人骨保护素N端片段在大肠杆菌中的表达及其活性分析  

Expression of N-terminal fragment of osteoprotegerin in E.coli and its bioactivity characterization

作　　者：戴芳[1] 王宝利[2] 邱明才[3] 

机构地区：[1]兰州大学功能有机分子化学国家重点实验室,甘肃兰州730000 [2]天津市内分泌研究所,天津300052 [3]天津医科大学总医院内分泌科,天津300070

出　　处：《兰州大学学报（自然科学版）》2006年第2期29-33,共5页Journal of Lanzhou University(Natural Sciences)

基　　金：国家自然科学基金(30300171)。

摘　　要：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,PCR 分别扩增骨保护素基因外显子2和外显子3,再以2种 PCR 产物的混合物作为模板,采用重组 PCR 法得到 N 端 D1～D4域编码序列,使该序列与载体 pET42a 部分序列相连,然后克隆入载体 pET42a 进行表达,SDS-PAGE 表明产物主要以包涵体形式存在,可被抗 OPG 多克隆抗体识别．Glutathione Sepharose 4B 亲和层析纯化融合蛋白 GST-hOPG_(D1-4),Xa 切割祛除担体蛋白及进一步分离纯化．采用破骨细胞样细胞诱导分化实验来检测重组蛋白的生物活性,证实该重组蛋白可以抑制 OLC 的生成．Osteoprotegerin is a cytokine that potently inhibits the formation and activation of osteoclasts and therefore blocks bone resorption and it might become an ideal strategy to deal with osteoporosis. It is reported that the bioactivity of osteoprotegerin is dependent on the presence of the N-terminal D1～D4 domain. We note that this region is encoded by only two exons, exon 2 and exon 3. In the present study, we amplified the sequence coding for this region by amplifying the two exons respectively and then linking them through a so-called overlap-extension method. The amplified coding sequence was followed by connection to a sequence in the vector pET42a between the cleavage site of factor Xa and Bgl Ⅱ site, which occurred before the Multiple Cloning Site（MCS） through another overlap extension PCR. Then the chimeric molecule was inserted into a prokaryocytic expression vector pET42a for expression and the expressed fusion protein existed mainly in the form of insoluble inclusion bodies, which were refolded through dialysis, followed by purification using Glutathione Sepharose 4B affinity-chromatography. Then the purified protein was cleaved by factor Xa and the recombinant protein was obtained. Finally, we confirmed that the recombinant protein was biologically active in that it inhibited ODF-induced formation of osteoclast-like ceils from bone marrow cells.

关 键 词：骨保护素 表达 大肠杆菌 

分 类 号：Q786[生物学—分子生物学]