Wang Hui, Dou Wenfang, Zhang Xiaomei, Xu Hongyu, Xu Zhenghong
Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Jul;27(7):983-9.
In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB 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 pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/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 GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees 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 GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.
为了大规模制备(GLP-1A2G)2-HAS (GGH)，将双质粒pPICZalphaB和pPIC9K共表达系统导入毕赤酵母GS115。首先，通过PCR扩增GGH融合基因以构建重组表达质粒pPICZalphaB-ggh，将其转化到已整合另一个重组表达质粒pPIC9K-ggh的毕赤酵母GS115/F2中。采用免疫学方法结合高浓度抗生素筛选能够高水平表达GGH蛋白的重组菌株毕赤酵母GS115/F3。在30℃、3%甲醇诱导80 h的表达条件下，GS115/F3表达的GGH融合蛋白比GS115/F2增加了49.7%。同时，定量PCR结果显示，GS115/F3中的GGH基因剂量相对于GS115/F2增加了26.7%。此外，蛋白质免疫印迹实验表明重组GGH具有GLP-1和HSA两种抗原性。
