产气荚膜梭菌α毒素C末端的三拷贝串联表达与免疫原性分析  

Three Copies Recombinant Expression and Immunogenicity Analysis of C-terminal of Clostridium perfringens α Toxin

作　　者：杜吉革 刘莹[1] 朱真[1] 薛麒[1] 张秀坤 李启红[1] 印春生[1] 康凯[1] 姚文生[1] 陈小云[1] DU Jige;LIU Ying;ZHU Zhen;XUE Qi;ZHANG Xiukun;LI Qihong;YIN Chunsheng;KANG Kai;YAO Wenshen;CHEN Xiaoyun(China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《中国畜牧兽医》2018年第11期3237-3245,共9页China Animal Husbandry & Veterinary Medicine

基　　金：科技部"十三五""牛羊重要疫病免疫防控新技术研究"重点专项子课题(2017WFD0500903);中国兽医药品监察所所级课题(201702)

摘　　要：本研究旨在获得产气荚膜梭菌α毒素(CPA)C末端(第247-370位氨基酸,CPA_C)三拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPAC编码基因(GenBank登录号:AY823400.1)进行优化设计,以同向串连的方式串联成三拷贝基因(GCPA_(C3)),片段之间用柔性氨基酸linker(GGGS)连接,经人工合成后克隆至原核表达载体pET-30a(+)中进行表达与纯化,获得CPA_C的三拷贝重组蛋白(rCPA_(C3))。利用Western blotting方法检测rCPA_(C3)与A型产气荚膜梭菌毒素抗血清的反应性。将rCPA_(C3)与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,检测一免及二免后兔血清的中和抗体效价。在二免21d后,对家兔经耳缘静脉注射1个家兔最低致死剂量(MLD)的A型产气荚膜梭菌毒素,检测rCPA_(C3)对家兔的免疫保护效果。结果显示,rCPA_(C3)主要以包涵体的形式表达,且能与A型产气荚膜梭菌毒素抗血清反应;每毫升的一免抗血清可中和30～50个、二免抗血清可中和70～100个小鼠MLD的A型产气荚膜梭菌毒素。采用1个家兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组家兔100%(4/4)死亡,免疫组得到了100%(4/4)的保护。以上结果说明,rCPA_(C3)具有良好的免疫原性,为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的试验数据。This study was aimed to obtain a tandem fusion protein of three copies of CPA C-terminal(CPA C),and evaluate its immunogenicity.Based on the known CPA sequence of Clostridium perfringens type A(GenBank accession No.:AY823400.1),CPA C gene was optimized,three copies of CPA C genes(GCPA C3)were tandemly linked in the same direction with the flexible amino acid linker(GGGS)gene and synthesized.In roder to get the recombinant protein of three copies of CPA C(rGCPA C3),GCPA C3 was cloned into prokaryotic expression vector pET-30a(+)for expression and purification.Reactivity of rGCPA C3 with antiserum of Clostridium perfringens type A was detected by Western blotting.Four rabbits were immunized with rGCPA C3 emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer.The results showed that rGCPA C3 was presented predominantly in an insoluble form(inclusion bodies),and it could react with the antiserum of Clostridium perfringen s type A.After the first immunization,sera from rabbits immunized with rGCPA C3 could neutralize 30 to 50 MLD/mL Clostridium perfringen s type A toxin,and 70 to 100 MLD/mL after twice immunization.Moreover,rabbits in rGCPA C3 immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringen s type A toxin challenge,whereas all of the rabbits died(100%,4/4)in control groups.These data suggested that rGCPA C3 was a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.

关 键 词：产气荚膜梭菌α毒素 C末端 串联表达 抗原性 

分 类 号：S852.616.3[农业科学—基础兽医学]