岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析

Purification,cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

Thrombin like enzyme has great medical application in treating thrombus. A thrombin like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion exchange chromatography on DEAE Sepharose and affinity chromatography on heparin sepharose. SDS polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32 000 dalton. Total RNAs were extracted from the venom gland of the G. saxatilis snake. Using degenerate primers, we amplified the cDNA of the thrombin like enzyme gene in the venom gland of G. saxatilis using the reverse transcription polymerase chainreaction (RT PCR) method. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Its open reading frame is composed of 774 nucleotides and codes a protein pre zymogen of 258 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed pro peptide of 6 amino acid residues. It contains 12 cysteine residues. The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin like enzyme genes of other snakes in the gene bank. The query sequence exhibits strong amino acid sequence homology of 88%, 88% and 86% to the serine proteas of T. gramineus , thrombin like defibrase Ⅰ of D. acutus and serine protease catroxase Ⅱ of C. atrox respectively. Based on the amino acid sequences of other thrombin like enzymes, the catalytic residues and disulfide bridges of this thrombin like enzyme are deduced as follows: catalytic residues, His 65 ,Asp 110 , Ser 204 ; and six disulfide bridges Cys 31 Cys 163 ,Cys 50 Cys 66 ,Cys 98 Cys 256 ,Cys 142 Cys 210 ,Cys 174 Cys 189 and Cys 200 Cys 225 . According to the possible linked glycosylation sites N X T (Asn X Thr) or N X S (Asn X Ser), its possible glycosylation sites are N 44 S 45 T 46 and N 251 T 252 T 253 residues.