Cloning And Expression Of The Gene Encoding Antiplatelet Peptide In Escherichia Coli

Platelets play a pivotal role in acute coronary syndromes . Plaque rupture exposes to highly thrombogenic components that induce platelet activation and initiate coagulation cascade. The recent development of a new class of drugs that allow direct inhibition of platelet GP IIb/IIIa receptors, the 'final common pathway' of platelet activation, has raised the possibility that these potent agents may reduce thrombotic complications after percutaneous coronary interventions or in acute coronary syndromes . In our study, a polypeptide expressing antiplatelet was produced using genetic engineering,and the activation of antiplatelet was assessed in vitro. Perhaps this will provide a direction for the development of antiplatelet drug. The major methods and results are as following:1. Construction of the gene encoding antiplatelet peptideThe recombinant peptide was constructed using glutathione S-transferate (GST) as a protein vector, fused via a cleavable linker to an antiplatelet peptide of 12 amino acids. The peptide was designed to include the Lys-Gly-Asp (KGD) amino acid sequence to prevent fibrinogen binding to platelets GPIIb/IIIa. The amino acid sequence of the 12 amino acid peptides was reversely translated, and the gene encoding the peptide contains 36bps, was assembled from 2 synthetic oligonucleotides, using BamHI and XhcoI restriction enzyme sites at the end of the gene. Two oligonucleotides were chemically synthesized in Beijing sbsbio co (2OD). The sample was identified with 10% polyacrylamidedel electrophoresis (PAGE) and a clear strap emerged which slightly over 30bps. Sequencing was performed using an automated sequencer. Sequencing result of PGEX-4T-1KGDX proved that objective DNA segment was inserted between the BamHⅠand XcoⅠof plasmid pGEX-4T-1 as cloning vector.2 Construction of the expression vector containing the gene encoding the antiplatelet peptides and its expression and purificationThe purified objective DNA segment was inserted between the BamHⅠand XcoⅠof plasmid pGEX-4T-1 as cloning vector and the gene was amplified from it, and E.coliDH 5αwas then transfected. The construct was sequenced to verify accurate gene synthesis. Subsequently the resultant gene was cloned between the BamHⅠand XcoⅠof expression plasmid pGEX-4T-1 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 37℃ heat induction after adding 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 purified using affinity chromatography by GST-agarose. This procedure was also followed in parallel to produce GST. Protein extracts and column fractions were analyzed on 15% SDS-PAGE gels and resulted in the appearance of an approximately 26 000Da和25 556Da. fusion protein were found to be soluble, abundant. Yields of 35mg/litre of cultures were obtained. The fued GST-KGDX protein was expressed in E.coli to a level of 48.02% of total celluar protein. 3. Measurement of antiplatelet activityBlood sample were collected from 8 healthy volunteer (no accepting any drugs influencing coagulate system. The experiment repeated 3 times and took the average value). Platelet rich plasmin (PRP) was produced with hypothermia centrifugal and platelet aggregation in response to ADP (measured at 4 min following addition of ADP) was turbidimetrically measured in a Platelet Aggregometer at room temperature (expressed by %). Inhibition of platelet Aggregation in platelet-rich plasma was determined for each of the peptides by a platelet aggreometer. A series of peptides with various dose were examined for their ability to inhibit the binding of fibrinogen to GPIIb/IIIa by flow cytometry greometer. In our s...