人源抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体的构建

目的构建以人HBsAg单链抗体(HBScFv)为导向作用、α干扰素为治疗作用的融合蛋白酵母表达载体pPICZ-αB/ScFv-IFN-α。方法通过重叠PCR构建目的蛋白质基因,再亚克隆到酵母表达载体pPICZαB中,DNA测序后,转化巴氏毕赤酵母宿主菌GS115 ,菌落PCR、高浓度Zeocin抗性筛选鉴定重组酵母,重组酵母经甲醇诱导后,通过SDS PAGE分析鉴定表达产物。结果DNA测序结果显示构建的目的基因片段(HBScFv IFN-α)的序列与原设计序列相符合;菌落PCR结果显示目的基因已整合到酵母菌中;诱导表达后,SDS PAGE结果显示在相对分子质量约4 4 0 0 0处可见目的蛋白质表达带。结论成功构建了抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体

Construction of a Pastoris expression vector for a fusion protein consisting of anti-HBsAg scfv and interferon-α

PurposeTo construct an Pastoris expression vector for anti-HBsAg ScFv and Interferon-а fusion protein.MethodsThe target protein gene was constructed using DNA overlap PCR technology, and then was cloned to an expression vector pPICZаB.After DNA sequencing,the resulting plasmid pPICZaB-HBScFv-IFN-а was linearized and transformed into P.Pastoris GS115.The recombinant Pichia strains,identified by direct PCR and zeocin-resistant screening of Pichia transformants,were cultured and induced with methanol,andthe expression production was detected by SDS-PAGE.ResultsDNA sequencing confirmed that the target gene has correctly cloned to the expression vector.The results of direct PCR showed that the target gene had correctly transformed into GS115,and a 44 000 recombinant protein was observed by SDS-PAGE after being induced with methanol.ConclusionA Pastoris expression vector for anti-HBsAg ScFv and Interferon-а fusion protein has been constructed successfully.