Park Jae Sung, Choi Bong Kum, Vijayachandran Lakshmi Sumitra, Ayyappan Vasudevan, Chong Chom-Kyu, Lee Ki-Sung, Kim Sei Chang, Choi Chang Won
Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Republic of Korea.
J Virol Methods. 2007 Dec;146(1-2):281-7. doi: 10.1016/j.jviromet.2007.07.021. Epub 2007 Sep 7.
The entire virion protein 2 (VP2) gene of Canine Parvovirus (CPV) was amplified by polymerase chain reaction (PCR) and engineered to be expressed by a bacterial expression vector pET-28a, under the control of the IPTG-inducible T7lac promoter. SDS-PAGE gel revealed that VP2 expressed as a 67kDa, and found mainly in the pellet of the bacterial lysates, suggesting that cytoplasmic expression is not preferred. The recombinant protein VP2 fused with His-tag was purified from Esherichia coli using Ni-NTA resin under denaturing conditions. SDS-PAGE analysis also showed the high expression of several lower molecular weight (LMW) bands. Western blot analysis showed that polyclonal antisera produced by rabbit against E. coli-VP2 protein reacted specifically with the purified VP2 protein as well as two other LMW bands. Some of the resulting LMW products failed to keep their antigenic site in the N-terminal region of the VP2. The degradation of recombinant VP2 protein in E. coli could be due to the action of host proteases. The immunodetection ability of the polyclonal antisera was compared with that of a commercial monoclonal antibody to test numerous clinical specimens by immuno-dot blot assays. There were distinctive differences in the degree of immunodetection ability of polyclonal antisera and monoclonal antibody to react with CPV antigens. The reaction time of polyclonal antisera was much faster in visual color appearance than that of monoclonal antibody during NBT/BCIP staining. The result from diagnostic PCR assay confirmed the presence of CPV in 44 out of 46 specimens collected, consistent with polyclonal antisera-positive result. Therefore, the polyclonal antisera can be used for CPV detection in the faeces of diarrhoeic dogs, which was found to be more rapid, sensitive, broad but less specific than the monoclonal antibody.
通过聚合酶链反应（PCR）扩增犬细小病毒（CPV）的全病毒粒子蛋白2（VP2）基因，并构建使其在异丙基-β-D-硫代半乳糖苷（IPTG）诱导型T7lac启动子控制下由细菌表达载体pET-28a表达。SDS-PAGE凝胶显示VP2以67kDa的形式表达，且主要存在于细菌裂解物的沉淀中，这表明胞质表达并非首选。在变性条件下，使用镍-亚氨基二乙酸（Ni-NTA）树脂从大肠杆菌中纯化融合有His标签的重组蛋白VP2。SDS-PAGE分析还显示有几条低分子量（LMW）条带高表达。蛋白质免疫印迹分析表明，兔抗大肠杆菌-VP2蛋白产生的多克隆抗血清与纯化的VP2蛋白以及另外两条LMW条带发生特异性反应。一些产生的LMW产物在VP2的N端区域未能保留其抗原位点。大肠杆菌中重组VP2蛋白的降解可能是由于宿主蛋白酶的作用。通过免疫斑点印迹试验比较了多克隆抗血清与商业单克隆抗体对众多临床标本的免疫检测能力。多克隆抗血清和单克隆抗体与CPV抗原反应的免疫检测能力程度存在明显差异。在NBT/BCIP染色过程中，多克隆抗血清在视觉颜色出现方面的反应时间比单克隆抗体快得多。诊断性PCR检测结果证实，在收集的46份标本中有44份存在CPV，这与多克隆抗血清阳性结果一致。因此，多克隆抗血清可用于检测腹泻犬粪便中的CPV，发现其比单克隆抗体更快速、敏感、宽泛但特异性较低。
