PHLD融合蛋白在毕赤酵母中的构建和表达  

Construction and Expression of PHLD Fusion Protein in Pichia pastoris

作　　者：张新国[1] 郭广君 李坤[1] 孙巧云 郭思佳 马小弟 马国艳 ZHANG Xin-guo;GUO Guang-jun;LI Kun;SUN Qiao-yun;GUO Si-jia;MA Xiao-di;MA Guo-yan(Key Laboratory of Drug Screening and Deep Processing for Traditional Chinese and Tibetan Medicine of Gansu Province, School of Life Science and Engineering,Lanzhou University of Technology,Lanzhou 730050,Gansu,China)

机构地区：[1]兰州理工大学生命科学与工程学院甘肃省中藏药筛选评价及深加工重点实验室,中国甘肃兰州730050

出　　处：《生命科学研究》2018年第6期475-482,共8页Life Science Research

基　　金：国家自然科学基金资助项目(31860004);甘肃省自然科学基金资助项目(17JR5RA128;1107RJZA225);甘肃省高校中藏药筛选评价及深加工重点实验室开放基金资助计划(20180802)

摘　　要：δ-睡眠肽(delta sleep inducing peptide, DSIP)的相对分子质量较小,在体内半衰期较短,因此本研究将蛋白质转导结构域(protein transduction domain, PTD)、人血清白蛋白(human serum albumin, HSA)、连接肽(linker)以及δ-睡眠肽(DSIP)进行融合表达,以获得重组PHLD睡眠肽融合蛋白。研究以pPIC9K/HSA为模板,利用PCR技术进行扩增,将获得的PHLD基因连接到表达载体pPIC9K上,然后转入组氨酸缺陷型毕赤酵母GS115,经G418筛选获得了高表达PHLD融合蛋白的酵母工程菌株,并进一步获得了高纯度的融合蛋白。本研究将为DSIP的深入研究和应用提供技术资料。Delta sleep inducing peptide (DSIP) has a low relative molecular mass and its metabolic half-life is short in vivo, which greatly limits its clinical application. Here the protein transduction domain (PTD), hu-man serum albumin (HSA), linker and DSIP were used to express the recombinant protein named PHLD. The recombination plasmid pPIC9K/PHLD was obtained by PCR amplification with the plasmid pPIC9K/HSA as a template. After PCR identification, restriction endonuclease analysis and DNA sequencing, the recombinant plasmid was transferred into the His-deficient mutant strain GS115. The high-expression strain named No.lut1211 was selected by screening on MM/MD plates with G418, and high-purity fusion proteins were obtained. This would lay the foundation for further study and application of DSIP.

关 键 词：δ-睡眠肽(DSIP) 人血清白蛋白(HSA) 连接肽 蛋白质转导结构域(PTD) 毕赤酵母 融合蛋白 

分 类 号：Q816[生物学—生物工程] R915[医药卫生—微生物与生化药学]