重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定

Expression and Identification of Recombinant Human Apolipoprotein C-I 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C-I具有相同结构和活性的重组人载脂蛋白C-I(rhApoC-I)。方法:RT-PCR法自人肝组织调取编码人ApoC-I的cDNA,构建真核分泌型表达载体pPICZα/hApoC-I。重组质粒线性化后转化毕赤酵母感受态细胞,甲醇诱导表达,建立rhApoC-I的毕赤酵母表达体系。对rhApoC-I进行Western blot分析和体外活性研究。结果:经PCR法克隆的hApoC-I cDNA序列与GenBank登录序列一致。SDS-PAGE和Western blot分析均在分子量约6.6kDa出现特异性条带,2L发酵条件下表达量达到80mg/L。结论:首次在毕赤酵母菌中高效分泌表达rhApoC-I,并确定其具有抑制血小板衍生生长因子诱导的平滑肌细胞增殖的抑制作用,为进一步研究其结构与功能提供物质基础。

Apolipoprotein C-I （ApoC-I） is a small, basic apolipoprotein which is mainly secreted by the liver as a component of triglyceride-rich lipoproteins and high density lipoproteins whose importance in plasma lipoprotein metabolism is increasingly evident. Therefore, a Pichia pastoris expression system was first used to obtain a high level expression of secreted, recombinant human ApoC-I （rhApoC-I）. The full-length ApoC-I encoding sequence, gained by RT-PCR, was inserted into the pPICZotC vector and transform Pichia pastoris strain X33 after sequencing. High expression transformants with zeocin resistance were detected by SDS-PAGE and Western blot analysis. The antiproliferation effects of rhApoC-I on rat coronary artery smooth muscle cells was analysed by the methylthiazoletetrazolium （MTY） assay. It was showed that there were rhApoC-I （6.6kDa） in the culture supernatant induced by methanol. The expression level of rhApoC-1 was 80mg/L. RhApoC-I can inhibit the proliferation which induced by PDGF remarkably. Ground on this, the composition and functions of rhApoC- I can be studied.