[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 (TLE). 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.
Zhang
Yan-Yu
YY
Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Ma
Ping
P
Lu
Yi-Ming
YM
Lü
Li-Ping
LP
Jiang
Sheng-Yang
SY
Zhou
Xi-Peng
XP
Sun
Shu-Han
SH
Xu
Jin-Bo
JB
D017836:Agkistrodon / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000096:biosynthesis / Q000201:enzymology* / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013917:Thrombin / Q000096:biosynthesis / Q000235:genetics*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013917:Thrombin; C530991:thrombin-like protein, Agkistrodon acutus; D008666:Metalloendopeptidases