【作者】 汪诚；

【导师】 徐志南；

【作者基本信息】 浙江大学 ， 微生物与生化药学， 2006， 硕士

【摘要】 无细胞蛋白表达系统又称为体外翻译系统,是一种相对胞内表达系统而言的开放表达系统。它以外源mRNA或DNA为模板,在细胞抽提物的酶系中补充底物和能量来合成蛋白质。随着生物组学时代的到来,无细胞蛋白质合成系统显示出快速、方便、易于高通量等优点,因而应用范围不断增加。 大肠杆菌无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板,利用大肠杆菌细胞抽提物的酶系,通过添加氨基酸、T7 RNA聚合酶和能量物质等来表达蛋白质的体外翻译系统。本论文开展大肠杆菌无细胞蛋白合成系统的构建工作,采用正交法对该系统进行优化,提高了大肠杆菌无细胞蛋白合成系统的合成目标蛋白质的能力。构建了6种HIV基因的大肠杆菌无细胞表达载体,并且选取HIV病毒感染因子(Vif)作为目标蛋白,在大肠杆菌无细胞蛋白质合成系统得到了表达,为将来高通量抗HIV药物得筛选奠定了一定的基础。最后,构建了适宜体外表达S-腺苷-L-甲硫氨酸合成酶和5-氨基乙酰丙酸合成酶的表达载体,为将来在无细胞体系中表达具有生物活性的酶开展了探索性工作。更多还原

【Abstract】 The cell-free expression system is an in vitro translation system, compare with the in vivo translation system. Cell-free synthesis system enables the direct in vitro expression of protein from extrinsic template DNA or mRNA. It synthesizes protein through complementing substrate and energy in the cell extraction. In the new era of "bio-omics", the cell-free expression system opens a new strategy for protem expression, and is used widely because it can produce proteins in a mode of rapid, convenient and high-throughput expression.E. coli cell-free expression system is one widely used system which can synthesize protein effectively through complementing substrate and energy. The E. coli cell-free expression system was constructed and optimized in the present paper. The orthogal design method was used to improve the productivity in this E. coli cell-free system. Then six vectors were constructed for the purpose of expressing six HIV genes in E. coli cell-free system. Among them, VIF gene can be expressed effectively in this cell-free system. This work may be helpful to further expression of HIV genes in the cell-free system. Finally, two more genes (one SAM biosynthetic gene and one ALA biosynthetic gene) were inserted into cell-free vectors for the further expression of these two enzymes in the cell-free systems.更多还原