Cloning and Bioinformation Analysis of a Novel Thrombin-like Enzyme Gene from Gloydius ussuriensis

Objective To obtain the thrombin-like enzyme gene of snake venoms.Methods Two primers were designed according to the snake venom thrombin-like enzyme(TLE) highly conserved regions of 5'and 3'.Primer 1 is 5'-CGCGAATTCATGGTCATTGGAGGTGTT-3' and Primer 2 is 5'-ATACTCGAGTCACTGGGGGCAGGTCG-3'.Total RNA was prepared from the venom gland of Gloydius ussuriensis and RT-PCR was conducted to amplify the TLE.A 716 bp DNA fragment was obtained,inserted into the pMD18-T vector and transferred into E.coli JM 109 competent cell,then identified by restriction enzyme and sequencing Results The results showed that the cDNA we got shared great sequence homology(95%) with the published snake TLE cDNA sequence,the amino acid sequence encoded by this cDNA was 237,which shared 94% homology with Gene Bank,it has very high homology with other snakes.Conclusion A novel cDNA encoding snake TLE was got by RT-PCR.