摘要
为获取DnaJ蛋白多克隆抗体,将截短的环形泰勒虫DnaJ蛋白C末端核酸序列构建在pET-30a(+)质粒并转化至Rosetta(DE3)感受态细胞,经终浓度1 mmol/L IPTG 37℃诱导表达,蛋白可溶性分析结果显示,该重组蛋白主要以包涵体的形式存在。SDS-PAGE结果显示,纯化后的蛋白分为2条,其中一条蛋白大小约为41.65 ku,与预期结果相符,另外一条蛋白可能因为翻译提前终止比预期条带位置偏低。将纯化后的重组蛋白与弗氏佐剂混匀乳化后,皮下分点注射BALB/c小鼠,制备小鼠源DnaJ蛋白多克隆抗体。通过Western-blot验证,发现截短后的环形泰勒虫DnaJ蛋白与抗His单克隆抗体、环形泰勒虫阳性血清和抗DnaJ多克隆抗体均能反应,表明该蛋白产物具有良好的反应原性。获得的多克隆抗体可用于研究环形泰勒虫DnaJ蛋白的功能。
In order to obtain polyclonal antibody against DnaJ protein,the C-terminal part of Theileria annulata DnaJ gene was constructed into pET-30a(+)plasmid and transformed into Rosetta(DE3)competent cells.The DnaJ protein was mainly expressed in inclusion bodies by induction of 1 mmol/L IPTG at 37℃.The purified protein showed two bands in SDS-PAGE gel,one of them was around 41.65 ku which was similar to the expected size.While the other protein band was migrated at 35 ku which might be caused by early termination of translation.The purified protein was mixed with Freund’s adjuvant at the ratio of 1∶1 in volume and was used to immunize BALB/c mice to prepare polyclonal antibody.With the Western-blot results,the recombinant DnaJ protein could react with the mouse-derived polyclonal antibody,T.annulata positive serum and monoclonal antibody against His tag,respectively.In conclusion,the DnaJ protein was expressed and purified,and mouse-derived polyclonal antibody against the DnaJ protein was obtained and had good reactionogenicity.The obtained materials will be helpful for further functions studies of T.annulata DnaJ protein.
作者
曹天行
李志
史康妍
石苗
刘爱红
马全英
关贵全
李有全
殷宏
罗建勋
刘军龙
CAO Tian-xing;LI Zhi;SHI Kang-yan;SHI Miao;LIU Ai-hong;MA Quan-ying;GUAN Gui-quan;LI You-quan;YIN Hong;LUO Jian-xun;LIU Jun-long(State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第12期1540-1547,共8页
Chinese Veterinary Science
基金
国家自然科学基金项目(31972706)。
