T7RNA聚合酶的合成与表达+文献综述.docVIP

T7RNA聚合酶的合成与表达+文献综述 摘要随着RNA广泛应用以及对生物研究的不断深入,人们对体外转录获得RNA的要求越来越迫切。根据已有研究知T7 RNA聚合酶对T7启动子的特异性识别能力极强，因此本论文采用T7 RNA聚合酶进行目标DNA的体外转录。本文主要通过构建带有T7 RNA聚合酶基因及6个his标签的质粒，将其导入大肠杆菌中扩大培养，后进行细胞破碎分离获得T7 RNA聚合酶，并利用6个his标签与Ni的络合作用进行T7 RNA聚合酶的提纯。在获得T7 RNA聚合酶后根据已有研究利用带有T7启动子的线性DNA进行体外转录，从而获得相应的RNA。最后本文通过蛋白质电泳及RNA电泳分别验证获得的T7 RNA聚合酶和RNA。8790 关键词T7启动子T7 RNA聚合酶体外转录 毕业设计论文外文摘要 TitleThe Tandem Affinity Purification (TAP) Method:A General Procedure of Protein Complex Purification Abstract Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration. M. Chamberlin 1970年第一次报道了T7 RNA 聚合酶，它是T7噬菌体基因Ⅰ的产物。在T7 RNA聚合酶的体外转录系统中，转录的产物常是模板的几倍甚至几百倍。由于T7 RNA聚合酶易于大量获得，价格比Sp6 RNA聚合酶便宜， 因此使用日趋广泛[4]。T7噬菌体的RNA聚合酶可特异识别T7噬菌体的启动子，并可作为唯一的酶蛋白成分独自催化T7基因的转录过程。基于T7噬菌体RNA聚合酶和T7启动子间特异性识别而建立起来的高效偶联表达系统已显示出了在真核生物基因工程中的巨大应用潜力[5]。 由于市场上可以买到的T7 RNA聚合酶的浓度比我们所需要的大规模合成工程要低，而且如果用于毫克水平上的RNA合成所需的费用也非常的高。所以，一般实验室中这些酶都是从含有带T7RNA聚合酶基因的质粒的大肠杆菌中分离得到的[6，7]。 根据已有研究，由