisal cytotoxicity of five of the recombination protein. Observethe death and pathological changes of the cell. The result shows that compare with thenatural PMT, toxicity of each recombination protein is to lower. In summary, in this study, we study the funtional of toxigenic Pasteurella multocida,cloned the whole toxA gene from toxigenic Pasteurella multocida, named HN-13, andexpressed it successfully. Authenticate immunoprotection and virulence with the animalexperiment and cell experiment. Experiment shows that the recombination protein havegood immunoprotection. This was useful for further primary study of T⁺Pm and theproduction of vaccine.更多还原