传染性支气管炎病毒N蛋白广谱单克隆抗体的制备与鉴定  

Preparation and Identification of Broad-spectrum Monoclonal Antibodies Against Infectious Bronchitis Virus N Protein

作　　者：杜旭彬 赵佳[1,2] 郭梦娇 李敏 张成成 吴艳涛 张小荣[1,2] DU Xubin;ZHAO Jia;GUO Mengjiao;LI Min;ZHANG Chengcheng;WU Yantao;ZHANG Xiaorong(College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏高校重要动物疫病与人兽共患病协同创新中心,江苏扬州225009

出　　处：《中国家禽》2021年第5期27-32,共6页China Poultry

基　　金：国家重点研发计划项目(2017YFD0500703-2);现代农业产业技术体系建设专项资金(CARS-40-K16);江苏省高校优势学科(2018年)项目;扬州大学“高端人才支持计划”(2016年)。

摘　　要：为制备能与多种基因型传染性支气管炎病毒(IBV)反应的广谱性单克隆抗体,反应谱能够覆盖当前在国内流行的主要优势基因型毒株。通过RT-PCR克隆了QX型IBV CK/CH/JS/2010/12株N基因,分别插入原核表达载体pET-32a(+)和pGEX-6P-1构建了重组质粒pET-N和pGEX-N,并在大肠杆菌中进行原核表达,通过亲和纯化获得His-N和GST-N两种重组融合蛋白。用GST-N融合蛋白免疫BALB/c小鼠,通过淋巴细胞杂交瘤技术取免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行融合,以重组蛋白His-N作为检测抗原建立间接ELISA方法筛选阳性杂交瘤细胞。经过筛选和亚克隆,共获得12株稳定分泌单克隆抗体的杂交瘤细胞株。Western blot结果显示8株单克隆抗体能与测试的所有基因型IBV发生特异性反应,另外4株可以与793B型4/91株以外的其他毒株发生反应。本研究成功获得了广谱性IBV单克隆抗体,为进一步研究建立通用性IBV抗原检测方法奠定了基础。The objective of this study was to prepare broad-spectrum monoclonal antibodies that could react with a variety of genotypes of infectious bronchitis virus(IBV), and the response spectrum could cover the dominant genotypes currently prevalent in China. The N gene of CK/CH/JS/2010/12 strain of QX-type was cloned by RT-PCR, and then inserted into prokaryotic expression vector pET-32 a(+) and pGEX-6 P-1, respectively, to construct recombinant plasmids pET-N and pGEX-N. Two recombinant fusion proteins, His N and GST-N, were prokaryotic expressed in Escherichia coli and purified by affinity chromatography method. Balb/c mice were immunized with recombinant protein GST-N.Spleen cells of immunized mice were harvested and fused with myeloma cells SP2/0 by lymphocyte hybridoma technique.The positive hybridoma cells were screened by indirect ELISA method established by using recombinant protein His-N as detection antigen. After screening and subcloning, 12 hybridoma cell lines were obtained. Western blot results showed that 8 of the monoclonal antibodies could react specifically with all the genotypes of IBV tested in this study,and the other 4 antibodies could react with all strains other than 4/91 of 793/B-type. In conclusion, broad-spectrum monoclonal antibodies against IBV were successfully obtained, which laid a foundation for further research to establish a universal method for detecting IBV antigen.

关 键 词：传染性支气管炎病毒 N蛋白 单克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]