Construction And Expression Of Procaryotic Expression Vector Of Tick Anticoagulant Peptide Mutant Containing KGDS Sequence

Thrombus is one of most dangerous diseases to people, especially the tissue infarction caused by cardiovascular embolism or cerebrovascular embolism may lead to serious threat to lives. All kinds of factors, which may cause thrombogenesis, can stimulate platelet adhesion, activation and aggregation. These factors may also activate the coagulation cascade through its intrinsic or extrinsic pathways, which results in the activation of thrombin. The thrombin catalyzed the conversion of the soluble fibrinogen to insoluble fibrin colt at last. Platelet and blood coagulation factor are usually activated at the same time and have synergistic effect between the two processes which terminate in thrombosis. The existing anticoagulants almost all target some or several sites in the two processes to achieve the goal of anti-coagulation.Activated blood coagulation factorⅩ(FⅩa)is the cross point between intrinsic and extrinsic pathways of coagulation cascade and also a participator of the prothrombinase complex. The inhibitors of FⅩa have potent antithrombotic effects with low bleeding side effect and are more effective in inhibiting thrombus formation induced by blood clot and keeping the artery from reocclusion after thrombolysis, comparing with the frequently-used anticoagulants hirudin and heparin. Tick anticoagulant peptide (TAP), isolated from the tick of Africa Ornithodoros moubata, is a potent, highly selective inhibitor of FⅩa. At present, recombinant TAP (rTAP) has been successfully expressed by Saccharomyces cerevisia. The structure and activity of rTAP have no distinction from the native inhibitor. TAP has similar anticoagulant effect and defect with other inhibitors of FⅩa, which have limited resistance to thrombus induced by fibrinogen.Binding between fibrinogen and adhesion molecule receptor glycoprotein(GP)Ⅱb/Ⅲa(αⅡbβ3)on active platelets would induce irreversible platelet aggregation and result in thrombogenesis. Some components found in the venene of snakes, containing KGD sequence or similar sequences(RGD,RGDS,KGDSW, etc) can inhibite platelet aggregation through comparable binding to fibrinogen receptorαⅡbβ3 to achieve the anticoagulant effect.Based on the structure analysis of TAP, We inserted a KGDS sequence into the peptide to construct a new protein with two expected functions of anticoagulation and resistance to platelet aggregation. The basic research will do some explorations to obtain a more effective anticoagulant.Methods:①The chimeric gene sequence of TAP-KGDS was designed by us and was synthesized by Shanghai Sangon Biological Engineering Technology&Services Co,Ltd. The destination gene was amplified by PCR,cloned directionally into prokaryotic expression vector pET32(a+), containing thioredoxin (Trx) gene , to construct expressing plasmid pET32a(+)-TAP-KGDS, which was transformed into Escherichia coli BL21(DE3). The positive bacteria clones were screened by PCR and restriction endonuclease digestion analysis.②The bacteria BL21 containing plasmid pET32a(+)-TAP-KGDS was induced by IPTG to express fusion protein Trx-TAP-KGDS. The expression product was identified by SDS-PAGE,and the expression time and concentration of IPTG were optimized. The harvested cells were crushed by ultrasonic methods. The supernatant and precipitation were collected respectively after centrifugation at 8500 rpm for 30 min to identify the expression form. The soluble fractions were used for the purification of Trx-TAP-KGDS using Ni2+-NTA agarose. The preliminary purified fusion protein was measured by Bradford and its effect of inhibition of platelet aggregation in vitro was tested.Results:①Correct construction of pET32a(+)-TAP-KGDS was identified by methods of PCR and restriction endonuclease digestion analysis. The fragments about 209bp were obtained. The sequence cloned into pET32a(+)was also proved to be correct by sequencing.②The fusion protein was about 24KD analyzed by SDS-PAGE, and most of the recombinant protein exited in the supernatant. When IPTG was 1mmol/L, after inducing about 3 hours, the engineering E.coli had the highest expression.③The preliminary purified fusion protein was about 4.02mg/ml and had activity of inhibition of platelet aggregation.Conclusions: The pET32a(+)-TAP-KGDS prokaryotic expression plasmid was successfully constructed and expressed to obtained a fusion protein Trx-TAP-KGDS. The fusion protein showed activity of inhibition of platelet aggregation which lays a basis to our further study.