嵌合口蹄疫病毒抗原表位的重组塞内卡病毒A的构建及其鉴定

doi:10.11843/j.issn.0366-6964.2020.03.016

摘要/Abstract

摘要： 塞内卡病毒A（SVA）与口蹄疫病毒（FMDV）引起猪的临床症状难以区分，并且以SVA作为载体插入FMDV抗原表位外源基因的研究，目前还没有报道。为了构建一种嵌合FMDV抗原表位的重组SVA毒株并进行鉴定，本研究利用基因克隆技术将O型FMDV的B细胞表位与白蛋白信号肽（human albumin signal peptide，HAS）基因串联插入到SVA基因组中，成功构建了重组质粒，转染细胞后拯救出重组病毒rSVA-HAS-OB。结果显示：RT-PCR扩增与基因测序结果表明目的基因正确插入；细胞间接免疫荧光和Western blot鉴定了嵌合抗原蛋白的有效表达；遗传稳定性分析表明重组病毒在25代次的传代过程中仍稳定存在B细胞表位；病毒蚀斑表型和一步生长曲线结果表明，重组病毒与亲本毒株具有相似的增殖特性。本研究成功构建并拯救出能够表达FMDV抗原表位的重组SVA毒株，为以SVA为基础的嵌合病毒及疫苗的研究提供了理论和实验基础。

关键词: 塞内卡病毒A, 口蹄疫病毒, 抗原表位, 嵌合病毒

Abstract: It is difficult to distinguish the clinical symptoms caused by Seneca virus A (SVA) and foot-and-mouth disease virus (FMDV), and the study of inserting foreign genes of FMDV epitope with SVA as avector has not been reported at present. This paper aims to construct a recombinant SVA strain with chimeric FMDV epitopes and identify it. The genes encoding B cell epitopes of type O FMDV and albumin signal peptide (Human albumin signal peptide, HAS) were inserted into the SVA genome by gene cloning technique. The recombinant plasmid was successfully constructed and the recombinant virus was rescued after transfection. The results of RT-PCR and gene sequencing showed that the target gene was inserted correctly, and the effective expression of chimeric antigen protein was identified by Western blot and cellular indirect immunofluorescence. Genetic stability analysis showed that the recombinant virus still had stable B cell epitopes during the passage of 25 generations. The results of plaque phenotype and one-step growth curve of the virus showed that the proliferation characteristics of the recombinant virus were similar to those of the parent strain. In this study, the recombinant SVA virus which can express FMDV epitopes was successfully constructed and rescued, which provides a theoretical and experimental basis for the research of chimeric virus and vaccine based on SVA.

Key words: Seneca virus A, foot-and-mouth disease virus, antigen epitope, chimeric virus

中图分类号:

S852.659.6

宋高媛, 杨帆, 郝荣增, 李郁, 郑海学. 嵌合口蹄疫病毒抗原表位的重组塞内卡病毒A的构建及其鉴定[J]. 畜牧兽医学报, 2020, 51(3): 565-573.

SONG Gaoyuan, YANG Fan, HAO Rongzeng, LI Yu, ZHENG Haixue. Construction and Identification of Recombinant Seneca Virus A of Chimeric Foot-and-mouth Disease Virus Antigenic Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 565-573.

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