Preparation and application of polyclonal antibody against a cysteine-rich protein encoded by Chinese wheat mosaic virus

doi:10.3785/j.issn.1008-9209.2023.02.011

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (5): 677-686 DOI: 10.3785/j.issn.1008-9209.2023.02.011

Special Topic: Major Bacterial and Viral Diseases in Crops

Preparation and application of polyclonal antibody against a cysteine-rich protein encoded by Chinese wheat mosaic virus

Yuanxing DAI^1,²(

),Liuming GUO²,Jing HE^1,²,Zhengrong SHEN^1,²,Yanfei GENG²,Mingfang Lü²,Zhengjie YUAN²,Jing LI²,Hengmu ZHANG²(

)

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

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Abstract

Chinese wheat mosaic virus (CWMV) is one of the most important pathogens causing mosaic disease in wheat and has threatened the yield and quality of wheat for a long time. Cysteine-rich protein (CRP) of CWMV plays important and complex roles in viral infection. To further study CRP functions and CWMV infection mechanisms, the CRP coding region was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from leaves of CWMV-infected wheat and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid pET-CRP was transformed into Escherichia coli BL21 (DE3) for inducible expression. The recombinant CRP was purified by nickel-column affinity chromatography and used as an antigen to immunize New Zealand white rabbits for polyclonal antibody preparation. A series of immunological assays, including Western blot, indirect enzyme-linked immunosorbent assay (ELISA) and dot ELISA, showed that the purified CRP antibody had high specificity, and its titer was as high as 1∶4 096 000, which was four times higher than that of the unpurified antibody. The antibody could recognize 0.5 ng antigen, showing its high sensitivity. In addition, the purified CRP antibody could specifically and sensitively recognize native CRP even at a 1∶120 000 dilution. In conclusion, the polyclonal antibody can be not only used for precise diagnosis of the CWMV-infected plant samples from fields, but also applied to detect CRP expressed transiently in plants, which lays a foundation for subsequent detection, quantification and subcellular localization of CRP.

Key words： Chinese wheat mosaic virus cysteine-rich protein prokaryotic expression protein purification polyclonal antibody

Received: 01 February 2023 Published: 03 November 2023

CLC:

S435.121.4

Corresponding Authors: Hengmu ZHANG E-mail: dai15979596433@163.com;zhhengmu@tsinghua.org.cn

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	Yuanxing DAI
	Liuming GUO
	Jing HE
	Zhengrong SHEN
	Yanfei GENG
	Mingfang Lü
	Zhengjie YUAN
	Jing LI
	Hengmu ZHANG

Cite this article:

Yuanxing DAI,Liuming GUO,Jing HE,Zhengrong SHEN,Yanfei GENG,Mingfang Lü,Zhengjie YUAN,Jing LI,Hengmu ZHANG. Preparation and application of polyclonal antibody against a cysteine-rich protein encoded by Chinese wheat mosaic virus. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 677-686.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2023.02.011 OR https://www.zjujournals.com/agr/Y2023/V49/I5/677

中国小麦花叶病毒富含半胱氨酸蛋白多克隆抗体的制备与应用

中国小麦花叶病毒（Chinese wheat mosaic virus, CWMV）是小麦花叶病的重要病原体之一，长期威胁小麦的产量和品质；CWMV富含半胱氨酸蛋白（cysteine-rich protein, CRP）在病毒侵染过程中具有重要而复杂的功能。为了深入研究CRP的功能和CWMV侵染机制，本研究采用反转录聚合酶链反应（reverse transcription-polymerase chain reaction, RT-PCR）从CWMV侵染的小麦叶片中获得CRP基因编码区，将其克隆至原核表达载体pET-32a上，并将重组质粒pET-CRP转化至大肠埃希菌BL21（DE3）感受态细胞中进行诱导表达；通过镍柱亲和层析法纯化CRP重组蛋白，并用作抗原免疫新西兰白兔，制备多克隆抗体。蛋白质印迹法、间接酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）和斑点ELISA分析结果显示：纯化的CRP抗体不仅具有高度的特异性，而且效价高达1∶4 096 000，是未纯化抗体效价的4倍；该抗体能识别0.5 ng抗原，显示出较高的灵敏度；在1∶120 000稀释条件下，该抗体能特异且灵敏地识别天然CRP。综上所述，本研究所制备的CRP抗体不但可用于田间CWMV病株样品的精准诊断，还可用于植物体内瞬时表达CRP的检测分析，为后续CRP检测、定量分析及其亚细胞定位等研究提供了依据。

