【作者】 赵娜；

【导师】 龙良启；

【作者基本信息】 华中农业大学 ， 水生生物学， 2006， 硕士

【摘要】 猪传染性胸膜肺炎（porcine contagious pleuropneumonia，PCP）是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的猪的一种高度传染性呼吸道疾病。本研究利用噬菌体随机12肽库对APP菌体及3种主要外毒素（APxⅠ、APxⅡ与APxⅢ）的抗原模拟表位进行了筛选，并对展示在噬菌体表面的模拟表位的免疫原性进行了初步鉴定。主要结果如下： 1．猪传染性胸膜肺炎菌体抗原模拟表位的筛选 利用猪传染性胸膜肺炎疫苗免疫日本大耳兔得到抗血清，将血清用饱和硫酸铵粗提后，再经低压层析系统纯化得到IgG，用此IgG为配体包被酶标板对噬菌体随机12肽库进行3轮不同条件的富集筛选。经过3轮筛选，噬菌体的回收率从5.76×10^-5增加到了1.69×10^-2，P／N值逐步提高，阳性克隆得到了富集；ELISA表明，随机挑取的10个噬菌体中有6个与纯化的IgG有较强的结合能力，且与无关抗体无交叉反应，特异性良好。梯度ELISA表明，阳性噬菌体克隆与抗血清的结合呈剂量依赖关系。在每一轮的筛选过程中，我们将洗脱下来的噬菌体再用正常的兔血清IgG进行吸附，去除了假阳性的噬菌体克隆，保证了阳性噬菌体克隆的特异性。DNA测序结果显示，所得到的6个噬菌体阳性克隆中，有5个阳性克隆递呈相同的氨基酸序列。 2．猪传染性胸膜肺炎3种主要外毒素抗原模拟表位的筛选与鉴定 将猪传染性胸膜肺炎3种主要外毒素（APxⅠ、APxⅡ与APxⅢ）的抗血清用饱和硫酸铵粗提后，再经低压层析系统得到纯化的IgG，用此IgG作配体包被酶标板对噬菌体随机12肽库分别进行不同条件的富集筛选。经过4轮筛选，噬菌体的回收率与P／N值逐步提高；ELISA表明，随机分别挑取的10个噬菌体中，有5个与纯化的IgG有较强的结合能力，且无交叉反应。在筛选过程中，我们采取外毒素蛋白抗原竞争洗脱的方法，而不是酸洗脱的方法，保证了阳性噬菌体克隆的特异性。DNA测序结果显示，所得到的噬菌体模拟表位与3种主要外毒素蛋白之间有较高的同源性。 3．噬菌体抗原模拟表位的免疫原性研究 利用筛选到的噬菌体阳性克隆、猪传染性胸膜肺炎全菌灭活苗与亚单位苗免疫昆明小鼠，利用间接血凝方法检测菌体抗体并用ELISA的方法检测外毒素抗体，显示有低水平的特异性抗体出现。更多还原

【Abstract】 Porcine contagious pleuropneumonia （PCP） caused by Actinobacillus pleuropneumoniae （APP） is a severe, contagious pulmonary disease of pigs. Mimotopes of APP and its 3 major Apx （Actinobacillus pleuropneumoniae RTX toxin） were explored from the phage display 12-mer random peptides library, and the immunogenicities of the different epitopes were identified after the screening. The results are as following:1. Screening mimotopes of Actinobacillus pleuropneumoniaeThe specific sera IgG were obtained by immunizing the rabbit with Actinobacillus pleuropneumoniae vaccine, and purified through low-pressure chromatography system after rude purification. This IgG was used as target to screen the phage display 12-mer random peptides library for 3 rounds in different conditions. After 3 rounds of screening, the yield ratio increased from 5.76×10^-5 to 1.69×10^-2, and the P/N increased gradually. There were 6 phage clones identified by ELISA and they did not have cross-reactions with irrespective antibodies. Gradient ELISA of the positive phage clones showed a dose independent relationship. In order to wipe of the fake positive, the eluted phages were absorbed by the negative rabbit sera. DNA sequence assays showed that 5 of the 6 positive clones displayed the same peptide sequences.2. Screening mimotopes of 3 major Actinobacillus pleuropneumoniae RTX toxins The specific sera IgG of 3 major Apx（Apx I ,Apx II ,and ApxIII） were purified throughlow-pressure chromatography system after rude purification. These IgG were used as targets to screen the phage display 12-mer random peptides library in different conditions respectively. After 4 rounds of screening, the yield ratios and the P/N increased gradually.5 phage clones were identified by ELISA respectively and they did not have cross-reactions. During the selection progresses, the competitional elution implemented by Apx protein was used but not acid elution to guarantee the specifities of the positive clones. DNA sequence assays showed the high homologous between the phage epitopes and 3 major Apx.3. Identification of the immunogenicities of the positive phage clones Kunming mice were immunized by the different positive clones of phage, inactivatedAPP and Subunit bacteria vaccine. Specific antibodies of APP and Apx were detected by IHA and ELISA.更多还原