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

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。

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.