嵌合型口蹄疫病毒样颗粒构建、表达及鉴定

Construction, expression and identification of chimeric foot-and-mouth disease virus-like particles

摘要:

为提高口蹄疫病毒 (Foot-and-mouth disease virus，FMDV) 病毒样颗粒 (Virus-like particles，VLPs) 的特异性识别和递呈，为靶向疫苗研究奠定基础，利用反向PCR技术，将卵清蛋白 (Ovalbumin，OVA) 第257–264位氨基酸 (Amino acids，aa) 的短肽嵌入FMDV结构蛋白VP3第171–172位aa或第173–174位aa，通过大肠杆菌表达FMDV结构蛋白VP0、VP1和嵌合型VP3，体外组装得到嵌合OVA257-264肽的病毒样颗粒 (VLPOVA)。用动态光散射、透射电镜检测VLPOVA大小和形态，免疫印迹、酶联免疫吸附试验和激光共聚焦显微镜检测短肽的嵌入情况。结果显示在VP3的第173–174位aa嵌入OVA，不影响蛋白表达和VLPs的组装且OVA位于VLPOVA的表面，VLPOVA粒径比VLPs稍大。

Abstract:

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA257-264 peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLPOVA) in vitro after purification. The VLPOVA was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA257-264 peptide into VP3 and its location. The results show that insertion of OVA257-264 into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLPOVA in size was larger than VLPs, and the OVA257-264 peptide was located on the surface of VLPOVA.