CAJ | 학술논문

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.pastoris GS115,对获得的高拷贝转化子P.pastoris GS115(pGAP9K-AS)进行组成型表达,同时以诱导型转化子P.pastoris GS115(pPIC9K-AS)作为对照.SDS-PAGE结果显示:组成型转化子于培养4 d后AS的表达水平已达到高峰,分泌量为58 mg/L;而诱导型转化子诱导4 d后表达的AS仅是组成型表达的70%,诱导6 d后达到高峰,表达量也只是组成型表达系统表达高峰时(4 d)的86%.CAM分析和抗癌实验结果显示:P.pastoris GS115(pGAP9K-AS)和P.pastoris GS115(pPIC9K-AS)表达的AS均具有抑制血管生成和C57BL/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90.61%和90.54%.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.
위탐색용GAP계동자(PGAP)취대AOX1계동자(PAOX1),재필절효모(P.pastoris)중조성형표체외원단백적가능성,응용PCR방법종P.pastoris염색체중확증료GAP계동자,이기취대유도형표체재체pPIC9K상적PAOX1,구건료조성형표체재체pGAP9K.장인혈관억제소(AS)기인중조우pGAP9K적다극륭위점,획득함AS기인적중조질립pGAP9K-AS.전화P.pastoris GS115,대획득적고고패전화자P.pastoris GS115(pGAP9K-AS)진행조성형표체,동시이유도형전화자P.pastoris GS115(pPIC9K-AS)작위대조.SDS-PAGE결과현시:조성형전화자우배양4 d후AS적표체수평이체도고봉,분비량위58 mg/L;이유도형전화자유도4 d후표체적AS부시조성형표체적70%,유도6 d후체도고봉,표체량야지시조성형표체계통표체고봉시(4 d)적86%.CAM분석화항암실험결과현시:P.pastoris GS115(pGAP9K-AS)화P.pastoris GS115(pPIC9K-AS)표체적AS균구유억제혈관생성화C57BL/6J실험소서적B16흑색소류적생장,기평균류중억제솔분별체도90.61%화90.54%.이상결과표명,이GAP계동자구건적조성형표체계통구유발효시간교단、표체수평교고、불용갑순유도、조작계통비교간단등우점,PGAP가이취대PAOX1재P.pastoris중표체AS급기타외원단백.
The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter( PAox1 )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. pasto-ris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the an-giostatin 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 PGAP can served as an effective alternative to the inductive promoter PAox1 to ex-press AS and other proteins in P. pastoris.