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

摘要/Abstract

摘要： 【目的】应用大肠杆菌表达系统表达非洲猪瘟病毒(African swine fever virus,ASFV) CD2v蛋白胞外域(N-端,CD2v-N)与胞内域(C-端,CD2v-C)并分析两者的抗原性,为ASFV检测方法的研发提供试验依据和指导。【方法】构建CD2v-N和CD2v-C的重组原核表达质粒,在体外表达并纯化CD2v-N和CD2v-C蛋白,通过Western blotting对表达蛋白进行鉴定;用表达的CD2v-N和CD2v-C蛋白分别肌内接种免疫新西兰大白兔,共免疫3次,每次间隔2周;每次免疫后14 d,应用ELISA方法测定CD2v-N或CD2v-C的特异性抗体水平;用纯化后的CD2V-N和CD2v-C蛋白作为包被抗原,通过ELISA方法检测95份ASFV感染猪的血清CD2v-N或CD2v-C特异性抗体,比较CD2v-N和CD2v-C在猪体内诱导的免疫反应水平。【结果】重组蛋白CD2v-N和CD2v-C分别以包涵体和可溶性形式表达,分子质量分别为22.9和34.0 ku,均能与猪抗ASFV血清特异性结合;用CD2v-N蛋白首免家兔后14 d,血清中未检测到特异性抗体,而在CD2v-C首免的家兔血清中检测到了高滴度的特异性抗体;三免后14 d,CD2v-N和CD2v-C蛋白免疫家兔的血清特异性抗体效价分别为1∶125 000和1∶10⁷;ELISA检测结果显示,ASFV感染猪的血清中CD2v-C特异性抗体水平显著高于CD2v-N (P＜0.05)。【结论】本研究表达了ASFV CD2v蛋白胞内域和胞外域,前者的抗原性比后者高,CD2v胞内域作为ASFV免疫学检测技术研究与开发的靶标更具优势。该研究结果对ASFV检测方法的研究与开发具有重要指导意义。

关键词: 非洲猪瘟病毒(ASFV); CD2v蛋白; 胞外域; 胞内域; 蛋白表达; 抗原性

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

中图分类号:

S852.65

李艳蕊, 任静, 崔锦蔷, 袁晨, 王云霄, 马亚娟, 刘志昌, 李永社, 宋勤叶. 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[J]. 中国畜牧兽医, 2024, 51(4): 1660-1670.

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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