Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (14): 2796-2804.doi: 10.3864/j.issn.0578-1752.2016.14.013

;

• VETERINARY SCIENCE • Previous Articles     Next Articles

Preparation of Monoclonal Antibodies Against DPV and Development of Colloidal Gold Strip for DPV Detection

ZHAO Dan-dan, YANG Guo-ping, DIAO You-xiang, CHEN Hao, TI Jin-feng, ZHANG Lu, ZHANG Ying, LI Chuan-chuan   

  1. College of Animal Science and Technology, Shandong Agricultural University, Tai’an 271000, Shandong
  • Received:2015-05-13 Online:2016-07-16 Published:2016-07-16

RichHTML

3

PDF

894

Cited

5

Abstract: 【Objective】 Duck plague（DP） is an acute, septic contagion, caused by duck plague virus（DPV）, with the characteristics of head and neck swelling, the mucosa of esophageal and cloacal bleeding and yellowish-white ulcer, head and neck skin has a yellowish-white gelatin sample. Once an outbreak of this disease manifested by with high morbidity and high mortality, it would cause serious harm to the duck industry. The aim of this assay is to establish a method of colloidal gold strip for the detection of duck plague virus (DPV) rapidly. 【Method】 The main antigenic domain of DPV was chosen and analyzed by using of the Protean Biology software to design a pair of primer to amplify the aim gene by PCR. Then the fragment was inserted into prokaryotic expression vector pET-28a to construct recombinant plasmid. Then it was transformed into Rosetta for expression. During the experiment, the authors have groped the concentration of the IPTG and the induction time. After purification, the concentration of the aim protein was tested and was also analyzed and identified by Western blotting. Hybridoma cell lines stably secreting monoclonal antibody against gB protein of DPV were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized mice, which used the gB protein of DPV, expressed and purified with prokaryotic, as the antigen. The monoclonal antibody-based colloidal gold immunochromatography strip was developed for the detection of DPV. The purified DPV-gB monoclonal antibody, named H6F6, was labeled with colloidal gold, with the appropriate pH between 8.0 and 8.5 and the concentration was 15 times dilution. The purified A8D7 monoclonal antibody, with the concentration of twice dilution, and the goat anti-mouse immunoglobulin G (IgG) antibody, with the concentration of ten times dilution, were blotted on nitrocellulose membrane as test line and control line, respectively. 【Result】 Hybridoma cell lines designated as A8D9, E6C3, H11F8, H6A10, stably secreting monoclonal antibody against gB protein of DPV. The titres of ascitic fluid was1:103, 1:103, 1:105, 1:103, respectively by indirect ELISA and the immunoglobulin subtype of the monoclonal antibodies was IgG2b, IgG2a, IgG2b, IgG1,with the light chain of kappa. The result of western blot showed that the four monoclonal antibodies were able to specifically recognize gB protein of DPV. The result of IFA showed that the four monoclonal antibodies were specific to DPV. The detection results indicated that the strip was specific to DPV and had no cross reaction with DRV, EDS-76V, AIV-H9N2, and TMUV. The detection limit of DPV were 50 times dilution. 38 clinical suspected samples were simultaneously detected by immunochromatography strip and PCR while the results showed 91.6 % accuracy between them. The monoclonal antibodies-based colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.【Conclusion】The colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.

Key words: duck plague virus, glycoprotein B, Prokaryotic expression, monoclonal antibody, colloidal gold strip

