产气荚膜梭菌重组ε毒素突变体的免疫保护力评价

摘要

旨在获得产气荚膜梭菌e毒素的重组突变体,并评价其毒力及免疫原性.对已知的D型产气荚膜梭菌ε毒素编码基因进行了优化设计和人工合成,同时引入3个氨基酸点突变:第30和196位酪氨酸突变为丙氨酸,第106位组氨酸突变为脯氨酸.将该基因片段克隆至原核表达载体pET30a-(+)中进行表达,并纯化.利用Westernblot方法检测纯化蛋白质与D型产气荚膜梭菌ε毒素抗血清的反应性,并检测其对小鼠的毒力.随后,以纯化的重组蛋白质免疫兔,根据《中华人民共和国兽药典》(2015年版)规定的方法检测兔血清的中和抗体效价.结果表明,e毒素的重组突变体为可溶性表达,通过灰度扫描,其表达量比例可达40％;该蛋白质能与D型产气荚膜梭菌ε毒素抗血清反应,小鼠安全试验显示,6.25×106 ng·kg-1的蛋白质仍无毒力;免疫兔血清对D型产气荚膜梭菌ε毒素的中和效价在一免后可达50～60最小致死量(MLD),二免后可达400～450 MLD;用1个MLD的D型产气荚膜梭菌毒素攻毒后,对照组4/4死亡,免疫组得到保护.由此表明,产气荚膜梭菌重组e毒素突变体无毒力且保留了良好的免疫原性,为D型产气荚膜梭菌病新型疫苗的研制提供了重要的实验数据.%This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin and subsequently evaluate the virulence and immunogenicity of the recombinant toxin.The ε toxin gene of C.perfringens type D strain using optimized codons was synthesized based on the sequence reported.At the same time,three amino acid mutations:Y30 and Y196 substituted with alanine,H106 substituted with proline,were introduced into this sequence synthesized.Then,this ε toxin gene was cloned into prokaryotic expression vector pET3a-(+) to construct a recombinant E.coli,followed by induction with IPTG to yield the recombinant protein,following with purification.The reactivity of the purified protein with antiserum of C.perfringens type D was determined by Western blot and the mouse was used to evaluate the virulence of purified protein.The rabbit antiserum against the recombinant proteins 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 expressed at a high level in a soluble form with a ratio of about 40％ bv grav scale scanning,And the protein could reAct with thE antiserum of C.perfringens type D.The protein with the injection volume of 6.25 × 106 ng · kg-1 still shows no virulence to mouse.The titer of rabbit antiserum against Clostridium perfringens type D could reach 50-60 and 400-450 Minimum Lethal Dose (MLD) after the first and second immunization and respectively.After challenge with 1MLD of C.perfringens type D toxin,all the rabbits immunized with recombinant protein were protected.The results suggest that the recombinant mutant of C.perfringens e toxin without virulence retains the good immunogenic antigen,which provides important experimental data for the development of novel C.perfringens vaccine.