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猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达
引用本文:邵美丽,刘思国,王春来,郭设平,宫强,佟恒敏. 猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达[J]. 中国兽医科学, 2006, 36(1): 25-28
作者姓名:邵美丽  刘思国  王春来  郭设平  宫强  佟恒敏
作者单位:1. 中国农业科学院,哈尔滨兽医研究所,黑龙江,哈尔滨,150001;东北农业大学,动物医学院,黑龙江,哈尔滨,150030
2. 中国农业科学院,哈尔滨兽医研究所,黑龙江,哈尔滨,150001
3. 东北农业大学,动物医学院,黑龙江,哈尔滨,150030
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。

关 键 词:猪胸膜肺炎放线杆菌  外膜蛋白基因  克隆  表达
文章编号:1673-4696(2006)01-0025-04
修稿时间:2005-08-18

Cloning and expression of OMP gene from Actinobacillus pleuropneumoniae
SHAO Mei-li,LIU Si-guo,WANG Chun-lai,GUO She-ping,GONG Qiang,TONG Heng-min. Cloning and expression of OMP gene from Actinobacillus pleuropneumoniae[J]. Chinese Veterinary Science, 2006, 36(1): 25-28
Authors:SHAO Mei-li  LIU Si-guo  WANG Chun-lai  GUO She-ping  GONG Qiang  TONG Heng-min
Abstract: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.
Keywords:Actinobacillus pleuropneumoniae  OMP gene  cloning  expression
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