[1]    GUO Y F, SHEN C J, CHENG A C, WANG M S, ZHANG N, CHEN S, ZHOU Y. Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosisin duck embryo fibroblast cultures. Veterinary Microbiology, 2009, 138: 258-265.
[2]    徐晓娟, 郭霄峰, 魏平华. 鸭瘟病毒研究进展. 养禽与禽病防治, 2012(10): 8-12.
XU X J, GUO X F, WEI P H. Research progress of duck plague  virus. Poultry Husbandry and Disease Control, 2012(10): 8-12. (in Chinese)
[3]    WANG Y H, X R LI, WANG G X, YIN H, CAI X P, FU B Q, ZHANG. D L. Development of an immunochromatographic strip for the rapid detection of Toxoplasmagondii circulating antigens. Parasitology International, 2011, 60: 105-107.
[4]    WANG H L, FENG N, YANG S T, WANG C Y, WANG T C, GAO Y W, SU J Q, ZHENG X X, HOU X Q, HUANG H N, YANG R M, ZOU X H, HUANG G, XIA X Z. A rapid immunochromatographic test strip for detecting rabies virus antibody. Journal of Virological Methods, 2010, 170: 80-85.
[5]    KUSANO N, IWANAMI T, NARAHARA K, TANAKA M. Production of monoclonal antibodies speci?c for the recombinantviral coat protein of Apple stem grooving virus-citrus isolate and theirapplication for a simple, rapid diagnosis by animmunochromatographicassay. Journal of Virological Methods, 2014, 195: 86-91.
[6]    王建科, 易立, 罗彬, 杨莘, 程世鹏. 胶体金免疫层析技术在动物病毒性传染病诊断中的应用. 动物医学进展, 2011, 32(1): 93-98.
WANG J K, YI L, LUO B, YANG S, CHENG S P. Application of gold immunochromatographic Technique in the diagnosis of animal viral infectious disease. Progress in Veterinary Medicine, 2011, 32(1): 93-98. (in Chinese)
[7]    LI Y M, HOU L D, YE J, LIU X Q, DAN H B, JING M L, CHEN H C, CAO S B. Development of a convenient immunochromatographic strip for diagnosis of infection with Japanese encephalitis virus in swine. Journal of Virological Methods, 2010, 168: 51-56.
[8]    LI J F, ZOU M Q, CHEN Y, XUE Q, ZHANG F, LI B B, WANG Y F, QI X H, YANG Y. Gold immunochromatographic strips for enhanced detection of Avian influenza and Newcastle disease viruses. Analytica Chimica Acta, 2013, 782: 54-58.
[9]    吴晓平, 吴异健, 韩典霖, 吴宝成, 黄一帆. 原核表达番鸭呼肠孤病毒σC与σB蛋白对雏番鸭的免疫保护. 中国农业科学, 2013, 46(14): 3040-3045.
WU X P, WU Y J, HAN D L, WU B C, HUANG Y F. Protection of musovy duck reovirus by immunization with the recombinant σC and σB protein expressed in E. coli. Scientia Agricultura Sinica, 2013, 46(14): 3040-3045. (in Chinese)
[10]   王洪海, 苏敬良, 曹振, 田克恭. 鸭肠炎病毒单克隆抗体的制备.中国兽医科技, 2004, 34(11): 13-17.
WANG H H, SU J L, CAO Z, TIAN K G. Preparation of monoclonal antibodies against duck enteritis virus. Chinese Journal of Veterinary Science and Technology, 2004, 34(11): 13-17. (in Chinese)  
[11]   周艳君, 华荣虹, 王云峰, 安同庆, 刘金霞, 杨金雨, 华玉卓, 童光志. SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定. 生物工程学报, 2005, 21(2): 211-215.
ZHOU Y J, HUA R H, WANG Y F, AN T Q, LIU J X, YANG       J Y, HUA Y Z, TONG G Z. Development of monoclonal    antibodies against SARS-CoV and identification of antigenic  epitopes. Chinese Journal of Biotechnology, 2005, 21(2): 211-215. (in Chinese)
[12]   孙建慧, 黄立平, 危艳武, 吴洪丽, 王一平, 张辉, 刘丹, 陈冬杰, 张智慧, 刘长明.猪细小病毒1型VP2蛋白的表达与单克隆抗体的制备及应用. 中国兽医科学, 2014, 44(12): 1279-1285.
SUN J X, HUANG L P, WEI Y W, WU H L, WANG Y P, ZHANG H, LIU D, CHEN D J, ZHANG Z H, LIU C M. Expression of porcine parvovirus type 1 VP2 protein and preparation and application of monoclonal antibody against VP2 protein. Chinese Veterinary Science, 2014, 44(12): 1279-1285. (in Chinese)
[13]   王莹, 郑浩, 何成华, 张爱华, 冯璐璐, 皇超英, 张海彬. 伏马菌素 B1单克隆抗体的制备及鉴定. 中国农业科学. 2011, 44(20): 4302-4308.
