关键词: 溶菌酶;毕赤酵母;聚合酶链反应;基因表达

    摘 要:目的 利用巴斯德毕赤(Pichia pastoris)酵母真核表达系统，构建真核表达载体pPIC9K与人溶菌酶基因(human lysozyme，hLY)的重组质粒pPIC9K-hLY，表达出具有活性的人溶菌酶. 方法 此项研究在本实验室构建的人溶菌酶(hLY)基因与pUC19的重组质粒pUC19-hLY的基础上，通过设计一对引物，用多聚酶链式反应(PCR)扩增出人溶菌酶全基因片段后，将其克隆到巴斯德毕赤酵母分泌型表达载体pPIC9K中，构建重组质粒pPIC9K-hLY，经Bg1Ⅱ酶切线性化后，用电穿孔法将其转入毕赤酵母细胞内.通过G418筛选，选到的高拷贝转化子经PCR检测人溶菌基因与毕赤酵母染色体是否稳定整合，阳性克隆经甲醇诱导进行表达. 结果 序列测定，经PCR扩增后的人溶菌酶基因与文献发表的序列相比，缺失4个碱基.我们决定采用重组PCR法予以纠正，经序列测定结果正确.用PCR检测，人溶菌酶基因与毕赤酵母染色体稳定整合.用甲醇诱导表达并以SDS-PAGE分析，在Mr为15000左右出现一条蛋白带，表达量约占上清总蛋白的0.47.Western Blot证实表达产物具有天然人溶菌酶的抗原性.用黄色小球菌平板溶圈法鉴定表达产物具有人溶菌酶的活性. 结论 成功的构建了重组质粒pPIC9K-hLY并在毕赤酵母中表达了人溶酶基因.

    Cloning and expression of human lysozyme gene in Pichia pastoris

    JIA Xiang-Zhi，YUAN Han-Ying，MA Wen-Yu，LI Yuan，LI Yu-Yang

    1 Department of Microbiology，Fourth Military Med-ical University，Xi'an710033，China，2 Institute of Genetics，Fudan University，Shanghai200433，Chi-na Keywords:muramidase;pichia;polymerase chain reaction;gene expression

    Abstract:AIM To construct a recombinant plasmid be-tween vector pPIC9K and human lysozyme gene.Active hu-man lysozyme was expressed.METHODS Recombinant plasmid pUC19-hLY was amplified by PCR.It was cloned in-to expression plasmid pPICPK and it's lined fragment was transformed into electroporated Pichia pastoris strains.Re-combinant of human lysozyme and Pichia pastoris was ob-tained.Was used G418to screen positive clone ......