【摘要】 目的 在毕赤酵母中高效分泌表达与天然人载脂蛋白A-Ⅱ具有相同结构和活性的重组人载脂蛋白A-Ⅱ(rhApoA-Ⅱ)。方法 通过逆转录聚合酶链反应(PCR)法从人肝组织钓取编码人ApoA-Ⅱ的cDNA,将其与pPICZα载体连接,构建真核分泌型重组质粒。重组质粒人ApoA-Ⅱ-pPICZα经过SacI酶切线性化,然后转化毕赤酵母感受态细胞。经过酵母基因组鉴定,筛选重组工程菌,建立rhApoA-Ⅱ的毕赤酵母分泌表达体系,对rhApoA-Ⅱ进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹实验(Western blot)分析。结果 经PCR法克隆的人ApoA-ⅡcDNA序列与GenBank登录序列一致。还原电泳条件下,SDS-PAGE和Westem blot分析均在分子量约8 700处出现特异性条带。结论 成功构建了人ApoA-Ⅱ-pPICZα表达载体,在毕赤酵母菌中高效分泌表达rhApoA-Ⅱ,为进一步研究其结构与功能提供了物质基础。更多还原

【Abstract】 Objective To induce recombinant human apolipoprotein A-Ⅱ(rhApoA-Ⅱ) expression and secretion in pichia pastoris.Methods Reverse transcription polymerase chain reaction(PCR) method was used to transfer the cDNA encoding ApoA-Ⅱ from human liver tissue,which was connected with pPICZa vector to construct recombinant plasmid.The recombinant plasmid ApoA-Ⅱ-pPICZa was purified by Sac Ⅰ enzyme and transformed into pichia pastoris competent cells.The rhApoA-Ⅱ expression system in pichia pastoris was screened through yeast genome identification.Expression of rhApoA-Ⅱ was tested by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot method.Results The ApoA-Ⅱ cDNA sequence cloned by PCR was consistent with GenBank sequence.SDS-PAGE and Western blot analysis showed specific bands at about8 700 molecular weights in electrophoresis analysis.Conclusion Rh ApoA-Ⅱ-pPICZa expression vector can be successfully constructed and expressed in pichia pastoris,providing the material basis for exploration on ApoA-Ⅱstructure and function.更多还原