Establishment of Indirect ELISA for Detection of Goose circovirus Based on ORF3 Protein Antibodies
FU Huan-Ru1,2,*, ZHAO Min1,2,*, JIANG Jin-Xiu1, CHEN Cui-Teng1, ZHANG Jing-Peng1, LI Zhao-Long3, WAN Chun-He1,3,**
1 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2 School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3 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
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.
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.
FU Huan-Ru, ZHAO Min, JIANG Jin-Xiu, CHEN Cui-Teng, ZHANG Jing-Peng, LI Zhao-Long, WAN Chun-He. Establishment of Indirect ELISA for Detection of Goose circovirus Based on ORF3 Protein Antibodies. 农业生物技术学报, 2024, 32(3): 730-738.
[1] 布日额, 李宝臣, 马波, 等. 2006. 应用GPV VP3基因重组原核表达产物建立检测抗体的ELISA方法研究[J]. 畜牧兽医学报, 37(2): 199-203. (Bu R E, Li B C, Ma B, et al.2006. Development of ELISA methods to detect antibody by prokaryotic recombinant expression product of GPV VP3 gene[J]. Acta Veterinaria et Zootechnica Sinica, 37(2): 199-203.) [2] 刘玲凤. 2017. 广东水禽圆环病毒全基因组序列分析及感染调查[D]. 硕士学位论文, 佛山科学技术学院, 导师: 司兴奎. pp. 6-35. (Liu L F.2017. Whole genome sequence analysis and infection investigation of Waterfowl circovirus in Guangdong province[D]. Thesis for M.S., Foshan University, Supervisor: Si X K, pp. 6-35.) [3] 孙敏华, 董嘉文, 李林林, 等. 2014. 鹅细小病毒NS1蛋白的原核表达及间接ELISA方法的建立[J]. 中国动物传染病学报, 22(6): 1-5. (Sun M H, Dong J W, Li L L, et al.2014. Development of an indirect ELISA assay using prokaryotically expressed NS1 protein of Goose parvovirus[J]. Chinese Journal of Animal Infectious Diseases, 22(6): 1-5.) [4] 万春和, 黄瑜. 2015. 鹅圆环病毒研究进展[J]. 中国家禽, 37(9): 46-48. (Wan C H, Huang Y.2015. Research progress in Goose circovirus[J]. China Poultry, 37(9): 46-48.) [5] 万春和, 黄瑜. 2020. 鹅圆环病毒JX1株ORF3基因序列分析[J]. 福建畜牧兽医, 42(6): 1-5. (Wan C H, Huang Y.2020. Genetic analysis the ORF3 gene of Goose circovirus[J]. Journal of Fujian Animal Husbandry and Veterinary Medicine, 42(6): 1-5.) [6] 万春和, 施少华, 程龙飞, 等. 2010. 朗德鹅圆环病毒全基因组序列测定和遗传演化分析[J]. 中国动物传染病学报, 18(4): 6-12. (Wan C H, Shi S H, Cheng L F, et al.2010. Genomic cloning and sequence analysis of circovirus from Landes geese[J]. Chinese Journal of Animal Infectious Diseases, 18(4): 6-12.) [7] 魏宗, 邹金峰, 雷战, 等. 2011. 鸭圆环病毒ORF3基因的克隆及序列分析[J]. 中国预防兽医学报, 33(9): 746-748. (Wei Z, Zou J F, Lei Z, et al.2011. Cloning and sequence analysis of ORF3 gene of Duck circovirus[J]. Chinese Journal of Preventive Veterinary Medicine, 33(9): 746-748.) [8] 吴方达. 2018. 鹅圆环病毒福建株全基因组的克隆及序列分析[J]. 畜牧与兽医, 50(3): 93-96. (Wu F D.2018. Cloning and sequencing the complete genome of the Fujian strain of Goose circovirus[J]. Animal Husbandry and Veterinary Medicine, 50(3): 93-96.) [9] 余旭平, 郑新添, 何世成, 等. 2005. 鹅圆环病毒浙江永康株全基因组的克隆及序列分析[J]. 微生物学报, 45(6): 860-864. (Yu X P, Zheng X T, He S C, et al.2005. Cloning and analysis of the complete genome of a Goose circovirus[J]. Acta Microbiologica Sinica, 45(6): 860-864.) [10] 张广芳. 2016. 猪圆环病毒2型ORF3基因的原核表达与间接ELISA检测方法的建立及初步应用[D]. 