[1]任春梅,杨柳,缪倩,等.小麦黄花叶病毒单克隆抗体的制备及ACP-ELISA检测方法的建立[J].江苏农业学报,2018,(01):34-40.[doi:doi:10.3969/j.issn.1000-4440.2018.01.005]
　REN Chun-mei,YANG Liu,MIAO Qian,et al.Production of monoclonal antibodies to wheat yellow mosaic virus and ACP-ELISA application in virus detection[J].,2018,(01):34-40.[doi:doi:10.3969/j.issn.1000-4440.2018.01.005]
点击复制

小麦黄花叶病毒单克隆抗体的制备及ACP-ELISA检测方法的建立()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2018年01期
页码:
34-40
栏目:
植物保护
出版日期:
2018-02-25

文章信息/Info

Title:
Production of monoclonal antibodies to wheat yellow mosaic virus and ACP-ELISA application in virus detection
作者:
任春梅; 杨柳; 缪倩; 程兆榜
（江苏省农业科学院植物保护研究所，江苏南京210014）
Author(s):
REN Chun-mei; YANG Liu; MIAO Qian; CHENG Zhao-bang
(Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
小麦黄花叶病毒; 单克隆抗体; ACP-ELISA
Keywords:
wheat yellow mosaic virus; monoclonal antibodies; ACP-ELISA
分类号:
S435.121
DOI:
doi:10.3969/j.issn.1000-4440.2018.01.005
文献标志码:
A
摘要:
为建立简便、有效、低成本小麦黄花叶病毒（WYMV）的检测方法，用提纯的WYMV免疫BALB/c小鼠后，利用杂交瘤细胞技术经细胞融合、筛选和克隆，共获得2D4、2D3和6F4 3株能稳定传代并分泌抗WYMV单克隆抗体的杂交瘤细胞，并分别注射小鼠腹腔制备其单抗腹水。3株单抗腹水的间接 ELISA 效价为10-6～10-7，抗体类型及亚类均为IgG1、κ链。特异性检测结果表明6F4和2D4这2株单抗仅与WYMV的32 kD外壳蛋白有特异性免疫反应。利用效价最高的2D4单抗建立了检测WYMV的抗原包被ELISA方法（ACP-ELISA），其灵敏度分析结果表明，当病叶以1∶25 600 (g/ml)倍稀释时仍能检测到病毒，特异性分析结果表明可有效区分小麦易感染的中国小麦花叶病毒（CWMV）。田间样品检测结果表明该方法可准确、可靠地用于小麦WYMV的大规模检测。
Abstract:
To develop a simple, effective and low-cost method for detecting wheat yellow mosaic virus (WYMV), the purified WYMV virus particles were used as an immunogen, after fusioned, selected and cloned，three hybridoma cell lines (2D4, 2D3 and 6F4) secreting monoclonal antibodies (MAbs) against WYMV were produced by fusing mouse myeloma cells with spleen cells from BALB/c, and injected intraperitoneally into BALB/c mice to produce ascetic fluids. The titers of three MAbs in ascites determined by an indirect-ELISA ranged from 10-6 to 10-7. Isotypes and subclasses of three MAbs belonged to IgG1, κlight chain. 6F4 and 2D4 MAbs could specifically react with coat protein (32 kD) of WYMV. Based on the most sensitive 2D4 MAb, an antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) for detecting WYMV was established. Sensitivity analysis results showed that the virus could be detected when the diseased leaves were diluted with 1∶25 600 (g/ml) times, specificity analysis results showed that the method could be used to distinguish WYMV and Chinese wheat mosaic virus(CWMV). The field sample detection results suggested that the ACP-ELISA could accurately and reliably detect WYMV in large-scale wheat samples.

