【作者】 宋辉；

【导师】 喻子牛； 黄友谊；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 硕士

【摘要】 DNA拓扑异构酶是生物体内的基础酶类,参与到所有涉及DNA拓扑结构变化的生命活动中,如DNA的复制,转录,重组。DNA旋转酶是类型Ⅱ拓扑异构酶的一种,也是唯一能够引入负超螺旋的拓扑异构酶。结核分枝杆菌的旋转酶是该菌体内唯一的类型Ⅱ拓扑异构酶,是抗生素的重要作用靶标。结核分枝杆菌旋转酶B亚基有四个主要的结构域：ATPase域、Transducer域、Toprim域和Tail域。本研究通过对结核分枝杆菌旋转酶B亚基的研究,构建了gyrB基因的缺失突变体gyrB463-714,gyrB1-631,gyrB1-482,gyrB1-270,gyrB271-480,gyrB485-714, gyrB278-714,gyrB1-542,gyrB1-519,gyrB1-500,将其连入表达载体pET28a,转入E.coli BL21中进行表达。表达并纯化野生型GyrB蛋白,缺失突变体GyrB1-462, GyrB463-714, GyrB1-631, GyrB1-482, GyrB1-270, GyrB271-480, GyrB485-714, GyrB278-714, GyrB1-542, GyrB1-519, GyrB1-500蛋白,将所得到的蛋白分别进行与单双链DNA结合的EMSA试验。试验结果发现,野生型GyrB蛋白能够结合单双链DNA；去除ATPase域后,GyrB278-714可以结合单双链DNA；去除ATPase和Transducer后的GyrB463-714可以结合单双链DNA；去除Toprim结构域和Tail结构域后的GyrB1-462,GyrB1-482可以结合单双链DNA；单独去除Tail结构域后的GyrB1-631可以结合单双链DNA；单独去除Toprim的GyrBΔtoprim能够结合单双链DNA。而单独克隆B亚基单个结构域的突变体亚蛋白不能得到表达,或表达后形成包涵体不能纯化。试验结果证实了结核分枝杆菌旋转酶B亚基是一个能够结合单双链DNA的蛋白；对旋转酶B亚基四个主要结构域分别进行缺失后,发现突变体蛋白保留了单双链DNA结合活性,表明这四个主要的结构域都具有不同程度的DNA结合活性,部分结构域的缺失对于DNA结合活性没有较大影响。更多还原

【Abstract】 DNA topoisomerase is an indispensable topoisomerase which plays an important role on DNA replication, transcription, and recombination, concerning DNA topology changes. DNA gyrase is the only topoisomerase that is able to introduce negative supercoils into DNA. Mycobacterium tuberculosis gyrase which is the only typeⅡtopoisomerase is a successful target for antibacterial.Four main domains can be found in Mtb, the ATPase domain, the Transducer domain, the Toprim domain and the Tail domain. All truncated mutants were constructed based on the structure of Mtb gyrase B subunit, gyrB1-462, gyrB463-714, gyrB1-631, gyrB1-482, gyrB1-270, gyrB271-480, gyrB485-714, gyrB278-714, gyrB1-542, gyrB1-519 and gyrB1-500, pET28a as its expression vector, and then transformed into E.coli BL21. After expression and purification of wild type GyrB and mutant protein GyrB1-462, GyrB463-714, GyrB1-631, GyrB1-482, GyrB1-270, GyrB271-480, GyrB485-714, GyrB278-714, GyrB1-542, GyrB1-519, GyrB1-500, all the proteins were bind with dsDNA and ssDNA with gel retardation assay. The experiments suggest results as follows. Wild type GyrB has the ability to bind with dsDNA and ssDNA; The deletion of ATPase domain (GyrB278-714) does not affect its binding with dsDNA and ssDNA; The deletion of ATPase domain and Transducer domain (GyrB463-714) has no effect on its binding with dsDNA and ssDNA; The deletion of Toprim domain and Tail domain (GyrB1-462, GyrB1-482) shows little effect on its binding activity with dsDNA and ssDNA; The deletion of C terminal domain (GyrB1-631) has little effect on the binding effect on ssDNA and dsDNA; The deletion of Toprim domain which lays in the CTD of B subunit (GyrBΔtoprim) shows no effect on its dsDNA and ssDNA binding activity. The truncations of single domain of GyrB tend to form inclusion body and can not be expressed or purified. It is suggested that Mtb gyrase B subunit is an ssDNA and dsDNA binding protein, the truncation of the four main domains retains the DNA binding activity, which give a clue that the four domains have different DNA binding activity independently, the deletion of different domain have little effect on its binding activity.更多还原