猪传染性胸膜肺炎放线杆菌毒素apxⅢA基因的克隆和表达  

Cloning and expressing of toxin apxⅢA gene of Actinobacillus pleuropneumoniae in E. coli

作　　者：邵美丽[1] 刘思国[1] 王春来[1] 郭洋[1] 张秀华[1] 郭设平[1] 佟恒敏[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2006年第2期139-141,共3页Chinese Journal of Preventive Veterinary Medicine

摘　　要：以猪传染性胸膜肺炎放线杆菌血清2型参考株基因组DNA为模板,PCR方法扩增出apxⅢA基因3.1kb的片段,扩增产物克隆于pMD18-T中,重组质粒经酶切鉴定后进行核苷酸序列测定,并与GenBank中不同血清型胸膜肺炎放线杆菌apxⅢA基因进行比较,结果显示核苷酸同源性均在96%以上。将该基因片段亚克隆到原核表达载体pGEX-6P-1的BamHⅠ/SalⅠ位点,成功构建了重组表达载体pGEX-apxⅢA,转化大肠杆菌BL-21(DE3)中并获得表达。SDS-PAGE结果显示,表达的融合蛋白分子量约为140Ku,与预测相符,Westernblot证明该融合蛋白具有免疫学活性。该融合蛋白的成功表达为猪传染性胸膜肺炎亚单位疫苗的研制奠定了基础。This study described the expression of Actinobacillus pleuropneumoniae （APP） Apx Ⅲ in E. coli. The DNA fragment ofapx ⅢA was amplified from the genomic DNA ofAPP serotype 2 reference strain by PCR and cloned into the pMD18-T vector. The sequence analysis showed that the cloned gene shared more than 96 % nucleotide homology with other serotype reference strains after blasted in GenBank. Then, the gene was subcloned into the expression vector pGEX-6P-1, yielding the recombinant plasmid pGEX-apx Ⅲ A. After transformation ofpGEX-apxⅢA into E. coli BL-21 （DE3） and subsequent induction with IPTG, an expected fusion protein was properly expressed. The fusion protein has been proved having immunogenicity when detected by Western blot, which demonstrates that it may be useful in the development of porcine pleuropneumonia subunit vaccine.

关 键 词：猪胸膜肺炎放线杆菌 apxⅢA基因 

分 类 号：S852.61[农业科学—基础兽医学] Q785[农业科学—兽医学]