【摘要】 旨在制备靶向牛A群轮状病毒（Group A Bovine Rotavirus, BRVA）VP7蛋白的单链抗体（single-chain fragment variable, scFv）.以项目组前期实验制备的BRVA VP7蛋白鼠源单克隆抗体C8杂交瘤细胞为模板，通过高通量测序获得其抗体可变区（variable region, V区）基因序列，并使用（Gly₄Ser）^×3作为linker构建scFv基因片段，并将其克隆至pET28a（+）载体，转化至大肠杆菌BL21（DE3）中表达.结果显示，C8-scFv在大肠杆菌BL21（DE3）中以包涵体形式表达，其相对分子质量约为26 kDa.免疫荧光试验结果表明，C8-scFv能够特异性识别BRVA,具有良好的反应活性.研究成功制备了靶向BRVA VP7蛋白的scFv,为开发基于免疫学的BRVA检测技术奠定了基础.更多还原

【Abstract】 This study aimed to generate an scFv targeting the VP7 protein of Group A Bovine Rotavirus（BRVA）.The VH and VL gene sequences of the mouse-derived monoclonal antibody hybridoma cell strain C8,which was previously generated against BRVA VP7 protein, were amplified via PCR.The（Gly₄Ser）^×3 linker was employed to connect the VH and VL domains, forming the scFv gene fragment, which was then cloned into the pET28a（+） expression vector and transformed into Escherichia coli BL21（DE3） cells for protein expression.The results showed that C8-scFv was expressed as inclusion bodies in e.coli, and a high-purity C8-scFv protein was obtained with a molecular weight of approximately 26 kDa.Immunofluorescence assays confirmed that C8-scFv could specifically recognize BRVA,demonstrating good reactivity.This work successfully developed an scFv targeting BRVA VP7 and provided a foundation for the development of immunological diagnostic technologies for BRVA.更多还原