Structure And Function Studies Of A Novel Snake Venom Thrombin-like Enzyme From Agkistrodon Acutus

Cardiovascular and cerebrovascular diseases such as stroke,coronary heart disease and peripheral vascular thrombosis embolism have seriously impacted on quality of people life,cause a heavy burden for families.As one precious nature resources,Snake venoms contain lots of biologically active proteins and peptides,including on the components of interacting vascular system.Using snake venom to the develop new thrombosis-treatment drugs has emerged as the important researches.In this study,we separated the tissue of Agkistrodon acutus venom gland,and constructed a phage display library of Agkistrodon acutus venom gland by use of Novagen's T7 Select(?) Phage Display System.After three rounds of panning,we obtained a new Agkistrodon acutus snake venom thrombin-like enzyme gene.Whereafter,we analyzed the gene structure of thrombin-like enzyme using bioinformatics.In order to express the thrombin-like enzyme,we constructed a prokaryon vector of pET-32a-TLE, then expressing induced by IPTG.After affinity purification and molecular sieve purification,we obtained the recombinant snake venom thrombin-like enzyme protein which keeped a goog biological activity.The main results of our study are as follws:1.Firstly constructed phage display library of Guangxi Agkistrodon acutus venom,after identifying,the titer of library reaching 2.5×10~6 pfu/ml.2.Set up approach to pan anticoagulant components using the ways of using fibrin/fibrinogen:Coat 100μl 10μg/m fibrin/fibrinogen on the solid-phase medium such as ELISA plate,add amplified phage library into coated plate,wash away the non-specific phage which is binded to the plate,after three rounds of biopanning "Adsorption -Elution-Amplificationion",enrich the specific phage which is binded to the plate.3.Obtaining a new thrombin-like enzyme gene from Agkistrodon acutus snake venom. Length is 783bp,it encodes 260 amino acids.Acquaired the GenBank Accession Number AY861138.1 after submission.It has 97%homology with the thrombin-like enzyme of Gen Bank accession nuber AF159060 through the Blast.we analysised the Hydrophilicity and hydrophobicity by the web of http://www.expasy.org/cgi-bin/protscale.pl,and found it obvious Hydrophilicity.Homology modeling analysis indicted finding that there are three structural domains,signal peptide,Tryp_Spc(Trypsin-like serine protease),and intrinsic disorder.4.Constructed the prokaryotic expression vector pET-32a-TLE,optimized expression conditions at 0.7 mmol/L of IPTG and 25℃.Protein inclusions expression was cut down under this condition,which laid the foundation for the simple and efficient purification of the thrombin-like enzyme.5.The biological activity of recombination Agkistrodon acutus snake venom thrombin-like was analyzed:plasma coagulation experiments in vitro showed that 4.4μg of recombinant thrombin-like enzyme could make 200μl plasma coagulation in 65 seconds at 37℃;Fibrinogen hydrolysis activity assay showed that 8μg thrombin-like enzyme could degrade the Aαchain of fibrinogen in 60 minutes throughly.