重组人巨噬细胞集落刺激因子受体胞外区蛋白的纯化及单抗的制备

PURIFICATION OF HUMAN MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR EXOCELLULAR PROTEIN AND PREPARATION OF ITS MONOCLONAL ANTIBODY

目的重组人巨噬细胞集落刺激因子受体 (macrophagecolony -stimulatingfactorreceptor,M -CSFR)胞外区蛋白的分离纯化及单抗的制备。方法用pET2 8(+)a构建的c -fms重组体转化大肠杆菌 ,IPTG诱导其表达 ,采用亲和层析进行复性纯化目的蛋白 ,利用细胞融合技术制备并筛选M -CSFR胞外区蛋白的单克隆抗体。结果经转化的大肠杆菌IPTG诱导后可大量表达目的蛋白 ,但形成不溶性的包涵体 ,亲和层析柱上复性可获得可溶性较纯的目的蛋白 ,经细胞融合技术筛选出 1株M -CSFR的单抗细胞。结论柱上复性可获得可溶性复性蛋白。

ObjectiveTo purify the human macrophage colony-stimulating factor receptor ( h- M-CSFR) exocellular protein and prepare its monoclonal antibody.MethodsThe recombinat pET28(+)a-fms was transformed into E coli. By affinity chromatography, the target protein was purified and renatured ,and its monoclonal antibody was prepared by cell hybridization technology.ResultsThe transformed E coli expressed M-CSFR exocellular protein, however, it produced issoluable inclusion bodies when induced by IPTG. After being lysised by denaturants such as 6 mol/L guanidine HCl and refolded on the column of affinity chromatography, purified and renatured target protein was obtained. By cell hybridization technology,the monoclonal antibody of target protein was available.ConclusionRefolding on the column obtained soluable renatured protein.