优化重组人C-反应蛋白在酵母中的分泌表达  

Optimizing the secretory expression of recombinant human C-reactive protein in Pichia pastoris

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

机构地区：[1]大连大学医学院,116622

出　　处：《实用医学杂志》2013年第19期3113-3116,共4页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:31070822);辽宁省教育厅高校科研计划项目(编号:L2010015)

摘　　要：目的：通过优化引导蛋白分泌的信号肽（signal peptide，SP），构建高效分泌表达具有免疫活性的重组人C-反应蛋白（recombinant human C-reactive protein，rhCRP）的毕赤酵母菌株。方法：通过重叠PCR和DNA重组技术，分别将信号肽MFαSP、CRNSP、SUC2SP和MFα-op SP的基因重组到rhCRP基因的5’端后克隆到毕赤酵母表达载体上．获得的4种重组质粒分别重组到毕赤酵母菌x．33中进行甲醇诱导分泌表达，Western blot法鉴定蛋白分泌情况;利用HisTrap HP柱纯化rhCRP后用间接ELISA法鉴定其免疫反应性。结果：成功构建了4种重组质粒。4种sP能引导表达出大小约为23kD的rhcRP，其中MFdSP引导rhCRP分泌的量约为另外3种信号肽的3-12倍。间接ELISA检测表明，纯化的rhCRP具有特异的免疫反应性。结论：成功筛选出了高效分泌表达rhCRP的重组毕赤酵母菌株并表达、纯化得到具有免疫反应性的rhCRP，为rhCRP相关的进一步临床及基础研究奠定了基础。Objective To construct the Pichiapastoris （P.pastoris） strains which can efficiently secret recombinant human C-reactive protein （rhCRP） with immunocompetence through optimizing the signal peptide. Methods The DNA fragments of 4 kinds of different signal peptides （MFα SP, CRN SP, SUC2 SP and MFα-op SP） were introduced at the 5＇ end of the rhCRP gene via overlapping PCR and DNA reconstruction technology, then these rhCRP genes were cloned into P.pastoris expression vectors respectively. And then, the recombinant plasmids were incorporated into P.pastoris X-33 respectively. The secreted rhCRP induced by methanol was tested by Western blot. After being purified by HisTrap HP column, the immunoreactivity of rhCRP was identified by indirect ELISA. Results The recombinant plasmids containing rhCRP gene with different secretion signals were constructed successfully. All of the strains which incorporated with the 4 kinds of plasmids could secret rhCRP with a respected 23 kD molecular size. And the yield of rhCRP by the strain carrying MFα SP was higher around 3 - 12 folds than others. Purified rhCRP had good immunoreactivity tested by indirect ELISA. Conclusion The P.pastoris strain which efficiently secreted rhCRP had been established successfully and the rhCRP with immunoreactivity was produced via the strain, which will be a promising way for the further clinical and foundation research on the rhCRP.

关 键 词：C-反应蛋白 毕赤酵母 信号肽 分泌表达 

分 类 号：R392.1[医药卫生—免疫学]