Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss
-
摘要:
目的 获得特异性识别新型番鸭细小病毒（New-genotype muscovy duck Parvovirus ，N-MDPV）Rep蛋白羧基端亚片段的多克隆抗体。 方法 通过蛋白序列分析，选取N-MDPV Rep羧基端亚片段区域487～627 aa，后全基因合成序列，并在其C末端添加His-tag标签，利用无缝克隆的方法，将该段基因克隆至pET-28a(+)载体，随后转化Rosetta（DE3）大肠杆菌，诱导表达得到重组蛋白。利用镍柱亲和层析技术纯化表达重组蛋白，将纯化的重组蛋白免疫新西兰白兔，制备针对Rep蛋白羧基端亚片段的多克隆抗体。 结果 构建了pET-28a-Rep-487-627原核表达质粒，纯化表达了该重组蛋白。SDS-PAGE结果表明该重组蛋白分子大小约24 kDa，主要以可溶性形式表达。间接免疫荧光和免疫印迹试验表明制备的多克隆抗体能与细胞内过表达的N-MDPV Rep蛋白特异性反应。 结论 制备的Rep多克隆抗体具有良好反应特异性，可识别Rep蛋白的构象表位和线性表位，满足进一步研究的需要。 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. -
图 1 Rep蛋白羧基端亚片段487～627 aa的表达与纯化
M：蛋白分子质量标准；1：未诱导 pET28-Rep-487-627；2：诱导 pET28-Rep-487-627；3：菌体裂解上清；4：菌体裂解沉淀；5～6：亲和纯化后的流出液；7～8：洗脱样；9：透析浓缩后的蛋白样。
Figure 1. Expression and purification of C-terminus subfragment region 487–627 aa of Rep protein
M: protein molecular weight standard; 1: uninduced PET28-Rep-487-627; 2: induced PET28-Rep-487-627; 3: mycelolytic supernatant; 4: mycelolytic precipitation; 5–6: affinity purified effluents; 7–8: elution sample; 9: protein sample after concentration by dialysis.
图 2 Rep 亚片段多克隆抗体介导的间接免疫荧光检测
A：转染 Rep 基因全长的真核表达质粒；B：转染 pcDNA3.1 空质粒。
Figure 2. Indirect immunofluorescence assay mediated by Rep subfragment polyclonal antibody
A: Eukaryotic expression plasmid transfected with full length Rep protein gene; B: transfected pcDNA3.1 empty plasmid.
图 3 Rep 亚片段多克隆抗体特异性识别质粒转染细胞中表达的Rep蛋白
1：转染 Rep 蛋白基因全长的真核表达质粒；2：转染 pcDNA3.1 空质粒。
Figure 3. Rep subfragment polyclonal antibody specifically recognizing Rep protein expressed in cells transfected by plasmid
1: Eukaryotic expression plasmid transfected with full length Rep protein gene; 2: transfected pcDNA3.1 empty plasmid.
-
[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.1007-5038.2020.02.001MIAO 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 -
下载:
点击查看大图
计量
- 文章访问数: 318
- HTML全文浏览量: 146
- PDF下载量: 42
- 被引次数: 0
