组成型表达重组人C-反应蛋白的毕赤酵母菌株构建  

Construction of Pichia pastoris Strains Constitutively Expressing Recombinant Human C-reactive Protein

作　　者：李军明[1] 林衡[2] 葛高顺[1] 张立超[1] 于昉[1] 胡学军[1] 

机构地区：[1]大连大学医学院医学研究中心,辽宁大连116622 [2]大连大学生命科学与技术学院,辽宁大连116622

出　　处：《生物技术》2013年第1期22-25,共4页Biotechnology

基　　金：国家自然基金项目("单链抗体在大肠杆菌体内定点糖基化及其通过糖链以共价键定向固定的研究";No.31070822/C080602);辽宁省高等学校科研计划项目("糖基化多肽聚合物的生物合成及其抑制毒素的活性研究";No.L2010015)资助

摘　　要：目的:构建组成型表达重组人C-反应蛋白(Recombinant human C-reactive protein,rhCRP)的毕赤酵母工程菌株,表达、纯化rhCRP并鉴定其免疫反应性。方法:将设计合成的rhCRP基因克隆到表达载体pGAPZαA上并转化至毕赤酵母X-33中进行组成型分泌表达,通过His亲和层析柱纯化rhCRP。分别采用SDS-PAGE、Western blot和间接ELISA法检测目的蛋白并鉴定其免疫反应性。结果:重组表达载体pGAPZαA/rhCRP经酶切及DNA测序鉴定构建成功。重组毕赤酵母工程菌株成功组成型表达23kDa的rhCRP,27h即达到最大表达水平,表达量约3mg/L。经一步分离纯化获得纯度为96.58%的rhCRP,经间接ELISA检测表明其具有免疫反应性。结论:成功构建了组成型分泌表达rhCRP的毕赤酵母菌株,为进一步自主研发人CRP检测试剂奠定了基础。Objective： This study was designed to engineer Pichia pastoris strains which can constitutively express recombinant human C - reactive protein, to express and purify rhCRP, and identify its immunity reactivity. Method：The synthetic DNA fragments encoding rhCRP were cloned into pGAPZotA vector. Recombinant plasmid was transformed into Pichia pastoris strain X - 33 which could secrete rhCRP con- stitutively. RhCRP was purified by Histamine affinity chromatography. The rhCRP was identified by SDS -PAGE and Western blot, and its immune reactivity was identified by indirect ELISA. Result：The pGAPZctA/rhCRP expression vector was successfully constructed, which was confirmed by restricting enzyme digestions and DNA sequencing. The rhCRP with a molecular weight of 23kDa was expressed success- fully by the recombinant Pichia pastoris strains and its expression level reached the maximum after about 27h, and the yield reached 3mg/ L. The rhCRP was purified by one step up to 96. 58% purity,and it was showed good immunity by indirect ELISA. Conclusion：The Pichia pastoris strains constitutively expressing recombinant human C - reactive protein were constructed successfully,which built up the founda- tion for developing testing CRP reagent.

关 键 词：C-反应蛋白 毕赤酵母 组成型表达 免疫反应性 

分 类 号：Q819[生物学—生物工程]