Intein融合表达鸡IL-2蛋白的多克隆抗体制备  

Generation and characterization of polyclonal antibodies against chicken IL-2 using IL-2-intein fusion protein

作　　者：邓晨[1] 郑海南[1] 于佩峰 张鑫[1] 吴山力[2] 王梦云[1] 吕岩[1] 艾永兴[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]吉林大学基础医学院,吉林长春130021

出　　处：《中国兽医学报》2015年第6期889-894,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272528);教育部新世纪优秀人才支持计划资助项目(NCET-12-0232);教育部留学回国人员科研启动基金资助

摘　　要：鸡白介素-2(chIL-2)是一种对T淋巴细胞、B淋巴细胞和自然杀伤细胞复制与分化起重要作用的细胞因子,并在机体天然免疫和获得性免疫的发生与维持中发挥重要作用。本研究中,将去除去了N-端信号肽的chIL-2基因(366bp)克隆连接到pTYB1原核表达载体,将chIL-2基因与1 362bp的Intein基因融合在同一开放阅读框内,编码相对分子质量约为71 000的chIL-2-Intein融合蛋白。诱导表达chIL-2-intein融合蛋白,应用chitin-sepharose亲和层析进行融合蛋白纯化。用纯化的chIL-2-intein融合蛋白免疫大白兔制备多克隆抗体。应用昆虫细胞表达系统纯化出的chIL-2-6His蛋白作为抗原,包被ELISA板用于抗体效价的检测,Western blot分析抗体特异性。结果,应用纯化的chIL-2-intein融合蛋白成功制备特异性chIL-2抗体,效价高达4 096 000,且该抗体也能用于Intein标签蛋白的检测。Chicken interleukin 2(chIL-2)plays an important role in the replication,differentiation and maturation of T lymphocytes,B lymphocytes and natural killer cells in the regulation of the immune system.In this study,366 bp chIL-2gene without N-terminal signal sequence was cloned into pTYB1 vector to fused with intein gene(1 362bp)in the same ORF which encodes a 71 000 fusion protein of chIL-2-Intein.The expression of chIL-2-intein was induced by 0.2 mmol/L IPTG.The fusion protein was purified on chitin-sepharose column.Rabbits were immunized with the purified chIL-2-intein fusion protein for the generation of polyclonal antibodies.The 6 Histagged chIL-2protein purified from baculo virus insect cells expression system was used as antigen to coat ELISA plates for polyclonal antibodies titer assay.Western blot was carried out for the testing of polyclonal antibody specificity against chIL-2.The results suggested chIL-2polyclonal antibodies were successfully generated with a high titer of 4.096 million.Moreover,the antibodies can specifically detect chIL-2fromE coli and insect cell,and recognize intein tag as well without visible non-specific band.

关 键 词：INTEIN CHICKEN IL-2 pTYB1 多克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]