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

doi:10.3969/j.issn.1004-1524.2021.01.13

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (1): 104-111.DOI: 10.3969/j.issn.1004-1524.2021.01.13

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

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.

Figures/Tables 8

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

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

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

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

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.

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.

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	-

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.

References 26

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

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