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(Gly_(4)Ser)^(×)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.
This study aimed to generate an seFv targeting the VP7 protein of Group A Bovine Rotavirus(BRVA).The VH and Vi,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(Gly_(4)Ser)^(×)3 linker was employed to connect the VH and VL domains,forming the selv 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-seFv was expressed as inclusion bodies in e.coli,and a high-purity C8-sel'v protein was obtained with a molecular weight of approximately 26 kDa.Immunofluorescence assays confirmed that C8-seFv could specifically recognize BRVA,demonstrating good reactivity.This work successfully developed an seFv targeting BRVA VP7 and provided a foundation for the development of immunological diagnostie technologies for BRVA.
作者
许博阳
陈曦
汤承
岳华
XU Boyang;CHEN Xi;TANG Cheng;YUE Hua(Sichuan Provincial Higher Educational Institutions Key Laboratory of Animal Medicine,Southwest Minzu University,Chengdu 610041,China)
出处
《西南民族大学学报(自然科学版)》
2025年第4期379-384,共6页
Journal of Southwest Minzu University(Natural Science Edition)
基金
“十四五”国家重点研发计划项目(2023YFD180250402)。
作者简介
通信作者:岳华(1963-),女,教授,博士,研究方向:动物病原分子生物学.E-mail:yhua900@163.com。
