重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

Expression and Protective Efficacy of Clostridium perfringensβtoxin Derivative

旨在获得产气荚膜梭菌β毒素(CPB)的重组突变体,并评价其毒力及免疫保护性。对已知的产气荚膜梭菌CPB编码基因进行优化设计,同时引入4个氨基酸突变位点,分别是第212位的精氨酸突变为谷氨酸,第268位的亮氨酸突变为甘氨酸,266位的酪氨酸和275位的色氨酸突变为丙氨酸。此外,在该基因5′端添加Th细胞表位(T)和鞭毛蛋白(flagellin)N末端的编码序列,经人工合成获得重组基因片段(GTF_(N)CPB_(m4))。将GTF_(N)CPB_(m4)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rTF_(N)CPB_(m4)。利用Western blot方法检测rTF_(N)CPB_(m4)与C型产气荚膜梭菌毒素抗血清的反应性,并检测rTF_(N)CPB_(m4)对小鼠的毒力。随后,以rTF_(N)CPB_(m4)免疫家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测家兔血清对C型产气荚膜梭菌毒素的中和抗体效价。结果表明,rTF_(N)CPB_(m4)主要以包涵体的形式表达且能与C型产气荚膜梭菌毒素抗血清反应。小鼠安全性试验显示,50μg的rTF_(N)CPB_(m4)对小鼠仍无致死性;免疫rTF_(N)CPB_(m4)后,每毫升家兔二免抗血清可中和10~20个小鼠最小致死量(MLD)的C型产气荚膜梭菌毒素;1个家兔MLD的C型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%(4/4)的保护。以上结果说明,rTF_(N)CPB_(m4)在丧失毒力的同时保留了良好的免疫原性,从而为C型产气荚膜梭菌病基因工程疫苗的研制提供了重要的数据。

This study was conducted to obtain the Clostridium perfringensβtoxin(CPB)derivative and to evaluate its virulence and immunoprotection.Based on the known sequence,four amino acid mutations,R212E,L268G,Y266A and W275A,were introduced into the gene of Clostridium perfringens CPB.Meanwhile,genes of Th cell and N-terminal of flagellin were added to 5′of the CPB gene,respectively.Then the gene GTF_(N)CPB_(m4) was optimized and synthesized,and was subsequently cloned into the prokaryotic expression vector pET-30a(+)for expression and purification to get the recombinant protein,rTF_(N)CPB_(m4).Reactivity of rTF_(N)CPB_(m4)with antiserum of Clostridium perfringens type C crude toxins was detected by Western blot.Meanwhile,the toxicity of rTF_(N)CPB_(m4) to mice was evaluated.According to the method prescribed in Chinese Veterinary Pharmacopoeia(2015),rabbits were immunized with rTF_(N)CPB_(m4) to prepare antiserum and detect the neutralizing titer against Clostridium perfringens type C crude toxins.Results showed that rTF_(N)CPB_(m4)was presented predominantly in an insoluble form(inclusion bodies),and it could react with the antiserum of Clostridium perfringens type C crude toxins.rTF_(N)CPB_(m4) with an injection volume of 50μg was still not fatal to mice.Sera from rabbits immunized with rTF_(N)CPB_(m4)can neutralize 10-20 mouse minimum lethal doses(MLD)of Clostridium perfringens type C crude toxins after twice immunization.Moreover,rabbits immunized with rTF_(N)CPB_(m4) can fully resist 1 rabbit MLD of Clostridium perfringens type C crude toxins challenge,whereas all of the rabbits died(4/4)in the control groups.These data suggest that rTF_(N)CPB_(m4) is a potential vaccine candidate for the subunit vaccine of Clostridium perfringens type C.