srhM-CSFR在昆虫细胞中的表达及其配基结合活性分析

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)胞外区的、具有全部配基结合活性区域的前三个免疫球样蛋白结构域的 DNA,将其克隆到杆状病毒载体 pbluebac4.5中 ,与杆状病毒 DNA一同转导昆虫细胞 Sf9.经过 2轮筛选 ,获得了纯化的重组病毒 ,再用重组病毒感染昆虫细胞 ,Western印迹检测证明 srh M- CSFR得到了表达 ,它是分泌到上清液中的糖基化蛋白 .Western印迹分析了不同时间点的 srh M- CSFR表达情况 ,结果表明 srh M- CSFR的表达在 96～ 1 2 0 h时达到最大 .srh M- CSFR的产量约为 1 mg/L,EIA法进行配基结合活性分析表明 ,srh M- CSFR与 M- CSF结合的解离常数为 5nmol/L.

Expression of srhM-CSFR in Insect Cells and Analysis of Binding Activity for its Ligand

Total RNA was extracted from human fresh placeta,and the cDNA fragment encoding the M CSFR extracellular region with the first three immunoglobulin(Ig) like encoding the ligand binding domain was amplified by RT PCR.The srhM CSFR was introduced into baculorvirus vector pbluebac4.5. The Sf9 insect cells were cotransfected with the constructed DNA and baculovirus DNA.The purified recombinant baculovirus was obtained with 2 cycles of screening and srhM CSFR was expressed in Sf9.srhM CSFR could be secreted into supernatants as glycoprotein.The yield of srhM CSFR reached the maximum in the cells cultured for 96～120 h and was assessed to be 1 mg/L.The binding ability of srhM CSFR to M CSF was subjected to EIA assay and the K d value of 5 nmol/L were obtained by Scatchard analysis.The K d value is similar to that of recombinant protein expressed in E.coli ,suggesting that M CSFR glycosylation is of no significance of its binding activity to M CSF.