Cloning Ecpression And Characterization Of A Multifunctional Anticoagulated Peptiede Gene In Escherichia.Coli

Platelet,fibrinogen and thrombin plays a key role in the process of angioplasty,rupture of arterial atheromatous plaque or other factors resulting in injury of the wall of blood vessel. The method preventing thrombosis is inhibiting either the activation of platelet and thrombin, or enhancing the fibrinolytic activation. In our study, a multifunctional hybrid molecule expressing antiplatelet and antithrombin simultaneously was produced using genetic engineering,and the activation of antiplatelet and antithrombin was assessed in vitro. Perhaps this will produce a direction for the development of antithrombin. This study was divided into three steps and briefed as follow: 1 Construction of the gene encoding multifounctional antithrombotic peptides. The recombinant hybrid molecule was constructed using glutathione S-transferate (GST) as a protein vector, fused via a cleavable linker to an antithrombotic peptide of 31 amino acids. The peptide was designed to include three inhibitory regions: (1) the Lys-Gly-Asp (KGD) amino acid sequence to prevent fibrinogen binding to platelets GPIIb/IIIa; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin ,a potent direct antithrombin. The amino acid sequence of the 31 amino acid peptides was reverse translated, and the gene encoding the multifounctional peptide contains 115bps, which include the restriction endonuclease in two sides. Two 70-mre oligonucleotides, designated primer 1 and primer 2 were chemically synthesized in Shanghai SongGong co (2OD). These two primers orerlap 25 base pairs and the lacking ends were filled in by PCR. The sample was identified with 10% polyacrylamidedel electrophoresis (PAGE) and a clear strap emerged which slightly over 100bps. 2 Construction of the expression vector containing the gene encoding the multifounctional antithrombotic peptides and its expression.The purified objective DNA segment was inserted between the BamHⅠand EcoRⅠof plasmid pUC18 as cloning vector and the gene was amplified from it, and E.coli DH 5αwas then transfected. The construct was sequenced to verify accurate gene synthesis. Subsequently the resultant gene was cloned between the BamHⅠand EcoRⅠof expression plasmid pGEX-5X-3 which linked with the 3′end of the gene encoding GST. Gene expression was under control of the strongly inducible tac promotor, which can be induced by the addition of 2mM (IPTG). Using E.coli DH5αas expression host and following transformation to it ,the cells were plated on LB culture medium containing ampicillin .Gene expression was subsequently induced by addition of 2mM IPTG to the culture. Following growth for the addition of IPTG, The cells were harvested and then breaked in iced bath. The fused proteins were isolated using affinity chromatography by GSH-agarose. This procedure was also followed in parallel to produce GST, which was also analyzed in all activity to ensure that the GST protein did not contribute to any antithrombotic or antiplatelet activity. Protein extracts and column fractions were analyzed on 15% SDS-PAGE gels and resulted in the appearance of an approximately 30 KD which larger than GST about 3 KD and purity over 95%.3 Measurement of the fused protein containing multifounctional antithrombotic peptide(1) Measurement of antithrombin activity Draw blood from 8 healthy volunteer (not accepting any drugs producing influence to coagulating system. The results repeated 3 times and toke the average.). Thrombin time (TT) and activated partial thromboplastin time (aPTT), measured with standard assay systems, were used to test for antithrombin activity. An amidolytic assay of thrombin activity, performed using chromozym TH as thrombin substrate, was also used. For the purpose of comparison, the effect of individual peptides of fibrinopeptide A and the tail of hirudin (residues 54-65) on TT, aPTT, and the amidolytic activity of thrombin were also tested. Results show that the purified fusion protein significantly lengthened the TT and aPTT and markedly inhibi...