摘要
【目的】制备抗猪轮状病毒(Porcine rotavirus,PoRV)VP6蛋白单克隆抗体,为PoRV检测方法的开发提供参考。【方法】以痘苗病毒启动子P28为VP6的启动子,以G3型PoRV核酸为模板扩增VP6基因,通过无缝克隆方法将VP6基因片段克隆至pSWE178质粒,构建重组质粒pSWE178-P28-VP6;利用同源重组和蚀斑纯化技术获得表达VP6蛋白的重组猪痘病毒rSWE178-P28-VP6,用SDS-PAGE鉴定VP6蛋白表达形式。以G9型PoRV为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞和SP2/0骨髓瘤细胞融合,以重组病毒表达的VP6蛋白为检测抗原,通过间接ELISA筛选分泌抗VP6单克隆抗体的杂交瘤细胞。通过间接免疫荧光试验(IFA)检测单克隆抗体与G3、G4、G5、G9型PoRV的反应原性,选择其中1株单克隆抗体对人工感染PoRV的猪肠道组织进行免疫组化(IHC)检测。【结果】在构建了表达PoRV VP6蛋白的重组质粒pSWE178-P28-VP6基础上,成功构建了表达PoRV VP6蛋白的重组猪痘病毒,表达的蛋白分子质量大小45 ku,为可溶性表达。采用杂交瘤技术制备单克隆抗体,筛选获得29株分泌抗VP6单克隆抗体的杂交瘤细胞株。IFA结果显示,26株单克隆抗体均能与G3、G4、G5、G9型PoRV反应,但荧光亮度存在差异。IHC检测结果显示,单克隆抗体能与感染PoRV的临床样品发生特异性免疫反应。【结论】本研究利用真核表达系统成功表达了PoRV VP6蛋白,采用杂交瘤技术筛选获得29株抗VP6单抗隆抗体,其中26株均能与G3、G4、G5、G9型PoRV反应。试验结果为PoRV免疫学检测方法的建立奠定了基础。
【Objective】The aim of this experiment was to prepare monoclonal antibody against Porcine rotavirus(PoRV)VP6 protein,and provide reference for the development of PoRV detection method.【Method】VP6 gene was amplified with Vaccinia virus promoter P28 as the promoter of VP6 and G3 type PoRV nucleic acid as the template.VP6 gene fragment was cloned into pSWE178 plasmid by In-Fusion cloning method,and the recombinant plasmid pSWE178-P28-VP6 was constructed.Recombinant Suipoxvirus rSWE178-P28-VP6 expressing VP6 protein was obtained by homologous recombination and plaque purification techniques.The expression of VP6 protein was identified by SDS-PAGE.BALB/c mice were immunized with G9 PoRV as the immunogener.Spleen cells of immunized mice were fused with SP2/0 myeloma cells.VP6 protein expressed by recombinant virus was used as the detection antigen,and hybridoma cells secreting anti-VP6 monoclonal antibody were screened by indirect ELISA.Indirect immunofluorescence assay(IFA)was used to detect the reactivity of monoclonal antibody with G3,G4,G5 and G9 PoRV.One of the monoclonal antibody strains was selected for immunohistochemical(IHC)detection of porcine intestinal tissue artificially infected with PoRV.【Result】On the basis of constructing the recombinant plasmid pSWE178-P28-VP6 expressing the PoRV VP6 protein,a recombinant Swinepox virus expressing the PoRV VP6 protein was successfully generated.The molecular weight of the expressed protein was 45 ku and which was soluble protein.Monoclonal antibody was prepared by hybridoma technique,and 29 hybridoma cell lines secreting anti-VP6 monoclonal antibody were screened.IFA results showed that 26 monoclonal antibodies could react with G3,G4,G5 and G9 PoRV,but the fluorescence brightness was different.IHC results showed that monoclonal antibody could produce specific immune response with PoRV-infected clinical samples.【Conclusion】In this study,PoRV VP6 protein was successfully expressed by eukaryotic expression system.The hybridoma technique was used to select 29 strains of anti-VP6 monoclonal antibodies,of which 26 strains could react with G3,G4,G5 and G9 PoRV.This results laid a foundation for the establishment of PoRV immunological detection method.
作者
魏黄思梧
张兴艺
黄校花
刘昌锦
武文杰
沈政乔
罗锋
邓舜洲
WEI Huangsiwu;ZHANG Xingyi;HUANG Xiaohua;LIU Changjin;WU Wenjie;SHEN Zhengqiao;LUO Feng;DENG Shunzhou(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;Lushan Botanical Garden,Jiangxi Province and Chinese Academy of Sciences,Jiujiang 332900,China;Jiangxi Jinyibo BiotechnologyCo.,Ltd.,Nanchang 330013,China)
出处
《中国畜牧兽医》
北大核心
2025年第6期2750-2761,共12页
China Animal Husbandry & Veterinary Medicine
基金
江西省现代农业产业技术体系建设专项基金资助项目(JXARS-03)。
