新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

doi:10.16431/j.cnki.1671-7236.2021.11.030

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (11): 4204-4212.doi: 10.16431/j.cnki.1671-7236.2021.11.030

新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

杨惠湖¹, 黄惠兰¹, 袁生¹, 李文俊¹, 姚鑫炎¹, 张玉倩¹, 梁昭平², 黄淑坚¹, 张雪莲¹

1. 佛山科学技术学院生命科学与工程学院, 佛山 528231;
2. 广州市华南农大生物药品有限公司, 广州 511300

收稿日期:2021-03-03 出版日期:2021-11-20 发布日期:2021-11-01
通讯作者: 黄淑坚, 张雪莲 E-mail:sjhuang.foshan@163.com;zxlsjw0312@163.com
作者简介:杨惠湖(1998-),男,广东惠州人,硕士,研究方向:水禽疫病防控,E-mail:yhh1369558045@163.com;黄惠兰(1996-),女,广东梅州人,硕士,研究方向:水禽疫病防控,E-mail:huanghl0609@163.com
基金资助:
广东省教育厅普通高校重点领域专项（乡村振兴）（2020ZDZX1004）；广东省教育厅青年创新人才项目（2018KQNCX277）；广东省基础与应用基础研究区域联合基金-青年基金项目（2019A1515110157）；佛山科学技术学院动物新发疫病防控重点实验室"开放基金"项目（KLPREAD201801-09、KLPREAD201801-08）；广东省现代农业产业技术体系创新团队（2019KJ137）

Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain

YANG Huihu¹, HUANG Huilan¹, YUAN Sheng¹, LI Wenjun¹, YAO Xinyan¹, ZHANG Yuqian¹, LIANG Zhaoping², HUANG Shujian¹, ZHANG Xuelian¹

1. College of Life Science and Engineering, Foshan University, Foshan 528231, China;
2. Guangzhou South China Agricultural University Biological Drug Co., Ltd., Guangzhou 511300, China

Received:2021-03-03 Online:2021-11-20 Published:2021-11-01

摘要/Abstract

摘要： 本研究旨在利用原核表达系统表达新型鸭呼肠孤病毒（NDRV） DH13株重组σB蛋白，并检测其免疫原性，为NDRV基因工程疫苗研制等提供物质材料。采用RT-PCR方法扩增获得NDRV σB基因，构建原核表达重组质粒pET-30a-σB和pET-32a-σB，将2个重组质粒转化大肠杆菌BL21（DE3）感受态细胞，利用IPTG诱导获得相应的重组蛋白，通过SDS-PAGE分析重组蛋白的表达形式。纯化无His的σB蛋白，并以此蛋白作为免疫原免疫新西兰白兔，制备兔源多克隆抗体。利用Ni²⁺柱亲和层析法纯化含His的σB蛋白，以此蛋白作为检测原，通过间接ELISA方法测定兔源多克隆抗体效价；Western blotting鉴定多克隆抗体与重组蛋白的特异性识别能力。结果显示，PCR扩增获得大小约为1 100 bp的σB基因片段。SDS-PAGE结果显示，高效表达出了2种重组σB蛋白，大小分别约为41和46 ku，均以包涵体形式存在。间接ELISA结果显示，制备的多克隆抗体效价达1∶204 800，能特异性地识别原核表达的重组蛋白，表明重组无His的σB蛋白具有良好的免疫原性。Western blotting特异性鉴定结果显示，含His的σB蛋白和无His的σB蛋白均与制备的兔源多克隆抗体发生特异性反应，表明原核表达得到的重组σB蛋白具备良好的反应原性。本试验利用原核表达系统成功地表达出了重组σB蛋白，并证实了重组NDRV σB蛋白具有良好的免疫原性及反应原性，该结果将有助于后续对NDRV σB蛋白生物学功能的鉴定、NDRV诊断用抗原的制备及NDRV新型疫苗的研制。

关键词: 新型鸭呼肠孤病毒(NDRV); σB蛋白; 原核表达; 免疫原性分析

Abstract: The purpose of this study was to express recombinant σB protein of DH13 strain of Novel duck reovirus (NDRV) using prokaryotic expression system, and detect its immunogenicity, so as to provide material basis for the development of NDRV gene engineering vaccine. σB gene of NDRV DH13 strain was amplified by RT-PCR and prokaryotic expression recombinant plasmids pET-30a-σB and pET-32a-σB were constructed and transformed into E. coli BL21 (DE3) and with IPTG induction corresponding recombinant proteins were obtained and analyzed by SDS-PAGE. The recombinant protein without His tag was purified by digestion and as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody. The recombinant protein with His tag was purified by Ni²⁺ affinity chromatography and as detection antigen to measure rabbit polyclonal antibody titer by indirect ELISA. It was determined whether the polyclonal antibody specifically recognize the recombinant protein by Western blotting. The results showed that σB gene with a size of about 1 100 bp was amplified. The results of SDS-PAGE showed that two recombinant σB proteins were efficiently expressed, the sizes of which were about 41 and 46 ku respectively, and both existed in the form of inclusion bodies. The indirect ELISA results showed the titer of the prepared polyclonal antibody was up to 1:204 800, it could specifically recognize the recombinant protein, which further reflected that the recombinant His σB protein had good immunogenicity. Western blotting results showed that the recombinant σB protein could reacted with the rabbit polyclonal antibody, indicating that the recombinant σB protein expressed by prokaryotic expression had good reactivity. This experiment successfully expressed the recombinant σB protein using the prokaryotic expression system, and confirmed that the NDRV σB protein had good immunogenicity and reactogenicity. This result would help the subsequent identification of the biological function of the NDRV σB protein, preparation of diagnostic antigens and development of new NDRV vaccines.

Key words: Novel duck reovirus (NDRV); σB protein; prokaryotic expression; immunogenicity analysis

中图分类号:

S852.65⁺7

杨惠湖, 黄惠兰, 袁生, 李文俊, 姚鑫炎, 张玉倩, 梁昭平, 黄淑坚, 张雪莲. 新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析[J]. 中国畜牧兽医, 2021, 48(11): 4204-4212.

YANG Huihu, HUANG Huilan, YUAN Sheng, LI Wenjun, YAO Xinyan, ZHANG Yuqian, LIANG Zhaoping, HUANG Shujian, ZHANG Xuelian. Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4204-4212.

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新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain

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