关键词： 中国小麦花叶病毒, 富含半胱氨酸蛋白, 原核表达, 蛋白质纯化, 多克隆抗体

引物名称 Primer name	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	限制性内切酶 Restriction enzyme
pET-CRP-F	GCTGATATCGGATCCATGACTACTGGTACTCATTCT	BamHⅠ
pET-CRP-R	TTGTCGACGGAGCTCTTACTCCACACGAGTCTTCTT	SacⅠ
CRP-F	ATGACTACTGGTACTCATTCT
CRP-R	TTACTCCACACGAGTCTTCTT
T7	TAATACGACTCACTATAGGG
T7-ter	GCTAGTTATTGCTCAGCGG
pCV-35S::CRP-F	GGTACCCGGGGATCCATGACTACTGGTACTCATTCT	BamHⅠ
TRV::CRP-F	GACAAGACCCTGCAGATGACTACTGGTACTCATTCT	PstⅠ
PVX::CRP-F	CCAGCTAGCATCGATATGACTACTGGTACTCATTCT	ClaⅠ
CRP-Flag-R	GGCGAATTGGTCGACCTCCACACGAGTCTTCTTC	SalⅠ

Table 1 Primers used for polymerase chain reaction amplification

Fig. 1 SDS-PAGE analysis of recombinant CRP (A) and specificity analysis of the antibody against CRP by Western blot (B, C)In the Fig. A, M: Protein marker; Lane 1: IPTG-induced host bacteria containing pET-32a; Lane 2: IPTG-uninduced host bacteria containing pET-CRP; Lanes 3-5: Host bacteria containing pET-CRP induced by IPTG for 1, 2, and 3 h, respectively; Lane 6: Purified recombinant CRP. In the Fig. B, M: Protein marker; Lane 1: IPTG-induced host bacteria containing pET-32a; Lane 2: Host bacteria containing pET-CRP induced by IPTG for 2 h. In the Fig. C, M: Protein marker; Lane 1: Healthy wheat leaves; Lane 2: CWMV-infected wheat leaves; Lane 3: BYDV-infected wheat leaves; Lane 4: ToMV-infected N. benthamiana leaves; Lane 5: TuMV-infected N. benthamiana leaves.

Fig. 2 Analysis of titers of unpurified and purified CRP antibodies by indirect ELISA

Fig. 3 Western blot assay of total protein samples extracted from CWMV-infected plant leaves using the gradient dilution antibodiesM: Protein marker; Lanes 1-5: Antibody dilution ratios of 1∶40 000/60 000/80 000/100 000/120 000, respectively.

Fig. 4 Sensitivity analysis of CRP antibodies by dot ELISA

Fig. 5 Sensitivity analysis of CRP antibodies by Western blotThe antibody dilution ratio is 1∶80 000. In the Fig. A, M: Protein marker; Lanes 1-5: 4, 2, 1, 0.5, 0.25 ng purified recombinant CRP samples, respectively. In the Fig. B, M: Protein marker; Lanes 1-5: Total protein samples extracted from CWMV-infected plant leaves, which are diluted by the ratios of 1∶4/6/8/10/12, respectively.

Fig. 6 Detection of the diseased wheat plants from fields by Western blot (A) and RT-PCR (B)In the Fig. A, M: Protein marker; Lanes 1-9: Samples of wheat leaves. In the Fig. B, M: DNA marker; +: Samples of CWMV-infected wheat leaves (positive control); －: Samples of healthy wheat leaves (negative control); Lanes 1-9: Samples of wheat leaves.

Fig. 7 Detection of CRP by transient expression in N. bentha-miana leaves using Western blotIn the Fig. A, CRP antibody is used as primary antibody; in the Fig. B, Flag tag antibody is used as primary antibody. M: Protein marker; Lanes 1 and 8: CWMV-infected leaves; Lanes 2 and 9: Empty pCV vector (control); Lanes 3 and 10: pCV-35S::CRP-3×Flag; Lanes 4 and 11: TRV-infected leaves; Lanes 5 and 12: Co-infiltration of TRV1 and TRV::CRP-3×Flag; Lanes 6 and 13: PVX-infected leaves; Lanes 7 and 14: PVX::CRP-3×Flag.


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