PDF《应用双质粒共表达体系提高融合蛋白 GGH 在毕赤酵母》

生物工程学报Chin J Biotech 2011, July 25;

27(7): 983?989 journals.

im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn ?2011 CJB, All rights reserved. Received: September 17, 2010;

Accepted: December 22,

2010 Supported by: National Key New Drug Creation of China (No. 2009ZX09102-221), National Natural Science Foundation of China (No. 31000016), Science and Technology Support Program of Jiangsu Province (No. BE2009629). Corresponding author: Zhenghong Xu. Tel: +86-510-85918206;

E-mail: zhenghxu@jiangnan.edu.cn 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. 基因工程 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 王慧

1 ,窦文芳

1 ,张晓梅

1 ,许泓瑜

1 ,许正宏 1,2

1 江南大学 医药学院制药工程研究室,无锡

214122 2 江南大学 工业生物技术教育部重点实验室,无锡

214122 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 mg/L,较GS115/F2 提高了 49.7%,通过荧光定量 PCR 发现 GGH 基因拷贝数在含有双质粒体系的 GS115/F3 中较出发菌株 GS115/F2 提高了 26.7%. Western blotting 杂交表明融合蛋白同时具有人血清白蛋白和人胰高血 糖素样肽-1 的抗原性. 关键词: 融合蛋白 GGH,双质粒,毕赤酵母,荧光定量 PCR,Western blotting High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system Hui Wang1 , Wenfang Dou1 , Xiaomei Zhang1 , Hongyu Xu1 , and Zhenghong Xu1,2

1 Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China

2 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China Abstract: 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

984 ISSN1000-3061 CN11-1998/Q Chin J Biotech July 25,

2011 Vol.27 No.7 Journals.im.ac.cn 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. Keywords: fusion protein GGH, double plasmid, Pichia pastoris, quantitative PCR, Western blotting 胰高血糖素样多肽