重组C型肉毒梭菌毒素的表达与免疫保护性分析

doi:10.16431/j.cnki.1671-7236.2021.04.025

摘要/Abstract

摘要： 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（CH_C）的编码基因序列进行优化和串联，获得基因片段GMBPCH_C。将GMBPCH_C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rMBPCH_C。将rMBPCH_C与Montanide ISA 201佐剂混合制备成疫苗，免疫4只家兔，剂量为100 μg/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，rMBPCH_C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，rMBPCH_C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。

关键词: C型肉毒梭菌毒素; C末端; 可溶表达; 免疫保护

Abstract: This study was aimed to obtain a recombinant Clostridium botulinum neurotoxin type C,and evaluate its virulence and immunogenicity.The fragment GMBPCH_Ccomposed of MBP and C-terminal of the Clostridium botulinum neurotoxin type C heavy chain (CH_C) was optimized and synthesized.Then,GMBPCH_C 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 rMBPCH_C expressed in a soluble form was then purified by Ni-IDA chromatograph.Then,4 rabbits were immunized with 100 μg of rMBPCH_C 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 rMBPCH_C 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 rMBPCH_C 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 rMBPCH_C 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 rMBPCH_C was a potential vaccine candidate for the subunit vaccine of Clostridium botulinum type C.

Key words: Clostridium botulinum neurotoxin type C; C-terminal; soluble expression; immunogenicity

中图分类号:

S852.61⁺6.3

杜吉革, 姚文生, 刘莹, 薛麒, 李倩琳, 朱真, 李启红, 印春生, 陈小云, 李旭妮. 重组C型肉毒梭菌毒素的表达与免疫保护性分析[J]. 中国畜牧兽医, 2021, 48(4): 1381-1387.

DU Jige, YAO Wensheng, LIU Ying, XUE Qi, LI Qianlin, ZHU Zhen, LI Qihong, YIN Chunsheng, CHEN Xiaoyun, LI Xuni. Expression and Immunogenicity of Recombinant Clostridium botulinum Neurotoxin Type C[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1381-1387.

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