| 广谱型鸡传染性支气管炎病毒N蛋白单克隆抗体的制备与鉴定 |
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| 引用本文： | 林丽婷,赵长润,张文,张愉,雍璐,唐金文,吕迪,韦平,磨美兰.广谱型鸡传染性支气管炎病毒N蛋白单克隆抗体的制备与鉴定[J].南方农业学报,2022,53(9):2674-2682. |
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| 作者姓名： | 林丽婷 赵长润 张文 张愉 雍璐 唐金文 吕迪 韦平 磨美兰 |
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| 作者单位： | 广西大学动物科学技术学院, 广西南宁 530004 |
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| 基金项目： | 广西重点研发计划项目(桂科AB21238003)广西自然科学基金项目(2019GXNS FAA245009,2020GXNSFDA297004) |
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| 摘 要： | 【目的】制备出广谱型鸡传染性支气管炎病毒(IBV)N蛋白单克隆抗体,为后续N蛋白保守区域表位鉴定及IBV通用型快速检测方法的建立打下基础。【方法】通过原核表达IBV广西优势血清型代表株GX-YL5的N蛋白,纯化后免疫BALB/c小鼠,选取血清效价在104以上的免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA筛选后通过小鼠腹水诱生法制备单克隆抗体,采用Western blotting、IFA及间接ELISA等进行效价、亚型、反应特异性、交叉反应谱鉴定,并将制备获得的单克隆抗体应用于免疫组化检测,检测SPF鸡人工感染4株IBV代表变异株(GX-NN130048、GX-NN160421、GX-QZ171023和GX-QZ170728)后不同时段内气管和肾脏中的带毒情况。【结果】制备获得的单克隆抗体N2D5效价大于217,其亚型为IgG2b;Western blotting和IFA鉴定结果表明单克隆抗体N2D5只与IBV发生阳性反应,与新城疫病毒(NDV)、传染性喉气管炎(ILTV)、禽偏肺病毒(a MPV)、传染性法氏囊病(IBDV)、禽白血病病毒(ALV)及马立克氏病毒(MDV)的反应结果均呈阴性;单克隆抗体N2D5能与国内流行的多种基因型及广西目前流行的7种血清型IBV发生交叉反应,包括常用标准株、疫苗株和野毒株。将制备获得的单克隆抗体N2D5应用于免疫组化检测,结果发现在3株鸡源IBV代表性变异株及1株鸭源分离株人工感染SPF鸡后不同时段内的气管和肾脏中均能检测到病毒信号,进一步证明其广谱性。【结论】基于杂交瘤技术制备获得的IBV单克隆抗体N2D5属于IgG2b亚型,具有特异性高、反应谱广的特点,且与IBV的结合能力强,可作为特异性诊断抗体应用于IBV临床诊断及病毒分布规律研究。
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| 关 键 词： | 鸡传染性支气管炎病毒(IBV) N蛋白 单克隆抗体 广谱型 杂交瘤技术 |
| 收稿时间： | 2022-03-14 |
Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus |
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| LIN Li-ting,ZHAO Zhang-run,ZHANG Wen,ZHANG Yu,YONG Lu,TANG Jin-wen,LU: Di,WEI Ping,MO Mei-lan.Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus[J].Journal of Southern Agriculture,2022,53(9):2674-2682. |
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| Authors: | LIN Li-ting ZHAO Zhang-run ZHANG Wen ZHANG Yu YONG Lu TANG Jin-wen LU: Di WEI Ping MO Mei-lan |
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| Affiliation: | College of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China |
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| Abstract: | 【Objective】To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus(IBV),so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method.【Method】N protein of GX-YL5,a representative strain of IBV dominant serotype in Guangxi,was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more,the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA,monoclonal antibody was prepared by ascites-induced method. Western blotting,IFA and indirect ELISA were used to identify the titer,subtype,reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants(GX-N130048,GX-N160421,GX-QZ171023 and GX-QZ170728).【Result】The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV,and were negative with Newcastle disease virus(NDV),infectious laryngotracheitis virus(ILTV),avian metapneumovirus(aMPV),infectious bursal disease virus(IBDV),avian leukosis virus(ALV) and Marek's disease virus(MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi,including commonly used standard strains,vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck,which further proved its broad spectrum.【Conclusion】The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity,wide response spectrum and strong binding ability with IBV.It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution. |
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