猪传染性胸膜肺炎放线杆菌ApxⅣ毒素基因的克隆与表达  

Cloning and expression of ApxⅣ of Actinobacillus pleuropneumoniae

作　　者：王方昆[1] 王一成[2] 袁秀芳[2] 徐丽华[2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]浙江省农业科学院畜牧兽医研究所,浙江杭州320021

出　　处：《中国兽医学报》2007年第5期657-660,共4页Chinese Journal of Veterinary Science

基　　金：浙江省科技计划重点资助项目(2004C22044)

摘　　要：以猪传染性胸膜肺炎放线杆菌(APP)血清2型标准菌株基因组DNA为模板,用PCR方法扩增ApxⅣ毒素基因特异片段1.5 kb左右,将PCR产物纯化后与pMD18-T连接并测序,结果该片段的碱基序列与GenBank中标准株序列的同源性为98%。随后将该片段亚克隆到原核表达载体pET-28a(+)的多克隆位点,经鉴定后得到重组质粒pET-ApxⅣ,将此重组质粒转化到受体菌BL21-DL3中,并用诱导剂乳糖进行诱导表达,5 h后表达达到高峰。经12%SDS-PAGE电泳检测,表达得到的融合蛋白约为61 000。经Western blotting分析,表达蛋白能与APP阳性血清发生特异性反应,而与阴性血清不反应。The ApxⅣ gene from serotype 2 strain of Actinobacillus pleuropneumoniae was amplified by PCR with a size about 1500 bp. The amplified DNA fragment was cloned into pMD18-T and sequenced. The result of sequencing showed that the consistency was 98%compared with that of standard strains. Inserted in pET-28a（ ＋ ） ,and transformed in Escherichia coli BL21（DL3）, The fragment was expressed after induction with lactose, The amount of expressed protein reached peak at 5 hours after the induction. The results of SDS-PAGE and Western blotting showed that the protein was 61 000 in size and specifically reacted with App-positive sera from pigs,but didn＇t with the negative sera. These results lay the foundation of studying novel vaccine and developing effective diagnostic method.

关 键 词：猪胸膜肺炎放线杆菌 ApxⅣ 克隆 表达 

分 类 号：S855.12[农业科学—临床兽医学]