High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody
High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody
复制标题
鸭甲型肝炎病毒VP1蛋白在大肠杆菌中的高产表达及多克隆抗体的制备和表征
DOI：
10.1016/j.jviromet.2013.04.004
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发表时间：
2013-07-01
影响因子：
3.1
通讯作者：
Liu, Guangqing
中科院分区：
文献类型：
--
作者：
Li, Chuanfeng;Chen, Zongyan;Liu, Guangqing
VP1蛋白是鸭甲型肝炎病毒(DHAV)的衣壳蛋白，含有诱导保护性免疫反应的关键表位。由于该蛋白在大肠杆菌中的表达水平较低，目前对其功能的研究较少。在本研究中，根据大肠杆菌中密码子使用的偏好性，化学合成了密码子优化的VP1基因，并将其亚克隆到pET32a(+)中以提高其表达。用Ni2+亲和层析His-Bind树脂从包涵体中纯化重组VP1融合蛋白，并用于制备兔抗DHAV-VP1多克隆抗体。与野生型VP1基因相比，密码子优化的VP1基因在大肠杆菌中的表达显著增加，至少增加了17倍。Western印迹分析表明，重组蛋白可被兔抗DHAV多克隆抗体识别。Western印迹结果表明，兔抗DHAV-VP1多克隆抗体能特异性识别纯化的VP1融合蛋白，在间接免疫荧光试验(IFA)中，该抗体能探测到DHAV-1感染细胞中的VP1蛋白。总之，密码子优化显著提高了DHAVVP1在E.cob中的表达，His标记的VP1融合蛋白具有良好的抗原性和免疫原性。(C)2013爱思唯尔B.V.保留所有权利。
VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni2+ affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. cob and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity. (C) 2013 Elsevier B.V. All rights reserved.