A kind of biological effect detection method of integrin GP IIb/IIIa specific binding molecules BatifeibanTechnical field
The invention belongs to biological technical field, relate in particular to a kind of biological effect detection method of integrin GP IIb/IIIa specific binding molecules Batifeiban.By building restructuring Chinese hamster ovary cell (the Chinese Hamster Ovary cell line of stably express integrin GP IIb/IIIa compound, Chinese hamster ovary celI), and by flow cytometer detection means, analyze the fibrinogen of Batifeiban and fluorescein isothiocynate (FITC) fluorescent material mark and compete in conjunction with integrin GP IIb/IIIa site, suppress thereby detect Batifeiban the ability that recombinant C HO is combined with fibrinogen.Can detect equally Batifeiban inhibition by the method and express the ability that the recombinant cell of integrin av β 3 compounds is combined with vitronectin.
Background technology
Hematoblastic adhesive attraction is mainly by quantity and the affinity control of acceptor.Hematoblastic activity factor is a lot, and wherein fibrin ferment (Thrombin), adenosine diphosphate (ADP) (ADP) can activate blood platelet.These factors can cause the transmission (transmission from inside to outside, inside-out Signaling) of cell within a cell information, thereby improve the affinity of glycoprotein receptor (GP IIb/IIIa acceptor).GP IIb/IIIa after activation can be combined with soluble ligand fibrinogen, together with making that blood platelet and soluble fibrin are former and interweaving, forms platelet aggregation.Wherein platelet aggregation is the main cause of coronary artery thrombosis.
GP IIb/IIIa(is also referred to as integrin alpha IIb β 3) be subordinated to cell surface receptor extended family, i.e. an integrin.Integrin receptor is the adhesion of mediated cell and viscous protein not only, and the transmission of mediation various kinds of cell information, and it,, in the physiology and pathologic process of cell, plays indispensable effect.Most of integrins are distributed in various kinds of cell, in general, a subunit can from multiple different β subunit combination, different β subunits can from different a subunit combinations, therefore can form multiple not homospecific acceptor.GP IIb(α IIb) be peculiar on blood platelet, therefore can form the distinctive GP IIb/IIIa of blood platelet acceptor.GP IIb/IIIa is the acceptor that platelet surface quantity is maximum (80,000 copies of about each blood platelet), accounts for 1%～2% of whole blood platelet albumens.And GP IIb/IIIa is only distributed in blood platelet and precursor thereof, the key effect with it in during platelet aggregation is consistent.
GP IIb/IIIa acceptor comprises Liang Ge subunit: GP IIb(α IIb), molecular weight is about 140 kDa; GP IIIa (or β 3), molecular weight is about 105 kDa.GP IIb is containing the heavy chain and the light chain that are connected by disulfide bond, and heavy chain is positioned at extracellular completely, and light chain is fixed on membrane plasmapheresis as foundation; GP IIIa is a single chain protein, is divided into film outskirt, permeable membrane district and intracellular region.GP IIb(α IIb) complex has 5 Ca2+ binding sites, wherein 4 in GP IIb subunit, one in GP IIIa subunit, under physiology Ca2+ concentration (1 μ M), all site is all combined, and this is essential for the 26S Proteasome Structure and Function that keeps GP IIb/IIIa.In the time that analyzing platelet aggregation as anti-coagulants, application sodium citrate finds, when Ca2+ concentration is during at 40～50 μ M, the combination rate of Ca2+ binding site is lower, and the ability of GP IIb/IIIa binding fiber proteinogen and mediation platelet aggregation also will weaken.Lower plasma C a2+ concentration (<1 μ M) will make GP IIb/IIIa dissociate and lose subunit structure.
Up to now, it is found that GP IIb can only combine with GP IIIa, and GP IIIa can also be combined with α v subunit form vitronectin (Vitronectin) acceptor (av β 3).To be only limited to blood platelet different from the Tissue distribution of GP IIb/IIIa, and α v β 3 is distributed in various kinds of cell, comprises vascular endothelial cell and vascular smooth muscle cell.α v β 3 is mainly distributed in vascular endothelial cell and vascular smooth muscle cell, with part vitronectin (Vitronectin) combination, stimulates vascular wall hyperplasia, causes and blocks.
