重组C型肉毒梭菌毒素的表达与免疫保护性分析

Expression and Immunogenicity of Recombinant Clostridium botulinum Neurotoxin Type C

本研究旨在获得重组C型肉毒梭菌毒素蛋白,并评价其免疫保护性。将麦芽糖蛋白(MBP)和C型肉毒梭菌毒素重链C末端(CHC)的编码基因序列进行优化和串联,获得基因片段GMBPCHC。将GMBPCHC克隆至pET28a-(+)后转化大肠杆菌BL21(DE3)感受态细胞,分别在15和37℃两种温度条件下诱导表达。利用Ni-IDA亲和层析方法对可溶性表达的目的蛋白进行纯化,从而获得重组蛋白rMBPCHC。将rMBPCHC与Montanide ISA 201佐剂混合制备成疫苗,免疫4只家兔,剂量为100μg/只。根据《中华人民共和国兽药典》(2015年版)规定的方法检测一免后21 d及二免后14 d家兔血清的中和抗体效价。同时,在二免后14 d对家兔进行攻毒。结果表明,rMBPCHC在37℃的诱导温度下,主要以包涵体的形式表达;在15℃的诱导温度下,可溶性表达的比例可达50%。一次免疫后,免疫组4只家兔血清对C型肉毒梭菌天然毒素(简称天然毒素)的中和效价均可达到1(0.1 mL血清中和1个小鼠最小致死量(MLD)的天然毒素)。二免后,家兔血清的中和抗体效价可达到4~8。用10个家兔MLD的天然毒素攻毒后,免疫组家兔得到了100%(4/4)的保护,而用1个家兔MLD的天然毒素攻毒后,对照组家兔100%(2/2)死亡。以上结果说明,rMBPCHC具有良好的免疫原性,从而为C型肉毒梭菌病基因工程亚单位疫苗的研制提供了重要的试验数据。

This study was aimed to obtain a recombinant Clostridium botulinum neurotoxin type C,and evaluate its virulence and immunogenicity.The fragment GMBPCHCcomposed of MBP and C-terminal of the Clostridium botulinum neurotoxin type C heavy chain(CHC)was optimized and synthesized.Then,GMBPCHC was cloned into pET28a-(+),and subsequently transformed into E.coli BL21(DE3)competent cells,followed with induction at 15 and 37℃,respectively.The recombinant protein rMBPCHC expressed in a soluble form was then purified by Ni-IDA chromatograph.Then,4 rabbits were immunized with 100μg of rMBPCHC emulsified with oil adjuvant of ISA 201.According to the method prescribed in Chinese Veterinary Pharmacopoeia(2015),serum samples were collected 21 d after the first immunization and 14 d after last immunization to detect the neutralizing titer against the Clostridium botulinum neurotoxin type C(crude toxin).At the same time,rabbits were challenged with crude toxin 14 days after the second immunization.Results showed that rMBPCHC was expressed predominantly in an insoluble form induced at 37℃and expressed as soluble form with a rate of 50%induced at 15℃.The neutralization titer against crude toxin of sera from 4 rabbits immunized with rMBPCHC could both reach 1(sera could neutralize 1 mice MLD crude toxin per 0.1 mL)after the first immunization.After the second immunization,neutralization titer could reach 4-8.Moreover,rabbits in rMBPCHC immunized group 100%(4/4)rabbits were survived at the dose of 10 rabbit MLD crude toxin challenge,whereas 100%(2/2)rabbits were died in control group.These data suggest that rMBPCHC was a potential vaccine candidate for the subunit vaccine of Clostridium botulinum type C.