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| Preparation of Monoclonal Antibody Based on VP1 Protein Against Senecavirus A (SVA) and Trial Production of Colloidal Gold Test Strips for SVA Detection |
| ZHANG Tao1,2, WANG Hui-Bao1,2, SUN Yan-Yan3, ZHAI Guo-Yuan2, CUI Yan1,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 China Agricultural Vet. Bio. Science and Technology CO., Ltd., Lanzhou 730046, China;
3 Lanzhou Animal Research Biotechnology Co., Ltd., Lanzhou 730046, China |
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Abstract Senecavirus A (SVA), an emerging pathogen in recent years, is posing a severe danger to the healthy development of pig (Sus domesticus) industry in China. Viral structural protein 1 (VP1), the main structural protein of SVA, can induce the body to produce specific antibodies. In order to prepare monoclonal antibodies (McAbs) against SVA VP1 and establish a rapid, efficient and simple method for the detection of SVA, the SVA VP1 gene was amplified by RT-PCR and cloned into the expression vector pET-28a (+). After induction, expression and purification, the recombinant SVA VP1 protein was obtained. BALB/c mice (Mus musculus) were immunized with recombinant SVA VP1 protein, 2 hybridoma cell lines (2E10 and 3E4) stably secreting monoclonal antibodies against SVA VP1 protein were obtained. Ascites type McAbs were obtained and purified by immunizing mice with cell lines 2E10 and 3E4. McAbs were identified by indirect immunofluorescence assay (IFA) and Western blot. The prepared 2E10 McAbs were used as capture antibody, Goat anti-mouse IgG and 3E4 McAbs were used as quality control band (C band) and detection band (T band) to produce SVA colloidal gold test strip. The results showed that the recombinant SVA VP1 protein was mainly expressed in the form of inclusion bodies, with a molecular weight of about 43 kD. The results of antibody subtype identification showed that the heavy chain subtypes of 2E10 and 3E4 were IgG2b and IgG2a, respectively, and the light chains were both κ chain. The results of IFA and Western blot showed that the obtained McAbs were specific bound to SVA. The test of strip showed that colloidal gold test strips could specifically detect SVA, and had no cross reaction with Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Classical swine fever virus (CSFV) and Porcine circovirus type 2 (PCV2); The minimum detected viral content was 5×101.13 TCID50/mL;The positive coincidence rate between this method and qPCR was 96.55%. The above results indicate that the prepared colloidal gold test paper based on VP1 monoclonal antibody in this study can provide a new method for the point-of-care testing of SVA.
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Received: 30 May 2022
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Corresponding Authors:
* cuiyan369@sina.com
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[1] 范慧, 曾洁, 李亮, 等. 2019. 塞内卡病毒A(SVA) VP1间接ELISA抗体检测方法的建立及应用[J]. 中国兽医学报, 39(10): 1907-1912.
(Fan H, Zeng J, Li L, et al.2019. Development of indirect ELISA assay for identification of antibody to Senecavirus A (SVA) in swine[J]. Chinese Journal of Veterinary Science, 39(10): 1907-1912.)
[2] 候吉超, 李忠鹏, 梁雨欣, 等. 2021. CP4-EPSPS蛋白抗原表位鉴定及其快速DAS-ELISA检测方法的建立[J]. 农业生物技术学报, 29(01): 159-168.
(Hou J C, Li Z P, Liang Y X, 2021. Mapping of antigenic epitopes on CP4-EPSPS protein and detection method establishment of rapid DAS-ELISA[J]. Journal of Agricultural Biotechnology, 29(01): 159-168.)
[3] 蒋韬, 梁仲, 陈涓, 等. 2008. 口蹄疫病毒O、A、Asia Ⅰ型定型诊断胶体金免疫层析方法的建立[J]. 中国农业科学, 41(11): 3801-3808.
(Jiang T, Liang Z, Chen J, et al.2008. Development of a rapid gold immunochromatographic strip test for the diagnosis of Foot-and-mouth disease virus O, A and Asia typeⅠ[J]. Scientia Agricultura Sinica, 41(11): 3801-3808.)
