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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.
Total RNA was extracted from human placenta and the first three immunoglobulin-like protein structures with full ligand-binding activity were amplified by RT-PCR from the extracellular domain of macrophage colony stimulating factor receptor (M-CSFR) Domain was cloned into the baculovirus vector pbluebac4.5 and transduced with the baculovirus DNA to Sf9 insect cells.After 2 rounds of screening, the purified recombinant virus was obtained, then the recombinant virus was used to infect insect cells and Western Western blot analysis of srh M-CSFR expression at different time points showed that the expression of srh M-CSFR was 96 ~ 120 h, and the yield of srh M-CSFR was about 1 mg / L. The ligand binding activity assay by EIA showed that the dissociation constant of srh M-CSFR binding to M-CSF was 5 nmol / L.