B型产气荚膜梭菌C58-1株ε毒素基因序列分析与可溶性表达  

Soluble Expression and Sequence Analysis of ε Toxin Gene of Clostridum perfringens Type B C58-1 Strain

作　　者：林初文[1,2] 张松林[2] 刘磊 马永彪[3] 沈志强[2,3] 韩文瑜[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]山东省滨州畜牧兽医研究院,山东滨州256600 [3]山东绿都生物科技有限公司,山东滨州256600

出　　处：《动物医学进展》2015年第5期29-32,共4页Progress In Veterinary Medicine

基　　金：山东省现代农业产业技术体系羊产业创新团队项目(SATS-201226-3)

摘　　要：利用PCR技术从B型产气荚膜梭菌扩增出ε毒素基因,然后用限制性核酸内切酶BamHⅠ和SalⅠ对其进行酶切,回收984bp的ε毒素基因片段,将其定向克隆在载体PET-32a中,获得重组质粒pETε984。将pETε984转化至受体菌BL21(DE3)中。重组菌株经IPTG诱导后,其表达产物经SDS-PACE分析。结果表明,重组目的蛋白在大肠埃希菌成功表达,融合蛋白大小为51ku,存在于细菌培养上清中,可溶性蛋白占菌体总蛋白相对含量的67.8%。序列分析表明C58-1株ε毒素蛋白序列与目前公布的所有B型和D型产气荚膜梭菌同源性均在99.7%以上,但ε毒素蛋白序列第321位出现了S→Y突变。研究结果为ε毒素蛋白的亚单位疫苗研究奠定了基础。In order to investigate the characteristics of prokaryotic expression ε toxin gene was amplified from Clostridum perfringens type B C58-1 strain by PCR, PCR products were digested with restriction endonucleases BamH I and Sal I , then the 984 bp gene fragment was recovered and inserted into the same site of PET-32a vector. The recombinant plasmid pETe984 was obtained and transfected into BL21（DE3）. The recombinant strain was induced with IPTG for expression. In SDS-PAGE analysis,the fusion protein was 51 ku as expected and distributed in ultrasonic lysis, and the expressed products were about 67.8% of total cellular protein. The ε sequence analysis showed that more than 99.7% homology was found in com- parison with that of Clostridum perfringens serotype 13 and D. However, S→Y mutation was determined in the 321th protein sequence. The results lay an important foundation for the further subunit vaccine research of epsilon toxin.

关 键 词：产气荚膜梭菌B型C58-1株 ε毒素基因 表达 序列分析 

分 类 号：S852.616.3[农业科学—基础兽医学]