应用Intein融合的鸡IL-2蛋白制备多克隆抗体  

Generation and Characterization of Polyclonal Antibodies against Chicken IL-2 Using IL-2-Intein Fusion Protein

作　　者：邓晨[1] 于佩峰 张鑫[1] 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,长春130062

出　　处：《吉林农业大学学报》2015年第3期368-374,共7页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(31272528);教育部新世纪优秀人才支持计划项目(NCET-12-0232);教育部留学回国人员科研启动基金项目[教外司留(2013)693号]

摘　　要：研究Intein标签与目的蛋白chIL-2融合作为抗原,进行高效价兔抗chIL-2蛋白多克隆抗体的制备,将去除去了N-端信号肽的chIL-2基因(366 bp)克隆连接到p TYB1原核表达载体,将chIL-2基因与1 362 bp的Intein基因融合在同1开放阅读框内,编码分子量约为71 ku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。结果表明: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 （chlL-2） plays an important role in replication, differentiation and maturation of T lymphocytes, B lymphocytes and natural killer ceils as well as in the regulation of the immune system. In this study, 366 bp chlL-2 gene without N-Terminal signal sequence was cloned into pTYB1 vector, and fused with Intein gene （1 362 bp） in the same ORF into a chlL-2- Intein fusion protein whose encoding molecular weight was about 71 ku. The expression of chlL-2-In- tein was induced by 0. 2 mmol/L IPTG. The fusion protein was purified on Chitin-Sepharose column. Rabbits were immunized with the purified chlL-2-Intein fusion protein for the generation of poly- clonal antibodies. The 6His-tagged chIL-2 protein purified from Baculovirus 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 chlL-2 polyclonal antibodies were successfully generated with a high titer of 4. 096 million. Moreo- ver, the antibodies can specifically detect chIL-2 from E. coli and insect cell, and recognize Intein tag as well without visible non-specific band.

关 键 词：内肽酶 鸡白介素-2 pTYB1 多克隆抗体 

分 类 号：Q785[生物学—分子生物学] S831.2[农业科学—畜牧学]