CN101619093B - Polypeptide with function of promoting activity of fibroblast growth factor receptor 3 and screening method and application thereof - Google Patents
Polypeptide with function of promoting activity of fibroblast growth factor receptor 3 and screening method and application thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide with function of promoting the activity of a fibroblast growth factor receptor 3 (FGFR3) and a screening method and an application thereof. The polypeptide is formed by an amino acid sequence shown as Val-Sel-Pro-Pro-Leu-Thr-Leu-Gly-Gln-Leu-Leu-Ser. The screening method comprises the following steps: incubating a bacteriophage 12 peptide library and an elisa plate coated by the FGFR3 together, washing non-specificity combined bacteriophage, then eluting specificity combined bacteriophage and amplifying to be used as input bacteriophage elutriated in the next turn, repeating the steps for performing elutriations in three turns, extracting DNA singly cloned by the bacteriophage elutriated in the third turn and sequencing, deducting the amino acid sequence of a foreign polypeptide shown on the bacteriophage, synthesizing the polypeptide, detecting the promotion of the polypeptide to the activity of the FGFR3, and obtaining the polypeptide with function of promoting the activity of the FGFR3. The polypeptide can be used for preparing medicine of resisting low FGFR3 function low diseases.
Description
Technical field
The present invention relates to a peptide species, particularly a kind of have promote the active polypeptide of fibroblast growth factor receptor3, also relate to the screening method and the application of this polypeptide.
Background technology
Fibroblast growth factor acceptor (fibroblast growth f actor receptors, FGFRs) be that a class has the active film tyrosine kinase receptor of striding of autophosphorylation, with its respective ligand fibroblast growth factor (fibroblast growth factors, FGFs) combination plays a significant role in histoorgan growth and injury repairing process.
4 kinds of FGFRs have been found at present, i.e. FGFR1, FGFR2, FGFR3 and FGFR4.Wherein, the negativity of FGFR3 mediation endochondral ossification is regulated molecule, has vital role in skeleton development.Previously studies show that, the transgenation of FGFR3 function acquisition type causes the propagation of chondrocyte in the epiphyseal growth plate and differentiation to be suppressed, cause multiple skeleton development defective genetic diseases, as fetal rickets (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD) etc.; And the transgenation of FGFR3 afunction type causes the delay of epiphyseal growth plate closing, loose cartilage area broadening and long bone hypertrophy etc.Recent study finds, FGFR3 also has vital role in the generation of skeletal diseases such as osteoarthritis, osteoporosis, fracture, development, promote the FGFR3 activity can prevent and treat skeletal diseases such as osteoarthritis, osteoporosis and fracture.
Though found 23 kinds of FGFs at present, only do not seen so far and FGFR3 specificity bonded FGFs.Because of polypeptide has advantages such as molecular weight is little, space structure is simple, immunogenicity is low,, be expected to develop the medicine that becomes anti-FGFR3 hypofunction disease so exploitation can combine and promote its active polypeptide that good prospects for application is arranged with the FGFR3 specificity.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of active polypeptide of the FGFR3 of promotion that has; Two of purpose is to provide the described screening method that promotes the active polypeptide of FGFR3 that has; Three of purpose is to provide the described application that promotes the active polypeptide of FGFR3 that has.
For achieving the above object, in a first aspect of the present invention, provide a kind of active polypeptide of the FGFR3 of promotion that has, form by the aminoacid sequence shown in the Val-Ser-Pro-Pro-Leu-Thr-Leu-Gly-Gln-Leu-Leu-Ser.
In a second aspect of the present invention, the described screening method that promotes the active polypeptide of FGFR3 that has is provided, may further comprise the steps:
The biological elutriation in a, phage 12 peptide storehouses
Phage 12 peptide storehouses and the enzyme plate that is coated with FGFR3 are hatched altogether, with the phage of TBST flush away non-specific binding, use glycine-hydrochloride buffer wash-out specificity bonded phage again, use in the Tris-HCl damping fluid and elutriant, get specificity bonded phage, measure titre; Specific amplification bonded phage in host bacterium ER2738 is measured titre, as the input phage of next round elutriation; Repeat above-mentioned steps and carry out the three-wheel elutriation altogether, enrichment can with FGFR3 specificity bonded phage;
DNA extraction of b, phage and order-checking
Picking phage mono-clonal at random on the titer determination flat board of the phage that goes out from the third round elutriation after the amplification, extracts DNA and checks order in host bacterium ER2738, derives the aminoacid sequence of the allogenic polypeptide of showing on the phage according to measured dna sequence dna;
Synthesizing of c, polypeptide
According to the aminoacid sequence of gained allogenic polypeptide, artificial synthetic polypeptide;
The short FGFR3 of d, polypeptide is active to be detected
Detect polypeptide to the active promoter action of FGFR3, promptly get and have the active polypeptide of the FGFR3 of promotion.
