摘要
为了制备猪白细胞介素21(porcine interleukin-21,pIL-21)的多克隆抗体,采用RT-PCR从猪外周血单个核细胞(PBMC)扩增获得pIL-21基因,然后将其克隆至大肠杆菌表达载体pET-32a(+)之中,转化至大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE和Western-blot分析结果显示,pIL-21以可溶性蛋白形式表达,分子质量为35ku,具有良好的反应原性。经过Ni-NTA镍离子亲和层析纯化,获得纯化的重组蛋白pIL-21。用其免疫BALB/c小鼠制备抗pIL-21血清抗体。Western-blot分析结果显示,该抗体可与杆状病毒表达的重组Cap-pIL-21、ConA和PHA共刺激的猪PBMC细胞发生特异性反应,ELISA抗体效价可达1∶12 800。上述研究结果为pIL-21的生物学功能研究奠定了重要基础。
In order to prepare polyclonal antibodies against porcine interleukin-21(pIL-21),apIL-21 gene was amplified from porcine peripheral blood mononuclear cell(PBMC)by RT-PCR and cloned into pET-32a(+)to construct a recombinant plasmid.The recombinant plasmid pET-32a(+)-pIL-21 was expressed in Escherichia coli BL21(DE3)after induction with IPTG.SDS-PAGE and Western-blot analyses showed that the recombinant pIL-21 protein was mainly soluble with a molecular weight of 35 ku and had a high reactionogenicity.The recombinant pIL-21 protein was purified through Ni-chelating affinity chromatography and the antibodies against pIL-21 were acquired after being immunized BALB/c mice.Westernblot analysis showed that the prepared antisera could specifically react with Cap-pIL-21 expressed in Sf9 and porcine PBMC.The antibody titer was up to 1∶12 800 by ELISA.The above-mentioned result laid the foundation for further studies on biological functions of pIL-21 protein.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第4期352-355,共4页
Chinese Veterinary Science
基金
教育部科技项目(313031
20120097110043)
国家自然科学基金项目(31230071)
国家生猪产业技术体系专项(CARS-36)
关键词
猪
白细胞介素21
原核表达
多克隆抗体
swine
interleukin-21
prokaryotic expression
polyclonal antibody
