Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss

HU Feifan; LIANG Qizhang; HUANG Yu

doi:10.19303/j.issn.1008-0384.2023.08.001

Volume 38 Issue 8

Aug. 2023

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Article Navigation > Fujian Journal of Agricultural Sciences > 2023 > 38(8): 889-893

HU F F, LIANG Q Z, HUANG Y. Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss [J]. Fujian Journal of Agricultural Sciences，2023，38（8）：889−893 doi: 10.19303/j.issn.1008-0384.2023.08.001

Citation:

HU F F, LIANG Q Z, HUANG Y. Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss [J]. Fujian Journal of Agricultural Sciences，2023，38（8）：889−893 doi: 10.19303/j.issn.1008-0384.2023.08.001

Citation:

HU F F, LIANG Q Z, HUANG Y. Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss [J]. Fujian Journal of Agricultural Sciences，2023，38（8）：889−893 doi: 10.19303/j.issn.1008-0384.2023.08.001

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Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss

doi: 10.19303/j.issn.1008-0384.2023.08.001

HU Feifan^{1, 2
,},
LIANG Qizhang^{2, 3
,
,},
HUANG Yu^{2, 3
,
,}

1.
College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural sciences, Fuzhou, Fujian　350013, China
3.
Fujian Key Laboratory for Avian Diseases Control and Prevention, Fuzhou, Fujian　350013, China

Received Date: 2023-04-25
Rev Recd Date: 2023-06-02

Available Online: 2023-08-16

Publish Date: 2023-08-28

Abstract

Abstract

Objective A polyclonal antibody specific to the carboxy-terminus subfragment of Rep protein of the new-genotype Muscovy duck parvovirus (N-MDPV) was prepared. Methods Based on the protein sequence, the C-terminus subfragment region 487–627 aa of Rep of N-MDPV was synthesized to add His-tag. Using seamless cloning method, the segment was introduced into pET-28a(+) vector, transformed into Rosetta (DE3) Escherichia coli, and induced to express the recombinant protein. The recombinant protein was then purified by nickel column affinity chromatography for immunization on New Zealand white rabbits to produce the polyclonal antibody. Results The prokaryotic expression plasmid pET-28a-Rep-487-627 was constructed, purified, and expressed. The recombinant protein was approximately 24 kDa and mainly expressed in a soluble form. The indirect immunofluorescence and western blot showed that the prepared Rep polyclonal antibody could react specifically with the overexpressed N-MDPV in cells. Conclusion The prepared Rep polyclonal antibody exhibited a reaction specificity that recognized the conformational epitopes and linear epitopes of Rep protein.
- New-genotype Muscovy duck parvovirus,
- Rep protein,
- indirect immunofluorescence assay,
- western blot

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References(14)

