一种新的五步蛇蛇毒类凝血酶Cdna的克隆和序列分析

目的：克隆出五步蛇的类凝血酶cDNA，为研究其结构和功能的关系。并为下一步基因表达开发抗血栓药物提供依据和基础。方法：从五步蛇蛇腺中提取了总RNA，通过反转录合成第一链cDNA，经PCR扩增出五步蛇毒腺中类凝血酶TLE1的cDNA片段，并将其连接到质粒载体扩增，然后进行DNA测序。结果：成功克隆出一种新的五步蛇类凝血酶的cDNA片段。此片段全长794bp，包括编码部分的全长为777bp。推导其编码的蛋白质序列为258个氨基酸，其中包括18个氨基酸的信号肽，6个氨基权的前肽和234个氨基酸的成熟氨基酸顺序。TLE1成熟肽与其它蛇毒类凝血酶相比，蛋白质一级结构具有较高的同源性。结论：从五步蛇中成功克隆出一 种新的类凝血酶cDNA，并确定了它的DNA顺序。

MOLECULAR CLONING OF THE NEW cDNA FOR THE THROMBIN-LIKE ENZYME FORM THE VENOM OF AGKISTRODON ACUTUS

Objective: To clone the cDNA for the thrombin like enzyme from the venom of Agkistrodon Acutus, and to study the relationship between its structure and function, so as to lay a good foundation for the development of the clinical medicines to cure the thrombotic diseases Methods: Total RNA extracted from the venom glands of Agkistrodon Acutus The cDNA fragment for the thrombin like enzyme named TLE1 was amplified by RT PCR and cloned into the PMD18 T vector The cDNA fragment sequence was determined Results: The length of the cDNA fragment is 794bp which includes 777bp of coding region The cDNA encoding a protein with 258 amino acids residues, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 residues and a mature peptide of 234 amino acids This protein is homologous for thrombin like enzyme from the snake venoms Conclusion: We have cloned and determined a new cDNA sequence of thrombin like enzyme from Agkistrodon Acutus