摘要
通过ProteinA亲和层析法分离纯化草鱼血清中的免疫球蛋白IgM,SDS-PAGE检测产物的纯度及分子量,用纯化产物免疫兔制备多克隆抗体,并用辣根过氧化物酶标记此免抗草鱼IgM抗体,用于检测经草鱼出血病病毒广东株(GCRV-GD108)衣壳蛋白VP4及VP5免疫草鱼后产生的抗体水平。结果表明,纯化后的草鱼IgM轻链及重链分子量分别在25 kD和75 kD左右,所制备的草鱼标记抗体可应用于草鱼血清IgM水平的快速检测。建立了标记抗体检测草鱼抗血清的方法,为进一步研究病原体入侵感染机制、草鱼的免疫防御机理和疫苗效果评价等提供便捷的血清学检测方法。
Protein A affinity chromatography was used to purify immunoglobulin IgM from grass carp serum, and the purity and molecular weight of the product was analyzed by SDS-PAGE. Rabbit polyclonal antibodies of the purified grass carp IgM were prepared and detected by ELISA. The results showed that molecular weights of the light chain and heavy chain of the purified grass carp IgM were about 25 kD and 75 kD, respectively, and high titers of rabbit anti-grass carp IgM antiserum was obtained. Horseradish peroxidase was used to label the rabbit anti-grass carp IgM antibody. The labeled antibody was used to detect the antibody levels in serum of grass carp after immunizing by capsid protein VP4 and VP5 of grass carp hemorrhage virus isolated from Guangdong province （ GCRV-GD108 ） . The results revealed that the labeled anti-grass carp IgM antibody could be used for the rapid detection of the IgM levels in grass carp serum. In this study, method to detect IgM levels in grass carp serum was established, which would provide a convenient method of serological testing for further study of pathogen invasion mechanism, grass carp immune defense mechanism and evaluation of grass carp vaccine effectiveness.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第4期194-200,共7页
Biotechnology Bulletin
基金
广东省重点科技项目(2008A020100016)
广东省海洋渔业科技项目(A200899F01,A201101G01)
广州市和荔湾区科技项目(2009J1-C021,20084411115)
