中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用

doi:10.3969/j.issn.1004-1524.20231119

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (9): 2042-2050.DOI: 10.3969/j.issn.1004-1524.20231119

中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用

沈峥嵘¹^,²(), 戴远兴², 郭留明², 汪芷瑶¹^,², 张恒木²^,^*()

1.浙江师范大学生命科学学院,浙江金华 321004
2.浙江省农业科学院病毒学与生物技术研究所,浙江杭州 310021

收稿日期:2023-09-18 出版日期:2024-09-25 发布日期:2024-09-30
作者简介:*张恒木,E-mail:zhhengmu@tsinghua.org.cn
沈峥嵘(1995—),男,浙江义乌人,硕士研究生,主要从事植物病理学研究。E-mail:18867182103@163.com
通讯作者: *张恒木,E-mail:zhhengmu@tsinghua.org.cn
基金资助:
浙江省万人计划人才培养项目(2019R52033)

Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)

SHEN Zhengrong¹^,²(), DAI Yuanxing², GUO Liuming², WANG Zhiyao¹^,², ZHANG Hengmu²^,^*()

1. College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-09-18 Online:2024-09-25 Published:2024-09-30

摘要/Abstract

摘要：

中国小麦花叶病毒(CWMV)是浙江省农业科学院发现并鉴定的一种土传病毒,其RNA2编码的外壳蛋白(CP)在病毒侵染过程中发挥重要功能。为了检测该蛋白表达并分析其生物学功能,该研究采用RT-PCR方法从感染CWMV小麦病叶中扩增获得CP基因,并通过In-Fusion技术构建了CWMV CP重组表达载体,将其导入BL21(DE3)菌株诱导表达;原核表达的重组CP蛋白经镍柱亲和层析纯化后注射免疫家兔,收集抗血清经亲和层析纯化获得CP多克隆抗体。Dot-ELISA、ID-ELISA、Western blot显示,该抗体不仅对CWMV具有高度的特异性,而且其效价高达1∶2.048×10⁷、灵敏度达6.25×10^-2 ng。应用该抗体采用Dot-ELISA、Western blot方法检测田间小麦样品显示其可用于CWMV的准确诊断,这为CWMV病毒病检测、CP定量分析及其功能研究奠定了基础。

关键词: 中国小麦花叶病毒, 外壳蛋白, 重组蛋白, 多克隆抗体

Abstract:

Chinese wheat mosaic virus (CWMV) is a soil-borne virus discovered and identified by Zhejiang Academy of Agricultural Sciences, whose genomic segment RNA2 encodes a major coat protein (CP) that plays an important role in virus infection. To detect its expression and analyze its biological function, the gene was amplified by RT-PCR from CWMV-infected wheat leaves. The recombinant expression vector was constructed with In-Fusion method and introduced into BL21(DE3) strain for the induction of CP expression. The recombinant CP protein was purified by nickel column affinity chromatography and injected into the rabbits for immunization. The antisera were purified by affinity chromatography and the polyclonal antibodies against CP were obtained. Dot-ELISA, ID-ELISA and Western blot assays showed that the purified antibodies were highly specific to CWMV with a titer of 1∶2.048×10⁷ and the sensitivity of 6.25×10^-2 ng. Dot-ELISA and Western blot assays showed that the antibody could be used for accurate diagnosis of CWMV, which laid the foundation for CWMV detection, CP quantitation and its functional research.

Key words: Chinese wheat mosaic virus, coat protein, recombinant protein, polyclonal antibody

中图分类号:

S435.12

沈峥嵘, 戴远兴, 郭留明, 汪芷瑶, 张恒木. 中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用[J]. 浙江农业学报, 2024, 36(9): 2042-2050.

SHEN Zhengrong, DAI Yuanxing, GUO Liuming, WANG Zhiyao, ZHANG Hengmu. Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2042-2050.

