摘要
利用PCR技术将鸭维甲酸诱导基因Ⅰ(RIG-Ⅰ)helicase区和RD区部分编码序列分别进行扩增,并分别命名为c、d段;克隆到p ET30a原核表达载体,构建了重组原核表达质粒p ET30a-c和p ET30a-d,通过转化BL21(DE3)感受态菌、IPTG诱导、镍柱纯化表达蛋白,将纯化的c、d蛋白免疫小鼠制备单克隆抗体(m Ab),采用ELISA、Western blot和间接免疫荧光试验对单克隆抗体进行鉴定分析。获得鼠抗鸭RIG-Ⅰ单克隆抗体有14株与原核分段表达的c、d蛋白有良好的反应性,其中3株与真核表达的RIG-Ⅰ蛋白有良好的反应性。制备的鼠抗鸭RIG-Ⅰ单克隆抗体为RIG-Ⅰ在抗病毒天然免疫信号转导途径的研究奠定了基础。
The coding regions of helicase domain and RD domain of duck retinoic acid inducible gene Ⅰ （RIG-Ⅰ ） were cloned into pET30a to construct the recombinant vectors pET30a-c and pET30a-d. Then the recombinant vectors were transformed into Escherichia coli BL21. The expressed proteins were induced by IPTG and purified with affinity Ni-eharged resin. Mouse were immunized with purified recombinant proteins to obtain the monoclonal antibody against duck RIG-Ⅰ. The titer of monoclonal antibody was detected by ELISA and the specificity was identified by Western blot and indirect immunofluorescence. The results showed that there were 14 hybridoma cell lines secreting mAb against c and d segment by prokaryotic expression,and 3 of 14 hybridoma cell lines secreting mAb against eukaryotic expressed RIG- Ⅰ were produced. As a conclusion,the preparation of monoclonal antibody against duck RIG- Ⅰ laid a foundation for studying antiviral innate immune signal transduetion pathways.
出处
《中国家禽》
北大核心
2014年第20期17-21,共5页
China Poultry
基金
地方科技攻关计划课题(PC13S16)
"十二五"农村领域国家科技计划课题(2011AA100305-2)
中央级公益性科研院所基本科研业务费(1610302014022)
关键词
鸭RIG-Ⅰ
helicase结构域
RD结构域
原核表达
单克隆抗体
duck RIG- blot and indirect immunofluorescence. The results showed that there were 14 hybridoma cell lines secreting mAb against c and d segment by prokaryotic expression,and 3 of 14 hybridoma cell lines secreting mAb against eukaryotic expressed RIG- Ⅰ were produced. As a conclusion,the preparation of monoclonal antibody against duck RIG- Ⅰ laid a foundation for studying antiviral innate immune signal transduetion pathways.
helicase domain
RD domain
prokaryotic expression
monoclonal antibody