WANG Y, ZHENG H, HE C H, ZHANG A H, FENG L L, HUANG C Y, ZHANG H B. Generation and identification of the monoclonal antibody against fumonisin B1. Scientia Agricultura Sinica, 2011, 44(20): 4302-4308. (in Chinese)
[14]   贠炳岭, 李德龙, 胡晓亮, 刘在斯, 秦立廷, 吴关, 刘文, 祁小乐, 王永强, 高宏雷, 高玉龙, 王笑梅. 禽白血病病毒群特异性抗原单克隆抗体的制备与鉴定. 中国预防兽医学报, 2012, 34(10): 782-785.
YUAN B L, LI D L, HU X L, LIU Z S, QIN L T, WU G, LIU W, QI X L, WANG Y Q, GAO H L, GAO Y L, WANG X M. Preparation and identification of monoclonal antibodies against the group specific antigen of avian leukosisvirus. Chinese Journal of Preventive Veterinary Medicine, 2012, 34(10): 782-785. (in Chinese)
[15]   许静, 李世军, 熊胜, 陈黎, 杨倩, 卢立志. 鸭胚成纤维细胞培养、传代及保存方法的研究. 中国家禽, 2012, 34(13): 9-13.
XU J, LI S J, XIONG S, CHEN L, YANG Q, LU L Z. Culture subculture and cryopreservation of embryo fibroblast of duck. China Poultry, 2012, 34(13): 9-13. (in Chinese)
[16]   李向梅, 王战辉, 肖希龙, 王照鹏, 温凯, 吴小平, 夏.曦, 武晋孝, 江海洋. 同时检测牛奶中喹诺酮类和庆大霉素残留的胶体金免疫层析方法研究. 中国农业科学, 2014, 47(19): 3883-3889.
LI X M, WANG Z H, XIAO X L, WANG Z P, WEN K, WU X P, XIA X, WU J X, JIANG H Y. Development of a colloidal gold immunochromatographic technique for simultaneous detection of quinolones and gentamicin in milk. Scientia Agricultura Sinica, 2014, 47(19): 3883-3889. (in Chinese)
[17]   蒋韬, 梁仲, 陈涓, 何继军, 吕律, 马维民, 刘在新, 刘湘涛. 口蹄疫病毒 O、A、AsiaⅠ型定型诊断胶体金免疫层析方法的建立. 中国农业科学 2008, 41(11): 3801-3808.
JIANG T, LIANG Z, CHEN J, HE J J, LÜ L, MA W M, LIU Z X, LIU X T. Development of a rapid gold immunochromatographic strip test for the diagnosis of Foot-and-Mouth disease virus O, A and AsiaⅠtype. Scientia Agricultura Sinica, 2008, 41(11): 3801-3808. (in Chinese)
[18]   宋万杰, 胡伯里, 金玉兰, 阮系真, 邵敬霞, 周继勇.猪圆环病毒2型抗体胶体金免疫层析方法的建立. 中国兽医科学, 2014, 44(7): 710-714.
SONG W J, HU B L, JIN Y L, RUAN X Z, SHAO J X, ZHOU J Y. Establishment of a method for detection of the antibody against porcine circovirus type 2 based on colloidal gold immunochromatographic assay. Chinese Veterinary Science, 2014, 44(7): 710-714. (in Chinese)
[19]   张鑫宇, 左伟勇, 朱善元, 夏晓莉, 孙怀昌. 非洲猪瘟病毒 p54 抗体胶体金试纸检测方法的建立. 中国预防兽医学报, 2014, 36(4): 281-285.
ZHANG X Y, ZUO W Y, ZHU S Y, XIA X L, SUN H C. Establishment of colloidal gold strip for detecting antibody against African swine fever virus. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(4): 281-285. (in Chinese)
[20] ZHOU P, LU Y T, ZHU J, HONG J B, LI B, ZHOU J, GONG D, MONTOYA A. Nanocolloidal gold-based immunoassay for the detection of the N-Methylcarbamate pesticide carbofuran. Journal of Agricultural and Food Chemistry, 2004, 52: 4355-4359.
[21]   JIANG T, LIANG Z, REN W W, CHEN J, ZHI X Y, QI G Y, LIU X T, CAI X P. A simple and rapid colloidal gold-based immunochromatogarphic strip test for detection of FMDV serotype A. Virologica Sinica, 2011, 26 (1): 30-39.
[22]   GRIFFIN A. M. The nucleotide sequence of the glycoprotein gB gene of infectious laryngotracheitisvirus: analysis and evolutionary relationship to the homologous gene from other herpesviruses. Journal of General Virology, 1991, 72(2): 393-398.
[23]   夏兴霞, 王永山, 孟祥升, 朱国强, 张雪寒, 何孔旺. 大肠杆菌0157: H7单克隆抗体胶体金免疫层析检测试纸条的研制. 中国动物传染病学报, 2010, 18(4): 47-53.
XIA X X, WANG Y S, MENG X S, ZHU G Q, ZHANG X H, HE K W. Development of a gold immunochromatogarphic strip for the detection of the E.Coli0157: H7. Chinese Journal of Animal Infectious Diseases, 2010, 18(4): 47-53.(in Chinese)
[24]   孙园园, 王云龙, 李玉林, 王继创, 程蕾, 邓黎黎.pH 值对胶体金标记单克隆抗体性能的影响. 细胞与分子免疫学杂志, 2014, 30(11): 1170-1173.
SUN Y Y, WANG Y L, LI Y L, WANG J C, CHENG L, DENG L L. Effects of pH on the properties of colloidal gold labeling monoclonal antibody. Chinese Journal of Cellular and Molecular Immunology, 2014, 30(11): 1170-1173. (in Chinese)
