【作者】 景巍；

【导师】 王转花；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 荞麦具有较高的营养品质和药用价值,并富含多种生物活性物质,在防治现代文明病,诸如糖尿病、高血压等心脑血管疾病和风湿性关节炎等方面具有一定的功效。然而荞麦中含有的过敏性成份又能使许多接触或食用它的人产生过敏症状。近些年来,一些国外的科学家对甜荞(common buckwheat CB)中的过敏成分进行了研究。但在苦荞(tartary buckwheat TB)过敏成分研究方面的报道却不多,有关苦荞过敏蛋白的基因克隆及表达调控的研究尚属空白。 根据先前分离纯化所得天然TBa蛋白经MALDI-TOF-MS(质谱法)测得的氨基酸序列和文献报道的甜荞过敏蛋白核苷酸序列设计引物,利用PCR的方法,并结合3′和5′-RACE方法,得到了编码苦荞过敏性贮藏蛋白(简称TBc)的全长cDNA序列。此序列共长1773bp,5′端有45bp富含GC的序列,在46-1593bp之间有一开放阅读框,共编码515个氨基酸,3′端包括200bp,其中C-端(TBa)包含195个氨基酸,N-端(TBb)包含320个氨基酸。本实验对测得的核苷酸序列和推导出的氨基酸序列进行了数据分析,包括保守序列分析、二硫键及转录后加工位点预测、信号肽预测、二级结构及3D结构预测、进化树分析、抗原表位预测。 利用盐析、阴离子交换层析、凝胶过滤层析及改进的电洗脱纯化等方法,分别对TBa、TBb、TBc进行了纯化,并将纯化得到的各蛋白进行了免疫活性分析。 根据以上实验结果,推测苦荞过敏性贮藏蛋白TBc由两个亚基构成,分别为TBa(约24kDa)和TBb(约35kDa),它们通过一个链间二硫键互相连接,其中TBb中还存在一个链内二硫键。ELISA分析表明,TBa和TBb的免疫活性相当,由此推测食用荞麦食品或接触荞麦产品引起的过敏反应可能主要与TBa和TBb有关。 以上内容对深入开展荞麦过敏蛋白结构与功能关系的研究及进一步寻找过敏性贮藏蛋白的抗原表位位点提供了重要的前期工作基础。更多还原

【Abstract】 The buckwheat has higher nutritional quality and medical value, and includes many kinds of bioactive material richly. These substances have shown significant efficient in preventing and curing the modern civilization diseases, cardiovascular diseases and rheumarthritis such as diabetes, hypertension etc. But the anaphylaxis compositions contained in the buckwheat cause a lot of person who contact or eat it produce allergy symptom. In the last few years, some foreign scientists have studied the allergic composition in common buckwheat (CB). But reports in allergic composition research of tartary buckwheat (TB) are rare, it still belongs to the blank that the research about tartary buckwheat allergenic storage protein.According to the amino acid sequence of TBa purified from tartary buckwheat seeds which was sequenced by MALDI-TOF-MS, and nucleic acid sequences of allergenic protein from common buckwheat, the primers were designed. Using method of PCR, 3’ and 5’- RACE, the full length cDNA sequence coding of an allergenic protein in tartay buckwheat (TBc) was amplified. The sequence consists of 1773 bp nucleic acids. 5’ end sequence of 45bp contain GC richly, there is a open reading frame among 46 to 1593bp, which encoding 515 amino acids, 3’ end include 200bp. TBc was made up of two subunits, TBa and TBb. TBa includes 195 amino acid residues, TBb includes 320 amino acid residues. Data analysis of nucleotide acids and deduced amino acids is performed, including conservation analysis of its amino acids, prediction of position of disulfide bridge and the location of post-translation, signal peptide prediction, second structure and 3D structure prediction, phylogenetic analysis, antigen sites prediction.TBa, TBb and TBc are purified respectively, with salting out, anion exchange chromatography, gel filtration and rapid efficient electrophoresis elution, and then immunological character of three proteins are analyzed.According to the above experimental result, we infer the tartary buckwheat allergenic storage protein (TBc) is composed by two subunits, Tba (MW更多还原