魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

doi:10.3969/j.issn.1004-1524.2022.11.16

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (11): 2471-2481.DOI: 10.3969/j.issn.1004-1524.2022.11.16

魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

唐国亮¹^,²^,³(), 张玉宝¹^,²^,^*(), 王若愚¹^,², 王亚军¹^,², 赵霞¹^,², 苏学思¹^,²^,³, 金卫杰¹^,²^,³

1.中国科学院西北生态环境资源研究院皋兰生态与农业综合试验站,甘肃兰州730000
2.甘肃省生态农业综合试验野外科学观测研究站,甘肃兰州730000
3.中国科学院大学,北京100049

收稿日期:2021-06-30 出版日期:2022-11-25 发布日期:2022-11-29
作者简介:*张玉宝,E-mail: zyubao@lzb.ac.cn
唐国亮(1994—),男,甘肃定西人,硕士研究生,主要从事植物病毒学研究。E-mail: tangguoliang@nieer.ac.cn
通讯作者: 张玉宝
基金资助:
国家重点研发计划(2018YFE0127200);甘肃省知识产权计划(20ZSCQ033);国防科工局核能开发项目(E19005040);兰州市人才创新创业项目(2019-HLJC-9)

Prokaryotic expression of Konjac mosaic virus capsid protein and preparation of polyclonal antibody

TANG Guoliang¹^,²^,³(), ZHANG Yubao¹^,²^,^*(), WANG Ruoyu¹^,², WANG Yajun¹^,², ZHAO Xia¹^,², SU Xuesi¹^,²^,³, JIN Weijie¹^,²^,³

1. Gaolan Ecological and Agricultural Comprehensive Test Station, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
2. Gansu Field Scientific Observation and Research Station for Agricultural Ecosystem, Lanzhou 730000, China
3. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2021-06-30 Online:2022-11-25 Published:2022-11-29
Contact: ZHANG Yubao

摘要/Abstract

摘要：

采用小RNA测序技术对甘肃省榆中县疑似感染病毒病的当归[(Angelica sinensis(Oliv.)Diels]样品进行测序鉴定,发现样品中含有魔芋花叶病毒(Konjac mosaic virus,KoMV),通过反转录PCR(RT-PCR)扩增KoMV衣壳蛋白(capsid protein,CP)的cp基因,克隆的KoMV cp基因连接原核表达载体pET-28a(+),导入E.coli Rosetta^TM(DE3)诱导表达蛋白,在Ni-NTA重力柱层析作用下纯化CP融合蛋白,并以此作为抗原免疫新西兰大耳白兔制备多克隆抗体。序列分析表明:KoMV cp基因片段大小为840 bp,编码280个氨基酸的外壳蛋白;与GenBank已注册同源性较高的KoMV分离物相比,核苷酸序列相似性为85.58%~99.41%,氨基酸序列相似性为89.32%~99.29%;KoMV的病毒种群分布存在明显的区域性和寄主差异。SDS-PAGE显示,不同温度诱导下KoMV CP融合蛋白在E.coli Rosetta^TM(DE3)中均以包涵体的形式大量表达,融合蛋白分子量为36 ku。间接ELISA和Western blot检测结果显示,制备的多克隆抗体效价达到32 000,能够与感染KoMV的当归叶片和纯化蛋白特异性结合,具有良好的特异性。本研究成功制备了当归KoMV CP融合蛋白的多克隆抗体IgG,为开发KoMV的血清学检测技术及CP蛋白的功能研究奠定了基础。

关键词: 当归, 魔芋花叶病毒, 外壳蛋白, 原核表达, 多克隆抗体

Abstract:

The samples of Angelica sinensis(Oliv.)Diels suspected to be infected with virus disease in Yuzhong County, Gansu Province were sequenced and identified by small RNA sequencing. It was found that Konjac mosaic virus(KoMV) was contained in the sample, and the cp gene of KoMV capsid protein was amplified by RT-PCR. The cloned KoMV cp gene connected to the prokaryotic expression vector pET-28a (+), and introduced into E.coli Rosetta^TM (DE3) to induce expression protein. The CP fusion protein was purified by Ni-NTA gravity column chromatography and immunized New Zealand white-eared rabbits with this protein as antigen to prepare polyclonal antibodies. Sequence analysis showed that the size of KoMV cp gene fragment was 840 bp, encoding a coat protein of 280 amino acids. Compared with KoMV isolates registered in GenBank, the nucleotide sequence similarity was 85.58%-99.41%, and the amino acid sequence similarity was 89.32%-99.29%. There were obvious regional and host differences in the distribution of KoMV virus populations. SDS-PAGE showed that the fusion protein of KoMV CP was expressed in large quantities in the form of inclusion bodies in E.coli Rosetta^TM (DE3) induced by different temperatures, and the molecular weight of the fusion proteins was 36 ku. The results of indirect ELISA and Western blot showed that the prepared polyclonal antibody had a titer of 32 000, which could specifically bind to the leaves of Angelica infected with KoMV and purified protein, showing good specificity. In this study, the polyclonal antibody IgG of KoMV CP fusion protein was successfully prepared, which laid a foundation for the development of serological detection technology of KoMV and the functional research of CP protein.

