【作者】 芦静；

【导师】 李庆伟；

【作者基本信息】 辽宁师范大学 ， 动物学， 2010， 硕士

【摘要】 内容摘要:酪氨酸激酶BTK是非受体酪氨酸家族的成员,它由PH结构域、TH结构域、SH3结构域、SH2结构域和催化结构域五部分组成。BTK参与多种信号通路,在免疫系统中发挥重要作用。近年来BTK参与B细胞信号通路、TLR信号通路和肥大细胞脱颗粒等信号通路的具体机制问题是科学研究的热点。BTK对细胞的增殖、分化和凋亡起着重要的调控作用,其突变可导致X连锁无丙种球蛋白血症,临床表现为反复的细菌感染和B细胞数量的显著下降。本文收集了日本七鳃鳗的白细胞并提取总RNA,利用紫外光分光光度计和琼脂糖凝胶电泳的方法检测提取的总RNA质量。通过RACE的实验方法从日本七鳃鳗白细胞中成功扩增BTK基因并克隆后到pET32a(+)载体。从一级结构比对、高级结构预测、保守基序的分布、构建进化树等方面分析了这段序列。主要的研究结果如下:1一级结构比对结果表明日本七鳃鳗白细胞的BTK基因的氨基酸序列与黑猩猩、人类、马、牛、小家鼠、犬属、鸭嘴兽、原鸡、斑马鱼、盲鳗和果蝇等其他物种的BTK基因的氨基酸序列同源性很高。2利用软件MEME预测日本七鳃鳗BTK氨基酸序列的高级结构,包括预测PH结构域的高级结构、SH3结构域的高级结构和激酶结构域的高级结构。预测结果表明日本七鳃鳗BTK的高级结构与模板BTK的高级结构十分相似。3利用MEME在线软件分析在不同物种的BTK氨基酸序列中发现的保守基序的类型和分布,结果表明日本七鳃鳗BTK的氨基酸序列拥有全部的10个保守基序。4基于邻接法构建的氨基酸序列进化树表明,所有的BTK氨基酸序列被分为两簇,一簇包括哺乳动物、鸟类、鱼类和七鳃鳗,另一簇包括盲鳗和昆虫类。综合上述的研究结果得出:在最原始的脊椎动物七鳃鳗中存在非受体酪氨酸激酶BTK,它的具体的免疫机制尚不明确。更多还原

【Abstract】 Content: Bruton’s agammaglobulinaemia tyrosine kinase belongs to a subfamily of non-receptor protein tyrosine kinases.It is characterized by five structural modules: Pleckstrin homology domain,Tec homology domain, Src homology3 domain,Src homology2 domain and Tyrosine kinase domain.BTK is thought to be a type of key signal molecules in various signal pathways, it plays a crucial role in immune system. Recently mechanism of how BTK plays an crucial role in B cell signaling pathway,TLR signaling pathway and mast cells degranulation is a hotspot.BTK plays important role in regulating cell proliferation, differentiation and apoptosis. Mutation of BTK could cause X-linked agammaglobulinemia, which clinically characterized by recurrent bacterial infections and markedly reduced numbers of B cells .In this work,we collected the leucocytes of lamprey, extracted the total RNA, measure concentration of RNA by ultraviolet spectrophotometer and agarose gel electrophoresis.We have successfully cloned BTK gene using RACE method and BTK gene was constructed into pET32a(+) vector. Then we study on primary structure alignment, advanced structure prediction,reconstructing a phylogenetic tree and conserved motifs distribution of this sequence.These mainly results were as follow:1 The intercomparison results of primary structure shows that its amino acid sequences are very conservative with other species,such as chimpanzee,human,horse,cattle,mouse,dog, ornithorhynchus,chicken,zebra fish,hagfish and drosophila and so on.2 We predicted the advanced structure of BTK amino acid from lamprey using the MEME system.It included the prediction result of PH domain advanced structure, the prediction result of SH3 domain advanced structure and the prediction result of kinase domain advanced structure.The prediction results show that advanced structure of Lamprey BTK is similar to Temple BTK.3 The MEME system shows that amino acid sequences of Lamprey BTK contains 10 conserved motifs totally.4 The phylogenetic tree contains two clusters:one including mammals, birds, fishes and lamprey,the other including hagfish and insects. These above results demonstrate that non-receptor protein tyrosine kinase BTK may exist in lampreys, the precise immune mechanism of lamprey remained to be elucidated.更多还原