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

作　　者：付环茹 赵敏 江锦秀[1] 陈翠腾[1] 张靖鹏[1] 李兆龙 万春和 FU Huan-Ru;ZHAO Min;JIANG Jin-Xiu;CHEN Cui-Teng;ZHANG Jing-Peng;LI Zhao-Long;WAN Chun-He(Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China;School of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention/Fujian Provincial Key Laboratory of Animal Genetics and Breeding/Fujian Animal Diseases Control Technology Development Center,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福州350013 [2]福建农林大学生命科学学院,福州350002 [3]福建省禽病防治重点实验室/福建省畜禽遗传育种重点实验室/福建省畜禽疫病防治工程技术研究中心,福州350013

出　　处：《农业生物技术学报》2024年第3期730-738,共9页Journal of Agricultural Biotechnology

基　　金：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);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)。

摘　　要：鹅圆环病毒(Goose circovirus,GoCV)是一种潜在的免疫抑制病原,主要感染宿主淋巴细胞,导致免疫功能下降,易造成其他病原继发感染,给养鹅(Anser cygnoides orientalis)业带来极大危害。为建立鹅圆环病毒血清学抗体的间接ELISA方法,本研究将GoCV的ORF3基因插入到原核表达载体pET-32a(+),构建重组质粒pET-32a-ORF3,经表达条件优化,以Ni柱亲和层析纯化目的蛋白,SDS-PAGE和Western blot分析表明,获得的重组ORF3蛋白具有良好的生物学活性。以纯化后的重组ORF3蛋白作为包被抗原,通过优化各反应条件建立了GoCV间接ELISA抗体检测方法。优化后的间接ELISA检测条件为:重组ORF3蛋白的最佳包被浓度为2.00μg/mL、待检血清稀释度为1∶80、酶标二抗最佳稀释度分别为1∶4000(羊抗鸡HRP-IgG)和1∶8000(羊抗鸭HRP-IgG)。采用建立的间接ELISA方法对鹅细小病毒(Goose parvovirus,GPV)、鹅源禽坦布苏病毒(Avian tembusu virus,ATmV)、鹅源H9N2亚型禽流感病毒(H9N2 subtype avian influenza virus,AIV)、鹅呼肠孤病毒(Goose reovirus,GRV)、新型鹅星状病毒(Goose astrovirus,GoAstV)、GoCV等鹅源阳性血清进行检测,结果显示,仅GoCV阳性血清为阳性结果,其他病原阳性血清检测结果均为阴性,特异性较强;重复性较好,批内和批间变异系数均小于5%。流行病学调查结果显示,福建和广东地区鸭(Anatinae)群中均存在GoCV跨种感染。本研究建立的检测GoCV血清抗体的间接ELISA方法特异性强、重复性好,可用于GoCV感染的诊断及流行病学调查研究。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 2.00μg/mL,the dilution of serum was 1∶80,and goat anti-duck IgG-HRP(or goat anti-chicken IgG-HRP)was 1∶4000(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 

分 类 号：S855.3[农业科学—临床兽医学]