应用双质粒共表达体系提高融合蛋白GGH在毕赤酵母GS115中的表达量
High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system
投稿时间:2010-09-17  
DOI:  
中文关键词:融合蛋白GGH,双质粒,毕赤酵母,荧光定量PCR,Western blotting
英文关键词:fusion protein GGH, double plasmid, Pichia pastoris, quantitative PCR, Western blotting
基金项目:国家重大新药创制专项 (No. 2009ZX09102-221),国家自然科学基金 (No. 31000016),江苏省科技支撑计划 (No. BE2009629) 资助。
作者单位E-mail
王慧 江南大学 医药学院制药工程研究室无锡 214122  
窦文芳 江南大学 医药学院制药工程研究室无锡 214122  
张晓梅 江南大学 医药学院制药工程研究室无锡 214122  
许泓瑜 江南大学 医药学院制药工程研究室无锡 214122  
许正宏 江南大学 医药学院制药工程研究室无锡 214122江南大学 工业生物技术教育部重点实验室无锡 214122 zhenghxu@jiangnan.edu.cn 
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中文摘要:
      为实现人胰高血糖素样肽-1-人血清白蛋白融合蛋白 ((GLP-1A2G)2-HSA,简称GGH) 的规模化制备,通过pPICZαB与pPIC9K双质粒共表达体系提高融合蛋白GGH在毕赤酵母中的表达量。首先运用PCR技术扩增出融合蛋白GGH的基因片段,构建了表达质粒pPICZαB-ggh,并电转至经载体pPIC9K-ggh异位整合的GGH分泌型菌株——毕赤酵母GS115/F2;然后采用免疫学方法并结合高浓度抗生素筛选获得高产菌GS115/F3,在30 ℃,3%甲醇诱导80 h后GGH的表达量达到了491 m
英文摘要:
      In order to make a large-scale preparation of (GLP-1A2G)2-HAS (GGH), the double-plamid pPICZαB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZαB-ggh, which was transformed into P. pastoris GS115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 °C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.
王慧,窦文芳,张晓梅,许泓瑜,许正宏.应用双质粒共表达体系提高融合蛋白GGH在毕赤酵母GS115中的表达量[J].生物工程学报,2011,27(7):983~989
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