猪胸膜肺炎放线杆菌毒素Ⅱ基因的克隆、表达及其免疫原性  

Cloning,Expression and Immunogenic Characterization of apxIIA Gene of Actinobacillus pleuropneumoniae

作　　者：严克霞[1] 刘建杰[2] 吴斌[1] 汤细彪[1] 蔡利军[1] 杨明柳[1] 陈焕春[1] 周锐[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室动物病原分室,武汉430070 [2]海口农工贸(罗牛山)股份有限公司,海口570125

出　　处：《农业生物技术学报》2006年第4期493-497,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.30530590);教育部新世纪优秀人才支持计划(No.NCET-04-0740)资助。

摘　　要：以本实验室分离的猪胸膜肺炎放线杆菌血清2型(Actinobacilluspleuropneumoniaeserotype2,APP-2)菌株HB08的基因组为模板,扩增了2871bp的APP毒素Ⅱ的结构基因apxIIA,并克隆到pET-28a原核表达载体中构建重组表达质粒pET28aIIA,转化到大肠杆菌(Escherichiacoli)BL21(DE3),经SDS-PAGE和Westernblot分析表明,表达的重组蛋白具有良好的反应活性。将表达的蛋白经或不经复性处理,与纯化的天然毒素Ⅱ分别免疫昆明小鼠,同时设PBS空白对照组,每组12只,间隔2周免疫2次,采血检测其抗体效价,二免后2周用致死剂量的APP血清7型(APP-7)菌株(1.08×108CFU(菌落形成单位,colonyformunit)/只)腹腔攻击。结果显示,复性蛋白组的保护率为83.3%,非复性蛋白组的保护率为58.3%,天然毒素蛋白对照组保护率为91.7%,空白对照组小鼠全部死亡,说明复性的重组毒素Ⅱ具有良好的免疫原性。The structural gene encoding Apx Ⅱ toxin （apxHA） was amplified from the genomic DNA of Actinobacillus pleuropneumoniae serotype 2 （APP-2） strain HB08 and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blotting analysis showed that the apx/L4 gene was expressed in Escherichia coli BL21 （DE3） and the expression products could react with Apx Ⅱ antibodies. The recombinant Apx Ⅱ was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice with an interval of two weeks using unfolded and refolded recombinant proteins, the native Apx Ⅱ extracted from the cultural supernatant of APP strain of serotype 7 （APP-7） and PBS, respectively. Serum antibody was examined by Apx Ⅱ -specific ELISA two weeks post every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 （1.08× 10^8CFU （colony form unit） per mouse）. The protection rate reached 91.7% in the native Apx Ⅱ group, 83.3 % in the refolded recombinant protein group and 58.3 % in the misfolded recombinant protein group, while all the mice in the PBS group died in 36 h post challenge. The data revealed that the refolded recombinant Apx Ⅱ had excellent immunogenicity and could elicit protection from the lethal challenge of APP.

关 键 词：胸膜肺炎放线杆菌 毒素Ⅱ 克隆 原核表达 免疫原性 猪 

分 类 号：S858.28[农业科学—临床兽医学]