重组人骨靶向干扰素γ-1b在大肠杆菌中的表达

Expression of Recombinant Human Bone Targeting Interferon Gamma-1b in E.coli

目的建立人骨靶向干扰素γ-1b的高效原核表达体系。方法从Drug Bank数据库下载人干扰素γ-1b氨基酸序列,在羧基端加上10个天冬氨酸标签作为骨靶向分子。采用大肠杆菌偏爱密码子,DNA Star软件分析获得骨靶向性干扰素γ-1b的基因序列。人工合成基因序列并克隆入pET3c质粒表达载体。将上述pET3c-rhIFNγ-1b-D10重组表达载体转入大肠杆菌BL21,筛选高效表达菌株。分离包涵体,8mol/L尿素溶解,利用镍柱亲和层析纯化重组蛋白。Western blot检测目的蛋白的特异性表达。结果测序结果显示所克隆的骨靶向rhIFNγ-1b基因序列正确,筛选出高效表达菌株。经SDS-PAGE显示在相对分子质量19KD处出现特异的目的蛋白表达带,表达量约占全菌蛋白的30%。表达产物以包涵体形式存在,8mol/L尿素变性后经镍柱亲和层析纯化,纯度大于90%,浓度为0.8mg/mL。Western检测显示在19KD处出现特异性目的条带。结论成功在大肠杆菌中表达了人骨靶向干扰素γ-1b,为研究其生物学活性及用于骨硬化症的治疗奠定基础。

Objective To construct a prokaryotic system for high expression of human bone-targeting interferon gamma-1b(rhIFNγ-1b-D10).Methods Download amino acid sequence of the human interferon gamma-1b from the Drug Bank database.Tag the C-terminal of IFNγ-1b with an acidic oligopeptide(a ten residue stretch of Asp)as bone-targeting molecules.Obtain gene sequence of bone-targeting interferon gamma-1b with DNA Star analysis software.Synthetic bone-targeting interferon gamma-1b gene sequence and clone it into the pET3 c plasmid.Transform the recombinant plasmid into E.coli and screen the bacterial strain in which rhIFNγ-1b-D10 is highly expressed.Separate the expressed protein existing in a form of inclusion body,dissolve with 8 mol/L urea,re-naturalize and purify by Nickel column affinity chromatography and detect for specificity by Western blot.Results The cloned rhIFNγ-1b-D10 gene showed a correct sequence,and an E.coli strain for high expression of rhIFNγ-1b-D10 was screened.SDS-PAGE showed a specific protein band with a relative molecular weight of 19 KD.The expressed product existed in a form of insoluble inclusion body and contained about 30%of total somatic protein.After purification,the purity of protein reached90%and the concentration of the targeted protein 0.8 mg/mL.Conclusion Bone-targeting rhIFNγ-1b-D10 protein was successfully expressed in E.coli.It laid a foundation of study on the biological activity of bone-targeting rhIFNγ-1b-D10 as well as treatment for Osteopetrosis.