硕士学位论文, 西北农林科技大学, 导师: 张彦明. pp. 16-26. (Zhang G F.2016. Establishment and preliminary application of indirect ELISA method baesd on ORF3 for Porcine circovirus 2 (PCV2)[D]. Thesis for M.S., Northwest A＆F University, Supervisor: Zhang Y M, pp. 16-26.) [11] 赵敏, 李家玉, 黄瑜, 等. 2022. 鹅圆环病毒TB Green实时荧光定量PCR方法的建立及初步应用[J]. 福建畜牧兽医, 44(4): 26-30. (Zhao M, Li J Y, Huang Y, et al.2022. Development of a TB Green real-time PCR assay for the detection of Goose circovirus[J]. Journal of Fujian Animal Husbandry and Veterinary Medicine, 44(4): 26-30.) [12] Alistair N J, Andrea B, Joan A S, et al.2006. Serological diagnosis of Goose circovirus infections[J]. Avian Pathology, 35(6): 495-499. [13] Biđin M, Lojkić I, Tišljar M, et al.2012. Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos[J]. Avian Pathology, 41(1): 91-97. [14] Chaiyakul M, Hsu K, Dardari R, et al.2010. Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic Porcine circovirus[J]. Journal of Virology, 84(21): 11440-11447. [15] Chen C L, Chang P C, Lee M S, et al.2003. Nucleotide sequences of Goose circovirus isolated in Taiwan[J]. Avian Pathology, 32(2): 165-1711. [16] Kozdruń W, Woźniakowski G, Samorek-Salamonowicz E, et al.2012. Viral infections in goose flocks in Poland[J]. Polish Journal of Veterinary Sciences, 15(3): 525-530. [17] Liu J, Chen I, Du Q, et al.2006. The ORF3 protein of Porcine circovirus Type 2 is involved in viral pathogenesis in vivo[J]. Journal of Virology, 80(10): 5065-5073. [18] Liu J, Zhu Y, Chen I, et al.2007. The ORF3 protein of Porcine circovirus type 2 interacts with porcine ubiquitin E3 ligase Pirh2 and facilitates p53 expression in viral infection[J]. Journal of Virology, 80(17): 9560-9567. [19] Meng X.2013. Porcine circovirus type 2 (PCV2): Pathogenesis and interaction with the immune system[J]. Annual Review of Animal Biosciences, 1(1): 43-64. [20] Soike D, Kohler B, Albrecht K.1999. A circovirus-like infection in geese related to a runting syndrome[J]. Avian Pathology, 28(2): 199-202. [21] Stenzel T, Farkas K, Varsani A.2015. Genome sequence of a diverse Goose circovirus recovered from greylag goose[J]. Genome Announcements, 3(4): e00767-15. [22] Todd D, Weston J H, Soike D.et al.2001. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon[J]. Virology, 286(2): 354-362. [23] Todd D.2004. Avian circovirus diseases: Lessons for the study of PMWS[J]. Veterinary Microbiology, 98(2): 169-174. [24] Wu Z, Zhang R, Li Y, et al.2018. C-terminal 20 residues of ORF3 protein of Duck circovirus genotype 2 regulates the nuclear localization and inhibits apoptotic activity of ORF3 protein[J]. Veterinary Microbiology, 214(1): 21-27. [25] Xiang Q, Wang X, Xie Z, et al.2012. ORF3 of Duck circovirus: A novel protein with apoptotic activity[J]. Veterinary Microbiology, 159(1-2): 251-256. [26] Zhang Y, Zhang X, Cheng A, et al.2021. Apoptosis triggered by ORF3 proteins of the circoviridae family[J]. Frontiers in Cellular and Infection Microbiology, 10(1): 609071.
