产气荚膜梭菌α毒素C末端与中和抗原表位的串联表达及免疫保护性分析

Immunogenicity of a Tandem Fusion Protein of C-terminal and a Neutralizing Epitope of Clostridium perfringens α Toxin

关键词:  产气荚膜梭菌α毒素  C末端  中和抗原表位  串联表达  抗原性

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

摘要:

为获得产气荚膜梭菌α毒素（CPA）的C末端（CPAC）与中和抗原表位NE（ARGFAK）的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及NE的编码基因进行优化设计，并将两个基因的三拷贝序列串联，经人工合成获得基因片段GCPAC3NE3。将该片段克隆至原核表达载体pET30a (＋)中进行表达与纯化，获得重组蛋白。利用Western blot方法检测重组蛋白与A型产气荚膜梭菌毒素抗血清的反应性。随后，以纯化的重组蛋白免疫家兔，根据《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清的中和抗体效价。在二免后21 d，以1个MLD的A型产气荚膜梭菌毒素对家兔进行攻毒。结果表明重组蛋白主要以包涵体的形式存在表达且能与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40个最小致死量（MLD）、二免抗血清可中和80个MLD的A型产气荚膜梭菌毒素；1个MLD的A型产气荚膜梭菌毒素攻毒后，对照组4/4死亡，免疫组得到了100%（4/4）的保护。以上结果说明，重组蛋白具有良好的免疫原性，从而为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的实验数据。

Abstract:

This study was conducted to obtain a tandem fusion protein of C-terminal (CPAC) of CPA and a neutralizing epitope, NE (ARGFAK), and to evaluate the immunogenicity of it. Based on the known CPA sequence of Clostridium perfringens type A, genes of CPAC and NE were optimized and three copies of CPAC and NE were tandemly linked, following with the GCPAC3NE3 being synthesized. Then，this sequence was cloned into prokaryotic expression vector pET30a (+) for expression and purification to get the recombinant protein. Reactivity of the protein with antiserum of Clostridium perfringens type A was detected by Western blot. The rabbit antiserum against the recombinant protein 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 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 recombinant protein could neutralize 40 minimum lethal dose (MLD) of Clostridium perfringens type A toxin per mL, and 80 mice MLD after twice immunization. Moreover, rabbits immunized by the recombinant protein fully survived at the dose of 1 MLD of Clostridium perfringens type A toxin challenge, whereas all of the rabbits died (4/4) in the control groups. These data suggest that the recombinant protein is a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.