论文部分内容阅读
为研究ｔＲＮＡＴｒｐ 与色氨酰ｔＲＮＡ合成酶（ＴｒｐＲＳ） 的相互识别及其结构、功能关系， 纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ 将ＴｒｐＲＳ固定化， 固定化ＴｒｐＲＳ的蛋白质回收率为９５ ．５ ％ ， 活力回收率为３１．３％ 。研究了固定化ＴｒｐＲＳ的酶学性质， 其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高， 最适温度、最适ｐＨ 均有一定程度的增大， 工作稳定性良好。以固定化ＴｒｐＲＳ为亲和层析介质， 对含有２０ 个核苷酸随机序列、长度为５６ 个核苷酸的单链ＲＮＡ 随机库进行了３ 轮筛选，ＲＮＡ 群体亲和固定化ＴｒｐＲＳ的比例从４ ．３ ％ 上升至１４ ．７ ％ 。筛选得到了与ｔＲＮＡＴｒｐ 氨基酸接受茎类似的ＲＮＡ二级结构。实验结果表明固定化ＴｒｐＲＳ可以作为ＳＥＬＥＸ 亲和层析介质， 进行模拟ｔＲＮＡＴｒｐ 分子的ＲＮＡ 随机库的ＳＥＬＥＸ 筛选。
In order to study the mutual recognition of tRNATrp and tryptophan-tRNA synthetase (TrpRS) and its structure and function, TrpRS was immobilized by Sepharose 4B, which was purified by Bacillus subtilis TrpRS and activated by cyanogen bromide. The protein recovery of immobilized TrpRS was 95. 5%, vitality recovery rate of 31.3%. The enzymatic properties of the immobilized TrpRS were studied. Compared with the liquid TrpRS, the thermostability and storage stability of the immobilized TrpRS were greatly improved. The optimal temperature and the optimum pH all increased to some extent, and the working stability was good . Using immobilized TrpRS as the affinity chromatography medium, three rounds of single stranded RNA random pool containing 20 nucleotide random sequences and a length of 56 nucleotides were screened. The ratio of RNA population affinity immobilized TrpRS was 4. 3% to 14. 7%. The RNA secondary structure similar to the stem of tRNATrp amino acids was screened. The experimental results show that immobilized TrpRS can be used as SELEX affinity chromatography medium to carry out SELEX screening to simulate RNA random pool of tRNATrp molecules.