On unprovoked blood platelet, GP IIb/IIIa is in inactivated state, not in conjunction with adhesive plasma proteins.After vessel endothelium and induced endothelial wound, blood platelet sticks with subendothelium immediately, and is activated (as fibrin ferment, collagen, ADP, adrenaline etc.) by one or more local activators.Every kind of activator will activate a hematoblastic independent signal transduction pathway, no matter and be which kind of activator, final each bars conduction path all will focus on GP IIb/IIIa acceptor, to the active state of convert.And the activator that some are strong, as fibrin ferment, collagen, can make GP IIb/IIIa storage pool in cell (be confined to α intragranular, sometimes contain the fibrinogen of combination in advance) transfer to platelet surface, increases the quantity of GP IIb/IIIa acceptor.The GP IIb/IIIa of activation can interact with several adhesive plasma proteinss, such as fibrinogen, vWF, vitronectin, fibronectin etc., thereby mediation platelet aggregation and adhesion.
Batifeiban is platelet glycoprotein acceptor repressor, and molecular weight is 818.95D[patent 03112798.3 and patent 200610083865.4].It can not only suppress blood platelet GP IIb/IIIa is combined with fibrinogen, can also suppress the combination of vitronectin and av β 3 integrins, thereby effectively suppress human blood platelets due to the caused aggegation of multiple activity factor, and can participate in suppressing the hyperplasia of cells of vascular wall.
The platelet aggregation method using is at present generally according to turbidimetry principle, do reference with platelet poor plasma (PPP), be rich in blood platelet blood plasma (PRP) under the stirring condition of particular device, add after derivant, due to platelet activation and then assemble, the turbidity of suspension declines, penetrability increases, photomultiplier (photoelectric cell) transfers turbidity signal to electric signal, and the variation of electric signal under instrument record can be calculated platelet aggregation degree and speed according to tracing curve.The biological effect of medicine platelet aggregation-against also can obtain by suppressing platelet aggregation, but the method for traditional platelet aggregation is utilized fresh blood, separates PRP and PPP and tests.The individual difference that is also subject to various factors, especially blood donor in experimentation, the common error ratio of experimental result is larger, is not suitable as the qualification of Batifeiban biological effector function.
Summary of the invention
The object of the invention is to for traditional existing shortcoming of platelet aggregation test method, a kind of biological effect detection method of integrin GP IIb/IIIa specific binding molecules Batifeiban is provided, can suppresses the ability that recombinant cell is combined with its part fibrinogen by Accurate Determining GP IIb/IIIa antagonist Batifeiban by flow cytometer.
First build the restructuring Chinese hamster ovary cell of stably express integrin GP IIb/IIIa or av β 3, then digest 5 ~ 500,000 cells/ reactions, and use respectively the Batifeiban [patent 03112798.3 and patent 200610083865.4] of 100ug/ml, 20ug/ml, 4ug/ml, 800ng/ml, 0ng/ml to hatch altogether in advance 30 minutes under 37 DEG C of condition of culture, then clean.Then use the FITC labelled fibrinogen (preferentially selecting concentration is 5ug/ml) of 0.1ug/ml ~ 10ug/ml to hatch altogether 1 hour, then clean, and carry out flow cytometer (BD Accuri C6) and detect.Owing to using in advance Batifeiban to hatch, occupied the binding site of integrin IIb/IIIa or av β 3, cause the follow-up fibrinogen adding (or vitronectin) can and the combination that is at war with of same site; Along with the reduction of Batifeiban working concentration, fluorescently-labeled fibrinogen (or vitronectin) increases progressively in conjunction with quantity thereupon, thereby indirect reaction goes out the affinity of Batifeiban to integrin IIb/IIIa or av β 3, according to the variation curve plotting of amount of fluorescence and Batifeiban molecular conecentration, thereby calculate the IC50 of the affinity of Batifeiban to integrin GP IIb/IIIa or av β 3.
