鼠隐藏管状线虫烯醇化酶基因的克隆与序列分析

CLONING AND SEQUENCE ANALYSIS OF ENOLASE GENE IN SYPHACIA OBVELATA

为获得鼠隐藏管状线虫(Syphacia obvelata)烯醇化酶结构及其功能,采用同源克隆方法结合RACE技术克隆获得了烯醇化酶基因c DNA序列,并对其进行了序列分析。测序结果显示,扩增获得的鼠隐藏管状线虫烯醇化酶基因大小序列为1603 bp,包含全长为1311 bp的开放阅读框(open reading frame,ORF)序列,共编码436个氨基酸,推导分子量为47.428 k Da。序列分析结果显示,鼠隐藏管状线虫烯醇化酶含有10个丝氨酸激酶磷酸化位点、3个苏氨酸激酶磷酸化位点和4个酪氨酸激酶磷酸化位点,1个潜在的跨膜螺旋结构,无信号肽剪切位点;在亚细胞水平,预测其主要定位于胞浆;二级结构主要以α-螺旋和无规则卷曲为主,其中α-螺旋占34.86%,无规则卷曲占30.50%,延伸链占24.08%,β-转角占10.55%。Swiss-Model模建的3D结构显示,鼠隐藏管状线虫烯醇化酶为一"哑铃"样结构,包含氨基端和羧基端两个结构域,结构域之间为Linker区。每个结构域由多个α-螺旋、β-折叠和卷曲所构成桶形结构,催化中心和Mg^(2+)结合位点位于桶形结构的中心。本研究结果为鼠隐藏管状线虫烯醇化酶基因的功能研究奠定基础。

To learn the structure and function of the enolase gene of Syphacia obvelata,we fi rst obtained the full-length cDNA of enolase by using homology-based cloning together with RACE technology.The cDNA sequence was then analyzed by bioinformatics databases and software.The full-length cDNA of enolase was1611bp in length,with a coding region of1311bp.This sequence encoded a protein of436amino acids with a molecular weight of47.428kDa.The enolase contained ten serine phosphorylation sites,three threonine phosphorylation sites,four tyrosine phosphorylation sites and a potential transmembrane helices region,but no signal peptide was found in the protein.At the subcellular level,the enolase protein was mainly localized to the cytoplasm,peroxisome,mitochondrial matrix space and lysosome.The secondary structure of enolase was mainly composed of alpha helix,consisting of34.86%helix,30.50%random coil,24.08%extended strand,and10.55%beta turn.Swiss-Model modulated predicted that3D structure was like dumbbell with a linker in the middle,each substrate unit was comprised of alternatively arrangedαhelix-βsheet and forms a barrel,the active sites,Mg2+binding sites closely located in the center of barrel.By cloning the enolase gene and predicting its structure,character and functions,we have obtained valuable information which can be used to study the relationship between parasite and host in the future.