猪流行性腹泻病毒S蛋白原核表达及多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2022.11.029

摘要/Abstract

摘要： 【目的】探索猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV） S蛋白的结构和功能，为建立PEDV感染的诊断方法和疫苗开发提供理论依据。【方法】以PEDV经典CV777株为模板，通过PCR扩增获得S、S1基因片段；PCR扩增产物分别克隆pET-30a （+）原核表达载体和pFLAG-CMV-3真核表达载体，构建原核表达质粒pET-30a-PEDV-S和真核表达质粒pFLAG-CMV-3-PEDV-S1；将pET-30a-PEDV-S转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导表达PEDV-S重组蛋白，通过变性、复性、浓缩纯化重组蛋白并进行Western blotting检测；将PEDV-S重组蛋白免疫C57BL小鼠制备多克隆抗体，获得的多克隆抗体经间接免疫荧光试验（IFA）检测特异性，通过间接ELISA法检测多克隆抗体效价；将pFLAG-CMV-3-PEDV-S1转染至HEK293T细胞，以制备的多克隆抗体为一抗，经IFA测定PEDV-S1蛋白的抗原性和多克隆抗体的反应性。【结果】成功克隆出PEDV S和S1基因，构建了可表达PEDV-S重组蛋白的原核表达质粒和在HEK293T细胞中高效表达S1蛋白的真核表达质粒；PEDV-S重组蛋白在IPTG为0.5 mmol/L、16 ℃诱导8 h条件下可获得最高表达量，主要以包涵体形式存在；Western blotting结果显示，PEDV-S重组蛋白成功表达；ELISA检测结果显示，制备的多克隆抗体效价达1：3 280 500；IFA结果显示，多克隆抗体有良好的特异性，可特异性识别PEDV-S和PEDV-S1蛋白。【结论】本研究获得了PEDV-S重组蛋白，并成功表达S1蛋白，制备出PEDV-S蛋白多克隆抗体，为研究S蛋白的结构和功能提供了条件，为揭示PEDV致病机制奠定了基础。

关键词: 猪流行性腹泻病毒(PEDV); S蛋白; 蛋白纯化; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to explore the structure and function of S protein of Porcine epidemic diarrhea virus (PEDV), so as to provide a theoretical basis for promoting the diagnosis of PEDV infection and vaccine development.【Method】 The classic CV777 strain of PEDV was used as the template, the S and the S1 genes fragments were amplified by PCR.The PCR amplification products were cloned into pET-30a (+) prokaryotic expression vector and pFLAG-CMV-3 eukaryotic expression vector respectively, and the prokaryotic expression plasmid pET-30a-PEDV-S and eukaryotic expression plasmid pFLAG-CMV-3-PEDV-S1 were constructed. pET-30a-pEDV-S was transformed into E.coli BL21(DE3) competent cells, and expressed PEDV-S recombinant protein induced by IPTG.The recombinant protein was purified by denaturation and renaturation, and then detected by Western blotting.The recombinant protein PEDV-S was immunized in C57BL mice to prepare polyclonal antibodies.The obtained polyclonal antibody specificity was detected by indirect immunofluorescence assay (IFA) and polyclonal antibody titer was detected by indirect ELISA. pFLAG-CMV-3-PEDV-S1 was transfected into HEK293T cells, and the prepared polyclonal antibody was used as the primary antibody to detect the antigenicity of PEDV-S1 protein and the reactivity of polyclonal antibody by IFA.【Result】 The PEDV S and S1 genes were successfully cloned, and a prokaryotic expression vector capable of expressing recombinant protein PEDV-S and a eukaryotic expression vector capable of efficiently expressing S1 protein in HEK293T cells were constructed.The highest expression of PEDV-S recombinant protein was obtained when IPTG was 0.5 mmol/L and induced at 16 ℃ for 8 h, mainly in the form of inclusion bodies.Western blotting showed that the recombinant protein of PEDV-S was successfully expressed.ELISA results showed that the titer of the polyclonal antibody was 1:3 280 500.IFA results showed that the polyclonal antibody had good specificity and could specifically recognize PEDV-S and PEDV-S1 proteins.【Conclusion】 In this study, PEDV-S recombinant protein and S1 protein were obtained, and polyclonal antibody against PEDV S protein was successfully prepared, which provided conditions for studying the structure and function of S protein and laid a foundation for revealing the pathogenic mechanism of PEDV.

Key words: Porcine epidemic diarrhea virus (PEDV); S protein; protein purification; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65^＋1

张天爱, 李婷婷, 陶晓莉, 李藤菲, 林家锋, 胡思漫, 刘文秀, 佟伟, 李永刚. 猪流行性腹泻病毒S蛋白原核表达及多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2022, 49(11): 4383-4391.

ZHANG Tianai, LI Tingting, TAO Xiaoli, LI Tengfei, LIN Jiafeng, HU Siman, LIU Wenxiu, TONG Wei, LI Yonggang. Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4383-4391.

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