胞红蛋白酵母表达载体构建与表达纯化  

Construction and Expression of Yeast Expression Vector for CYGB

作　　者：陶伟娟[1] 贾晓健[1] 钱杰[1] 张玉彬[1] 吴梧桐[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2011年第4期304-307,共4页Pharmaceutical Biotechnology

基　　金：江苏省六大人才项目;先声药业研究生创新项目资助

摘　　要：胞红蛋白是脊椎动物珠蛋白超家族的一员,具有重要的生物学功能,已在大肠杆菌中成功表达。酵母表达系统作为常用的真核表达系统,具有外源蛋白表达蛋白量大且活性高等优点。该研究通过PCR扩增技术获得胞红蛋白编码基因并通过DNA重组技术构建并鉴定了胞红蛋白毕赤酵母分泌型表达载体CYGB-pGAPZαA。将重组载体经单酶切线性化后通过电转化法转入毕赤酵母GS115,利用梯度浓度的Zeocin抗生素和PCR技术筛选得到阳性转化子。工程菌经发酵培养后,发酵上清液经镍柱分离得到较高纯度的目的蛋白,并经电泳等方法验证鉴定。CYGB（cytoglobin） is a new member of globin superfamily with an important role in vertebrate. It has already been expressed in E. coli. Pichia Yeast is mostly used protein expression system with the advantages such as lager quantity product and high activity. The gene of CYGB was obtained from cDNA by PCR, and then expression vector CYGB-pGAPZαA was constructed using recombinant DNA technology. The linear DNA vector digested by restriction enzyme XbaI and Xho was transformed into P. pastoris GSl15. The positive recombinant strain was screened with gradient concentration of Zeocin. The engineering strain was identified by digestion of restriction endonuclease and PCR technology. Then the engineering strain was cultivated under the best condition. CYGB was purified from formentation though Ni-NTA, and analyzed by SDS- PAGE. The results demonstrated that the yeast expression system for CYGB had been successfully constructed.

关 键 词：胞红蛋白 基因重组 酵母表达 分离纯化 

分 类 号：Q51[生物学—生物化学]