摘要
本研究通过RT-PCR从正常鸡外周血淋巴细胞总RNA中扩增chβ2m基因全长片段,然后将其克隆至原核表达载体pET-32a(+),转化大肠杆菌BL21(DE3),用IPTG对其进行诱导表达,利用HiTrap亲和柱对表达产物进行纯化。结果显示,采用RT-PCR扩增出chβ2m基因全长约360 bp,编码120个氨基酸,与基因库公布的相一致,同源性为100%;经0.8 mM IPTG诱导表达后,SDS-PAGE分析发现chβ2m融合蛋白主要以可溶性形式存在于细菌裂解上清;利用His标签纯化该蛋白,SDS-PAGE分析仅见约34 kDa大小的chβ2m融合蛋白条带;Western-blot分析表明,纯化后的chβ2m融合蛋白与特异单克隆抗体反应呈现特异性的条带。以上结果证实,本研究成功表达并获得了纯化的可溶性chβ2m融合蛋白,为进一步对chβ2m结构及其功能研究奠定基础。
chβ2m gene was amplified from normal chicken peripheral blood lymphocytes by RT-PCR. It was cloned into pET-32a（＋） vector and induced by IPTG, then purified with HiTrap affinity column. The results showed that the sequence of chβ2m gene amplified from chickens by RT-PCR was the same as that previously reported （M84767, AY989898, Z48921） and consists of 360 bp encoding 120 amino acids （aa）. Most of the soluble recombinant chβ2m proteins existed in the bacterial supernatant induced with 0.8 M IPTG. Only one band of molecular weight of 34 kDa was purified by ion-exchange chromatography. Western blotling assay demonstrated that anti-His-tag monoclonal antibody could specifically recognize the recombinant protein. This work would provid us the basis materials for further study of the structure and functions of chβ2m.
出处
《中国动物传染病学报》
CAS
2014年第1期63-68,共6页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金项目(31272560
31072135)
教育部创新团队项目(IRT0978)
江苏省优势学科和江苏省教育厅重大基础研究(12KJA23001)项目资助