Further, the concrete grammar of described step a is: be the FGFR3 solution coated elisa plate of 20 μ g/mL with concentration, 4 ℃ of bags of temperature are spent the night, discard coating buffer, adding concentration is the BSA solution of 5mg/mL, 37 ℃ of sealings of temperature 1 hour, discard confining liquid, use the TBST thorough washing, add phage 12 peptide storehouses, concussion was hatched 1 hour under the room temperature, discard the phage of non-specific binding, use the TBST thorough washing, adding concentration is 0.2M, pH is glycine-hydrochloride buffer of 2.2, and the concussion wash-out is 8 minutes under the room temperature, draw elutriant, adding concentration is 1M, pH is 9.1 Tris-HCl damping fluid neutralization, gets specificity bonded phage, measures titre; The ER2738 bacterium is seeded in the LB substratum, and 37 ℃ of concussions of temperature are cultured to mid-log phase, add specificity bonded phage, 37 ℃ of concussions of temperature are cultivated, 4 ℃ of temperature are centrifugal, get supernatant liquor, and are centrifugal again, get supernatant liquor, the PEG/NaCl that adds 1/6 volume, 4 ℃ of standing over night precipitations of temperature phage, 4 ℃ of temperature are centrifugal, abandoning supernatant, add the resuspended precipitation of TBS, centrifugal again, get supernatant liquor, the PEG/NaCl that adds 1/6 volume, ice bath precipitation 90 minutes, 4 ℃ of temperature are centrifugal, abandoning supernatant, add and contain the resuspended precipitation of TBS that concentration is the sodium azide of 0.2g/L, centrifugal again, collect supernatant liquor, the specificity bonded phage that must increase, measure titre, as the input phage of next round elutriation; Repeat above-mentioned steps and carry out the three-wheel elutriation altogether, enrichment can with FGFR3 specificity bonded phage, wherein, during first round elutriation among the TBST concentration of Tween-20 be 1mL/L, the concentration of Tween-20 is increased to 5mL/L among second TBST when taking turns with the third round elutriation;
Further, the concrete grammar of described step b is: the ER2738 bacterium is seeded in the LB substratum, 37 ℃ of concussions of temperature are cultured to mid-log phase, the phage mono-clonal of picking at random on the titer determination flat board of the phage that adding goes out from the third round elutriation, 37 ℃ of concussions of temperature are cultivated, centrifugal, get and contain the phage supernatant liquor, add PEG/NaCl, room temperature is placed, centrifugal, abandon supernatant liquor, precipitation is resuspended in the iodide damping fluid, add ethanol, incubated at room, centrifugal, abandon supernatant liquor, precipitation is the washing with alcohol of 700mL/L with concentration, be resuspended among the TE after the of short duration vacuum-drying, get this solution and carry out determined dna sequence, derive the aminoacid sequence of the allogenic polypeptide of showing on the phage according to measured dna sequence dna.
In a third aspect of the present invention, provide the described application of the active polypeptide of promotion FGFR3 in the medicine of the anti-FGFR3 hypofunction disease of preparation that have.
Beneficial effect of the present invention is: polypeptide of the present invention can combine and promote its activity with the FGFR3 specificity, and have advantages such as molecular weight is little, preparation is simple, quality controllable, immunogenicity is low, can be used to prepare the medicine of anti-FGFR3 hypofunction disease, prevention or treatment FGFR3 hypofunction disease such as osteoarthritis, osteoporosis, fracture etc. have good potential using value.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 shows that Western blot method detects the variation that polypeptide P1 handles ATDC5 cell ERK phosphorylation level after 0.5,1.5,2.5 hours;
Fig. 2 shows that Western blot method detects the variation that polypeptide P2 handles ATDC5 cell ERK phosphorylation level after 0.5,1.5,2.5 hours;
Fig. 3 shows that Western blot method detects the variation that polypeptide P1 handles ATDC5 cell P38 phosphorylation level after 0.5,1.5,2.5 hours;
Fig. 4 shows that Western blot method detects the variation that polypeptide P2 handles ATDC5 cell P38 phosphorylation level after 0.5,1.5,2.5 hours;
Fig. 5 shows that the polypeptide P1 of different concns handles the cell proliferation situation of ATDC5 cell after 24 hours;
Fig. 6 shows that the polypeptide P1 of different concns handles the cell proliferation situation of C3H10T1/2 cell after 24 hours;
Fig. 7 shows the cell proliferation situation after polypeptide P1 or P2 continue processing ATDC5 cell.