Concrete steps of the present invention are:
(1) first build the restructuring Chinese hamster ovary cell of stably express integrin GP IIb/IIIa or av β 3;
(2) then by the detection means such as cell Elisa or flow cytometer checking Batifeiban molecule can with recombinant cell generation specific binding;
(3) next use fibrinogen (or vitronectin) competition of Batifeiban and fluorescent material mark in conjunction with the binding site of the integrin GP IIb/IIIa above recombinant cell or av β 3;
(4) by fibrinogen albumen (or vitronectin) concentration of fixing fluorescent material mark, change Batifeiban molecular conecentration, measure the amount of fluorescence of the binding site that is finally combined in integrin GP IIb/IIIa above recombinant cell or av β 3, according to the variation curve plotting of amount of fluorescence and Batifeiban molecular conecentration, thereby calculate the IC50 of the affinity of Batifeiban to integrin GP IIb/IIIa or av β 3.
Brief description of the drawings
The restructuring Chinese hamster ovary cell (flow cytometer detects figure) of Fig. 1 stably express integrin GP IIb/IIIa
Negative control is CHO(DG44) cell, sample is recombinant cell, primary antibodie is mouse-anti IIb/IIIa(Abcam company, lot number: ab7116, model: GR36679-2), two resist for sheep anti mouse FITC mark two anti-(ProteinTech company, lot number: SA00003-1)
The restructuring Chinese hamster ovary cell (flow cytometer detects figure) of Fig. 2 stably express integrin alpha v beta 3
Negative control is CHO(DG44) cell, sample is recombinant cell, primary antibodie is mouse-anti α v-β 3(Abcam company product, lot number: ab38431, model: GR61269-3), two resist for sheep anti mouse FITC mark two anti-(ProteinTech company, lot number: SA00003-1)
Fig. 3 Batifeiban and the competition of fluorescently-labeled fibrinogen are in conjunction with the integrin GP IIb/IIIa site above recombinant cell (flow cytometer detects figure)
Negative control is CHO(DG44) cell, sample is recombinant cell, primary antibodie is the fibrinogen (5ug/ml) of FITC mark
The fibrinogen working concentration 5ug/ml of FITC mark, Batifeiban competition concentration is 100ug/ml, 20ug/ml, 4ug/ml, 800ng/ml, 0ng/ml.
Fig. 4 Batifeiban and the competition of fluorescently-labeled vitronectin are in conjunction with integrin av β 3 sites above recombinant cell (flow cytometer detects figure)
Negative control is CHO(DG44) cell, sample is recombinant cell, primary antibodie is the vitronectin (5ug/ml) of FITC mark.
The vitronectin working concentration 5ug/ml of FITC mark, Batifeiban competition concentration is 100ug/ml, 33ug/ml, 11ug/ml, 3.7ug/ml, 1.2ug/ml, 0.4ug/ml, 0ug/ml.
Fig. 5 A is Batifeiban and fluorescently-labeled fibrinogen competition binding curve figure.
Fig. 5 B is Batifeiban and fluorescently-labeled vitronectin competition binding curve figure.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The restructuring Chinese hamster ovary cell of embodiment 1 construction expression integrin GP IIb/IIIa or integrin alpha v beta 3
By the expression vector pCDNA3-IIb that contains IIb and IIIa genes of interest with pCDNA3-IIIa(clones respectively genes of interest IIb by genetic engineering and IIIa enters in pCDNA3 plasmid, routine operation is referring to " molecular cloning "), and contain respectively the expression vector pCDNA3-av of av and β 3 genes of interest and pCDNA3-β 3(clones respectively genes of interest av by genetic engineering and β 3 enters in pCDNA3 plasmid, routine operation is referring to " molecular cloning "), use respectively restriction enzyme Pvu I(NEB company) linearization electric turning in the Chinese hamster ovary cell CHO (DG44) (ATCC Number:CRL-9096) that enters adhere-wall culture.Wherein cell number can be 1 ~ 5 × 106, the each 5 ~ 40ug of the pCDNA3-IIb after linearization and pCDNA3-IIIa, p α v and the each 5 ~ 40ug of P β 3 plasmid cotransfection CHO (DG44) respectively.Then be laid in 96 orifice plates with 5000cells/well respectively, cultivating system is maintained DMEM+10%FBS+650ug/mlG418(Merck company).Wait cloning when longer, choose in 24 orifice plates, continue to maintain 650ug/ml G418 screening and press.After covering with in 24 orifice plates, forward in T75 culture flask, after covering with, part carries out flow cytometer detection (BD Accuri C6).
