抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定

doi:10.11843/j.issn.0366-6964.2021.05.018

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (5): 1328-1336.doi: 10.11843/j.issn.0366-6964.2021.05.018

抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定

王丽娟, 石正旺, 杨波, 麻园, 罗俊聪, 万颖, 宋锐, 田宏, 郑海学*

中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室 OIE/国家口蹄疫参考实验室, 兰州 730046

收稿日期:2020-10-15 出版日期:2021-05-23 发布日期:2021-05-22
通讯作者: 田宏,主要从事动物病毒分子生物学及疫病诊断技术研究,E-mail:xibeitian0931@163.com;郑海学,主要从事动物传染病学与流行病学研究,E-mail:zhenghaixue@caas.cn
作者简介:王丽娟(1994-),女,河南许昌人,硕士生,主要从事兽医学研究,E-mail:792815782@qq.com
基金资助:
国家重点研发计划项目（2018YFC0840400）

Construction, Expression and Activity Identification of Anti-ASFV Single-Chain Antibody

WANG Lijuan, SHI Zhengwang, YANG Bo, MA Yuan, LUO Juncong, WAN Ying, SONG Rui, TIAN Hong, ZHENG Haixue*

State Key Laboratory of Veterinary Etiological Biology and OIE/National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2020-10-15 Online:2021-05-23 Published:2021-05-22

摘要/Abstract

摘要： 制备抗非洲猪瘟病毒（ASFV）猪源单链抗体（ScFv），并对其生物学活性进行鉴定，筛选出具有ASFV反应活性的猪源单链抗体（ScFv），为ASFV的诊断提供新的材料。采集ASFV感染康复猪的外周血，分离淋巴细胞，提取淋巴细胞总RNA，以此为模板通过PCR扩增得到猪源IgG重链可变区与轻链可变区基因，利用SOE-PCR技术扩增拼接得到猪源ScFv基因；构建pET-30a-ScFv表达载体，进行蛋白的表达与纯化，用ELISA和IFA鉴定ScFv抗体的反应活性。结果显示，成功扩增出猪源ScFv（VH-VLκ、VH-VLλ）基因，鉴定到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体。结果表明，成功筛选到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体，为ASFV诊断和防控提供了新型原材料。

关键词: 非洲猪瘟病毒, SOE-PCR, 单链抗体, 诊断与防控

Abstract: In this study, we aimed to prepare anti-ASFV pig-derived single-chain antibody (ScFv), and identify its biological activity, screen out the pig-derived ScFv with ASFV reactivity, and provide a new diagnosis for ASFV material. the peripheral blood of ASFV-infected and recovered pigs were collected to separate lymphocytes, then lymphocyte total RNAs were extracted and used as a template to obtain pig-derived IgG heavy chain variable region and light chain variable region genes by PCR amplification, using SOE-PCR technology to expand. The pig-derived ScFv gene was obtained by splicing; the pET-30a-ScFv expression vector was constructed, and the protein was expressed and purified. The reactivity of the ScFv antibody was identified by ELISA and IFA. The results showed that the pig-derived ScFv (VH-VLκ, VH-VLλ) genes were successfully amplified, and an scFv (VH-VLλ₁₁) antibody that was reactive with ASFV was identified. The results showed that an ScFv (VH-VLλ₁₁) antibody that was reactive with ASFV was successfully screened, providing new raw material for ASFV diagnosis, prevention and control.

Key words: ASFV, SOE-PCR, ScFv, diagnosis and prevention

中图分类号:

S852.659.1

王丽娟, 石正旺, 杨波, 麻园, 罗俊聪, 万颖, 宋锐, 田宏, 郑海学. 抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定[J]. 畜牧兽医学报, 2021, 52(5): 1328-1336.

WANG Lijuan, SHI Zhengwang, YANG Bo, MA Yuan, LUO Juncong, WAN Ying, SONG Rui, TIAN Hong, ZHENG Haixue. Construction, Expression and Activity Identification of Anti-ASFV Single-Chain Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1328-1336.

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抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定

Construction, Expression and Activity Identification of Anti-ASFV Single-Chain Antibody

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