非洲猪瘟病毒CD2v蛋白胞内域与胞外域的原核表达及其抗原性分析

doi:10.16431/j.cnki.1671-7236.2024.04.034

Abstract

Abstract: 【Objective】 The extracellular domain (N-terminal fragment,CD2v-N) and intracellular domain (C-terminal fragment,CD2v-C) of African swine fever virus (ASFV) CD2v protein were expressed by Escherichia coli expression system,and the antigenicity of the two proteins was analyzed,so as to provide experimental basis and guidance for the development of ASFV detection methods.【Method】 Recombinant prokaryotic expression plasmids of CD2v-N and CD2v-C were constructed,the CD2v-N and CD2v-C proteins were expressed and purified in vitro,and the expressed proteins were identified by Western blotting.New Zealand White rabbits were inoculated with the expressed CD2v-N and CD2v-C proteins intramuscularly for three times,each time at an interval of 2 weeks 14 d after each immunization,the specific antibody levels of CD2v-N or CD2v-C were determined by ELISA.The purified CD2V-N and CD2v-C proteins were then used as coated antigens to detect CD2v-N or CD2v-C specific antibodies in 95 sera of ASFV-infected pigs by ELISA,and the levels of immune response induced by CD2v-N or CD2v-C in pigs were compared.【Result】 Recombinant proteins CD2v-N and CD2v-C were expressed in inclusion bodies and soluble forms,respectively,with relative molecular masses of 22.9 and 34.0 ku,and could bind specifically to porcine anti-ASFV serum.14 d after the primary immunization with the CD2v-N,no specific antibodies were detected in the serum of rabbits,whereas the specific antibodies with high titer were detected in the rabbits after the primary immunization with CD2v-C. 14 d after the third immunization,the titers of CD2v-N and CD2v-C were 1∶125 000 and 1∶10⁷,respectively.The level of CD2v-C-specific antibodies in the serum of ASFV-infected pigs was significantly higher than that of CD2v-N (P＜0.05).【Conclusion】 The intracellular and extracellular domains of CD2v were expressed,and the antigenicity of the former was higher than that of the latter.The intracellular domain of CD2v was more advantageous as a target for the research and development of ASFV immunological detection techniques.The results of this study had important guiding significance for the research and development of ASFV detection methods.

Key words: African swine fever virus (ASFV); CD2v protein; extracellular domain; intracellular domain; protein expression; antigenicity

CLC Number:

S852.65

LI Yanrui, REN Jing, CUI Jinqiang, YUAN Chen, WANG Yunxiao, MA Yajuan, LIU Zhichang, LI Yongshe, SONG Qinye. Prokaryotic Expression and Antigenicity Analysis of Intracellular and Extracellular Domains of African Swine Fever Virus CD2v Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1660-1670.

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Prokaryotic Expression and Antigenicity Analysis of Intracellular and Extracellular Domains of African Swine Fever Virus CD2v Protein

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