Expression Of Hirudin Fusion Protein And Its Conjugation With Fibrin Aptamer

Cardio cerebro vascular diseases(CCVD)is one of major threats to human health,because of its high incidence,disability rate and mortality,it has been a serious impact on human health and life quality.With the change of environment and living habits,CCVD in our country has high incidence,even has trend that onset of younger.So the prevention and treatment for CCVD is one of the emphasis in medical research.Thrombosis plays the core role during the pathological process of CCVD,which severely affects its development and prognosis.Many factors can lead to the thrombosis.So it is the indirect method of reducing the onset of CCVD and improving its prognosis by prevention and treatment thrombus.However,for the shortage of the drugs used clinically,heparin and warfarin are common drugs and have certain clinical effects,there are some disadvantages such as the slow onset,inconvenient,easily bleeding,need monitoring as well as easily interact with foods and drugs.So,a new,convenient,specifically safe anticoagulants has been the focus of drug development.Leech is the representative drug of activating blood circulation and removing blood stasis,including efficacy of invigorating the circulation of blood,removing blood stasisand emmenagogue.It is commonly used in various diseases caused by stagnation of blood stasis.The extracts of Leech salivary gland,which is the strongest natural thrombin inhibitors,have some shortcomings too,such as small molecular weight,short half-life span,single function,easily bleeding.So its gene was recombined.First,RGD sequence was inserted into the gene to make it has the antiplatelet and anticoagulant function.Meanwhile,streptavidin(SA)was connected to the sequence for increasing the molecular weight and prolonging the half-life span.pET-44b-SA-H-RGD plasmid was constructed in previous experiment,fusion protein SA-H-RGD will be induced by IPTG.Then,the fusion protein will be identified and purified by SDS-PAGE and Western-blot.Next,the BCA method will be used to detect the concentration of purified protein and the antithrombin and antiplatelet activity of SA-H-RGD will be determined.On the other hand,the fibrin aptamer G81-2 labelled with biotin at the 5'end will be conjugated with SA-H-RGD.This novel,targeted,multi-functional anticoagulant drugs may promote the process of controlling and prevention thrombotic disease,so as to reduce the incidence of CCVD,even improve its prognosis.Objective:The incidence of CCVD that caused by thrombus has been increasing year by year,preventing the formation of thrombus is the key point to the treatment of CCVD.In this study,the recombinant plasmid pET-44b-SA-H-RGD is confirmed by sequencing,then IPTG was used to induct the expression of fusion protein SA-H-RGD.And the function of SA-H-RGD were verified.Then,the biotin-labelled fibrin aptamer G81-2 will be coupled with SA-H-RGD,which will lay foundation for the development of new anticoagulants.Methods:1.Inoculating glycerol bacteria containing pET-44b-SA-H-RGD plasmid into the LB medium without antibiotics,then adding it in LB medium with antibiotics(Ampicillin 100mg/L)and chloramphenicol(34 mg/L)to cultivate.2.Using the SSCS methods respectively to prepare Rosetta-gami competent cells.3.Extracting pET-44b-SA-H-RGD plasmid with the plasmid extraction kit and sequencing to verify its sequence.Comparing the sequence with theoretical sequence byBioEdit software.4.Transformation of the pET-44b-SA-H-RGD plasmid into Rosetta-gami competent cells.Adding the IPTG to induct when the OD value between 0.4~0.6.Meanwhile,it has designed different concentration gradient and time gradient,as concentration gradient including 0.1,0.3,0.5,0.7,0.9,1.0 mmol/L,the time gradient was 0,1,2,3,4,5,6h respectively.So as to optimize the induction conditions.5.Ultrasound pyrolysis bacteria liquid on ice,then the lysate was identified by SDS-PAGE and Western-blot.6.His Gravi Trap purified protein.Cleaning pillars with Binding buffer,then deal with the supernatant after ultrasound with the filter that has a diameter of 0.45 mm.Next,adding His GraviTrap pillars to purify and continue to add Binding buffer.Using the Elution buffer to elute protein at last.7.SDS-PAGE.Preparing the 12% separating gel and 5% concentrated gel.Then dyeing with Coomassie blue and rinsing with bleaching liquid,sweeping the gel finally.8.Western-blot.Dyeing with Ponceaux after transmembrane Then sealing it in blocking buffer for 1 hour and adding anti-H under the condition of 4 ? for a night.On the second day,it is continue to add second antibody for 1 hour,finally scan the gel.9.Applying the BCA method to detect the concentration of the purified protein.At first,according to the number of standard products and protein samples,calculate the working fluid volume,then prepare the 9 standard substance and dilute the sample protein according the certain concentration.Finally,adding standard substance and protein samples in 96-well plates and testing them with BCA.10.By detecting the activated partial thromboplastin time(APTT),prothrombin time(PT),thrombin time(TT)values of different concentrations SA-H-RGD.11.By detecting platelet aggregation rate after adding different concentrations of SA-H-RGD to verify the antiplatelet activity of fusion protein.12.Modifying the fibrin aptamer G81-2 with biotin at 5' end,coupling with SA-H-RGD and detecing its function with electrophoretic mobility shift assay(EMSA).Results:1.Obtained Rosetta-gami competent cells successfully by the method of SSCS.2.Got pET-44b-SA-H-RGD plasmid.3.Plasmid sequences was consistent with the theoretical sequence by BioEdit software.4.Obtained the optimal inducing conditions of IPTG 0.9 mmol/L for 5 hour.5.Solubility analysis showed that the SA-H-RGD protein of induced was soluble protein.6.SDS-PAGE and Western-blot showed SA-H-RGD fusion proteins was induced successfully.7.The concentration of fusion protein was 7.8mg/mL by the method of BCA.8.In the anticoagulant experiment,APTT,PT and TT values were extended after SA-H-RGD was added,and there were concentration-response relationship between them,with increasing concentration of SA-H-RGD.9.In the antiplatelet experiment,SA-H-RGD significantly inhibited platelet aggregation,and with the increase of concentration of SA-H-RGD,inhibition for platelet aggregation was becoming more obvious.10.EMSA results show that the protein expressed in this experiment can combine the fibrin aptamer G81-2 that selected in previous screening.Conclusion:Fusion protein SA-H-RGD was successfully expressed and its anticoagulant and antiplatelet aggregation function were successful validated.Meanwhile,SA-H-RGD was confirmed the activity of conjugating with fibrin aptamer labeled with biotin.This lays substantial basis for new anticoagulation drug development that targeted to thrombus.