Study Of Recombinant Neutrophil Inhibitory Factor And Hirulog Hybrid On Clinical New Target

Recombinant neutrophil inhibitory factor and hirulog chimeric protein (TNHH)joined a section of the crosslinked peptides, leukocyte inhibitory factor (NIF) and thehirulog (Hirulog) intermediate with the clot of fibrin binding oriented achieved areduction NIF and Hirulog fragment of risk, the purpose of increasing theeffectiveness, can effectively inhibit leukocyte activation, migration, adhesionfunction, inhibition of thrombin activity, the blood cells with the fibrin binding. Thisproduct has multiple effects in acute ischemic stroke animal model can effectivelypromote the protection and repair of brain tissue damage, improve the localanticoagulant microcirculation and blood flow reperfusion, inhibition ofthrombin-induced brain edema, promote brain hematoma absorption.Objective：Explore the clinical new target of recombinant neutrophil inhibitory factor andhirulog chimeric protein (TNHH).Methods：To take immunoblot identification test and combine respectively HPLC analysis,antithrombin III (AT-III) activity analysis,analysis judgment of d-dimer contentmeasurement of d-dimer transformation experiments.Above the target based of theabove-mentioned experimental determination,to use CADD (computer aided drugdesign) make molecular docking(DOCK) with fibrinogen (FIB), antithrombin III(AT-III) and plasminogen (PLG) of possible new target,to use PSIPRED, Sybyl8.0,and Discovery Studio2.5software make results of the target validation.Results：Recombinant neutrophil inhibitory factor and hirulog chimeric protein (TNHH)make a western blot identification testrespectively with fiber fibrinogen (FIB),antithrombin III (AT-III) and plasminogen (PLG),Western blot results of the rabbitanti TNHH multi-antibody with TNHH+FIB and TNHH+AT-III experimental groupwere negative, with TNHH+PLG experimental group is positive.Western blot resultsof mouse anti-fibrinogen (FIB) monoclonal antibody, mouse anti-thrombin III (AT-III)monoclonal antibody with TNHH+FIB and TNHH+AT-III experimental group arenegative,Western blot results of mouse anti-plasminogen monoclonal antibody with TNHH+PLG was positive.This shows that TNHH does not contain fibrinogen (FIB),or antithrombin III (AT-III), and TNHH contains plasminogen (PLG), expressedTNHH with FIB or AT-III without binding,and have binding with PLG.The resultsshow: TNHH retention time of the standard control was16.335min, and the peak areawas33,578,168.000fibrinogen (FIB) and TNHH make vitro binding assay by HPLCanalysis;The positive control retention time of TNHH+rabbit anti TNHHmulti-antibody is16.095min,and peak area is13,985,716.804; TNHH the retentiontime of the experimental group is15.655min,and peak area is34,964,292.010;Thisshows that TNHH peak area of the the standard control and TNHH peak area ofexperimental group are no significant difference, indicated that the FIB and TNHHare no combination.Antithrombin III (AT-III) and TNHH make vitro binding assay ofAT-III: A activity analysis,results show that: the standard control of AT-III activity is104.7%, and the positive control activity of AT-III is48%, and the experimental groupactivity of the AT-III is109.3%.This indicates that AT-III activity of the standardcontrol and the experimental group in between is no significant differences, indicatingthe anti-AT-III and TNHH was no binding, can not inhibit the activity of antithrombinIII. The plasminogen (PLG) and TNHH make D-dimer transformation experimentsAnalysis of vitro binding experiments, results show that: the positive control ofD-dimer is generated, and the content is0.246mg/L. The experimental group has theD-dimer generated, and the content is0.068mg/L. This shows that plasminogen(PLG) the TNHH have combined to produce plasmin, the plasmin catalyzed fibrindegradation to generate D-dimer. PSIPRED, Sybyl8.0and Discovery Studio2.5software analyze by computer-aided drug design (CADD), the C-terminal153Arg to175Tyr of plasminogen (PLG) is a conserved sequence RNPDNDPQGPWCYTTDPEKRDY with the C-terminal133Lys to140Tyr of recombinant neutrophil inhibitoryfactor and hirulog chimeric protein (TNHH) is a conserved sequence KEGEGVLYhave interaction in the spatial structure of protein, and core function regional offibrinogen (FIB) and antithrombin III (AT-III) have no interaction with TNHH.Conclusions：Through the western blot identification experiment, HPLC experiment, AT-III: Aactive experiment and d-dimer transformation experimental analysis,and combinePSIPRED, Sybyl8.0and Discovery Studio2.5software make target validationanalysis.The preliminary conclusion is: Clinical new target of TNHH is plasminogen (PLG).