摘要
从人胎盘中提取总RNA,利用RT-PCR技术扩增出巨噬细胞集落刺激因子受体(M-CSFR)胞外具有结合活性区域的cDNA,经平端连接将其克隆到原核表达载体pET-28a的His-Tag下游,转化大肠杆菌BL21(DE3)后,经IPTG诱导,重组的人可溶型M-CSFR(rhM-CSFsR)在宿主菌中获得高效表达,表达量约占菌体总蛋白的38%.重组蛋白经Ni2+组氨酸结合树脂螯合层析柱纯化,获得了纯化的rhM-CSFsR,经SDS-PAGE显示为单一区带,其表观分子量为34kD.用酶联免疫吸附分析(enzyme-linkedimmunosorbentassay,ELISA)证明rhM-CSFsR有明显的M-CSF专一结合活性,Kd值为7.8nmol/L,只有一个M-CSF结合位点.本实验结果显示原核表达的rhM-CSFsR具有明显的配基结合活性,提示M-CSFR的糖基化程度对于其配基结合活性不是必不可少的。
Total RNAs were extracted from human fresh placenta, and the cDNA fragment of the M CSFR extracellular region, which encoded the ligand binding domains, was amplified by RT PCR. The cDNA was then cloned into the expression vector downstream to the His Tag site. After transformation into E.coli BL21 (DE3) host bacteria, the recombinant rhM CSFsR protein was expressed efficiently in inclusion bodies with the yield of 38% total bacteria proteins. The recombinant protein was purified by affinity chromatogaphy using a His Bind Resin column charged with nickel. The rhM CSFsR showed a single band in SDS PAGE and the apparent molecular weight was about 34 kD. Enzyme linked immunosorbent assay showed that rhM CSFsR had obviously specific binding activity for its ligand, the K d for dissociation was 7 8 nmol/L, and rhM CSFsR had one binding site. It suggests that glycosylation may be not essential for M CSFR. This throws a light on the studies of biological function and potential clinical application of rhM CSFsR .
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第5期709-713,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
86 3高技术项目资助!( 10 2 110 10 3 )
