检测鸡TRIM25蛋白的免疫组化方法  

Immunohistochemistry Assay for Detecting Chicken TRIM25 in Different Tissues

作　　者：禇颖 马文杰 胡卫国 陈茹 邱建华 郭慧君 李宏梅 CHU Ying;MA Wen-jie;HU Wei-guo;CHEN Ru;QIU Jian-hua;GUO Hui-jun;LI Hong-mei(Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University Tai＇an 271018, China)

机构地区：[1]山东农业大学动物科技学院山东省动物生物工程与疾病防治重点实验室山东省畜禽疫病防制工程技术研究中心,泰安271018

出　　处：《畜牧兽医学报》2018年第4期865-870,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划专项(2016YFD0500800);山东省重点研发计划项目(2016CYJS05A02;2017CXGC0308);山东省"双一流"奖补资金

摘　　要：为建立检测鸡TRIM25蛋白表达的免疫组化方法,根据发表的鸡TRIM25基因序列,设计引物,从海兰褐鸡脾中扩增TRIM25基因,构建重组原核表达质粒,通过诱导表达获得重组TRIM25蛋白,经纯化后免疫接种兔和小鼠,获得高效价血清抗体,用该抗体建立免疫组化方法,对鸡肺、脾、肝组织中TRIM25蛋白进行检测。结果显示,克隆的鸡TRIM25抗原基因大小为1 012bp,构建的重组表达质粒能够在大肠杆菌中稳定表达,经纯化后的重组蛋白质能够诱导兔和小鼠产生效价为1∶106以上的血清抗体,该血清抗体免疫组化检测发现鸡肺、脾和肝组织中均有不同程度的TRIM25蛋白表达,在肺组织中主要存在肺泡周围的内皮细胞、淋巴细胞等的细胞质内;脾组织主要出现在脾淋巴细胞和血管内皮细胞等的细胞质中;肝组织中主要出现在肝小叶肝细胞、淋巴细胞以及血管内皮细胞细胞质中。该研究为深入研究鸡TRIM25蛋白的组织分布、动态表达和生物学活性提供物质基础和方法依据。The experiment was conducted to set up the immunohistochemitry assay to detect chicken TRIM25 expression in the chicken’s tissues.Chicken TRIM25 gene was amplified from chicken’s spleen and the gene was cloned into a prokaryotic expression vector pEASY-Blunt E1 to construct recombinant plasmid named as pEASY-Blunt E1-TRIM25.The recombinant plasmid was expressed in E.coli and then identified by SDS-PAGE and Western blot.After being purified,the fusion protein was inoculated to rabbits and BALB/c mice as antigen for preparing polyclonal antibody.The titer of the antibody was detected by ELISA,and the sera antibodies were used to setup immunohistochemistry assay;the tissue sections of chicken’s lung and spleen and liver were assayed.The results showed that TRIM25 was expressed as fusion protein with 48 kD.The titers of the antibody in rabbits’ and mice’s sera were above 1：106.The results of immunohisto-chemistry assay indicated that TRIM25 mainly exists in the cytoplasm of lymphocytes,hepatocyte,and epithelial cells in chicken’s lung,spleen and liver.In conclusion,the immunohistochemitry assay mediated with polyclonal antibody against chicken TRIM25 were successfully set up in this study and TRIM25 distribution in some tissues of chickens was firstly reported.

关 键 词：鸡 TRIM25 多克隆抗体 免疫组化 

分 类 号：S852[农业科学—基础兽医学]