福建农业学报2025，Vol.40Issue(10)：1014-1021,8.DOI:10.19303/j.issn.1008-0384.2025.10.005
猪圆环病毒4型Cap蛋白间接ELISA检测方法的建立及临床应用
Indirect ELISA for detecting PCV 4 Capsid Protein
摘要
Abstract
[Objective]A serological detection method targeting the capsid protein of porcine circovirus type 4(PCV4)was developed for epidemiological studies on the viral disease.[Method]The genome of PCV4 FJ2010001 was used as the PCR amplification template with specific primers designed for the viral capsid protein.Full-length coding sequence of the gene was obtained by PCR to clone it into a pET30a(+)prokaryotic expression system.After purification by affinity chromatography,DS-PAGE and western blotting were used to verify antigenicity of the recombinant protein.A capsid protein detection system applying the optimized conditions of 1.25 μg·mL-1 for antigen encapsulation concentration,1:200 for serum dilution,and 1:10 000 for working concentration of secondary antibody was established.Retrospectively,1 195 serum specimens collected from the large-scale pig farms in Fujian Province between 2020 and 2024 were tested using the newly developed assay.Cases of PCV4 co-infecting with PCV2 and/or PCV3 were recorded.[Result]Efficient soluble expression induced at 15℃for 16 h was shown by the SDS-PAGE analysis of the recombinant capsid protein with a molecular weight of approximately 28.3 kDa.Western blotting confirmed the single band protein to have specific immunoreactivity with PCV4-positive serum.The methodological validation data proved the detection had no cross-reactivity with sera positive for common swine pathogens(e.g.,PRV,PRRSV,etc.).The assay provided a minimum detection threshold at 1:3 200 dilution with coefficients of variation less than 10%both within and between batches.The seroepidemiologic surveys for the past 5 years in the province indicated a total positive rate of PCV4 antibodies of 7.62%(91/1 195),which peaked in 2021.The rate of co-infection between PCV4 and PCV2 was 26.37%(24/91),while that between PCV4 and both PCV2 and PCV3 was 14.29%(13/91).[Conclusion]The newly established indirect ELISA was highly efficient and applicable for epidemiological studies on the viral diseases caused by PCV2,PCV3,and PCV4.关键词
猪圆环病毒4型/衣壳蛋白/原核表达/间接ELISAKey words
porcine circovirus 4/Capsid protein/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
陈如敬,吴学敏,吴仁杰,严山,陈秋勇,何冰,周伦江..猪圆环病毒4型Cap蛋白间接ELISA检测方法的建立及临床应用[J].福建农业学报,2025,40(10):1014-1021,8.基金项目
福建省科技计划公益类专项(2025R1024002) （2025R1024002）
福建省自然科学基金项目(2024J01333) （2024J01333）
