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

doi:10.16431/j.cnki.1671-7236.2018.11.031

摘要/Abstract

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CPA_C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21 d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。

关键词: 产气荚膜梭菌α 毒素; C末端; 串联表达; 抗原性

Abstract:

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_C3was presented predominantly in an insoluble form (inclusion bodies),and it could react with the antiserum of Clostridium perfringens type A.After the first immunization,sera from rabbits immunized with rGCPA_C3could neutralize 30 to 50 MLD/mL Clostridium perfringens type A toxin,and 70 to 100 MLD/mL after twice immunization.Moreover,rabbits in rGCPA_C3immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge,whereas all of the rabbits died (100%,4/4) in control groups.These data suggested that rGCPA_C3was a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.

Key words: Clostridium perfringens α toxin; C-terminal; recombinant expression; antigenicity

中图分类号:

S852.61⁺6.3

杜吉革, 刘莹, 朱真, 薛麒, 张秀坤, 李启红, 印春生, 康凯, 姚文生, 陈小云. 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[J]. 《中国畜牧兽医》, 2018, 45(11): 3237-3245.

DU Jige, LIU Ying, ZHU Zhen, XUE Qi, ZHANG Xiukun, LI Qihong, YIN Chunsheng, KANG Kai, YAO Wensheng, CHEN Xiaoyun. Three Copies Recombinant Expression and Immunogenicity Analysis of C-terminal of Clostridium perfringens α Toxin[J]. , 2018, 45(11): 3237-3245.

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