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