酵母表达的长角血蜱谷氨酰胺转移酶及其活性分析

发布时间：2018-03-12 19:36

本文选题：分子特征　切入点：长角血蜱　出处：《畜牧兽医学报》2017年02期 　论文类型：期刊论文

【摘要】：谷氨酰胺转移酶(TGase)通过催化蛋白质的谷氨酰胺残基与赖氨酸残基之间形成ε-(γ-谷氨酰基)赖氨酸异肽键,或在与肽结合的谷氨酰胺残基处掺入伯胺,促进蛋白质分子内和分子间的交联,形成网状的高分子聚合物,参与多种重要的生物学活动。本研究旨在克隆和表达长角血蜱(Haemaphysalis longicornis)的谷氨酰胺转移酶基因(HlTGase,GenBank登录号为KX59300),并分析重组蛋白质的活性,评估其可能的应用价值。首先从长角血蜱上海株的成虫提取总RNA,根据表达序列标签的序列信息,设计引物扩增并克隆HlTGase基因,以质粒pPICZC为表达载体,将该基因在毕赤酵母中重组表达,进而对其编码蛋白质的分子特征及可能的应用价值进行评估。结果显示HlTGase基因的开放阅读框为2 262bp,编码了一条756aa的多肽链,该多肽链具有四个谷氨酰胺转移酶结构域,理论相对分子质量为84.6ku;系统发育树显示其与果蝇的谷氨酰转移酶亲缘关系最近;在毕赤酵母中成功表达的重组蛋白质具有谷氨酰胺转移酶的活性,能催化酪蛋白交联成较大的分子。本研究成功地用酵母表达了长角血蜱谷氨酰胺转移酶,重组蛋白质有催化蛋白质交联的活性,具有一定的应用前景。
[Abstract]:Transglutaminase (TGase) catalyzes the formation of 蔚-(纬 -glutamyl) lysine isopeptide bonds between glutamine residues and lysine residues of proteins, or the incorporation of primary amines at the sites of glutamine residues that bind to peptides. Promoting intramolecular and intermolecular crosslinking of proteins to form a network of polymer polymers, The aim of this study was to clone and express the glutamine transferase gene of Haemaphysalis longicornis, and to analyze the activity of recombinant protein by using GenBank accession number KX59300. To evaluate its potential application value. Firstly, total RNAs were extracted from adults of Haemaphysalis longicornis Shanghai strain. Primers were designed to amplify and clone the HlTGase gene according to the sequence information of the expressed sequence tags, and the plasmid pPICZC was used as the expression vector. The gene was expressed in Pichia pastoris, and the molecular characteristics and potential application value of the protein were evaluated. The results showed that the open reading frame of HlTGase gene was 2262 BP, encoding a 756aa polypeptide chain. The polypeptide chain has four glutamine transferase domains and the theoretical relative molecular weight is 84.6 ku.The phylogenetic tree shows that the phylogenetic tree has the closest relationship with the glutamyl transferase of Drosophila melanogaster. The recombinant protein successfully expressed in Pichia pastoris has the activity of transglutaminase and can catalyze the cross-linking of casein into larger molecules. The recombinant protein has the activity of catalyzing the cross-linking of proteins and has a certain application prospect.
【作者单位】：福建师范大学生命科学学院福建省发育与神经生物学重点实验室;
【基金】：福建省自然科学基金(2014J01120) 福建师范大学生命科学学院本科生拔尖人才培养项目
【分类号】：S852.746

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