农业生物技术学报2024，Vol.32Issue(3)：730-738,9.DOI:10.3969/j.issn.1674-7968.2024.03.022
基于ORF3蛋白抗体的鹅圆环病毒间接ELISA检测方法的建立
Establishment of Indirect ELISA for Detection of Goose circovirus Based on ORF3 Protein Antibodies
摘要
Abstract
Goose circovirus(GoCV)is a potential immunosuppressive pathogen that mainly infects host lymphocytes,leading to a decline in immune function,and easily causes secondary infection by other pathogens,which greatly harms the goose(Anser cygnoides orientalis)breeding industry.To establish an indirect ELISA method for the detection of GoCV antibodies,the ORF3 gene from GoCV was synthesized and cloned into the pET32a(+)vector.The recombinant plasmid pET-32a-ORF3 was constructed and used to express ORF3 protein in a prokaryotic expression system.After conditional optimization,the recombinant ORF3 protein was purified by NI-NTA affinity chromatography and showed good biological activity after SDS-PAGE and Western blot analysis.An indirect ELISA to detect GoCV antibodies was established using the obtained ORF3 protein.The optimized reaction conditions were as follows:the best coating antigen(the obtained ORF3 protein)concentration was 2.00 μg/mL,the dilution of serum was 1∶80,and goat anti-duck IgG-HRP(or goat anti-chicken IgG-HRP)was 1∶4 000(or 1∶8000).Specificity results showed that only GoCV-positive sera were positive,with other goose origin pathogen-positive sera(such as GPV,ATmV,AIV,GRV,GoAstV)being negative,indicating good specificity.The results of repeatability tests within and between batches showed that the coefficients of variation were both less than 5%,indicating good repeatability.The optimized indirect ELISA conditions were used to carry out epidemiological investigation,and the data showed that GoCV sera positivity was found in ducks(Anatinae)collected in Fujian and Guangdong,indicating GoCV cross-species infection from geese to ducks.In conclusion,the established indirect ELISA method for the detection of GoCV antibodies in this study has good reproducibility and specificity and provides a practical method for serological investigation and surveillance of Goose circovirus infection.关键词
鹅圆环病毒(GoCV)/ORF3蛋白/原核表达/间接ELISA/流行病调查Key words
Goose circovirus(GoCV)/ORF3 protein,Prokaryotic expression/Indirect ELISA/Serological分类
农业科技引用本文复制引用
付环茹,赵敏,江锦秀,陈翠腾,张靖鹏,李兆龙,万春和..基于ORF3蛋白抗体的鹅圆环病毒间接ELISA检测方法的建立[J].农业生物技术学报,2024,32(3):730-738,9.基金项目
财政部和农业农村部 国家现代农业产业技术体系(水禽)(CARS-42-18) （水禽）
福建省科技计划(2020J06029 （）
2020R1026009 （）
2021R1026001 （）
2021R1026006 （）
2023R1076 （）
2023R1077) （）
福建省农业科学院科技计划(CXPT202108 （）
CXTD2021005-1) （）
