白唇竹叶青蛇毒类凝血酶基因的克隆和序列分析  

Cloning and Sequencing of the Thrombin-like Enzyme Gene from the Venom Glands of Chinese Green Pit Viper(Trimeresurus albolabris)

作　　者：宋锡迅[1] 余晓东[1] 林奕心[1] 和七一[1] 李恒[1] 

机构地区：[1]重庆师范大学生命科学学院重庆市动物生物学重点实验室重庆市生物活性物质工程研究中心,重庆400047

出　　处：《重庆师范大学学报（自然科学版）》2009年第4期38-42,共5页Journal of Chongqing Normal University:Natural Science

基　　金：重庆市自然科学基金(No.CSTC2006BA503)

摘　　要：通过分析多种已知的毒蛇蛇毒类凝血酶（SVTLEs）基因序列同源性，以保守的N和C末端氨基酸序列设计引物，克隆得到白唇竹叶青（Trimeresurus albolabris）SVTLE基因并分析其序列。以毒腺总RNA为模板进行RT—PCR，纯化并克隆至pMD18-T。结果表明，该酶基因大小为777bp；推测出其相应氨基酸序列含258个氨基酸残基，分子量为28．02kD，pI值为6．07；其3个可能糖基化位点为NDT103～105、NNS121～123和NRT251～253；与其它毒蛇的已知SVTLEs基因比较分析，推测出其含6对二硫键即Cys31—162、Cys50-66、Cys98．256、Cys142—210、Cys174—189和Cys200—225；其催化活性中心氨基酸残基为His65、Asp110和Ser204。分子进化树分析表明，毒蛇SVTLEs一级结构的进化具有一定种属特征，可为蛇的系统分类提供参考。In order to get the SVTLE gene of Trimeresurus albolabris and analyze its sequence, two primers are designed with reference to the known conservative N and C-termiuns amino acid sequences of SVTLEs from other toxic snakes. Total RNA is isolated from venom glands of T. albolabris, as amplified by one step RT-PCR, the product is purified and cloned into pMD18-T. The corresponding amino acid sequence is got by screeneing positive clones and sequencing. The results show that the encoding gene of SVTLE gene of T. albolabris is successfully cloned and apart from the primers, the length of encoding cDNA is 777 bp ;its deduced amino acid sequence has 258 acid residues , its molecular weight is about 28.02 kD and its pI is 6.02. Three possible sites of glycosylation are NDT103 - 105, N NS121 - 123 and NRT251 -253. With the comparative analysis of the known encoding gene of SVTLE from other snakes, the multiple alignments reveal that the SVTLE of T. albolabris had six disulfide bonds Cys31-162, Cys50-66, Cys98-256, Cys142-210, Cys174-189 and Cys200-225. It has three amino acid residues （His65 ,Asp110 and Ser204） at its catalytic active center. The molecular phylogenetie tree shows that some differences has oeeured at the primary structure among SVTLEs from toxic snakes, which may contribute to snkae taxonomy.

关 键 词：白唇竹叶青 蛇毒类凝血酶(SVTLEs) 克隆 序列分析 

分 类 号：Q785[生物学—分子生物学]