坏死梭杆菌白细胞毒素基因BSBSE片段的克隆及原核表达  

Cloning and Expression of the Leukotoxin BSBSE Gene from Fusobacterium necrophorum

作　　者：陈立志[1] 冯书章[2] 张秀华[3] 王克坚[3] 刘晓颖[3] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]解放军军事医学科学院军事兽医研究所,长春130062 [3]中国农业科学院特产研究所,吉林132109

出　　处：《吉林农业大学学报》2007年第5期568-571,共4页Journal of Jilin Agricultural University

基　　金："十五"国家科技攻关子课题(2002BA518A04);中国农业科学院特产研究所科研基金项目(Tcs2005-03)

摘　　要：以牛源坏死梭杆菌FNn株为试材,根据GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列设计1对引物,利用PCR技术扩增出1 131 bp的坏死梭杆菌白细胞毒素BSBSE基因。将PCR产物插入pGEM-T Easy vector中,经双酶切鉴定正确后进行序列测定。分析表明该BSBSE序列与GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列的核苷酸同源性为99%,推导出的氨基酸序列同源性为98%。为研究BSBSE的免疫原性,构建了原核表达载体pMAL-p2X-BSBSE,用IPTG诱导在大肠杆菌中表达。结果表明,BSBSE基因在大肠杆菌中进行了高效特异性融合表达,融合蛋白分子量约为84.5×103,其中41.5×103为BS-BSE基因表达的蛋白质,43.0×103为MBP融合标签,Western-blotting检测表明该表达产物有免疫原性。According to the sequence of announced lktA gene in Fusobacterium necrophorum, a pair of primers were designed. The BSBSE gene was amplified by PCR. The product was cloned into pGEM-T Easy vector. When nucleotide sequence and deduced amino acid sequence were compared with homologous sequence of the FN AF312861 lktA of GenBank, the homologne of the mucleotide sequence is 99% and the homologne of the amino acid sequence is 98 %. The BSBSE fragment was inserted into expression vector pMAL-p2X and the plasmid pMAL-p2X-BSBSE were expressed in E. coli BL21 by IPTG induction. The SDS-PAGE analysis indicated the weight of the fusion protein was about 84.5.0× 10^3, which ineluded the 41.5 x 10^3 protein expressed from BSBSE gene and 43.0 ×10^3 fusion MBP tag.The recombinant BSBSE-pMAL-p2X production has Immunogenicity with western-blotting. The cloning and expression of the BSBSE gene established the foundation of further research on the function and application of the BSBSEgene.

关 键 词：坏死梭杆菌 白细胞毒素 BSBSE 克隆 表达 

分 类 号：Q78[生物学—分子生物学] S852.61[农业科学—基础兽医学]