中国兽医科学2018，Vol.48Issue(9)：1124-1130,7.DOI:10.16656/j.issn.1673-4696.2018.0166
腐败梭菌α毒素重组突变体的免疫保护力评价
Evaluation of protective efficacy of recombinant mutant of Clostridium septicum a toxin
摘要
Abstract
This study was conducted to obtain a recombinant mutant of Clostridium septicum a toxin and to evaluate its virulence and immunogenicity. Based on the known sequence of Clostridium septicum a toxin gene,the gene fragment Gcsam4 with four amino acid mutations,C86L,N296A,H301A and W342,was optimized and synthesized. Then.Gcsam4 was cloned into prokaryotic expression vector pET-30a(+ ) for expression and purification. Reactivity of the purified protein(rcsam4) with antiserum of Clostridium septicum was detected by Western-blot. Meanwhile,rcsam4 was incubated with Vero cells and injected into mice to detect the virulence of rcsam4. According to the method prescribed in Chinese Veterinary Pharmacopoeia(2015) ,four rabbits were immunized with rcsam4 emulsified with oil adjuvant ISA 206 to prepare antiserum and detect the neutralizing titer of antiserums after the primary and second immunization. Moreover,rabbits were challenged with 1 rabbit MLD Clostridium septicum toxin through the ear marginal veins on day 21 after the second immunization to detect the protective efficacy of rcsam4. In result ,rcsam4 was presented predominantly in an insoluble form(inclusion bodies) and it could react with the antiserum of Clostridium septicum. For Vero cell,there were no apparent cytopathic effect (CPE) incubated with rcsam4 of 9. 85 μg/mL. At the same time, rcsam4 with the injection volume of 5. 5 × 103 μ g/kg was still not fatal to ICR mice. After the first immunization, sera from rabbits immunized with rcsam4 could neutralize 50-70 mice MLD Clostridium septicum toxin per mL,and 110—130 mice MLD after twice immunization. Moreover,rabbits the in rcsam4 immunized group fully survived at the dose of 1MLD of Clostridium septicum toxin challenge, whereas all of the rabbits died (4/4) in the control groups. These results suggest that the recombinant protein rcsam4 is a potential vaccine candidate for future research.关键词
腐败梭菌α毒素/突变/重组表达/毒性/抗原性Key words
Clostridium septicum α toxin/mutation/recombinant expression/virulence/antigenicity分类
农业科技引用本文复制引用
杜吉革,薛麒,朱真,李启红,印春生,彭小兵,姚文生,康凯,陈小云..腐败梭菌α毒素重组突变体的免疫保护力评价[J].中国兽医科学,2018,48(9):1124-1130,7.基金项目
科技部"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资助项目(201702) （201702）
