几种病毒蛋白的晶体学相关研究-生物化学与分子生物学专业论文.docxVIP

摘 摘 要 病毒的调控蛋白在病毒的基因表达与调控过程中具有重要的作用。Borf一1 是牛泡沫病毒编码的唯一调控蛋白，能够反式激活位于长末端重复序列和env 基因内部的启动子。JDV TAT是Jembrana Disease Virus编码的反式调控因子 可以通过结合RNA的TAR序列提高转录效率。ICPO和BICPO分别是人疱疹病毒 和牛疱疹病毒编码的调节蛋白可以促进病毒基因的表达。 本文以这上述四种蛋白为材料，经PeR构建表达质粒，并利用大肠杆菌进行 表达。通过对诱导条件的优化获得可溶性表达蛋白，结果Borf-i和ICPON端片断 在37℃即能够可溶性表达，而jDv TAT和BICPO需要进行低温诱导在18。C进行 可溶性表达。在此基础上根据不同蛋白的特性利用亲合层析，离子交换层析和分 子排阻层析等方法对蛋白进行纯化，通过优化条件，四种蛋白均获得较高纯度。 对于纯化效果理想的Borf一1进行结晶相关研究，运用动态光散射和圆二色 谱的方法对分子的均一性和二级结构进行测定。发现在溶液中Borf—l是以多聚 体的形式存在的，其二级结构中a螺旋的含量较低。利用气相扩散法进行Borf一1 的晶体生长，摸索晶体生长的条件。实验发现Borf一1在以聚y．--醇(PEG)作为 沉淀剂时，生成了类似晶体的沉淀，并在此基础上进行晶体生长条件的优化。 关键词：病毒调控蛋白，蛋白表达，蛋白纯化，气相扩散法，结晶 AbstractThe Abstract The regulatory proteins of viruses are important in the progress of viral gene expression and regulation．Borf—l，encoded by the Bovine foamy virus·can transactivate the promoters within the 5’long terminal repeat and 3’end of CZ／V gene．Jembrana disease virus Tat protein is a transcriptional activator，which can binds to RNA site TAR and increases the efficiency of transcription．ICPO and BICPO are viral regulatory proteins of HSV and BHV that promote the expression of viral genes． The plasmids expressing these four proteins are constructed by PCR with materials of oar laboratory．These proteins then are expressed in ￡coil．By changing the condition of induction such as temperature，the method to get soluble expressed proteins are found：Borf—l and ICPON can be induced at 37℃：while JDV Tat and BICPO should be induced at 18℃．The proteins then are purified by affinity／ion exchange／gel filtration chromatography． Borf—1 which is purified well is analyzed by Dynamic Light Scatter and circular dichroism．The results are as following：Borf—l is oligomer in solution and the percentage of q—helix in secondary structure iS low． Using the Vapour diffusion method，the trial of Borf一1 crystallization was performed．A regular granular precipitate iS found in the eondition with the precipitant of Polyethylene Glycol(PEG)．In order to get protein crystal，we optimized the crysta