基因1型鹅星状病毒VP27蛋白的原核表达及其多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2024.06.032

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (6): 2576-2584.doi: 10.16431/j.cnki.1671-7236.2024.06.032

• 预防兽医 • 上一篇

基因1型鹅星状病毒VP27蛋白的原核表达及其多克隆抗体的制备与鉴定

向勇¹, 李林林¹, 张俊勤¹, 董嘉文¹, 黄允真¹, 翟颀¹, 徐志宏¹, 廖明^1,2, 孙敏华¹

1. 广东省农业科学院动物卫生研究所, 农业农村部禽流感等家禽重大疾病防控重点实验室, 广东省畜禽疫病防治研究重点实验室, 农业农村部兽用药物与诊断技术广东科学观测实验站, 广州 510640;
2. 仲恺农业工程学院动物科技学院, 广州 510225

收稿日期:2023-11-29 发布日期:2024-06-01
通讯作者: 孙敏华 E-mail:smh2002smh@163.com
作者简介:向勇,E-mail:xiangyong@gdaas.cn。
基金资助:
"十四五"国家重点研发项目(2022YFD1801000);广东省现代农业产业技术体系创新团队(2023KJ137);广东省重点领域研发计划项目(2020B0202090004);广东省畜禽疫病防治研究重点实验室(2023B1212060040);广东省动物疫病野外科学观测研究站项目(2021B1212050021)

Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody

XIANG Yong¹, LI Linlin¹, ZHANG Junqin¹, DONG Jiawen¹, HUANG Yunzhen¹, ZHAI Qi¹, XU Zhihong¹, LIAO Ming^1,2, SUN Minhua¹

1. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China;
2. College of Animal Science & Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

Received:2023-11-29 Published:2024-06-01

摘要/Abstract

摘要： 【目的】制备可特异性识别基因1型鹅星状病毒(Goose astrovirus genotype 1,GAstV-1)且与GAstV-2不发生交叉反应的多克隆抗体,以期为后续GAstV-1免疫检测技术的建立提供材料。【方法】通过比对GAstV-1与GAstV-2衣壳蛋白的氨基酸序列以寻找其差异较大的区域作为靶基因。对GAstV-1 GDYJ-21-01株VP27基因密码子进行偏嗜性优化合成,构建原核表达质粒pCZN1-GAstV-1 VP27,转化大肠杆菌Top10感受态细胞以诱导表达VP27蛋白;将纯化后的VP27蛋白免疫新西兰白兔后收获抗血清以制备GAstV-1 VP27多克隆抗体,通过间接ELISA和SDS-PAGE分析该纯化抗体的效价和纯度,并利用间接免疫荧光试验(IFA)检测多克隆抗体的特异性。【结果】试验成功构建重组质粒pCZN1-GAstV-1 VP27;SDS-PAGE显示其表达的重组蛋白分子质量约为35 ku,主要以包涵体形式存在。间接ELISA试验结果显示,纯化后的GAstV-1 VP27多克隆抗体效价高达1∶9 841 500,纯度高于85%。IFA结果显示,GAstV-1 VP27多克隆抗体可特异性识别GAstV-1,且与GAstV-2无交叉反应。临床病料组织检测结果显示,GAstV-1核酸阳性的病料组织均可出现特异性荧光,而GAstV-2核酸阳性且GAstV-1核酸阴性的病料组织未出现荧光。【结论】本研究获得了高效价且可特异性识别GAstV-1(与GAstV-2无交叉反应)的VP27多克隆抗体,为建立鉴别诊断GAstV-1的抗原免疫检测技术奠定了基础。

关键词: 1型鹅星状病毒(GAstV-1); VP27; 原核表达; 多克隆抗体

Abstract: 【Objective】 The aim of this study was to obtain a polyclonal antibody that could specifically recognize Goose astrovirus genotype 1 (GAstV-1) and did not cross-react with GAstV-2,so as to provide a material for the subsequent establishment of GAstV-1 immunoassay.【Method】 By comparing the amino acid sequences of GAstV-1 and GAstV-2 capsid proteins,the regions with large differences were found as target genes.According to VP27 gene sequence of GAstV-1 GDYJ-21-01,the gene codons were optimized and synthesized,the prokaryotic expression plasmid pCZN1-GAstV-1 VP27 was constructed and transformed into Escherichia coli Top10 competent cells to induce VP27 protein expression.The purified VP27 protein was used to immunize New Zealand White rabbits,and the antiserum was harvested to prepare GAstV-1 VP27 polyclonal antibody.The titer and purity of the purified antibody were analyzed by indirect ELISA and SDS-PAGE,and the specificity of the polyclonal antibody was evaluated by indirect immunofluorescence assay (IFA).【Result】 The recombinant plasmid pCZN1-GAstV-1 VP27 was successfully constructed.SDS-PAGE results showed that the molecular weight of the recombinant protein was about 35 ku,which mainly existed in the form of inclusion bodies.The results of indirect ELISA showed that the titer of purified GAstV-1 VP27 polyclonal antibody was 1∶9 841 500,and the purity was higher than 85%.IFA results showed that GAstV-1 VP27 polyclonal antibody could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2.The detection results of clinical samples showed that the tissues with GAstV-1 nucleic acid positive exhibited specific fluorescence,while the tissues with GAstV-2 nucleic acid positive but GAstV-1 nucleic acid negative did not show fluorescence.【Conclusion】 In this study,a high titer of VP27 polyclonal antibody was obtained,which could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2,laying a foundation for the establishment of antigen immunoassay for the differential diagnosis of GAstV-1.

Key words: Goose astrovirus genotype 1 (GAstV-1); VP27; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺9.6

向勇, 李林林, 张俊勤, 董嘉文, 黄允真, 翟颀, 徐志宏, 廖明, 孙敏华. 基因1型鹅星状病毒VP27蛋白的原核表达及其多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2024, 51(6): 2576-2584.

XIANG Yong, LI Linlin, ZHANG Junqin, DONG Jiawen, HUANG Yunzhen, ZHAI Qi, XU Zhihong, LIAO Ming, SUN Minhua. Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2576-2584.

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基因1型鹅星状病毒VP27蛋白的原核表达及其多克隆抗体的制备与鉴定

Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody

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