Key words: Angelica sinensis, Konjac mosaic virus, capsid protein, prokaryotic expression, polyclonal antibody

中图分类号:

S435.67

唐国亮, 张玉宝, 王若愚, 王亚军, 赵霞, 苏学思, 金卫杰. 魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2022, 34(11): 2471-2481.

TANG Guoliang, ZHANG Yubao, WANG Ruoyu, WANG Yajun, ZHAO Xia, SU Xuesi, JIN Weijie. Prokaryotic expression of Konjac mosaic virus capsid protein and preparation of polyclonal antibody[J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2471-2481.

图/表 7

图1 健康(A)及感染Konjac mosaic virus的田间当归植株(B, C)

Fig.1 Healthy Angelica sinensis plants (A) and Angelica sinensis plants infected by Konjac mosaic virus (B and C)

图2 KoMV cp基因PCR扩增 M,DNA 2000 marker;1,阴性对照;2、3,KoMV cp基因

Fig.2 PCR amplification of KoMV cp gene M, DNA marker; 1, Negative control; 2-3, KoMV cp gene

图3 基于KoMV cp基因核苷酸序列系统进化树的构建

Fig.3 The phylogenetic tree of KoMV isolates based on the nucleotide sequences of cp genes

图4 KoMV CP重组蛋白的SDS-PAGE分析 A,KoMV CP蛋白的原核表达:M,蛋白质marker;1,pET-28a空载体对照;2,未经IPTG诱导的重组载体;3~5,16、28、37 ℃分别诱导20、12、4 h目标蛋白表达产物的上清;6~8,16、28、37 ℃分别诱导20、12、4 h目标蛋白表达产物的沉淀。B,KoMV CP蛋白的纯化:M,蛋白质marker;1,pET-28a空载体对照;2,未经IPTG诱导的重组载体;3,16 ℃诱导20 h目标蛋白表达产物的上清;4~5,纯化的重组KoMV CP蛋白。

Fig.4 SDS-PAGE analysis of recombinant KoMV CP A, Prokaryotic expression of recombinant KoMV CP:M, Protein marker; 1, Negative control of pET-28a without cp gene inserted; 2, Recombinant plasmid not induced by IPTG; 3-5, The supernatant of target protein expression product induced at 16, 28 and 37 ℃ for 20, 12 and 4 h, respectively; 6-8, The pellet of target protein expression product induced at 16, 28 and 37 ℃ for 20, 12 and 4 h, respectively. B, Purification of recombinant KoMV CP:M, Protein marker; 1, Negative control of pET-28a without cp gene inserted; 2, Recombinant plasmid not induced by IPTG; 3, The supernatant of target protein expression product induced at 16 ℃ for 20 h; 4-5, Purified recombinant KoMV CP protein.

图5 KoMV CP蛋白SDS-PAGE所分离蛋白的质谱图谱 A,a蛋白质谱鉴定。B,b蛋白质谱鉴定。

Fig.5 Mass spectrum of proteins isolated from KoMV CP protein by SDS-PAGE A, Identification of protein a by mass spectrometry. B, Identification of protein b by mass spectrometry.

表1 ELISA测定KoMV CP抗血清效价

Table 1 Titer determination of prepared antiserum against recombinant KoMV CP by ELISA

血清稀释度 Dilution of serum	抗血清D₄₅₀ D₄₅₀ of antiserum	阴性血清D₄₅₀ D₄₅₀ of negative serum	P/N>2.0
32 000	3.237	0.253	+
16 000	3.306	0.260	+
8 000	3.339	0.268	+
4 000	3.408	0.269	+
2 000	3.413	0.318	+
1 000	3.506	0.339	+
500	3.519	0.387	+

图6 Western blot鉴定KoMV CP多克隆抗体 M,蛋白质marker;1,健康当归样品;2~4,分别感染LSV、CMV和PLAMV的百合样品;5,感染LNYV的生菜样品;6,感染LMoV的百合样品;7,感染PVY的马铃薯样品;8,感染KoMV的当归样品;9,纯化的KoMV CP蛋白。

Fig.6 Western blot analysis of KoMV CP using rabbit polyclonal antibody M, Protein marker; 1, Negative control of healthy Angelica sinensis; 2-4, Extracts from lily samples infected with LSV, CMV and PLAMV, respectively; 5, Extracts from lettuce samples infected with LNYV; 6, Extracts from lily samples infected with LMoV; 7, Extracts from potato samples infected with PVY; 8, Extracts from Angelica sinensis samples infected with KoMV; 9, Purified KoMV CP protein.

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魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

Prokaryotic expression of Konjac mosaic virus capsid protein and preparation of polyclonal antibody

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