中国畜牧兽医2025，Vol.52Issue(6)：2750-2761,12.DOI:10.16431/j.cnki.1671-7236.2025.06.027
猪轮状病毒VP6蛋白的真核表达及单克隆抗体制备和应用
Eukaryotic Expression of Porcine Rotavirus VP6 Protein and Preparation and Application of Its Monoclonal Antibody
摘要
Abstract
[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.VP 6 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.关键词
猪轮状病毒(PoRV)/VP6蛋白/真核表达/单克隆抗体Key words
Porcine rotavirus(PoRV)/VP6 protein/eukaryotic expression/monoclonal antibody分类
农业科技引用本文复制引用
魏黄思梧,张兴艺,黄校花,刘昌锦,武文杰,沈政乔,罗锋,邓舜洲..猪轮状病毒VP6蛋白的真核表达及单克隆抗体制备和应用[J].中国畜牧兽医,2025,52(6):2750-2761,12.基金项目
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) （JXARS-03）
