Preparation And Gene Screening Of Snake Venom Thrombin-like Enzymes

ObjectiveVascular injury hemostasis plays an important role in clinic. Compared with otherhemostatic reagents, snake venom thrombin-like enzymes are better in clinic.No serious sideeffects are found in clinical treatment. There are many publications ans patents about snakevenom thrombin-like enzymes, but there is no core patent for hemostatic protein in China. Thereis also a limit of. In the past eight years, we have found a new hemostatic protein-Agacutse.Because Agacutase is very low in the snake venom and the venom raw material is in limit. Soour objective is to purify the protein and clone the gene. By gene recommendating production,the production cost will be reduced and the production technique will be updated.METHODSA novel thrombin-like enzyme-Agacutase was purified from Agkistrodon acutus venom drypowder by Sephadex G-75, DEAE-Sepharose Fast Flow, and Sephadex G-25culumnchromatography. In the experiment, A280was used to detect the eluation gradients. The puritywas analyzed by using HPLC and SDS-PAGE methods. The accurate molecular weight andclotting activity were obtained by using MORDI-TOF and human plasma cloting methods.The sense primer was designed by using the N-terminal44amino acid sequences ofAgacutase. The antisense primer is M13（-48）. The gene products were amplified by RT-PCR technique. The products were cloned into T-vector and sequenced. The amino acid sequenceBLAST analysis reveal the novel9genes cloned preliminary functional information.RESULTSThe SDS-PAGE results indicated a single band and the the rever-phase HPLC did was90%purity. The MALDI-TOF MS result indicated that the accurate molecular weight is31084Da. The fibrinogen substrate hydrolysis activity showed that the α-subunit wasgradually hydrolyzed by Agcutase with the time. The hydrolysis peak occurs after3hour.In the experiment, porcine thrombin as control obviously hydrolyzes α-and β-subunits ofthe fibrinogen. The hydrolysis peak occurred in1hour. The degradation of β-subunit wasobviously faster than α-subunit.The different lengths of upstream primers were designed according to the N-terminalamino acid sequence. By using RT-PCR method, the9gene fragments were successfullyamplified respectively. Three fragments1200bp,800bp,600bp were obtained by using No.Iprimer pairs. A500bp fragment was obtained by using No.II primer pairs; two fragments1500bp,750bp were obtained by using No.III primer pairs; a750bp fragment was obtainedby using No.IV primer pair1200bp and750bp fragments were obtained by using No.Vprimer pairs. After the PCR products were cloned into T-vector, respectively. Genesequencing and BLAST comparison on NCBI web were determined homology:Gene-II has the maximum homology of21%with the receptor for activated protein kinaseC which has hydrolysis of the alpha chain of fibrinogen; and Gene-III has the maximumhomology of78%with the snake venom serine protease Da-36which has the cleavage offibrinogen Aa, Bβ and γ chain; Gene-IV has the maximum homology of95%with theNADH dehydrogenase subunit4; the gene-V has the maximum homology of77%withrCG20216protein; Gene-VI has the maximum homology g of80%with Snake venomserine protease Dav-PA which has the cleavage of fibrinogen Aα and Bβ chain function;Gene-VII has the maximum homology of95%with Thrombin-like enzyme acutobin whichhas the hydrolysis function of the alpha chain of fibrinogen; Gene-VIII has maximumhomology of60%; Gene-IX has the the maximum homology of55%with Agkistrodon acutus metalloprotease Aahivwhich has the function of metalloproteinases.CONCLUSIONSA high purity and activity of Agacutase was obtained. The gene screening studydiscovered6new snake venom gene fragments.According to the result of protein BLAST,we discovered that six of them have more than50%homology with known thrombin-likeenzymes, some even up to95%.