参考文献/References:

[1]陈剑平,阮义理,董玛佳. 我国一些地区发生的小麦土传病毒病原研究[J]. 病毒学杂志, 1989(2):176-181.
[2]雷利娟,陈炯,陈剑平,等. 我国真菌传线状小麦花叶病毒病病原初步鉴定为小麦黄花叶病毒(WYMV) [J]. 中国病毒学, 1998,13(1): 89-96.
[3]SAWADA E. Wheat yellow mosaic prevention[J]. Journal of Plant Protection (Byochugai-Zasshi), 1927, 14: 444-449.
[4]缪倩,季英华,任春梅,等. 一种快速同步检测小麦花叶病毒和中国小麦花叶病毒的方法[J]. 麦类作物学报, 2013, 33(3): 595-599.
[5]孙炳剑,羊健,孙丽英,等. 禾谷多黏菌传小麦病毒病的分布及变化动态[J]. 麦类作物学报, 2011,31(5): 969-973.
[6]燕飞,孙丽英,尚佑芬,等. 我国粮食作物病毒病发生与防控现状[J]. 植物保护, 2013,39(5): 33-37.
[7]李鹏，崔正勇，高国强，等. 小麦黄花叶病研究进展[J]．山东农业科学，2017,49(8):168-172.
[8]叶荣,郑滔,徐磊,等. 山东烟台小麦土传病毒病由小麦黄花叶病毒和土传小麦花叶病毒相关病毒复合侵染所致[J]. 病毒学报, 2000,16(1): 80-82.
[9]XING Y, SU N, LI D. Over-expression of 72 kDa protein of wheat yellow mosaic virus in E.coli and preparation of its antiserum[J]. Chinese Science Bulletin, 2000, 45(6):525-528.
[10]向荣,孙丽英,孙炳剑,等. 小麦黄花叶病毒P1蛋白原核表达、抗血清制备及其检测[J]. 浙江农业学报, 2011, 23(2):324-328.
[11]韩成贵,李大伟,于嘉林,等. 小麦黄色花叶病毒外壳蛋白基因E.coli表达产物特异性抗血清的制备及其应用[J]. 农业生物技术学报, 2002, 10(4):373-376.
[12]尚巧霞,韩成贵,杨莉莉. 小麦黄花叶病毒的血清学检测[J]. 北京农学院学报, 2002, 17(2):15-17.
[13]耿波,韩成贵,翟亚锋,等. 利用异种动物抗血清双抗夹心法检测小麦黄花叶病毒[J].中国病毒学, 2003, 18(1):76-78.
[14]丛倩倩,许斐斐,李向东,等. 山东小麦黄花叶病毒的分子检测与鉴定[J].山东农业科学, 2012, 44(11): 8-11.
[15]TAO Y, MAN J, WU Y. Development of a multiplex polymerase chain reaction for simultaneous detection of wheat viruses and a phytoplasma in China[J]. Archives of Virology， 2012, 157(7):1261-1267.
[16]ZHANG Z Y, LIU X J, LI D, et al. Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification[J]. Virology Journal, 2011, 8(1):550.
[17]FUKUTA S, TAMURA M, MAEJIMA H, et al. Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction[J]. Journal of Virological Methods, 2013,189(2):348-354.
[18]任春梅,程兆榜,朱慧,等. 3种麦类土传花叶病毒的多抗制备及检测应用[J]. 江苏农业科学, 2014, 42(11):143-146.
[19]SHANG H, XIE Y, ZHOU X, et al. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus[J]. Virology Journal, 2011, 8(1):228-236.
[20]WU J X, NI Y Q, LIU H, et al. Development and use of three monoclonal antibodies for the detection of Rice black-streaked dwarf virus in field plants and planthopper vectors[J]. Virology Journal, 2013, 10（1）:114.
[21]汪谦. 现代医学实验方法[M], 北京: 人民卫生出版社, 1997：730-738.
[22]刘欢,倪跃群,饶黎霞,等. 南方水稻黑条矮缩病毒和水稻黑条矮缩病毒的单抗制备及其检测应用[J]. 植物病理学报, 2013,43(1):27-34.
[23]孔君,刘箐,韩跃武,等. 单克隆抗体制备技术的最新进展及应用前景[J]. 免疫学杂志, 2011, 27(2):170-173.
[24]陈剑平. 中国禾谷多黏菌传麦类病毒研究现状与展望[J]. 自然科学进展, 2015,15(5):524-533.
[25]HAN C G, LI D W, XING Y M, et al. Wheat yellow mosaic virus widely occurring in wheat(Triticum aestivum) in China[J]. Plant Disease, 2000,84(6):627-630.

