人骨保护素N端片段在大肠杆菌中的表达及其活性分析

骨保护素 (OPG)成熟肽N端D1～D4结构域仅由 2个外显子编码 .以人基因组DNA作为模板 ,采用重叠延伸PCR得到N端D1～D4域编码序列 ,并在其上游引入 2×His密码子序列 ,然后克隆入载体pQE 30进行表达 ,SDS PAGE表明 8×His融合蛋白主要以包涵体形式存在 ,可被抗OPG抗体识别 .变性条件下通过Ni NTA金属螯合亲和层析对表达产物进行纯化后再经梯度透析进行复性 ,采用破骨细胞样细胞 (osteoclast likecell ,OLC)诱导分化实验来检测重组蛋白的生物活性 ,证实单核 巨噬细胞集落刺激因子 (M CSF)和破骨细胞分化因子 (ODF)可协同促进多核OLC的生成 ,但加入重组OPG片段后 ,OLC生成显著减少 .

Expression of N-terminal Fragment of Osteoprotegerin in E.coli Using a New Strategy and Its Bioactivity Characterization

Osteoprotegerin is a cytokine that potently inhibits the formation and activation of osteoclasts and therefore blocks bone resorption.It is reported that the bioactivity of osteoprotegerin is dependent on the presence of the N\|terminal D1～D4 domain,which is encoded by only two exons(exon 2 and exon 3).The sequence coding for this region was amplified through overlapping extension method.The amplified sequence was added with 2×His sequence at 5′ end,followed by ligation to a sequence in the vector pQE\|30 before the 6×His sequence through another overlapping extension PCR.Then the chimeric molecule was inserted into prokaryotic expression vector pQE\|30 for expression.The expressed 8×His fusion protein existed mainly in the form of insoluble inclusion bodies.The inclusion bodies were washed,followed by purification using Ni\|NTA affinity\|chromatography under denaturing conditions.Then the purified protein was refolded through gradient dialysis.Finally,bioactivity analysis was performed and found that osteoclast differentiation factor and M\|CSF synergistically induced the formation of osteoclast\|like cells(OLC) from bone marrow cells,and the addition of recombinant fusion protein to the culture significantly reduced the OLC number.