摘要
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。
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-6P-1, 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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第1期25-28,共4页
Chinese Veterinary Science