E型产气荚膜梭菌iota毒素的截短表达及细胞毒性鉴定  

Truncated expression and cytotoxicity identification of iota toxin of Clostridium perfringens type E

作　　者：白静[1] 史燕敏 付璇 赵宝华[1] BAI Jing;SHI Yan-min;FU Xuan;ZHAO Bao-hua(College of Life Science,Hebei Normal University,Shijiazhuang 050024,China)

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024

出　　处：《中国兽医学报》2020年第5期960-965,共6页Chinese Journal of Veterinary Science

基　　金：病原微生物生物安全国家重点实验室开放课题资助项目(SKLPBS1529);2018年河北省研究生创新资助项目(CXZZBS2018097)。

摘　　要：E型产气荚膜梭菌产生的iota(ι)毒素能够引起多种动物坏死性肠炎、肠毒血症及人的食物中毒等。目前该病在临床上尚无有效治疗措施,严重威胁牲畜及人类的健康。目前活化的ι毒素多提取自产气荚膜梭菌培养液,或将异源表达的原毒素经酶切后获得,过程繁琐,毒素产量低。本试验对ι毒素基因(ia和ib)进行截短突变,构建异源表达载体,进行截短突变体蛋白的可溶性表达;以犬肾上皮细胞(MDCK细胞)为宿主细胞进行细胞毒性试验。结果显示纯化的ι毒素截短突变体蛋白具有较强的细胞毒性作用,细胞坏死特征明显:细胞形态不规则,无明显清晰的细胞膜,胞质液泡化,细胞核肿胀占据大部分空间,核内异染色质絮状化,线粒体出现空泡结构。因此,本试验制备的ι毒素截短突变体蛋白无需额外酶切即具备生物活性,节省了时间及成本,为ι毒素致病机理的深入研究奠定了基础。The iota(ι) toxin produced by Clostridium perfringens type E causes many kinds of animal necrotizing enteritis,enterotoxemia and human food poisoning.At present,there is no effective treatment for the disease in clinic,which seriously threatens the health of livestock and human beings.Most of the activated ι toxins are extracted from Clostridium perfringens culture medium or obtained by enzymatic digestion of heterologous expressed prototoxins.The process is cumbersome and the yield of toxins is low.In this study,truncated mutants of ι toxin genes(ia and ib) were used to construct heterologous expression vectors for soluble expression of truncated mutant proteins.Cytotoxicity tests were carried out with canine renal epithelial cells(MDCK cells) as host cells.The results showed that the purified truncated mutant protein had strong cytotoxicity and obvious necrosis characteristics including irregular cell morphology,no clear cell membrane,vacuolation of cytoplasm,swelling of nucleus occupying most of the space,flocculation of heterochromatin in nucleus,vacuole structure of mitochondria.Therefore,the truncated mutant protein of ι toxin prepared in this study has biological activity without additional digestion,which saves time and cost,and provides material guarantee for further study of the pathogenesis of ι toxin.

关 键 词：E型产气荚膜梭菌 iota毒素 截短突变体蛋白 细胞毒性 

分 类 号：S852.61[农业科学—基础兽医学]