图/表 7

表1 用于PCR 扩增的引物

Table 1 Primers used for PCR amplification

引物名称 Primer name	引物序列 Primer sequences (5'→3')	限制性内切酶 Restriction enzyme
CP-F	ATGGCCGTGAAATCTGG	—
CP-R	GCTCGAACCTTCCCACTTA	—
CP-F'	GCTGATATCGGATCCATGGCCGTGAAATCTGG	BamH I
CP-R'	TTGTCGACGGAGCTCGCTCGAACCTTCCCACTTA	Sac I
T7	TAATACGACTCACTATAGGG	—
T7-ter	GCTAGTTATTGCTCAGCGG	—

图1 CWMV CP原核表达载体构建及鉴定 M为DNA Marker;第1泳道为扩增成功的CWMV CP基因;第2泳道为pET-32a空载体PCR产物(作为阴性对照);第3~8泳道为pET-CP重组载体PCR产物;第9~11泳道为3个PCR阳性克隆双酶切产物。

Fig.1 Construction and identification of recombinant vector pET-CP for expression in prokaryotic cells M, DNA Marker; Lane 1, Amplified fragment of CWMV CP gene; Lane 2, PCR products of empty pET-32a vector as negative control; Lanes 3-8, PCR products of recombinant pET-CP vectors; Lanes 9-11, Restriction enzyme digestion of PCR-positive pET-CP recombinant vectors with BamHⅠ/SacⅠ.

图2 重组CWMV CP蛋白诱导表达及其亲和纯化分析 A:M为蛋白 Marker,第1泳道为未诱导的含pET-32a载体的宿主菌,第2泳道为IPTG诱导的含pET-32a载体的宿主菌,第3泳道为未诱导的含pET-CP载体的宿主菌,第4~6泳道分别为IPTG诱导1~3 h的含pET-CP载体的宿主菌;B:M为蛋白 Marker,第1泳道为从含pET-CP并诱导3 h的宿主菌中纯化的CP重组蛋白。

Fig.2 Assays for induced expression and affinity purification of recombinant CWMV CP protein A: M, Protein Marker; Lane 1, Uninduced host bacteria containing pET-32a; Lane 2, IPTG induced host bacteria containing pET-32a; Lane 3, Uninduced host bacteria containing pET-CP; Lanes 4-6, Host bacteria containing pET-CP induced by IPTG for 1-3 h. B: M, Protein Marker; Lane 1, Recombinant CP protein purified from host bacteria that containing pET-CP and induced by IPTG for 3 h.

图3 CWMV CP抗体特异性分析 A:M为蛋白Marker,第1泳道为诱导的含pET-32a的宿主菌,第2泳道为IPTG诱导的含 pET-CP的宿主菌;B:M为蛋白Marker,第1泳道为健康小麦样品,第2泳道为CWMV侵染的小麦样品,第3泳道为BYDV侵染的小麦样品,第4泳道为WYMV侵染的小麦样品,第5泳道为ToMV侵染的本氏烟样品,第6泳道为TuMV侵染的本氏烟样品。

Fig.3 Specificity analysis of antibody against CWMV CP A: M, Protein Marker; Lane 1, Induced host bacteria containing pET-32a; Lane 2, IPTG induced host bacteria containing pET-CP. B: M, Protein Marker; Lane 1, Healthy wheat sample; Lane 2, CWMV-infected wheat sample; Lane 3, BYDV-infected wheat sample; Lane 4, WYMV-infected wheat sample; Lane 5, ToMV-infected N. benthamiana sample; Lane 6, TuMV-infected N. benthamiana sample.

图4 间接ELISA法分析CP抗体效价

Fig.4 Titer of purified CP antibodies analyzed by indirect ELISA

图5 Dot-ELISA法分析CP抗体的灵敏度

Fig.5 Sensitivity of CP antibody detected by Dot-ELISA

图6 应用Dot-ELISA(A)、Western blot(B)和RT-PCR(C)检测田间小麦病株 A:M为DNA Marker,第1 泳道为阳性对照,第2 泳道为阴性对照,第3~11 泳道为小麦病株样品;B:M 为蛋白 Marker,第 1~9 泳道为小麦病株样品;C:第1 泳道为阳性对照,第2 泳道为阴性对照,第3~11 泳道为小麦病株样品。

Fig.6 Detection of the diseased wheat plants from fields by Dot-ELISA (A), Western blot (B) and RT-PCR(C) A: M, DNA Marker; Lane 1, Positive control; Lane 2, Negative control; Lanes 3-11, Samples of diseased wheat plants. B: M, Protein Marker; Lanes 1-9, Samples of diseased wheat plants. C: M, DNA Marker; Lane 1, Positive control; Lane 2, Negative control; Lanes 3-11, Samples of diseased wheat plants.

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中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用

Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)

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