尖吻蝮蛇类凝血酶基因的克隆及生物信息学分析

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

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。

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＇ CTCCTCTTAACTITTTCAAAAGTTT 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 DH5α, 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 783bp 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.