[4] 廖迎新, 范锦戴, 张梦茹, 等. 2021. A型塞内卡病毒研究进展[J]. 中国兽医学报, 41(8): 1645-1650.
(Liao Y X, Fan J D, Zhang M R, et al.2021. Research progress of Senecavirus A[J]. Chinese Journal of Veterinary Science, 41(8): 1645-1650.)
[5] 林彤, 邵军军, 丛国正, 等. 2009. Asia1型口蹄疫病毒胶体金免疫层析检测方法的建立[J]. 生物工程学报, 25(5): 767-772.
(Lin T, Shao J J, Cong G Z, et al.2009. Establishment of colloidal gold-immunochroma-togrphy assay strip for detection of type Asia1 Foot-and-mouth disease virus[J]. Chinese Journal of Biotechnology, 25(5): 767-772.)
[6] 彭宛清, 钱炳旭, 廖凯, 等. 2022. 塞内卡病毒A VP1蛋白的原核表达及其单克隆抗体的制备与初步应用[J]. 中国兽医科学, 52(07): 867-874.
(Peng W Q, Qian B X, Liao K, et al.2022. Prokaryotic expression of Senecavirus A VP1 protein and preparation and application of its monoclonal antibody[J]. Chinese Veterinary Science, 52(07): 867-874.)
[7] 赵丹丹, 杨国平, 刁有祥, 等. 2016. 鸭瘟病毒单抗的制备及胶体金试纸条检测方法的建立[J]. 中国农业科学, 49(14): 2796-2804.
(Zhao D D, Yang G P, Diao Y X, et al.2016. Preparation of monoclonal antibodies against DPV and development of colloidal gold strip for DPV detection[J]. Scientia Agricultura Sinica, 49(14): 2796-2804.)
[8] Armson B, Walsh C, Morant N, et al.2019. The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca valley virus 1[J]. Transboundary and Emerging Diseases, 66(1): 497-504.
[9] Bracht A J, O’Hearn E S, Fabian A W, et al.2016. Real-time reverse transcription PCR assay for detection of Senecavirus A in swine vesicular diagnostic specimens[J]. PLOS ONE, 11(1): e0146211.
[10] Buckley A, Lager K.2022. Efficacy of an inactivated Senecavirus A vaccine in weaned pigs and mature sows[J]. Vaccine, 40(12): 1747-1754.
[11] Cai P, Wang R, Ling S, et al.2021. A high sensitive platinum-modified colloidal gold immunoassay for tenuazonic acid detection based on monoclonal IgG[J]. Food Chemistry, 360: 130021.
[12] Dall Agnol A M, Otonel R A A, Leme R A, et al.2017. A TaqMan-based qRT-PCR assay for Senecavirus A detection in tissue samples of neonatal piglets[J]. Molecular and Cellular Probes, 33: 28-31.
[13] Dvorak C M, Akkutay-Yoldar Z, Stone S R, et al.2017. An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine[J]. BMC Veterinary Research, 13(1): 50.
[14] Feronato C, Leme R A, Diniz J A, et al.2018. Development and evaluation of a nested-PCR assay for Senecavirus A diagnosis[J]. Tropical Animal Health and Production, 50(2): 337-344.
[15] Fowler V L, Ransburgh R H, Poulsen E G, et al.2017. Development of a novel real-time RT-PCR assay to detect Seneca valley virus-1 associated with emerging cases of vesicular disease in pigs[J]. Journal of Virological Methods, 239: 34-37.
[16] Gimenez-Lirola L G, Rademacher C, Linhares D, et al.2016. Serological and molecular detection of Senecavirus A associated with an outbreak of swine idiopathic vesicular disease and neonatal mortality[J]. Journal of Clinical Microbiology, 54(8): 2082-2089.
[17] Goolia M, Vannucci F, Yang M, et al.2017. Validation of a competitive ELISA and a virus neutralization test for the detection and confirmation of antibodies to Senecavirus A in swine sera[J]. Journal of Veterinary Diagnostic Investigation, 29(2): 250-253.
[18] Guo B, Piñeyro P E, Rademacher C J, et al.2016. Novel Senecavirus A in swine with vesicular disease, United States, July 2015[J]. Emerging Infectious Diseases, 22(7): 1325-1327.
