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.
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。