猪A群轮状病毒VP6单克隆抗体的制备和初步鉴定  

Preparation and preliminary characterization of monoclonal antibodies against VP6 of porcine group A rotavirus

作　　者：李科茫 周金柱 周俊明[2] 牛贝贝 武琦 郭容利[2] 张雪寒[2] 朱雪蛟 李彬[2] 粟硕[1] LI Kemang;ZHOU Jinzhu;ZHOU Junming;NIU Beibei;WU Qi;GUO Rongli;ZHANG Xuehan;ZHU Xuejiao;LI Bin;SU Shuo(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所,江苏南京210014

出　　处：《畜牧与兽医》2023年第4期79-85,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2022YFD1800601);国家自然科学基金(31872481);江苏省杰出青年基金(BK20190003)。

摘　　要：旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blot验证该重组蛋白能够与小鼠抗轮状病毒血清反应。将该重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性杂交瘤细胞,Western blot和间接免疫荧光验证阳性杂交瘤细胞分泌的单克隆抗体与RVA的反应性。经3轮亚克隆最终获得一株能稳定分泌针对VP6蛋白单克隆抗体的阳性杂交瘤细胞株5G9,其分泌的单抗亚类为IgG1,轻链类型为Kappa,制备的小鼠腹水效价可达1∶4096000,能够与不同基因型的RVA反应,同时不与常见的猪腹泻相关病毒反应,为未来猪RVA相关基础研究、临床血清学检测方法的开发奠定了基础。In this study,the coding sequence of the VP6 gene segment of porcine group A rotavirus(RVA)was amplified by RT-PCR and was cloned into the pET28a-SUMO plasmid to construct a prokaryotic expression vector pET28a-SUMO-VP6.E.coli BL21(DE3)transformed with the recombinant vector was induced by IPTG.And the expressed recombinant protein SUMO-VP6 was purified by nickel column affinity chromatography.Western blot verified that the recombinant protein could react with mouse anti-rotavirus serum.BALB/c mice were immunized with the recombinant protein.And the spleen cells of the immunized mice were fused with SP2/0 myeloma cells.The positive hybridomas were screened by indirect ELISA,and the reactivity with RVA of the monoclonal antibodies secreted by the positive hybridomas was verified by Western blot and Indirect Immunofluorescence.After 3 rounds of subcloning,a positive hybridoma cell line 5G9,which stably secreted the monoclonal antibody against VP6,was finally obtained.The subclass of the monoclonal antibody was IgG1,and the light chain type was Kappa.The titer of the mouse ascites was 1∶4096000.The monoclonal antibody 5G9 reacted with different genotypes of RVA,and did not react with common swine diarrhea-related viruses.This study laid a foundation for future basic research on porcine RVA and the development of clinical serological detection methods for porcine RVA.

关 键 词：猪A群轮状病毒 单克隆抗体 VP6蛋白 

分 类 号：S852.659[农业科学—基础兽医学]