重组人载脂蛋白A-Ⅱ在毕赤酵母中的分泌表达及鉴定

Expression and identification of recombinant human apolipoprotein A-Ⅱ in pichia pastoris

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Sac Ⅰ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-Ⅱ cDNA序列与GenBank登录序列一致。还原电泳条件下，SDS-PAGE和Western 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。

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 pPICZα vector to construct recombinant plasmid. The recombinant plasmid ApoA-Ⅱ-pPICZα 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 about 8 700 molecular weights in electrophoresis analysis. Conclusion Rh ApoA-Ⅱ-pPICZα expression vector can be successfully constructed and expressed in pichia pastoris, providing the material basis for exploration on ApoA-Ⅱ structure and function.