人破骨细胞形成抑制因子活性域片段在大肠杆菌中的融合表达

Fusion Expression of Gene Fragment at Active Domain of Human Osteoclastogenesis Inhibitory Factor in E.coli

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 (Osteoclastogenesisinhibitoryfactor,OCIF)活性域片段。方法 从中国人正常肝细胞系LO2中提取总RNA ,利用RT PCR得到OCIF活性域编码基因cDNA ,将其与pMD18 T连接 ,转化大肠杆菌K80 2 ,筛选得到阳性重组克隆载体pMD18 OCIFAD ,经双酶切得到OCIF活性域编码片段 ,将其插入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活性结构域编码序列片段经测序与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 6 0 0 0 0处出现一条特殊条带 ,与预期的相对分子质量一致。Westernblot证实了表达产物能与抗人OCIF单抗发生特异性反应。结论 已成功地获得了人OCIF活性结构域编码片段的克隆 ,并实现了其在大肠杆菌中的融合表达。表达产物对体外培养破骨细胞的骨吸收功能有明显的抑制作用。

Objective To highly express the gene fragment at active domain of human osteoclastogenesis inhibitory factor (OCIF) in E.coli .Methods Amplify the cDNA encoding the active domain of OCIF by RT-PCR,using normal Chinese liver cell line LO2 as a template.Clone the PCR pr oduct into plasmid pMD18-T and transform to E.coli K802.Screen recombinant plasmid pMD18-OCIF AD and digest with EcoR I and Sal I to obtain the gene fragment encoding the active domain of OCIF.Insert the obtained gene fragment in to pMAL-c2 vector and transform to E.coli TB1.Screen the positive recombina nts for expression under induction of IPTG.Identify the expressed product by SDS -PAGE and Western blot. Results The sequence of obtained gene fragment at active domain of OCIF was completely consistent with that reported in GenBank.The expressed produ ct,containing about 20% of total somatic protein,showed a band with a relative m olecular weight of 60 000 on SDS-PAGE profile,which was consistent with that ex pected.Western blot proved that the expressed fusion protein reacted specificall y with McAb against human OCIF. Conclusion The gene fragment encoding active domain of OCIF was success fully cloned and highly expressed in E.coli.The expressed product showed sig nificant inhibiting effect on the bone adsorption function of osteoclast culture d in vitro. [