猪传染性胸膜肺炎放线杆菌毒素Ⅰ抗原模拟表位的筛选与鉴定  

Screening and Identifying Mimotopes of Actinobacillus pleuropneumoniae RTX Toxin Ⅰ

作　　者：何玉龙[1] 赵娜[1] 伍晓雄[1] 王安华[1] 龙良启[1] 刘平 

机构地区：[1]华中农业大学,武汉430070 [2]武汉成长动物营养研究所,武汉430070

出　　处：《畜牧兽医学报》2006年第10期1073-1077,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：湖北省自然科学基金(2004ABA133)

摘　　要：将猪传染性胸膜肺炎放线杆菌毒素Ⅰ(APP ApxⅠ)抗血清用饱和硫酸铵分级粗提后,再经DEAE-sepha-dex-A50低压层析系统纯化得到IgG,用此IgG作为配基对噬菌体随机十二肽库进行4轮不同条件的富集筛选。通过改变筛选条件,噬菌体的回收率从1.44×10-6增加到了8.9×10-5,P/N值逐步提高,阳性噬菌体得到了富集。ELISA结果表明,随机挑取的10个噬菌体克隆中,有5个与纯化的IgG有较强的特异性结合能力。对筛选到的5个阳性克隆提取ssDNA进行测序,并对其递呈的氨基酸序列进行分析。结果有3个克隆的4个连续的氨基酸序列(RVDV)相同,并与APP ApxⅠ蛋白的原始序列具有较高的同源性。The specific serum IgG was purified through DEAE-sephadex-A50 low pressure chromatography system after rude purification. This IgG was used as target molecular to screen the phage display 12-mer random peptide library in different conditions for 4 rounds. By altering the condition of screening, the yield ratio increased from 1.44×10^-6 to 8.9× 10^- 5, and the P/N value increased gradually, which meant that specific enrichment had been achieved. 5 phage clones were identified as positive by ELISA and they did not have cross reactions with irrespective antibodies, ssDNA of 5 positive clones were purified for DNA sequencing and the sequences of peptides displayed on the positive clones were analyzed. 4 continuous amino acid （RVDV） of 3 clones were identical, and they were homologous to the original peptide of Actinobacillus pleuropneumoniae RTX toxin Ⅰ

关 键 词：噬菌体随机肽库 模拟表位 猪胸膜肺炎放线杆菌 外毒素Ⅰ 

分 类 号：S852.61[农业科学—基础兽医学]