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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。
Objective To isolate and purify the extracellular region of recombinant human macrophage colony-stimulating factor receptor (M-CSFFR) and to prepare monoclonal antibodies. Methods Recombinant c -fms constructed by pET2 8 (+) a was transformed into E. coli and induced by IPTG. The recombinant protein was purified by affinity chromatography and the extracellular domain of M-CSF was prepared by cell fusion Monoclonal antibody. Results After induced by IPTG, the recombinant protein could express large amount of target protein, but insoluble inclusion body was formed. On the affinity column, the target protein could be obtained by renaturation on IPTG. One M-CSFFR was screened by cell fusion technique Of mAb cells. Conclusion Soluble refolding protein can be obtained by refolding the column.