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

Abstract

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

CLC Number:

S852.61⁺6.3

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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Expression and Immunogenicity of Recombinant Clostridium botulinum Neurotoxin Type C

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