作　　者： ; ; ; ; ; ; ; ; ; ; ;

机构地区： 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处： 《中国预防兽医学报》 2008年第2期153-156,共4页

摘　　要： 本研究应用限制性内切酶MboⅠ消化胸膜肺炎放线杆菌血清7型的基因组DNA,回收500bp～3000bp的片段,插入到真核表达载体pCDNA3.1(+)的BamHⅠ酶切位点构建重组质粒,电转化大肠杆菌TG1,获得胸膜肺炎放线杆菌的基因组表达文库(大约含有106个克隆)。将该文库随机分为10个亚文库(10个Clone pool),分别提取每个亚文库的重组质粒免疫小鼠(n=130只),免疫剂量为100μg,免疫3次后以1型胸膜肺炎放线杆菌进行攻毒,通过相对保护率、肺组织荷菌数、病理组织学3个指标测定其对小鼠的免疫保护作用。试验结果表明,Clone pool3、Clone pool2、Clone pool6和Clone pool7的免疫效果明显优于其他免疫组,尤其是Clone pool3的免疫保护效果最佳。本研究成功构建了7型胸膜肺炎放线杆菌的基因组表达文库,从而为筛选和获得新的保护性抗原基因奠定基础。 The genomic DNA of Actinobacillus pleuropneumoniae （APP） serotype 7 was digested with Mbo Ⅰ to generate genomic expression library, The DNA fragments of 500 bp to 3 000 bp were ligated to the eukaryotic expression vector pCDNA3.1 （＋） and transformed into competent Escoherichia coli TG1 by electroporation, The resultant genomic expression library （about 10^6 clones） was subdivided into ten pools of clones （about 10^5 clones/pool） and DNA extracted from each pool was used to inoculate the mice at 100 μg per dose, Mice were booster immunized three times and then challenged with virulent APP1, Bacteria in lungs was isolated and pulmonary tissue damage were examined, Significant protection was induced by DNA vaccination from four clone pools. The result demonstrated that the genomic expression library of APP7 generated in this work could be used to select the protective antigen genes against APP7.

关 键 词： 胸膜肺炎放线杆菌 基因组表达文库 构建 保护作用

领　　域： [农业科学] [农业科学] [农业科学] [农业科学] [农业科学] [农业科学]