优化合成有机磷水解酶opd p基因在毕赤酵母中的表达  

The expression of optimized and synthesized organic phosphorus hydrolase opd p gene in Pichia pastoris

作　　者：王颖 黄灿 孙其濛 马立新[1] 严红[1] WANG Ying;HUANG Can;SUN Qimeng;MA Lixin;YAN Hong

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2018年第1期7-11,23,共6页Journal of Hubei University：Natural Science

基　　金：国家自然科学基金(31300074)资助

摘　　要：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1 149 bp(base pair).基因克隆入p HBM905BDM毕赤酵母表达型质粒,成功构建重组表达质粒p HBM905BDM-opd p,以及多拷贝表达质粒p HBM905BDM-opd p-2C,p HBM905BDM-opd p-3C,p HBM905BDMopd 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/m L.但是随着拷贝数的进一步增加表达量呈现下降的趋势.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 was1149bp(base pair).The recombinant plasmids of pHBM905BDM opd p,pHBM905BDM opd p2C,pHBM905BDM opd p3C,pHBM905BDM opd p4C were constructed and transformed into Pichia pastoris GS115competent 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 of2copies was improved compared with1copy,was increased to0.2U/mL.But the expression level did not increase with the increase of copy number.

关 键 词：毕赤酵母 有机磷水解酶 opd基因 分泌表达 

分 类 号：Q78[生物学—分子生物学] Q933