畜牧兽医学报2018，Vol.49Issue(4)：777-785,9.DOI:10.11843/j.issn.0366-6964.2018.04.015
产气荚膜梭菌重组ε毒素突变体的免疫保护力评价
Evaluation of Protective Efficacy of Recombinant Mutant of Clostridium perfringens ε Toxin
摘要
Abstract
This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin and subsequently evaluate the virulence and immunogenicity of the recombinant toxin.The ε toxin gene of C.perfringens type D strain using optimized codons was synthesized based on the sequence reported.At the same time,three amino acid mutations:Y30 and Y196 substituted with alanine,H106 substituted with proline,were introduced into this sequence synthesized.Then,this ε toxin gene was cloned into prokaryotic expression vector pET3a-(+) to construct a recombinant E.coli,followed by induction with IPTG to yield the recombinant protein,following with purification.The reactivity of the purified protein with antiserum of C.perfringens type D was determined by Western blot and the mouse was used to evaluate the virulence of purified protein.The rabbit antiserum against the recombinant proteins was prepared and the neutralizing titer was measured according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015).The results showed that recombinant protein was expressed at a high level in a soluble form with a ratio of about 40％ bv grav scale scanning,And the protein could reAct with thE antiserum of C.perfringens type D.The protein with the injection volume of 6.25 × 106 ng · kg-1 still shows no virulence to mouse.The titer of rabbit antiserum against Clostridium perfringens type D could reach 50-60 and 400-450 Minimum Lethal Dose (MLD) after the first and second immunization and respectively.After challenge with 1MLD of C.perfringens type D toxin,all the rabbits immunized with recombinant protein were protected.The results suggest that the recombinant mutant of C.perfringens e toxin without virulence retains the good immunogenic antigen,which provides important experimental data for the development of novel C.perfringens vaccine.关键词
产气荚膜梭菌ε毒素/突变/重组表达/毒力/抗原性Key words
Clostridium perfringens ε toxin/mutation/recombinant expression/virulence/antigenicity分类
农业科技引用本文复制引用
杜吉革,朱真,薛麒,李启红,印春生,彭小兵,姚文生,康凯,陈小云..产气荚膜梭菌重组ε毒素突变体的免疫保护力评价[J].畜牧兽医学报,2018,49(4):777-785,9.基金项目
科技部十三五“牛羊重要疫病免疫防控新技术研究”重点专项课题(2017WFD0500903) （2017WFD0500903）
中国兽医药品监察所所级课题(201702) （201702）
