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

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pMD18-T连接,转化大肠杆菌K802,筛选得到阳性重组克隆载体pMD18-OCIFm,双酶切重组克隆质粒pMD18-OCIFm得到OCIF成熟肽基因,将其定向插入pMAL-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GenBank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80000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(＞120ng/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.

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

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.