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

doi:10.3969/j.issn.1004-1524.2022.11.16

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (11): 2471-2481.DOI: 10.3969/j.issn.1004-1524.2022.11.16

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S435.67

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.

Figures/Tables 7

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

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

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

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.

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.

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	+

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

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