刺苋花粉泛过敏原profilin基因的克隆与表达

发布时间：2018-08-08 17:52

【摘要】： 前纤维蛋白(profilin)作为一种泛过敏原(pan-allergen)广泛存在于树木、杂草等花粉中。刺苋(Amaranthus spinosus L．)为苋科、苋属一年生草本植物。目前，刺苋花粉已经成为我国华南地区一种重要的吸入性过敏原，对该花粉中泛过敏原基因克隆表达有着重要的理论与实际意义。本文主要采用生物信息学方法，从数据库中获得已知植物花粉泛过敏原profilin的氨基酸序列，通过多重序列比对和构建进化树，获得核心代表序列。经同源性比较，以序列保守区设计合成引物，采用递减式PCR(touchdown PCR)技术从刺苋花粉的cDNA池(cDNA pool)中进行基因克隆，结果获得两个不同的基因，分别命名为prf7和prf23。通过序列测定及比对分析，初步确定所克隆基因为一种泛过敏原基因。之后将prf23基因连接到表达载体pET-44a上转化大肠杆菌进行诱导表达，经SDS-PAGE证实，prf23基因能够编码一种蛋白。最后结合生物信息学从蛋白质三维空间结构和抗原性高低角度对这两个致敏蛋白进行分析比较。本研究的重要意义在于：通过对过敏原基因的获得和蛋白质水平功能预测分析，，为临床应用重组过敏原治疗和过敏原的基因工程改造奠定基础。
[Abstract]:Prefibrin (profilin), as a panallergen (pan-allergen), is widely found in pollen such as trees, weeds and so on. (Amaranthus spinosus L.) is an annual herbaceous plant of the genus Amaranthus. At present, amaranth pollen has become an important inhaled allergen in South China, which has important theoretical and practical significance for the cloning and expression of panallergen gene in this pollen. In this paper, the amino acid sequences of plant pollen panallergen profilin were obtained from the database by bioinformatics, and the core representative sequences were obtained by multiple sequence alignment and phylogenetic tree construction. By homology comparison, primers were designed and synthesized with sequence conserved region. The gene was cloned from cDNA pool (cDNA pool) of purslane pollen by decreasing PCR (touchdown PCR) technique. Two different genes were obtained, named prf7 and prf23, respectively. The cloned gene was identified as a panallergen gene by sequence determination and alignment analysis. Then the prf23 gene was ligated to the expression vector pET-44a and transformed into Escherichia coli for expression, and the SDS-PAGE confirmed that the gene could encode a protein. Finally, the two sensitized proteins were analyzed and compared in terms of their three-dimensional spatial structure and antigenicity with bioinformatics. The significance of this study lies in: through the acquisition of allergen genes and the analysis of protein level function prediction, we can lay a foundation for clinical application of recombinant allergen therapy and genetic engineering of allergens.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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