摘要
为探索用GAP启动子 (PGAP)取代AOX1启动子 (PAOX1) ,在毕节酵母 (P .pastoris)中组成型表达外源蛋白的可能性 ,应用PCR方法从P .pastoris染色体中扩增了GAP启动子 ,以其取代诱导型表达载体 pPIC9K上的PAOX1,构建了组成型表达载体 pGAP9K。将人血管抑制素 (AS)基因重组于pGAP9K的多克隆位点 ,获得含AS基因的重组质粒 pGAP9K AS。转化P .pastorisGS115 ,对获得的高拷贝转化子P .pastorisGS115 (pGAP9K AS)进行组成型表达 ,同时以诱导型转化子P .pastorisGS115 (pPIC9K AS)作为对照。SDS PAGE结果显示 :组成型转化子于培养 4d后AS的表达水平已达到高峰 ,分泌量为 5 8mg/L ;而诱导型转化子诱导 4d后表达的AS仅是组成型表达的 70 % ,诱导 6d后达到高峰 ,表达量也只是组成型表达系统表达高峰时 (4d)的 86 %。CAM分析和抗癌实验结果显示 :P .pastorisGS115 (pGAP9K AS)和P .pastorisGS115 (pPIC9K AS)表达的AS均具有抑制血管生成和C5 7BL/ 6J实验小鼠的B16黑色素瘤的生长 ,其平均瘤重抑制率分别达到 90 6 1%和 90 5 4 %。以上结果表明 ,以GAP启动子构建的组成型表达系统具有发酵时间较短、表达水平较高、不用甲醇诱导、操作系统比较简单等优点 ,PGAP可以取代PAOX1在P .pastoris中表达AS及其他外源蛋白。
The GAP gene promoter was amplified from P.pastoris GS115 and used to replace the AOX1 promoter(P_(AOX1)) on pPIC9K resulting in plasmid pGAP9K.The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K.pGAP9K-AS was then transformed into P.pastoris GS115.The multi-copy integration transformant P.pastoris GS115(pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P.pastoris.The expression of angiostatin reached its peak after 4 d of culture in P.pastoris GS115(pGAP9K-AS) while the angiostatin expressed in P.pastoris GS115(pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P.pastoris GS115(pGAP9K-AS).The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system.The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%,respectively.The above data indicates that the constitutive promoter P_(GAP) can served as an effective alternative to the inductive promoter P_(AOX1) to express AS and other proteins in P.pastoris.
基金
国家自然科学基金 (编号 :3 9670 0 13 )
广州市"2 2 5"科技工程项目 (编号 :99 Z 0 0 4 0 0 1)资助~~
