尖吻蝮蛇类凝血酶基因的克隆及生物信息学分析

为了获得蛇毒类凝血酶基因，本研究根据不同蛇毒类凝血酶cDNA5’和3’保守性序列设计了引物；通过RT—PCR从尖吻蝮蛇毒腺总RNA中扩增到1个808bp特异性片段，将该cDNA重组到pMD18-T载体中，利用生物信息学的方法对测序结果进行了分析。结果表明：该特异性片段中有一个783bp的开放阅读框架．其编码蛋白质序列与蛇的类凝血酶序列有98．5％的同源性，也具有蛇毒类凝血酶的典型特征。结论：本实验获得了一个新的尖吻蝮蛇类凝血酶基因。

Cloning and Bioinformatics Analysis of a Thrombin-like Enzyme Gene from Agkistrodon Acutus

The venoms of Viperidae and Crotalidae snakes contain a large variety of proteins and peptides affecting the hemostatic system, which classified as coagulant, anticoagulant and fibrinolytic factors. To obtaind the thrombin-like enzyme gene of snake venoms, primers 1 5' ATGGTGCTGATCAGAGTGCTAGC 3' and 2 5' CTCCTCTTAA-CTTTTTCAAAAGTTT 3' were designed according to the snake venom thrombin-like enzyme highly conserved regions of 5' and 3'. Total RNA was prepared from the venom glands of a D. acutus specimen collected from Guangxi province of China, RT-PCR was conducted to amplify the gene of the venom thrombin-like enzyme (TIL). A 0.8 kb DNA fragment was specifically amplified, inserted into the pMD18-T vector and transformed into Escherichia coli strain DH5alpha, then identified by PCR and sequencing. The results showed that this cDNA shared great sequence homology (98.5%) with the published snake TLE cDNA sequence, the deduced amino acid sequence of this TLE encoded by the 783 bp consisted of 260 amino acids, which included a signal peptide of 24 amino acids and a matured peptide of 236 amino acids. In conclusion, a new cDNA encoding snake TLE was obtained by amplificantion.