References

[1]	林世棠, 郁晓岚, 陈炳钿, 等. 一种新的雏番鸭病毒性传染病的诊断 [J]. 中国畜禽传染病, 1991, 13(2):25−26. LIN S T, YU X L, CHEN B (D /T), et al. Diagnosis of a new viral infectious disease in Muscovy duck [J]. Chinese Journal of Preventive Veterinary Medicine, 1991, 13(2): 25−26.(in Chinese)
[2]	胡奇林, 吴振兖, 周文谟, 等. 雏番鸭细小病毒病的流行病学调查 [J]. 中国兽医杂志, 1993, 29(6):7−8. HU Q L, WU Z Y, ZHOU W M, et al. Epidemiological investigation of parvovirus disease in Muscovy ducks [J]. Chinese Journal of Veterinary Medicine, 1993, 29(6): 7−8.(in Chinese)
[3]	黄瑜, 万春和, 傅秋玲, 等. 新型番鸭细小病毒的发现及其感染的临床表现 [J]. 福建农业学报, 2015, 30(5):442−445. HUANG Y, WAN C H, FU Q L, et al. The identity and clinic infectious symptoms of the new genotype Muscovy duck parvovirus [J]. Fujian Journal of Agricultural Sciences, 2015, 30(5): 442−445.(in Chinese)
[4]	WANG J Y, HUANG Y, ZHOU M X, et al. Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30 [J]. Virology Journal, 2016, 13: 104. doi: 10.1186/s12985-016-0564-9
[5]	TEWARY S K, ZHAO H Y, DENG X F, et al. The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA [J]. Virology, 2014, 449: 297−303. doi: 10.1016/j.virol.2013.11.031
[6]	LE GALL-RECULÉ G, JESTIN V. Biochemical and genomic characterization of Muscovy duck parvovirus [J]. Archives of Virology, 1994, 139(1): 121−131.
[7]	POOLE B D, ZHOU J, GROTE A, et al. Apoptosis of liver-derived cells induced by parvovirus B19 nonstructural protein [J]. Journal of Virology, 2006, 80(8): 4114−4121. doi: 10.1128/JVI.80.8.4114-4121.2006
[8]	万春和, 傅秋玲, 陈翠腾, 等. 基因重组型番鸭细小病毒FJM3的全基因组特征 [J]. 中国兽医学报, 2016, 36(11):1836−1841. WAN C H, FU Q L, CHEN C T, et al. Molecular characterization of the genome for recombinant Muscovy duck parvovirus strain FJM3 [J]. Chinese Journal of Veterinary Science, 2016, 36(11): 1836−1841.(in Chinese)
[9]	JAMEIE F, DALIMI A, PIRESTANI M, et al. Development of a multi-epitope recombinant protein for the diagnosis of human visceral leishmaniasis [J]. Iranian Journal of Parasitology, 2021, 16(1): 1−10.
[10]	ZHANSAYA A, MALIKA N, BORIS D, et al. Expression of recombinant CTLA-4 and PD-L1 proteins fused with thioredoxin, and determination of their ligand-binding activities [J]. Reports of Biochemistry & Molecular Biology, 2022, 11(2): 310−319.
[11]	王志仙, 凌珏怡, 贾婧宇, 等. 番鸭细小病毒Rep1蛋白的原核表达及多克隆抗体制备 [J]. 中国家禽, 2018, 40(6):15−18. WANG Z X, LING J Y, JIA J Y, et al. Prokaryotic expression and polyclonal antibody preparation of Rep1 protein of Muscovy duck parvovirus [J]. China Poultry, 2018, 40(6): 15−18.(in Chinese)
[12]	苗碧琛, 陈松彪, 张秀娟, 等. 猪细小病毒NS基因原核表达与多克隆抗体制备 [J]. 动物医学进展, 2020, 41(2):1−8. doi: 10.3969/j.issn.1007-5038.2020.02.001 MIAO B C, CHEN S B, ZHANG X J, et al. Prokaryotic expression of porcine parvovirus NS gene and preparation of polyclonal antibody [J]. Progress in Veterinary Medicine, 2020, 41(2): 1−8.(in Chinese) doi: 10.3969/j.issn.1007-5038.2020.02.001
[13]	廖健淇, 谢芝勋, 张民秀, 等. 鸡细小病毒NS1和VP2蛋白的原核表达及多克隆抗体制备 [J]. 西南农业学报, 2023, 36(2):419−426. LIAO J Q, XIE Z X, ZHANG M X, et al. Prokaryotic expression of chicken parvovirus NS1 and VP2 proteins and preparation of polyclonal antibodies [J]. Southwest China Journal of Agricultural Sciences, 2023, 36(2): 419−426.(in Chinese)
[14]	LI L, QIU J M, PINTEL D J. The choice of translation initiation site of the rep proteins from goose parvovirus P9-generated mRNA is governed by splicing and the nature of the excised intron [J]. Journal of Virology, 2009, 83(19): 10264−10268. doi: 10.1128/JVI.01255-09