重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

doi:10.11843/j.issn.0366-6964.2020.11.018

Abstract

Abstract: This study was conducted to obtain the Clostridium perfringens β toxin (CPB) derivative and to evaluate its virulence and immunoprotection. Based on the known sequence, four amino acid mutations, R212E, L268G, Y266A and W275A, were introduced into the gene of Clostridium perfringens CPB. Meanwhile, genes of Th cell and N-terminal of flagellin were added to 5' of the CPB gene, respectively. Then the gene GTF_NCPB_m4 was optimized and synthesized, and was subsequently cloned into the prokaryotic expression vector pET-30a (＋) for expression and purification to get the recombinant protein, rTF_NCPB_m4. Reactivity of rTF_NCPB_m4with antiserum of Clostridium perfringens type C crude toxins was detected by Western blot. Meanwhile, the toxicity of rTF_NCPB_m4 to mice was evaluated. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), rabbits were immunized with rTF_NCPB_m4 to prepare antiserum and detect the neutralizing titer against Clostridium perfringens type C crude toxins. Results showed that rTF_NCPB_m4 was presented predominantly in an insoluble form (inclusion bodies), and it could react with the antiserum of Clostridium perfringens type C crude toxins. rTF_NCPB_m4 with an injection volume of 50 μg was still not fatal to mice. Sera from rabbits immunized with rTF_NCPB_m4can neutralize 10-20 mouse minimum lethal doses (MLD) of Clostridium perfringens type C crude toxins after twice immunization. Moreover, rabbits immunized with rTF_NCPB_m4 can fully resist 1 rabbit MLD of Clostridium perfringens type C crude toxins challenge, whereas all of the rabbits died (4/4) in the control groups. These data suggest that rTF_NCPB_m4 is a potential vaccine candidate for the subunit vaccine of Clostridium perfringens type C.

Key words: Clostridium perfringens CPB, mutation, Th cell epitope, flagellin, virulence, immunoprotection

CLC Number:

S851.616.3

DU Jige, ZHU Zhen, XU Zhongqing, LI Qianlin, YAO Wensheng, LI Qihong, YIN Chunsheng, YANG Liu, FU Lizhi, CHEN Xiaoyun, LIU Ying, XUE Qi. Expression and Protective Efficacy of Clostridium perfringens β toxin Derivative[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2794-2801.

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Expression and Protective Efficacy of Clostridium perfringens β toxin Derivative

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