猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达

以猪传染性胸膜肺炎放线杆菌(APP)血清7型25-4株基因组DNA为模板,用PCR扩增外膜蛋白(OMP)基因特异片段,并克隆于pMD 18-T中,经酶切及核苷酸序列分析鉴定后,亚克隆于原核表达载体pGEX-6P-1,成功构建了重组表达载体pGEX-omp;以此转化大肠埃希氏菌BL21(DE3),经SDS-PAGE鉴定,表达的可溶性融合蛋白分子质量约为61 ku,命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解,获得切掉标签的OMP。经ELISA检测,该OMP蛋白能够与兔抗APP的阳性血清反应,具有很好的免疫活性。GST-OMP蛋白的成功表达为APPOMP相关分子生物学功能的研制奠定了基础。

Cloning and expression of OMP gene from Actinobacillus pleuropneumoniae

The DNA fragment of OMP gene was amplified from genomic DNA of Actinobacillus pleuropneumoniae(APP) 25-4 strain by PCR and the amplicons were cloned into a pMD 18-T vector.After being confirmed by sequencing,the gene was subcloned into the expression vector pGEX-6P1,and the recombinant plasmid was named as pGEX-omp.An expected fusion protein(GST-OMP) was expressed properly in E.coli BL21(DE3) transfected with the pGEX-omp and induced with IPTG.The OMP cut tag was obtained by using GST purification kit and Factor Xa digestion.The OMP protein could react with rabbit sera against APP 25-4 strains,as confirmed by ELISA.Results showed that the expressed GST-OMP may be useful for the studies of molecular biological functions of APP OMP.