禽白血病P27蛋白单克隆抗体的制备
Monoclonal antibody preparation to Avian Leukosis P27 protein
李洁琼 1罗明展 2王雄 2胡小敏 1张梦杨 3姚伦广4
作者信息
- 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;南阳师范学院 生命科学学院,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;南阳师范学院 生命科学学院,河南 南阳 473061
- 3. 南阳师范学院 生命科学学院,河南 南阳 473061
- 4. 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;河南省畜禽保健品工程技术研究中心,河南 南阳 473061;南阳师范学院 生命科学学院,河南 南阳 473061
- 折叠
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.
Abstract
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 pre-pared 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,mon-oclonal 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 iden-tified 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 re-act 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Key words
poultry farming/Avian Leukosis Virus/monoclonal antibodies/baculovirus expression system引用本文复制引用
出版年
2025
国家科技期刊平台
NSTL
万方数据