细粒棘球绦虫三磷酸甘油醛脱氢酶基因的克隆、表达及其分子特征的生物信息学分析

doi:10.11843/j.issn.0366-6964.2015.09.019

摘要/Abstract

摘要：

三磷酸甘油醛脱氢酶（GAPDH）是糖酵解途径中的关键酶，现已发现该酶可作为一些寄生虫的疫苗候选分子和药物靶标。然而，有关绦虫三磷酸甘油醛脱氢酶的研究报道较少。为探究细粒棘球绦虫GAPDH（EgGAPDH）的分子特征与应用价值，克隆细粒棘球绦虫的GAPDH基因，并以生物信息学方法对其进行系统的分析。结果显示：EgGAPDH基因全长1 011 bp，编码336个氨基酸。EgGAPDH是由4个结构相同的单体蛋白（O、P、Q、R）组成的一个同源四聚体，每个单体蛋白质含有3个活性中心，即NAD+绑定区域（aa 4-151 和aa 315-336）、催化结合区域（aa 156-314）和一个酶活性位点（aa 149-156）。通过对EgGAPDH的氨基酸序列的抗原表位预测得到了16个B细胞抗原表位和9个T细胞结合位点；Western blot显示原核表达的重组EgGAPDH能与感染细粒棘球蚴的小鼠血清发生特异性结合；以纯化的重组EgGAPDH蛋白为抗原通过ELISA法检测17份细粒棘球蚴小鼠阳性血清，阳性检出率为100％。试验结果表明：成功克隆并表达细粒棘球绦虫的EgGAPDH基因，EgGAPDH蛋白具有3个酶功能区域和多个免疫结合位点，Western blot和ELISA结果显示，该蛋白质可能具有作为抗细粒棘球蚴的药物靶标和疫苗候选分子的潜力。

关键词: 细粒棘球绦虫, 3-磷酸甘油醛脱氢酶(GAPDH), 分子特征

Abstract:

As a key enzyme in the glycolytic pathway，glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has wide applications (vaccine candidate and drug target) against some parasites，but until now，few studies have been carried out in tapeworms.In order to determine its molecular character and practical value，Echinococcus granulosus GAPDH (EgGAPDH) was cloned，expressed and analyzed.The EgGAPDH gene isolated from E.granulosus cDNA was 1 011 bp in length，encoding 336 amino acids.The quaternary structure of this protein consisted of four chemically identical subunits，O，P，Q，and R.Each subunit consisted of an NAD+-binding domain (residues 4-151 and 315-336)，a catalytic domain (residues 156-314) and one enzyme active site (residues 149-156).Recombinant EgGAPDH was expressed and purified from Escherichia coli.Specific immunogenicity of EgGAPDH was shown by Western blot，ELISA analysis，and epitope prediction (16 B cell epitopes and 9 cytotoxic T lymphocyte epitopes).EgGAPDH gene was successfully amplified，cloned and expressed，EgGAPDH has three functional areas and a number of epitope prediction，combing with the results of Western blot and ELISA，it may be serve as a vaccine candidate and drug target against larval of E.granulosus.

Key words: Echinococcus granulosus, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), molecular characterization

中图分类号:

S852.734

王家海，王凝，胡丹丹，钟秀琴，宋星桔，古小彬，汪涛，杨光友. 细粒棘球绦虫三磷酸甘油醛脱氢酶基因的克隆、表达及其分子特征的生物信息学分析[J]. 畜牧兽医学报, 2015, 46(9): 1629-1637.

WANG Jia-hai，WANG Ning，HU Dan-dan，ZHONG Xiu-qin，SONG Xing-ju，GU Xiao-bin，WANG Tao，YANG Guang-you. Cloning，Expression and Bioinformatical Analysis of Echinococcus granulosus Glyceraldehyde-3-phosphate Dehydrogenase Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(9): 1629-1637.

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