一种新的五步蛇蛇毒类凝血酶cDNA的克隆和序列分析

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

目的 :克隆出五步蛇的类凝血酶 c DNA,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 c DNA,经 PCR扩增出五步蛇毒腺中类凝血酶 TL E1的 c DNA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 c DNA片段。此片段全长 794 bp,包括编码部分的全长为 777bp。推导其编码的蛋白质序列为 2 5 8个氨基酸 ,其中包括 18个氨基酸的信号肽 ,6个氨基酸的前肽和 2 34个氨基酸的成熟氨基酸顺序。 TL E1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 c DNA,并确定了它的

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