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

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

关键词:融合蛋白GGH，双质粒，毕赤酵母，荧光定量PCR，Western blotting

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 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.

Key words:fusion protein GGH, double plasmid, Pichia pastoris, quantitative PCR, Western blotting