猪繁殖与呼吸综合征病毒GP3蛋白单克隆抗体制备及抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2023.08.027

摘要/Abstract

摘要： 旨在制备猪繁殖与呼吸综合征病毒（PRRSV） GP3蛋白单克隆抗体及解析其抗原表位。本研究构建了PRRSV类NADC30毒株FJ1402的GP3大肠杆菌表达质粒，制备获得重组GP3蛋白。经过免疫BALB/c小鼠和间接ELISA方法筛选获得阳性杂交瘤细胞。利用间接免疫荧光试验和蛋白印迹鉴定单克隆抗体特异性。通过构建GP3蛋白基因截短体和合成多肽ELISA鉴定单克隆抗体识别的表位区域。结果显示，成功筛选了7株稳定分泌GP3蛋白抗体的杂交瘤细胞株，7株单克隆抗体均可与PRRSV FJ1402产生特异性反应。鉴定出GP3蛋白4个抗原表位，分别为⁵⁵PLCPTRQAAAEILE⁶⁸、⁶⁹PGKSFWCRI⁷⁷、⁷⁸GHDRCSESDH⁸⁷和⁸⁸DELGFMVPPGLSS¹⁰⁰。2E12单抗与FJ1402、BB0907和S1三个谱系毒株均可发生IFA和Western blot特异反应，4H9、9F3和6B3单抗只与FJ1402毒株反应，而不与BB0907和S1毒株反应。本研究研制成功7株GP3蛋白单克隆抗体，并鉴定出4个GP3蛋白抗原表位，为PRRSV检测和GP3蛋白功能研究提供了重要工具。

关键词: 猪繁殖与呼吸综合征病毒, GP3蛋白, 单克隆抗体, 抗原表位

Abstract: This experiment was conducted to obtain monoclonal antibodies against the GP3 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and analyze its antigenic epitopes. The prokaryotic expression plasmid of PRRSV glycoprotein 3(GP3) from NADC30-like strain FJ1402 was constructed and the recombinant GP3 protein was obtained. Positive hybridoma clones were obtained through BALB/c mouse immunization and indirect ELISA. The specificity of monoclonal antibodies was further identified by indirect immunofluorescence assay and Western blot. The recognizing epitope regions of monoclonal antibodies on GP3 protein were identified by constructing truncated GP3 protein and antigenic epitope ELISA. The results showed that seven hybridoma cell lines stably secreting antibodies against the GP3 protein were successfully selected,Moreover, all monoclonal antibodies can specifically react with PRRSV FJ1402 strain. And four antigenic epitopes were identified as following,⁵⁵PLCPTRQAAAEILE⁶⁸,⁶⁹PGKSFWCRI⁷⁷, ⁷⁸GHDRCSESDH⁸⁷, and ⁸⁸DELGFMVVPPGLSS¹⁰⁰. And 2E12 mAb can also react with PRRSV BB0907 and S1 strains by using IFA and Western blot, but 4H9、9F3 and 6B3 cannot. This study successfully obtained monoclonal antibodies against GP3 protein of PRRSV, and identified four GP3 protein epitopes, laying an important foundation for the diagnosis of PRRSV and the study of the function of the viral GP3 protein.

Key words: PRRSV, GP3 protein, monoclonal antibody, epitope

中图分类号:

S852.659.6

袁丽, 孙杨杨, 张路捷, 张杰, 孙海凤, 白娟, 姜平. 猪繁殖与呼吸综合征病毒GP3蛋白单克隆抗体制备及抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3424-3434.

YUAN Li, SUN Yangyang, ZHANG Lujie, ZHANG Jie, SUN Haifeng, BAI Juan, JIANG Ping. Production and Epitope Characterization of Monoclonal Antibodies against GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3424-3434.

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