车前草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.1004-1524.2021.01.13

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 104-111.DOI: 10.3969/j.issn.1004-1524.2021.01.13

车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

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

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.中国科学院大学,北京100049

收稿日期:2020-08-07 出版日期:2021-01-25 发布日期:2021-01-25
通讯作者: 张玉宝
作者简介:*张玉宝,E-mail:zyubao@lzb.ac.cn
苏学思(1995—),男,河南新乡人,硕士研究生,主要从事植物病毒学研究。E-mail:suxuesi18@mails.ucas.ac.cn
基金资助:
陇原青年创新创业人才(团队)项目(2020-339);兰州市人才创新创业项目(2019-HLJC-9);兰州市城关区科技计划(2019-6-2)

Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody

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

1. Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2020-08-07 Online:2021-01-25 Published:2021-01-25
Contact: ZHANG Yubao

摘要/Abstract

摘要：

为制备车前草花叶病毒(Plantago asiatica mosaic virus,PlAMV)衣壳蛋白(capsid protein,CP)及其多克隆抗体,采用RT-PCR方法从甘肃省种植的切花百合上克隆了PlAMV cp基因的部分序列,连接表达载体pET-28a(+),转化Escherichia coli BL21(DE3)菌株,经IPTG诱导和Ni-NTA重力柱层析获得了大量纯化的CP融合蛋白,将其作为抗原免疫家兔制备了多克隆抗体。序列分析结果表明,PlAMV cp基因片段大小为621 bp,编码207个氨基酸;与已注册的PlAMV分离物相比,核苷酸序列相似性为74.7%~100%,氨基酸序列相似性达到83.6%~100%;不同植物PlAMV的分离物序列差异明显,病毒种群分布呈现寄主差异。SDS-PAGE结果表明,CP蛋白在E. coli BL21(DE3)中大量表达,蛋白相对分子质量为24 ku。Western blot结果显示,抗体能够特异结合天然PlAMV病毒蛋白,可用于检测感病材料中PlAMV蛋白的表达量。本研究可以为PlAMV的血清学检测技术开发和致病机理研究提供一定的参考。

关键词: 百合, 车前草花叶病毒, 衣壳蛋白, 原核表达, 多克隆抗体

Abstract:

The cp gene of Plantago asiatica mosaic virus (PlAMV) was cloned from an isolate of PlAMV, obtained from lily growing in Gansu Province, China. The cp gene fragment of 621 bp was constructed into the prokaryotic expression vector pET-28a (+), and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to express the fusion CP protein. After IPTG induction and Ni-NTA gravity column chromatography, purified CP fusion protein was obtained and used as immunogen to prepare a rabbit-derived anti-CP polyclonal antibody. BLAST result showed that the similarity of nucleotide and amino acid sequences of cp gene was up to 74.7%-100% and 83.6%-100% between the cloned PlAMV isolate and the known PlAMV isolates, respectively. We noted that cp sequences from different hosts were significantly different, which suggested the distribution of PlAMV population was influenced by species of host. SDS-PAGE indicated that CP protein was highly expressed in E. coli BL21 (DE3), and had a relative protein molecular weight of 24 ku. Western blot result demonstrated that the prepared polyclonal antibody could successfully capture the natural PlAMV capsid proteins, which was used to check the expression level of PlAMV protein in susceptible plant tissue. This work provided a reference for the study of the pathogenic mechanism of PlAMV and the development of serological detection techniques for the virus.

Key words: lily, Plantago asiatica mosaic virus, capsid protein, prokaryotic expression, polyclonal antibody

中图分类号:

苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草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, 2021, 33(1): 104-111.

SU Xuesi, ZHANG Yubao, WANG Ruoyu, WANG Yajun, TANG Guoliang, JIN Weijie. Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 104-111.

图/表 8

图1 感染Plantago asiatica mosaic virus的百合

Fig.1 Lily infected with Plantago asiatica mosaic virus exhibiting symptoms of rust-brown necrotic strips and spots

图2 PlAMV cp基因的PCR扩增结果 M,DNA 2 000 marker;1,阴性对照;2,PlAMV cp基因。

Fig.2 PCR amplification result of PlAMV cp gene M, DNA 2 000 marker; 1, Negative control; 2, PlAMV cp gene.

图3 基于PlAMV cp基因核苷酸序列构建的系统进化树

Fig.3 Phylogenetic tree of PlAMV isolates based on the nucleotide sequences of their cp genes

图4 PlAMV CP蛋白的二级结构、亲水性、柔韧性和抗原性预测

Fig.4 Prediction of secondary structure, hydrophilicity, flexibility and antigenicity of PlAMV CP protein

图5 pET-28a重组质粒的双酶切鉴定结果 M,DNA Marker;1,未经酶切的pET-28a重组质粒;2~3,pET-28a重组质粒的双酶切结果。

Fig.5 Identification of recombinant plasmid pET-28a by double enzyme digestion M, DNA Marker; 1, Recombinant plasmid pET-28a without enzyme digestion; 2-3, Double enzyme digestion of recombinant plasmid pET-28a.

图6 PlAMV CP重组蛋白的SDS-PAGE分析 A,PlAMV CP蛋白的原核表达:M,蛋白质分子质量标准;1为pET-28a空载体对照;2~6 分别为0、0.5、1.0、1.5和2.0 mmol·L-1 IPTG诱导表达的PlAMV CP蛋白。B,PlAMV CP蛋白的纯化:M,蛋白质分子质量标准;1~2为纯化的重组CP蛋白。

Fig.6 SDS-PAGE analysis of recombinant PlAMV CP A, Prokaryotic expression of recombinant PlAMV CP: M, Protein marker; 1, Negative control of pET-28a without cp gene inserted; 2-6, Expression of PlAMV CP induced by 0, 0.5, 1.0, 1.5 and 2.0 mmol·L-1 IPTG, respectively. B, Purification of recombinant PlAMV CP: M, Protein marker; 1-2, Purified recombinant PlAMV CP protein.

表1 ELISA测定CP抗血清效价

Table 1 Titer determination of prepared antiserum against recombinant 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
1∶500	2.907	0.232	+
1∶2 000	2.292	0.206	+
1∶8 000	1.133	0.191	+
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
1∶32 000	0.493	0.171	+
1∶128 000	0.230	0.153	-

图7 Western blot鉴定PlAMV CP多克隆抗体 M,蛋白质分子质量标准;1,健康百合样品;2~4,分别感染LSV、CMV和LMoV的百合样品;5,感染PVX的马铃薯样品;6,感染PlAMV的百合样品。

Fig.7 Western blot analysis of PlAMV CP using rabbit polyclonal antibody M, Protein marker; 1, Negative control of healthy lily; 2-4, Extracts from lily samples infected with LSV, CMV and LMoV, respectively; 5, Extracts from potato samples infected with PVX; 6, Extracts from lily samples infected with PlAMV.

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车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody

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