猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析

doi:10.16431/j.cnki.1671-7236.2020.06.004

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (6): 1668-1676.doi: 10.16431/j.cnki.1671-7236.2020.06.004

猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析

刘磊^1,2, 秦毅斌¹, 卢冰霞¹, 蒋冬福¹, 陈忠伟¹, 何颖¹, 赵硕³, 周英宁¹, 李斌¹, 段群棚¹, 梁家幸¹, 赵武¹

1. 广西兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001;
2. 广西农业职业技术学院, 南宁 530007;
3. 广西大学, 南宁 530005

收稿日期:2019-12-04 出版日期:2020-06-20 发布日期:2020-06-20
通讯作者: 赵武 E-mail:zhaowu168866@163.com
作者简介:刘磊(1989-),男,安徽宣城人,硕士,助理研究员,研究方向:动物传染病病原与分子生物学,E-mail:gxullei@163.com;秦毅斌(1983-),男,广西临桂人,硕士,研究方向:动物传染病病原与分子生物学,E-mail:qinyibin5188@163.com
基金资助:
广西创新驱动发展专项资金项目（桂科AA17204057）；广西自然科学基金项目（2017GXNSFBA198092）；广西兽医生物技术重点实验室开发基金课题（17-259-36-B-2、16-380-45-B-3）；广西基本科研业务费专项（桂科专项17-2、桂科专项19-2）；柳州市科学技术研究与技术开发计划项目（2018BH20501）；南宁市西乡塘区科学研究与科技开发计划项目（201810204）

Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain

LIU Lei^1,2, QIN Yibin¹, LU Bingxia¹, JIANG Dongfu¹, CHEN Zhongwei¹, HE Ying¹, ZHAO Shuo³, ZHOU Yingning¹, LI Bin¹, DUAN Qunpeng¹, LIANG Jiaxing¹, ZHAO Wu¹

1. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China;
2. Guangxi Agricultural Vocational and Technical College, Nanning 530007, China;
3. Guangxi University, Nanning 530005, China

Received:2019-12-04 Online:2020-06-20 Published:2020-06-20

摘要/Abstract

摘要： 本研究旨在了解猪流行性腹泻病毒（PEDV）S2蛋白的抗原性，为下一步诊断试剂盒及亚单位疫苗的研究奠定基础。试验通过反转录PCR的方法扩增PEDV CH/GX/2015/750A株S2基因部分片段（S2A），将其克隆后插入原核表达载体pET-32a（+）中，构建原核表达质粒pET32a-S2A。将原核表达质粒转化入大肠杆菌BL21感受态细胞中，IPTG诱导表达重组蛋白，Ni柱亲和层析法纯化重组蛋白，Western blotting检测重组蛋白S2A的反应原性。纯化复性的重组蛋白S2A免疫昆明小鼠制备多克隆抗体，用间接ELISA法检测获得的多克隆抗体效价，间接免疫荧光法验证制备的多克隆抗体的特异性。结果显示，重组S2A蛋白在IPTG终浓度为0.2 mmol/L时，37 ℃诱导表达3 h可获得最高表达量，该重组蛋白主要以包涵体的形式存在；Western blotting结果显示，纯化复性后的重组蛋白S2A能够与PEDV阳性血清发生特异性结合，具有良好的反应原性。制备的多克隆抗体效价可达1：32 000，间接免疫荧光结果表明，制备的多克隆抗体能够特异性识别和结合PEDV。结果表明，PEDV S2A蛋白具有良好的抗原性，可作为诊断试剂盒或亚单位疫苗的候选抗原。

关键词: 猪流行性腹泻病毒(PEDV); S2蛋白; 原核表达; 抗原性分析; 多克隆抗体制备

Abstract: The purpose of this study was to understand the antigenicity of porcine epidemic diarrhea virus (PEDV) S2 protein and lay a foundation for the further study of diagnostic kit and subunit vaccine.In this study,the partial fragment of S2 gene (S2A) of PEDV CH/GX/2015/750A strain was amplified by RT-PCR and inserted into prokaryotic expression vector pET-32a(+) to construct prokaryotic expression plasmid pET32a-S2A.The prokaryotic expression plasmid pET32a-S2A was transformed into E.coli BL21(DE3).The recombinant protein S2A was induced by IPTG and purified by affinity chromatography on Ni column.The activity of S2A protein was detected by Western blotting.The purified recombinant protein S2A was used to immunize Kunming mice to prepare polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA,and the specificity of the polyclonal antibody was verified by indirect immunofluorescence.The results showed that the optimal induction condition of recombinant S2A protein was that when the final concentration of IPTG was 0.2 mmol/L,the induction temperature was 37 ℃,and the induction time was 3 h.The recombinant protein mainly existed in the form of inclusion body.Western blotting results showed that the purified and renatured recombinant protein S2A could specifically bind to PEDV positive serum and had good react ogenicity.The titer of the polyclonal antibody was 1:32 000.IFA results showed that the polyclonal antibody could specifically recognize and bind PEDV virus.The results of this study showed that PEDV S2A protein had good antigenicity and could be used as a candidate antigen of diagnostic kit or subunit vaccine.

Key words: porcine epidemic diarrhea virus(PEDV); S2 protein; prokaryotic expression; antigenicity analysis; polyclonal antibody preparation

中图分类号:

S852.6

刘磊, 秦毅斌, 卢冰霞, 蒋冬福, 陈忠伟, 何颖, 赵硕, 周英宁, 李斌, 段群棚, 梁家幸, 赵武. 猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析[J]. 中国畜牧兽医, 2020, 47(6): 1668-1676.

LIU Lei, QIN Yibin, LU Bingxia, JIANG Dongfu, CHEN Zhongwei, HE Ying, ZHAO Shuo, ZHOU Yingning, LI Bin, DUAN Qunpeng, LIANG Jiaxing, ZHAO Wu. Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1668-1676.

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猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析

Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain

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