[25]   顾菲菲, 高海岗, 陆建荣.不同粒径大小胶体金的制备. 中国畜牧兽医文摘. 2013, 29(10): 45-46.
GU F F, GAO H G, LU J R. Different sizes of colloidal gold preparation. Chinese Abstracts of Animal Husbandry and Veterinary Medicine. 2013, 29(10): 45-46.(in Chinese)
[1] ZHANG FengXi,XIAO Qi,ZHU JiaPing,YIN LiHong,ZHAO XiaLing,YAN MingShuai,XU JinHua,WEN LiBin,NIU JiaQiang,HE KongWang. Preparation and Identification of Monoclonal Antibodies to P30 Protein and Establishment of Blocking ELISA to Detecting Antibodies Against African Swine Fever Virus [J]. Scientia Agricultura Sinica, 2022, 55(16): 3256-3266.
[2] WEI Tian,WANG ChengYu,WANG FengJie,LI ZhongPeng,ZHANG FangYu,ZHANG ShouFeng,HU RongLiang,LÜ LiLiang,WANG YongZhi. Preparation of Monoclonal Antibodies Against the p30 Protein of African Swine Fever Virus and Its Mapping of Linear Epitopes [J]. Scientia Agricultura Sinica, 2022, 55(15): 3062-3070.
[3] Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids [J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.
[4] YuXin LIANG,JianXiang WU,XiaoYu LI,ChunYu ZHANG,JiChao HOU,XuePing ZHOU,YongZhi WANG. Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein [J]. Scientia Agricultura Sinica, 2021, 54(6): 1154-1162.
[5] XiaoHe LIU,GuiSheng QIU,ZhaoGuo TONG,HuaiJiang ZHANG,WenTao YAN,Qiang YUE,LiNa SUN. Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii [J]. Scientia Agricultura Sinica, 2021, 54(5): 945-958.
[6] LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie. Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody [J]. Scientia Agricultura Sinica, 2021, 54(20): 4478-4486.
[7] QIN JianHui,LI JinQiao,ZHAO Xu,LI KeBin,CAO YaZhong,YIN Jiao. Expression, Purification and Functional Analysis of Odorant Binding Protein 11 (OBP11) in Anomala corpulenta [J]. Scientia Agricultura Sinica, 2021, 54(14): 3017-3028.
[8] XIE KunLun,LIU LiMing,LIU Mei,PENG Bin,WU HuiJie,GU QinSheng. Prokaryotic Expression of dsRNA of Zucchini yellow mosaic virus and Its Control Efficacy on ZYMV [J]. Scientia Agricultura Sinica, 2020, 53(8): 1583-1593.
[9] HU XiaoFei,LI QingMei,YAO JingJing,HU SiYu,SUN YaNing,XING YunRui,DENG RuiGuang,ZHANG GaiPing. Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs [J]. Scientia Agricultura Sinica, 2020, 53(5): 1071-1080.
[10] BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein [J]. Scientia Agricultura Sinica, 2019, 52(23): 4429-4436.
[11] Ling LI,Yao TAN,XiaoRong ZHOU,BaoPing PANG. Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica [J]. Scientia Agricultura Sinica, 2019, 52(20): 3705-3712.
[12] LI Du, NIU ChangYing, LI FengQi, LUO Chen. Binding Characterization of Odorant Binding Protein OBP56h in Drosophila suzukii with Small Molecular Compounds [J]. Scientia Agricultura Sinica, 2019, 52(15): 2616-2623.
[13] WANG Hui,CHAI ZhiXin,ZHU JiangJiang,ZHONG JinCheng,ZHANG ChengFu,Xin JinWei. Cloning and Identification of Long-Chain Non-Coding RNA Linc24063 and Its Correlation with the Expression Level of miRNAs in Yak [J]. Scientia Agricultura Sinica, 2019, 52(14): 2538-2547.
[14] YI Min,LÜ Qing,LIU KeKe,WANG LiJun,WU YuJiao,ZHOU ZeYang,LONG MengXian. Expression, Purification and Localization Analysis of Polar Tube Protein 2 (NbPTP2) from Nosema bombycis [J]. Scientia Agricultura Sinica, 2019, 52(10): 1830-1838.
[15] ZHANG Kui,LI ChongYang,SU JingJing,TAN Juan,XU Man,CUI HongJuan. Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2019, 52(1): 181-190.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd