小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (4): 588-595.doi:

小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

龙云凤1，周晓黎2，杨俊兴3，王琼4，祝贺 2，叶玲玲2，董俊2，吕建强3，
王金萍5，陈朝银1*，花群义3，杨仕标5，尹尚莲2，徐维佳2，艾军2*

1.昆明理工大学，昆明 650500； 2.云南出入境检验检疫局，昆明 650228；3.深圳出入境检验检疫局，深圳 518045； 4. 中国科学院昆明动物研究所，昆明 6500223; 5.云南省畜牧兽医科学院，昆明 650224

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-04-25 发布日期:2012-04-25
通讯作者: 陈朝银，艾军

Preparation and Preliminary Application of Monoclonal Antibodies against Nucleoprotein of Peste des Petits Ruminants Virus

ONG Yunfeng1, ZHOU Xiaoli2, YANG Junxing3, WANG Qiong4, ZHU He2, YE Lingling2, DONG Jun2, LV Jianqiang3, WANG Jinping5, CHEN Chaoyin1*, HUA Qunyi3, YANG Shibiao5, YIN Shanglian2,XU Weijia2, AI Jun2*

1.Kunming University of Science and Technology, Kunming 650500, China; 2.Yunnan EntryExit Inspection and Quarantine Bureau, Kunming 650228, China; 3. Shenzhen EntryExit Inspection and Quarantine Bureau, Shenzhen 518045, China; 4. Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; 5. Yunnan Animal Science and Veterinary Institute, Kunming 650224, China

Received:1900-01-01 Revised:1900-01-01 Online:2012-04-25 Published:2012-04-25
Contact: CHEN Chaoyin，AI Jun

摘要/Abstract

摘要： 针对小反刍兽疫病毒核蛋白制备特异性的单克隆抗体,并对其进行生物学特性鉴定和初步应用。以纯化的BacmidPPRVN重组蛋白为抗原免疫BALB/c小鼠，取免疫小鼠的致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合，获得单克隆抗体，并通过染色体技术等方法研究其生物学特性，将其作为竞争单抗，BacmidPPRVN重组蛋白作为检测抗原建立竞争ELISA检测方法。结果表明：经克隆和间接ELISA筛选，获得了2株能稳定分泌抗小反刍兽疫病毒N蛋白抗体的杂交瘤细胞株，分别命名为5B11和3H103B8。生物学特性鉴定试验表明：5B11和3H103B8抗体类型和亚类均为IgG2b；5B11单抗腹水的效价达1∶819 200，3H103B8达1∶12 800；血清学试验证明2株单抗均能与BacmidPPRVN重组蛋白抗原结合，具有高度的特异性；相加ELISA试验结果显示，5B11和3H103B8 2株单克隆抗体分别识别N蛋白上不同的抗原位点；2株杂交瘤细胞的染色体均为99～104。应用建立的cELISA检测方法对222份血清样品进行PPRV抗体的检测，与参考试剂盒比较得到98.20%的符合率。本研究获得了2株能稳定分泌抗PPRV N蛋白单克隆抗体的杂交瘤细胞株，以单抗5B11作为竞争抗体建立了PPRV的cELISA检测方法。

Abstract: The objective of this study was to prepare specific monoclonal antibodies (mAbs) against nucleoprotein of peste des petits ruminants virus (PPRV) , characterize its properties, and use it in ELISA. After immunization of BALB/c mice with purified recombinant protein BacmidPPRVN, two monoclonal antibodies against PPRV nucleoprotein were successfully prepared by polyethyleneglycol (PEG)mediated fusion of sensitized lymphocytes and myeloma cells and named 5B11 and 3H103B8, respectively. The specificity and biological characterization of the mAbs were identified. A competitive ELISA using PPRV recombinant nucleoprotein as coating antigen and mAb 5B11 as competitive antibody was established. Our results about isotypes and subclasses indicate that 5B11 and 3H103B8 belong to IgG2b, the titres of ascitic fluids of 5B11 was up to 1:819 200 and 3H103B8 was 1:12 800. These mAbs could specifically recognize recombinant protein BacmidPPRVN antigens in a serologic test. Antibodies additivity assay demonstrated that 5B11 and 3H103B8 recognized the different epitopes of PPRV nucleoprotein. The number of chromosome of the hybridoma cell lines was 99 to 104. A total of 222 serum samples were detected in parallel by cELISA and reference cELISA kit.The coincidence rate of the two assaya was 98.15%. We successfully prepared two specific monoclonal antibodies against PPRV nucleoprotein and established the competitive immunofluorescent method for detecting PPRV.

龙云凤;周晓黎;杨俊兴;王琼;祝贺;叶玲玲;董俊;吕建强;王金萍;陈朝银;花群义;杨仕标;尹尚莲;徐维佳;艾军. 小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用[J]. 畜牧兽医学报, 2012, 43(4): 588-595.

ONG Yunfeng;ZHOU Xiaoli;YANG Junxing;WANG Qiong;ZHU He;YE Lingling;DONG Jun;LV Jianqiang;WANG Jinping;CHEN Chaoyin;HUA Qunyi;YANG Shibiao;YIN Shanglian;XU Weijia;AI Jun. Preparation and Preliminary Application of Monoclonal Antibodies against Nucleoprotein of Peste des Petits Ruminants Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(4): 588-595.

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