mNUDC基因在巴斯德毕赤酵母中表达载体的构建及表达  

Construction and Expression of mNUDC Gene in the Pichia Pastoris

作　　者：陈彦球[1] 魏明旭[2] 张磊[2] 杨燕[3] 

机构地区：[1]中山大学第一附属医院,广东广州510008 [2]中山大学教育部基因工程重点实验室,广东广州510275 [3]中山大学公共卫生学院,广东广州510008

出　　处：《现代生物医学进展》2009年第10期1805-1808,共4页Progress in Modern Biomedicine

基　　金：广东省自然科学基金博士启动资助项目(06300645);教育部博士点基金新教师资助项目(20070558275);中山大学2007实验室开放基金项目(KF200707)

摘　　要：目的:探索小鼠核迁移蛋白C(mNUDC)在毕赤酵母分泌表达的方法。方法:应用PCR扩增本实验室所构建的重组质粒PET28b-his-mNUDC中的mNUDC基因,使用基因重组方法构建毕赤酵母真核表达载体pPICZαA-his-mNUDC,电击转化酵母菌株KM71后,经摇瓶发酵和甲醇诱导,SDS-PAGE和Westernblot分析鉴定上清中重组mNUDC蛋白表达量。结果:经过PCR方法,有效扩增了mNUDC基因,构建了pPICZαA-his-mNUDC酵母表达质粒,序列分析表明所构建的含mNUDC基因的质粒与设计相同,mNUDC蛋白得到正确表达。使用SDS-PAGE和Western blot方法可以检测到mNUDC的稳定、高效地分泌表达。结论:成功地构建了mNUDC基因的毕赤酵母表达载体pPICZαA-his-mNUDC,并在毕赤酵母中实现分泌型离表达,为进一步研究mNUDC蛋白对小鼠的生物活性奠定了实验基础。Objective： To explore the method of secretory expression of recombinant murine nuclear distribution C （mNUDC） in Pichia pastoris. Methods： The murine gene mNUDC was amplified from PET28b-his-mNUDC by PCR. The expression vector pPICZoα A-his-mNUDC was constructed with DNA recombination method. The constructed plasmid was transformed into yeast KM71 by electroporation. The recombinant with a high copy number of the plasmid was harvested. The expression of mNUDC protein was induced by methanol. SDS-PAGE and Western blot was used to analyze the expression of protein. Results： The mNUDC gene was amplified effectively. The expression vector pPICZα A-his-mNUDC was constructed, and the result of DNA sequence showed that the constructed mNUDC gene was the same as designed. The result of SDS-PAGE and Western blot showed that the mNUDC was induced by methanol in the culture supernatant and expressed in Pichia pastoris. Conclusions： The mNUDC gene has been successfully cloned and expressed, which provides a new protein mNUDC for further experiments on murine effects.

关 键 词：mNUDC 毕赤酵母 分泌表达 

分 类 号：R392.12[医药卫生—免疫学]