破骨细胞抑制性凝集素胞外段可抑制骨髓干细胞向破骨细胞分化

Inhibition of Osteoclast-Like Cell Formation by hOCIL Extracellular Domain

目的:在大肠杆菌中表达人破骨细胞抑制性凝集素(OCIL)胞外段重组蛋白,体外检测表达的重组蛋白活性。方法:通过修改OCIL胞外段编码的碱基序列和重组PCR方法得到连续DNA片段;将表达载体pMAL-c2x/hOCIL转化入BL21(DE3)plysS宿主菌,IPTG诱导表达。利用直链淀粉亲和层析柱分离纯化,Western blotting鉴定重组蛋白。体外培养小鼠股骨骨髓细胞,在巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配基(RANKL)诱导骨髓细胞向破骨细胞分化的基础上,加入不同浓度的重组蛋白,观察其对破骨细胞样细胞生成的影响。结果:重组菌中确有融合蛋白MBP-hOCIL表达,其中目的蛋白占纯化液总蛋白的91.36%,纯化后重组融合蛋白产率为24.3mg/L培养基;所制备的重组蛋白对小鼠骨髓破骨细胞样细胞的形成有抑制作用。结论:本研究通过基因工程的方法成功获得了hOCIL胞外段重组蛋白,此蛋白在体外可以明显抑制破骨细胞样细胞的形成。

Objective： To investigate the effect of the osteoclast inhibitory lectin （OCIL） on the formation of osteoclast-like cells in vitro. Methods： Based on the codon preference and degeneration, the codons of OCIL extracellular domain was changed into those preferred by E.coli coding and ligated the fragments with recombinant PCR. By induction of IPTG, the recombinant cells BL21 （DE3） plysS with transfection of pMAL-c2x/hOCIL produced the soluble recombinant protein in the cell lysates. Then the combination activity between maltose binding protein （MBP） and amylose was used to purify the fusion protein. The biological action of the recombinant protein was analyzed by its effect on osteoclast-like cell formation in murine bone marrow cell culture under the stimulation of macrophage-colony stimulating factor （M-CSF） and receptor activator of nucleic factor κB ligand （RANKL）. Results： The productivity of purified recombinant protein was 24.3 mg/L medium. The osteoclast-like cell （OLC） formation was significantly inhibited by the recombinant protein.Conclusion：The recombinant protein hOCIL was successfully obtained from E.coli with biological activity that may inhibit the osteoclast-like cell formation in vitro.