鸭瘟病毒单抗的制备及胶体金试纸条检测方法的建立

（College of Animal Science and Technology, Shandong Agricultural University, Tai’an 271000, Shandong）
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
关键词：鸭瘟病毒；gB 蛋白；原核表达；单克隆抗体；胶体金试纸条
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
收稿日期：2015-05-13；接受日期：2016-03-10 基金项目：国家现代农业产业技术体系项目（CARS-43-10） 联系方式：赵丹丹，Tel：17863963319；E-mail：zhaodandan_0925@。通信作者刁有祥，Tel：13605386443；E-mail：yxdiao@
14 期
赵丹丹等：鸭瘟病毒单抗的制备及胶体金试纸条检测方法的建立
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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
0 引言
【研究意义】鸭瘟（duck plague，DP）是由鸭瘟 病毒（duck plague virus，DPV）引起的一种急性、败 血性传染病[1]，以头颈肿胀、食道黏膜和泄殖腔黏膜 出血、头颈部皮下有淡黄色胶冻样渗出为特征。该病 一旦发生，发病急、死亡快、死亡率高，对养鸭业危 害严重。快速诊断是控制鸭瘟的重要措施之一，可以 及时确定病原，以便采取有效防制手段。【前人研究 进展】目前已建立了 PCR、ELISA、间接免疫荧光技 术、微量中和试验等检测鸭瘟的方法[2]，但上述检测 方法均需特殊的仪器设备，且耗时长，不适于在基层 推广应用。胶体金免疫层析技术是近年来发展起来的 集胶体金标记技术、蛋白层析技术于一体的新型快速 免疫检测技术[3-4]。该技术操作简便、耗时短，不需要 特殊的仪器设备[5-6]，特别适合在基层推广应用[7-8]。 【本研究切入点】鸭瘟对养鸭业危害严重，目前已建 立了 PCR、ELISA、间接免疫荧光技术、微量中和试 验等检测鸭瘟的方法，但上述检测方法均需特殊的仪 器设备，且耗时长，不适于在基层推广应用。胶体金 免疫层析技术是近年来发展起来的集胶体金标记技 术、蛋白层析技术于一体的新型快速免疫检测技术。 该技术操作简便、耗时短，不需要特殊的仪器设备，
中国农业科学 2016,49(14):2796-2804 Scientia Agricultura Sinica