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

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.