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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
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邵美丽1，2，刘思国1，王春来1，郭设平1，宫强1，佟恒敏2
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(1. 中国农业科学院 哈尔滨兽医研究所，黑龙江 哈尔滨150001；
2. 东北农业大学 动物医学院，黑龙江 哈尔滨150030)
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摘要：
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以猪传染性胸膜肺炎放线杆菌(APP)血清7型254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 18T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pGEX6P1，成功构建了重组表达载体pGEXomp；以此转化大肠埃希氏菌BL21(DE3)，经SDSPAGE鉴定，表达的可溶性融合蛋白分子质量约为61ku，命名为GSTOMP。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。GSTOMP蛋白的成功表达为APP OMP相关分子生物学功能的研制奠定了基础。 |
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关键词：
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猪胸膜肺炎放线杆菌； 外膜蛋白基因； 克隆； 表达 |
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中图分类号：S 852.619: Q 786 文献标识码：
A 文章编号：1673-4696(2006)01-0025-04 |
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Cloning and expression of OMP gene from
Actinobacillus pleuropneumoniae |
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SHAO Meili1,2, LIU
Siguo1, WANG Chunlai1, GUO
Sheping1, GONG Qiang1, TONG
Hengmin2 |
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(1.Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;
2. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China ) |
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Abstract: The DNA fragment of OMP gene was amplified from genomic DNA of Actinobacillus pleuropneumoniae (APP) 254 strain by PCR and the amplicons were cloned into a pMD 18T vector. After being confirmed by sequencing, the gene was subcloned into the expression vector pGEX6P1, and the recombinant plasmid was named as pGEXomp. An expected fusion protein(GSTOMP) was expressed properly in E.coli BL21(DE3) transfected with the pGEXomp 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 254 strains, as confirmed by ELISA. Results showed that the expressed GSTOMP may be useful for the studies of molecular biological functions of APP OMP. |
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Key words: Actinobacillus pleuropneumoniae; OMP gene; cloning; expression |
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