禽白血病P27蛋白单克隆抗体的制备  

Monoclonal antibody preparation to Avian Leukosis P27 protein

作　　者：李洁琼 罗明展 王雄 胡小敏 张梦杨 姚伦广 LI Jieqiong;LUO Mingzhan;WANG Xiong;HU Xiaomin;ZHANG Mengyang;YAO Lunguang(Henan Provincial Field Scientific Observation and Research Station for Wetland Ecosystem at the Head of the South-North Water Diversion Middle Line,Nanyang 473061,China;Henan Animal and Poultry Health Products Engineering Technology Research Center,Nanyang 473061,China;School of Life Science,Nanyang Normal University,Nanyang 473061,China)

机构地区：[1]河南省南水北调中线渠首湿地生态系统野外科学观测研究站,河南南阳473061 [2]河南省畜禽保健品工程技术研究中心,河南南阳473061 [3]南阳师范学院生命科学学院,河南南阳473061

出　　处：《南阳师范学院学报》2025年第1期46-52,共7页Journal of Nanyang Normal University

基　　金：河南省中原科技创新领军人才项目(244200510040);河南省本科高校研究性教学改革研究与实践项目(2022SYJXLX079)。

摘　　要：为提高禽类养殖场对禽白血病病毒(Avian Leukosis Virus,ALV)的检测效率,采用原核表达系统制备J亚群禽白血病病毒(Avian Leukosis Virus Subgroup J,ALV-J)的P27蛋白,并基于该蛋白制备了高特异性的单克隆抗体。首先,通过BABL/c小鼠抗原免疫反应,获得杂交瘤细胞株诱生的小鼠腹水。随后,利用辛酸-硫酸铵沉淀法和Protein G琼脂凝胶亲和层析柱法对腹水中的单克隆抗体进行纯化。通过酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)对纯化抗体进行效价和特异性鉴定。最后,利用杆状病毒表达系统表达的P27蛋白检测制备的抗体的特异性。研究结果显示,本实验成功获得了4株杂交瘤细胞(3C9、1H15、1H6和7G2)。其中,作为酶标抗体的7G2表现出最佳性能,而3C9则为最佳抗原。间接免疫荧光法(Indirect Immunofluorescence Assay,IFA)的结果显示,单克隆抗体能够与表达禽白血病P27的杆状病毒发生特异性反应。这表明禽白血病病毒P27蛋白单克隆抗体制备成功。In order to improve the detection efficiency of Avian Leukosis Virus(ALV)in poultry farms,the P27 protein of Avian Leukosis Virus Subgroup J(ALV-J)was prepared by a prokaryotic expression system,and prepared a highly specific monoclonal antibody based on this protein in this study.Firstly,mouse ascites induced by hybridoma cell lines were obtained by immunological reaction with BABL/c mouse antigens.Subsequently,monoclonal antibodies were purified from ascites using octanoic acid-ammonium sulfate precipitation and Protein G agar gel affinity chromatography column method.The potency and specificity of the purified antibodies were identified by Enzyme Linked Immunosorbent Assay(ELISA).Finally,the baculovirus expression system was used to express the P27 protein,and the specificity of the prepared antibody was tested.The results showed that four strains of hybridoma cells(3C9,1H15,1H6 and 7G2)were successfully obtained in this experiment.7G2,which was used as an enzyme-labelled antibody,showed the best performance,while 3C9 was the best antigen.The results by Indirect Immunofluorescence Assay(IFA)showed that the monoclonal antibodies were able to react specifically with baculovirus expressing avian leukaemia P27.This indicated that the monoclonal antibody to avian leukaemia P27 protein was successfully prepared.

关 键 词：家禽养殖 禽白血病病毒 单克隆抗体 杆状病毒表达系统 

分 类 号：Q812[生物学—生物工程]