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Abstract：
Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences.
In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.
Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.
In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.
In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.
Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.
In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.
摘要：
背景：牛细小病毒（BPV）是一种自主的DNA病毒，具有较小的分子大小和其结构蛋白的细微差异，与其他动物细小病毒不同。更重要的是,这种病毒有可能在畜牧业中产生无声的经济灾难，尽管很少受到关注。细小病毒病毒样颗粒(VLP)作为疫苗和作为疫苗部署的后勤平台已被充分研究。然而,没有关于VP1在BPV-VLP组装和稳定性中的作用的单一实验报告。此外，自组装，与使用任何表达方法的VP1VP2Cap蛋白相比,重组BPVVP2的VLP的完整性和稳定性先前尚未研究。在这项研究中,我们通过实验评估了从单个结构蛋白（VP2）合成BPV病毒样颗粒（VLP）并整合VP2和VP1氨基酸序列的自组装能力。
方法:进行计算机模拟和实验克隆方法。克隆了带His标签和无His标签的VP2和V1VP2编码氨基酸序列，并将其插入pFastbacdual中，并通过SDS-PAGE和蛋白质印迹评估了昆虫细胞产生的重组蛋白。通过IFA确定感染期和表达水平。通过透射电子显微镜评估BPVVLP的完整性和稳定性。通过圆二色性分析来自VP2和V1VP2的BPVVLP的二级结构。
结果：我们的发现表明，VP2单独表达并纯化为可检测的蛋白质，两种VLP在不同温度和pH值下的稳定性没有明显差异。此外，BPV-VP2VLP因其比BPV-VP1VP2VLP更高的纯度和完整性而受到赞誉，如SDS-PAGE所示。因此,我们的研究表明，VP1的功能与BPV-VLP的稳定性或完整性无关。
结论：总之，已经发现令人难以置信的物理化学稳定的BPVVP2衍生的VLP是开发针对肠道病毒的多价疫苗和免疫诊断试剂盒的有希望的候选物,并且携带用于各种经济上重要的家畜疾病的异质表位。
方法:进行计算机模拟和实验克隆方法。克隆了带His标签和无His标签的VP2和V1VP2编码氨基酸序列，并将其插入pFastbacdual中，并通过SDS-PAGE和蛋白质印迹评估了昆虫细胞产生的重组蛋白。通过IFA确定感染期和表达水平。通过透射电子显微镜评估BPVVLP的完整性和稳定性。通过圆二色性分析来自VP2和V1VP2的BPVVLP的二级结构。
结果：我们的发现表明，VP2单独表达并纯化为可检测的蛋白质，两种VLP在不同温度和pH值下的稳定性没有明显差异。此外，BPV-VP2VLP因其比BPV-VP1VP2VLP更高的纯度和完整性而受到赞誉，如SDS-PAGE所示。因此,我们的研究表明，VP1的功能与BPV-VLP的稳定性或完整性无关。
结论：总之，已经发现令人难以置信的物理化学稳定的BPVVP2衍生的VLP是开发针对肠道病毒的多价疫苗和免疫诊断试剂盒的有希望的候选物,并且携带用于各种经济上重要的家畜疾病的异质表位。
