人破骨细胞形成抑制因子成熟肽基因在大肠杆菌中的融合表达  

Cloning and expression of human osteoclastogenesis inhibitory factor mature peptide gene in E.coli

作　　者：张兴群[1] 王梁华[1] 袁勤生2 缪辉南[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础医学部生物化学与分子生物学教研室,上海200433 [2]华东理工大学国家生物反应器中心,上海200237

出　　处：《第二军医大学学报》2004年第10期1086-1089,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 0 0 0 0 0 78) ;国家高新技术发展规划 ("863"计划 )课题 ( 2 0 0 1AA2 15 44 1)

摘　　要：目的 :在大肠杆菌中高效融合表达人破骨细胞形成抑制因子 (osteoclastogenesis inhibitory factor,OCIF)成熟肽基因。方法 :利用 PCR从人肝细胞文库中扩增得到 OCIF成熟肽编码基因序列 ,将其与 p MD18- T连接 ,转化大肠杆菌 K80 2 ,筛选得到阳性重组克隆载体 p MD18- OCIFm,双酶切重组克隆质粒 p MD18- OCIFm得到 OCIF成熟肽基因 ,将其定向插入p MAL- c2载体中 ,转化大肠杆菌 TB1。筛选得到阳性重组表达子后进行诱导表达。 结果 :所获得的 OCIF成熟肽基因编码序列经测序与 Gen Bank报道的完全一致。所表达的产物经 SDS- PAGE分析可见在相对分子质量 80 0 0 0处出现一条特殊条带 ,与预期的相对分子质量一致。Western印迹证实了表达产物的正确。表达产物在高剂量 (>12 0 ng/L)时可诱导体外培养小鼠成熟破骨细胞的凋亡。 结论 :成功获得了人 OCIF成熟肽基因的克隆并实现了其在大肠杆菌中的融合表达。Objective:To clone and express human osteoclastogenesis inhibitory factor (OCIF) mature peptide gene in E.coli.Methods:Using human liver cell cDNA library as a template, human OCIF mature peptide gene was amplified by PCR and cloned into pMD18-T vector and sequenced. After digested with appropriate restriction enzyme, the gene encoding OCIF mature peptide (OCIFm) was inserted into prokaryotic expression vector pMAL-c2 and transformed into E.coli TB1 for expression by IPTG. OCIFm-MBP fusion protein was identified by SDS-PAGE and Western blot analysis. The bioactivity of fusion protein was identified by osteoclast-like cell apoptosis-inducing assay.Results:The sequence of obtained human OCIFm was completely identical to that of OCIFm in GenBank.SDS-PAGE and Western blot analysis proved that the relative molecular weight of the fusion protein was about 80 000.Fusion protein accounted for about 20% of total bacteria protein and reacted specifically with anti-human OCIF monoclone antibody. The fusion protein induced the osteoclast-like cell apoptosis at higher concentration(>120 ng/L) in vitro.Conclusion:Human OCIFm gene is successfully cloned and expressed in E.coli.

关 键 词：破骨细胞形成抑制因子 基因克隆 融合蛋白 

分 类 号：Q78[生物学—分子生物学]