Collect negative control cell CHO(DG44) and cell line to be checked, PBS cleans 2 times.Respectively toward IIb/IIIa and α v β 3 cell lines to be checked and the middle corresponding primary antibodie mouse-anti IIb/IIIa(abcam company that adds of negative control cell strain CHO (DG44), lot number: ab7116, model GR36679-2) and mouse-anti α v-β 3(abcam company, lot number: ab38431, model: GR61269-3), final concentration is 2 μ g/ml, 200 μ l/ pipes, mix, hatch on ice 1 hour.PBS cleans after 2 times, adds sheep anti mouse FITC mark two anti-(ProteinTech company, lot number: SA00003-1), and final concentration is 1:500,200 μ l/ pipes, and lucifuge is hatched 30 minutes on ice.PBS washes 3 times, is finally resuspended in 200 μ l PBS, carries out flow cytometer (BD Accuri C6) and detects.Pick out positive recombinant cell strain and carry out second and take turns subclone, be laid in 96 orifice plates respectively with 1 ~ 5 cells/well, cultivating system is maintained DMEM+10%FBS+650ug/mlG418.Wait cloning when longer, repeat second and take turns clone's step, detect until carry out flow cytometer (BD Accuri C6).The final restructuring Chinese hamster ovary cell of picking out respectively stably express integrin GP IIb/IIIa or integrin alpha v beta 3.(Fig. 1, Fig. 2).
Embodiment 2 Batifeibans and fluorescently-labeled fibrinogen (or vitronectin) are competed in conjunction with the integrin GP IIb/IIIa site above recombinant cell or integrin alpha v beta 3 site
First use the fibrinogen (or vitronectin of FITC mark) of FITC mark to carry out the pre-enclosed experiment in cell surface site.The restructuring Chinese hamster ovary cell of trypsinization stably express integrin GP IIb/IIIa or integrin alpha v beta 3 respectively, then with 2 × 105packing is carried out in cells/ reaction, and fibrinogen (or vitronectin) variable concentrations (20ug/ml, 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 0 ug/ml) that uses FITC mark carries out fluorescent dye and flow cytometer (BD Accuri C6) detects, then calculate according to the variation of protein concentration and fluorescence numerical value, extrapolate fibrinogen (or vitronectin) concentration of the FITC mark that can occupy integrin site corresponding to recombinant cell surface, and for next step experiment.
Then according to reckoning, digest respectively 5 ~ 50 × 104the stably express integrin GP IIb/IIIa of reaction or the restructuring Chinese hamster ovary cell of integrin alpha v beta 3 (preferentially select 2 × 105cells/ reaction), and use respectively 100ug/ml, 20ug/ml, 4ug/ml, 800ng/ml, 0ng/ml(5 doubly to dilute or 3 times of dilutions) Batifeiban hatch altogether on ice 30 minutes, then PBS cleans 2 times.Then use 0.1ug/ml ~ 10ug/ml(preferentially to select 5ug/ml) FITC labelled fibrinogen (or vitronectin) hatch altogether 1 hour on ice, then PBS clean 2 times, carry out flow cytometer (BD Accuri C6) detect.(Fig. 3, Fig. 4)
The affinity of embodiment 3 Batifeibans to integrin IIb/IIIa or av β 3
Use in advance the restructuring Chinese hamster ovary cell of Batifeiban and stably express integrin GP IIb/IIIa or integrin alpha v beta 3 to hatch (seeing embodiment 2), to occupy the binding site of integrin IIb/IIIa or av β 3, the fibrinogen (or vitronectin) that impact adds is below combined with integrin IIb/IIIa or av β 3 sites; Along with the reduction of Batifeiban working concentration, the fibrinogen (or vitronectin of FITC mark) of FITC mark increases progressively with the combination quantity of integrin thereupon, go out the affinity of Batifeiban to integrin IIb/IIIa or av β 3 from the variation indirect reaction of combined with fluorescent quantity, and can carry out curve plotting to the concentration of fluorescence numerical value and Batifeiban, be that 0.6ug/ml(is 0.733uM thereby calculate Batifeiban for the affinity IC50 of integrin IIb/IIIa on recombinant cell); Batifeiban is that 0.539ug/ml(is 0.658uM for the affinity IC50 of integrin av β 3 on recombinant cell) (Fig. 5 A and Fig. 5 B).