【摘要】 为制备抗A型塞内卡病毒(SVA)VP1蛋白的单克隆抗体，并初步应用于A型塞内卡病毒的检测，本研究构建了SVA VP1原核重组表达质粒pET30a-GST-VP1(528 bp)。将重组质粒转化大肠埃希菌BL21感受态细胞并大量表达VP1重组蛋白，利用纯化的VP1重组蛋白免疫BALB/c小鼠，取其脾细胞与骨髓瘤细胞(SP2/0细胞)进行融合，经过细胞亚克隆筛选，获得杂交瘤细胞。将其选为腹水生产细胞株，最后纯化单克隆抗体，通过Western blotting和间接免疫荧光试验对其进行鉴定。结果显示，本研究获得1株能够稳定分泌抗VP1重组蛋白单克隆抗体的杂交瘤细胞，其亚型重链为IgG2a、轻链为Kappa链；Western blotting和间接免疫荧光检测显示，该单克隆抗体能够特异性识别SVA病毒粒子。本研究成功制备的抗SVA VP1蛋白单克隆抗体可为进一步研究A型塞内卡病毒VP1蛋白的生物学功能及A型塞内卡病毒致病机制提供有效途径。更多还原

【Abstract】 This study aimed to prepare and identify monoclonal antibody against Senecavirus A(SVA) VP1 protein for diagnostic application. A prokaryotic recombinant expression plasmid pET30 a-GST-VP1(528 bp) was constructed and transformed into E. coli BL21 competent cells for VP1 expression. BALB/c mice were immunized with purified recombinant VP1 protein and hybridoma cells were obtained by fusion of spleen cells and myeloma cells(SP2/0 cells) and clonal screening. Monoclonal antibodies from selected clones were purified and identified by Western blotting and indirect immunofluorescence assay(IFA). A hybridoma cell line that stably secreted monoclonal antibody against VP1 protein was generated. This antibody was of IgG2 a subclass with Kappa light chain, and specifically recognized the SVA in Western blotting and IFA. This anti-SVA VP1 monoclonal antibody thus provides the opportunity to further investigate the biological function of Senecavirus A VP1 protein and the pathogenesis of Senecavirus A.更多还原