优化合成有机磷水解酶opd p基因r在毕赤酵母中的表达

摘要

In this study, code-optimized organophosphorus hydrolase gene ( opd) and bio-brick method were used to construct opd multi-copy vector to increase the expression in Pichia pastoris.The original opd gene was designed to synthesize the new organophosphatase gene according to Pichia pastoris and six His-Tags were added at the C-terminus.The total length of the synthesized opd p gene was 1149 bp( base pair) .The recombinant plasmids of pHBM905BDM-opd p, pHBM905BDM-opd p-2C, pHBM905BDM-opd p-3C, pHBM905BDM-opd p-4C were constructed and transformed into Pichia pastoris GS115 competent cells.The results showed that OPD P protein was secreted and expressed under the AOX1 ( alcohol oxidase gene ) promoter control, which was induced by methanol.Western Blot showed that the target protein was expressed in the four transformed strains, and the expression level of 2 copies was improved compared with 1 copy, was increased to 0.2 U/mL.But the expression level did not increase with the increase of copy number.%拟验证通过密码子优化有机磷水解酶(opd)基因,和生物砖方法构建opd多拷贝载体,以提高在毕赤酵母中的表达量.将原始的opd基因依照毕赤酵母偏爱密码子设计合成新的有机磷水解酶基因,并在C端添加了6个His-Tag融合蛋白标签.所合成的opd p基因全长1149 bp(base pair).基因克隆入pHBM905BDM毕赤酵母表达型质粒,成功构建重组表达质粒pHBM905BDM-opd p,以及多拷贝表达质粒pHBM905BDM-opd p-2C,pHBM905BDM-opd p-3C,pHBM905BDM-opd p-4C,转化毕赤酵母感受态细胞GS115.实验结果表明,经甲醇诱导后,在AOX1(乙醇氧化酶基因)启动子调控下,获得OPD P蛋白分泌表达,Western Blot检测4个转化的菌株中都表达了目的蛋白,且两拷贝表达量较一拷贝有提高,达到0.2 U/mL.但是随着拷贝数的进一步增加表达量呈现下降的趋势.