禽多瘤病毒VP4蛋白单克隆抗体的制备及其间接免疫荧光检测方法的初步建立  

Preparation of monoclonal antibody against avian polyomavirus VP4 protein and preliminary establishment of an indirect immunofluorescence detection method

作　　者：汪溪 翟天舒 许冠龙 王嘉[2] 孔冬妮[2] 邓永[2] 闫佳佳 薛青红[2] 印春生[2] 何后军[1] 陈小云[2] 毛娅卿[2] WANG Xi;ZHAI Tianshu#;XU Guanlong;WANG Jia;KONG Dongni;DENG Yong;YAN Jiajia;XUE Qinghong;YIN Chunsheng;HE Houjun;CHEN Xiaoyun;MAO Yaqing(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang,330045,China;China Institute of Veterinary Drug Control,Beijing 100081,China;Faculty of Animal Medicine,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]中国兽医药品监察所,北京100081 [3]山西农业大学动物医学学院,山西太谷030801

出　　处：《中国兽医科学》2024年第5期584-594,共11页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32002273);中国兽医药品监察所公益性专项(GY202105);兽药行业公益性重点专项(GY202104)。

摘　　要：为制备禽多瘤病毒(avian polyomavirus,APV)VP4蛋白单克隆抗体,初步建立APV的间接免疫荧光检测方法,本研究通过原核表达APV VP4蛋白,纯化后免疫BALB/c小鼠制备单克隆抗体,以制备的VP4单克隆抗体为一抗,FITC-山羊抗小鼠IgG为二抗,优化反应条件。结果表明,筛选获得15株阳性细胞株,经亚型鉴定,其中2株单克隆抗体的重链为IgG2b类型,13株重链为IgG1类型,轻链均为κ型。选择3株细胞制备单克隆抗体,这3株抗VP4单克隆抗体浓度分别为2.9、2.6、3.2 mg/mL;亲和常数分别为1.06×10^(10)、2.95×10^(9)、9.60×10^(9)。其中两株效价均为1∶204800,另一株的效价为1∶51200。经Western-blot和间接免疫荧光试验(IFA)鉴定,这3株单克隆抗体均能与APV发生特异性反应;本研究建立的间接免疫荧光检测方法的最适条件为:Anti-A-VP4-151∶1000倍稀释,4℃条件下过夜孵育,二抗1∶200稀释,37℃条件下孵育1 h。以建立的方法检测细胞中的减蛋综合征病毒(EDSV)、网状内皮组织增生症病毒(REV)、禽白血病病毒(ALV)、新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)和APV,只有APV的检测结果显示阳性,检测其他病毒的结果均为阴性。应用该单克隆抗体建立的间接免疫荧光方法在细胞上能检出至少5 TCID50 APV感染。本研究制备的单克隆抗体和建立的间接免疫荧光方法为APV感染的流行病学调查和实验室诊断奠定了基础。In order to prepare a monoclonal antibody against VP4 protein of avian polyomavirus(APV),and to initially establish an indirect immunofluorescence detection method for APV.the monoclonal antibody was prepared by prokaryotic expression of APV VP4 protein after purification and immunising BALB/c mice,and the reaction conditions were optimized with the prepared against VP4 monoclonal antibody as the primary antibody and FITC-goat anti-mouse IgG as the secondary antibody.The results showed that 15 positive cell lines were screened and identified by subtype.The heavy chain of 2 monoclonal antibodies was IgG2b type,the heavy chain of 13 monoclonal antibodies was IgG1 type,and the light chain wasκtype.Three cell strains were selected to prepare monoclonal antibodies.The concentrations of against VP4 monoclonal antibodies of the 3 strains were 2.9,2.6,and 3.2 mg/mL,respectively.The affinity constants were 1.06×10^(10),2.95×10^(9),and 9.60×109,respectively.The titer of two strains was both 1:204800,and the titer of the other strain was 1:51200.Western-blot and indirect immunofluorescence assay(IFA)showed that all 3 monoclonal antibodies could react specifically with APV.The optimal conditions for the indirect immunofluorescence detection method establi shed in this study were as follows:1∶1000 dilution of Anti-A-VP4-15,overnight incubation at 4℃,1∶200 dilution of secondary antibody,incubation at 37℃for 1 h.The established method was used to detect egg drop syndrome virus(EDSV),reticuloendotheliosis virus(REV),avian leukosis virus(ALV),Newcastle disease virus(NDV),avian infectious bronchitis virus(IBV)and APV in the cells.Only the results for APV were positive,and tests for other viruses were negative.The indirect immunofluorescence method established using this monoclonal antibody was able to detect at least 5 TCID50 APV infections on cells.The monoclonal antibody and the indirect immunofluorescence method developed in this study provide a basis for epidemiological investigation and laboratory diagnosis of APV

关 键 词：禽多瘤病毒 VP4蛋白 原核表达 单克隆抗体 间接免疫荧光试验 

分 类 号：S852.659.2[农业科学—基础兽医学]