[19] Halesl M, Knowles N J, Reddy P S, et al.2008. Complete genome sequence analysis Seneca valley virus-001, a novel oncolytic picrnavirus[J]. Journal of General Virology, 89(5): 1265-1275.
[20] Hou W, Wang S, Wang X, et al.2015. Development of colloidal gold immunochromatographic strips for detection of Riemerella anatipestifer[J]. PLOS ONE, 10(3): e0122952.
[21] Houston E, Temeeyasen G, Piñeyro P E.2020. Comprehensive review on immunopathogenesis, diagnostic and epidemiology of Senecavirus A[J]. Virus Research, 286: 198038.
[22] Leme R A, Alfieri A F, Alfieri A A.2017. Update on Senecavirus in pigs[J]. Viruses, 9(7): 170.
[23] Leme R A, Oliveira T E, Alfieri A F, et al.2016. Pathological, immunohistochemical and molecular findings associated with Senecavirus A-induced lesions in neonatal piglets[J]. Journal of Comparative Pathology, 155(2-3): 145-155.
[24] Lin L, Wu X, Cui G, et al.2020. Colloidal gold immunochromatographic strip assay for the detection of azaperone in pork and pork liver[J]. ACS Omega, 5(3): 1346-1351.
[25] Liu F, Wang Q, Huang Y, et al.2020. A 5-year review of Senecavirus A in China since its emergence in 2015[J]. Frontiers in Veterinary Science, 7: 567792.
[26] Resende T P, Marthaler D G, Vannucci F A.2017. A novel RNA-based in situ hybridization to detect Seneca valley virus in neonatal piglets and sows affected with vesicular disease[J]. PLOS ONE, 12(4): e0173190.
[27] Segales J., Barcellos D., Alfieri A., et al.2017. Senecavirus A: An emerging pathogen causing vesicular disease and mortality in pigs?[J]. Veterinary Pathology, 54: 11-21.
[28] Tao Y, Hao H W, Li J, et al.2019. Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae[J]. Chinese Journal of Biotechnology, 35(5): 901-909.
[29] Wang H, Dong J, Zhang T, et al.2022. A novel rapid detection of Senecavirus A using recombinase polymerase amplification (RPA) coupled with lateral flow (LF) dipstrip[J]. Analytical Biochemistry, 646: 114627.
[30] Wang H B, Tian B, Lv H L, et al.2017. Emergence and complete genome of Senecavirus A in pigs of Henan province in China, 2017[J]. Polish Journal of Veterinary Sciences, 22(1): 187-190.
[31] Wu Q, Zhao X, Bai Y, et al.2017. The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J]. Transboundary and Emerging Diseases, 64(5): 1633-1640.
[32] Yang F, Xiao Y, Chen B, et al.2020. Development of a colloidal gold-based immunochromatographic strip test using two monoclonal antibodies to detect H7N9 Avian influenza virus[J]. Virus Genes, 56(3): 396-400.
[33] Yang G, Chen K, Guo W, et al.2022. Development of a test card based on colloidal gold immunochromatographic strips for rapid detection of antibodies against Theileria equi and Babesia caballis[J]. Microbiology Spectrum, 10(1): e0241121.
[34] Yang M, Bruggen R, Xu W.2012. Generation and diagnostic application of monoclonal antibodies against Seneca valley virus[J]. Journal of Veterinary Diagnostic Invstigation, 24(1): 42-50.
[35] Yu J, Lin Y, Cao Y, et al.2020. Development and application of a colloidal gold test strip for the rapid detection of the infectious Laryngotracheitis virus[J]. Poultry Science, 99(5): 2407-2415.
[36] Zhang Z, Zhang Y, Lin X, et al.2019. Development of a novel reverse transcription droplet digital PCR assay for the sensitive detection of Senecavirus A[J]. Transboundary and Emerging Diseases, 66(1): 517-525.
[37] Zhu Z,Yang F,Chen P,et al.2017. Emergence of novel Seneca valley virus strains in China[J]. Transboundary and Emerging Diseases, 64(4): 1024-1029. |
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