产气荚膜梭菌α毒素C末端的二拷贝串联表达与免疫原性分析

Expression and Immunogenicity of a tandem fusion protein of two copies of C-terminal of Clostridium perfringens αtoxin

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)二拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPA_C编码基因进行优化设计,将其以同向串连的方式串联成二拷贝基因(GCPA_(C2)),经人工合成获得基因片段GCPA_(C2)。将GCPA_(C2)克隆至原核表达载体pET-30a (+)中进行表达与纯化,获得重组蛋白rCPA_(C2)。利用Western Blot方法检测rCPA_(C2)与A型产气荚膜梭菌毒素抗血清的反应性。随后,用rCPA_(C2)免疫家兔,并检测一免及二免后兔血清的中和抗体效价。在二免21 d后,经耳缘静脉注射1个家兔MLD的A型产气荚膜梭菌毒素,检测rCPA_(C2)对家兔的免疫保护效果。结果表明,rCPA_(C2)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。每毫升的一免和二免抗血清分别可中和30个和80个小鼠MLD的A型产气荚膜梭菌毒素。1个兔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,rCPA_(C2)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。

This study was conducted to obtain a tandem fusion protein of two copies of CPA C-terminal(CPAC)and to evaluate the immunogenicity of it.Based on the known CPA sequence of Clostridium perfringens type A,gene of CPAC was optimized.Two copies of CPAC genes,GCPAC2 were tandemly linked in the same direction and synthesized.Then,GCPAC2 was cloned into prokaryotic expression vector pET-30 a(+)for expression and purification to get the recombiant protein,rGCPAC2.Reactivity of rGCPAC2 with antiserum of Clostridium perfringens type A was detected by Western blot.Rabbits were immunized with rGCPAC2 to prepare antiserum and detect the neutralizing titer.The results showed that rGCPAC2 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 rGCPAC2 could neutralize 30 mice MLD Clostridium perfringens type A toxin per ml,and 80 mice MLD after twice immunization.Moreover,rabbits in rGCPAC2immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge,where as all of the rabbits died(4/4)in the control groups.These data suggest that rGCPAC2 is a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.