重组产气荚膜梭菌α毒素对家兔的安全性和免疫效力评价

Safety andimmune efficacy of recombinant Clostridium perfringensα-toxin in rabbit

为探究重组产气荚膜梭菌α毒素对家兔的安全性和免疫保护效力,本研究构建了含产气荚膜梭菌α毒素基因的重组质粒pVL1393-CPA,并通过BD BaculoGoldTM转染试剂盒转染Sf9细胞后,经噬斑纯化获得单克隆重组病毒,将其感染High 5细胞进行表达,表达的重组蛋白经SDS-PAGE和western blot检测,结果显示均获得了45 ku的目的蛋白,即为重组α毒素。将重组α毒素用0.1%甲醛溶液灭活脱毒后与ISA 206 VG佐剂乳化制备亚单位疫苗(抗原含量为20μg/mL),以40μg/只肌肉注射体质量1.5 kg~2.0 kg的家兔,同时设PBS对照组,注射后连续观察7 d,结果显示实验兔临床观察无异常,该疫苗对家兔的安全性良好。随后以20μg/只肌肉注射家兔,免疫21 d后以相同剂量加强免疫一次,同时设佐剂对照组和α毒素对照组,首免21 d、二免14 d时采血分离血清,采用ELISA方法检测免疫兔血清IgG抗体效价,同时进行二免血清小鼠体内毒素中和试验以及家兔攻毒免疫保护试验,以评价该疫苗对家兔的免疫保护效力。结果显示,疫苗免疫组家兔血清IgG抗体效价比佐剂对照组和毒素对照组均极显著增高(P<0.001),二免血清对A型产气荚膜梭菌毒素小鼠体内的中和效价为32,疫苗免疫组家兔攻毒后均健活,攻毒保护率达100%,佐剂对照组和毒素对照组家兔攻毒后死亡率则为100%。上述结果表明制备的重组产气荚膜梭菌α毒素疫苗对家兔具有良好的安全性和免疫保护效力,本研究为开发其他各型梭菌亚单位疫苗提供了重要借鉴。

To investigate the safety and immune efficacy of recombinant Clostridium perfringensα-toxin in rabbits,the recombinant plasmid pVL1393-CPA based on Clostridium perfringensα-toxin gene was constructed in this study,and was co-transfected into Sf9 cells by BD BaculoGoldTM kit.The monoclonal recombinant virus was purified by plaque phagocytosis and the target protein of 45ku was expressed in High 5 cells and then detected by SDS-PAGE and western blot.The recombinantα-toxin was inactivated with 0.1%formaldehyde solution and emulsified with ISA 206 VG adjuvant to prepare the subunit vaccine(antigen content was 20μg/mL).Rabbits(1.5-2.0kg)were injected intramuscularly with the vaccine containing 40μg of the protein,and PBS control group was set up,all experiment rabbits were observed for 7 days post injection.The results showed that the vaccine was safe for rabbits.In another experiment,groups of rabbit(n=10)were immunized twice with 20μg in 21 day's internal.Meanwhile,the adjuvant control group or toxin control group were set up.At 21 days after prime and 14 days after boost,serum was collected for the detection of IgG titers by ELISA.The toxin neutralization test in mice and the protection test in rabbits were used to evaluate the immune protection efficacy of this candidate vaccine.The results showed that the titer of serum IgG antibody in the vaccine immunized group was significantly different from that in the adjuvant control group and the toxin control group(P<0.001).The neutralizing titer of two immunized serum against Clostridium perfringens type A mice was 32,and the rabbits in the vaccine immunized group were healthy after challenge,and the protection rate was 100%.The mortality of rabbits in adjuvant and toxin control groups was 100%.These results indicated that the recombinant Clostridium perfringensα-toxin vaccine prepared in this study has good safety and immune protective efficacy in rabbits,which provides an important reference for the development of other clostridium subunit vaccines.