相似文献/References:

[1]张帅,齐颖颖,张红星,等.快速检测生鲜肉中三种食源性致病菌[J].江苏农业学报,2016,(04):939.[doi:10.3969/j.issn.100-4440.2016.04.036]
　ZHANG Shuai,QI Ying-ying,ZHANG Hong-xing,et al.Rapid detection of three foodborne pathogens in raw meat[J].,2016,(01):939.[doi:10.3969/j.issn.100-4440.2016.04.036]
[2]王怡雯,齐颖颖,张红星,等.福氏志贺氏菌2a单克隆抗体的制备及ELISA方法的建立[J].江苏农业学报,2017,(02):450.[doi:doi:10.3969/j.issn.1000-4440.2017.02.033]
　WANG Yi-wen,QI Ying-ying,ZHANG Hong-xing,et al.Preparation of Shigella flexneri 2a monoclonal antibodies and establishment of ELISA for its detection[J].,2017,(01):450.[doi:doi:10.3969/j.issn.1000-4440.2017.02.033]
[3]李盼,张晓帅,邱雨楼,等.有机磷农药广谱特异性单克隆抗体的制备及特性分析[J].江苏农业学报,2017,(06):1415.[doi:doi:10.3969/j.issn.1000-4440.2017.06.031]
　LI Pan,ZHANG Xiao-shuai,QIU Yu-lou,et al.Production and characterization of broad-specific monoclonal antibody for organophosphorus pesticides recognization[J].,2017,(01):1415.[doi:doi:10.3969/j.issn.1000-4440.2017.06.031]
[4]何方洋,杨昌松,韩深,等.应用酶联免疫法测定环境水样中雌三醇的残留量[J].江苏农业学报,2017,(06):1422.[doi:doi:10.3969/j.issn.1000-4440.2017.06.032]
　HE Fang-yang,YANG Chang-song,HAN Shen,et al.Enzyme-linked immunosorbent assay（ELISA） for determination of estriol in environmental water sample[J].,2017,(01):1422.[doi:doi:10.3969/j.issn.1000-4440.2017.06.032]
[5]祁光宇,刘斌,赵兴绪.猪流行性腹泻病毒HB2015分离株N基因表达及单克隆抗体的制备[J].江苏农业学报,2018,(01):76.[doi:doi:10.3969/j.issn.1000-4440.2018.01.011]
　QI Guang-yu,LIU Bin,ZHAO Xing-xu.Expression of N gene of porcine epidemic diarrhea virus（PEDV） HB2015 strain and preparation of monoclonal antibodies against PEDV[J].,2018,(01):76.[doi:doi:10.3969/j.issn.1000-4440.2018.01.011]
[6]杨柳,任春梅,缪倩,等.黄瓜绿斑驳花叶病毒的双抗夹心ELISA检测[J].江苏农业学报,2018,(04):769.[doi:doi:10.3969/j.issn.1000-4440.2018.04.008]
　YANG Liu,REN Chun-mei,MIAO Qian,et al.DAS-ELISA detection against cucumber green mottle mosaic virus[J].,2018,(01):769.[doi:doi:10.3969/j.issn.1000-4440.2018.04.008]
[7]刘蓓蓓,韦艳娜,陈蓉,等.半固体培养法制备非洲猪瘟病毒pA104R蛋白的单克隆抗体[J].江苏农业学报,2024,(04):682.[doi:doi:10.3969/j.issn.1000-4440.2024.04.012]
　LIU Bei-bei,WEI Yan-na,CHEN Rong,et al.Preparation of monoclonal antibody against pA104R protein of African swine fever virus by semi-solid culture method[J].,2024,(01):682.[doi:doi:10.3969/j.issn.1000-4440.2024.04.012]

备注/Memo

备注/Memo:
收稿日期：2017-07-25 基金项目：国家转基因重大专项（2016ZX08002） 作者简介：任春梅（1981-），女，江苏大丰人，副研究员，硕士，主要从事植物病毒研究。 通讯作者：程兆榜,（E-mail）onlyone8501@126.com
更新日期/Last Update: 2018-03-06