Embodiment
The present invention adopts the display technique of bacteriophage screening to have the active polypeptide of the FGFR3 of promotion.Display technique of bacteriophage is the appropriate location that the dna sequence dna of foreign protein or polypeptide is inserted into bacteriophage coat protein structure gene, foreign gene is expressed with the expression of coat protein, simultaneously, foreign protein or polypeptide re-assemblying and be shown to the biotechnology of phage surface with phage.The foreign protein or the polypeptide that are expressed in phage surface can combine with the target molecule specificity.The target molecule bag is carried out the screening of phage display peptide library behind solid phase carrier, just expression can be had phage separation and purification from numerous phages of corresponding foreign protein or polypeptide to come out, and foreign protein or amino acid sequence of polypeptide can obtain by dna sequencing to phage.
The screening of phage display peptide library is biological elutriation (biopanning) process of an affinity purification.Its detailed process is: phage peptide library and target molecule were interacted after for some time, the phage of non-specific combination is removed in washing, again specificity bonded phage is eluted and increase, drop into the next round elutriation then, repeat 3～4 and take turns elutriation, can obtain and target molecule specificity bonded phage.
Below with reference to accompanying drawings, preferred embodiment is described in detail.
The FGFR3 that uses in the preferred embodiment is available from U.S. sigma company, and phage 12 peptide storehouse screening reagent boxes (comprising the ER2738 bacterial classification) are available from U.S. New England Lab company.
One, from phage 12 peptide storehouses the screening can with FGFR3 specificity bonded polypeptide
May further comprise the steps:
The biological elutriation in a, phage 12 peptide storehouses
With concentration is that (concentration is 0.1M for the FGFR3 solution of 20 μ g/mL, pH is that 8.6 sodium bicarbonate is a solvent) 150 μ L bag is by 96 holes sterilization polystyrene enzyme plate, 4 ℃ of bags of temperature are spent the night, discard coating buffer, (concentration is 0.1M to the BSA solution that full hole adding concentration is 5mg/mL, pH is that 8.6 sodium bicarbonate is a solvent), 37 ℃ of sealings of temperature 1 hour, discard confining liquid, with the TBST (TBS that promptly contains Tween-20, the concentration of Tween-20 is 1mL/L during first round elutriation, the concentration of second Tween-20 when taking turns with the third round elutriation is increased to 5mL/L) washing 6 times, add 2 * 10
11The phage of the pfu (mixed solution that adds phage former storehouse 10 μ L in 12 peptide storehouses and TBST 1mL during first round elutriation, second adds the specificity bonded phage of previous round amplification when taking turns with the third round elutriation), gentle concussion was hatched 1 hour under the room temperature, discard the phage of non-specific binding, with TBST washing 10 times, adding concentration is 0.2M, pH is glycine-hydrochloride buffer 200 μ L of 2.2, gentle concussion wash-out is 8 minutes under the room temperature, draw elutriant, adding concentration is 1M, pH is 9.1 Tris-HCl damping fluid 150 μ L neutralization, get specificity bonded phage, measure titre; The ER2738 bacterium is seeded among the LB substratum 20mL, 37 ℃ of concuss of temperature are cultured to mid-log phase (OD600 value about 0.5), add specificity bonded phage 100 μ L, 37 ℃ of concuss of temperature were cultivated 4.5 hours, 4 ℃ of temperature, rotating speed 10, centrifugal 10 minutes of 000rpm, get supernatant liquor, centrifugal again 5 minutes, get about 80% supernatant liquid (lower floor contains sedimentary clear liquid and discards), the PEG/NaCl (is that the PEG8000 of 200g/L and NaCl that concentration is 2.5M form by concentration) that adds 1/6 volume, 4 ℃ of standing over night precipitations of temperature phage, 4 ℃ of temperature, rotating speed 10, centrifugal 15 minutes of 000rpm, abandoning supernatant, add the resuspended precipitation of TBS 1mL, centrifugal again 5 minutes, get supernatant liquor, the PEG/NaCl that adds 1/6 volume, ice bath precipitation 90 minutes, 4 ℃ of temperature, rotating speed 10, centrifugal 10 minutes of 000rpm, abandoning supernatant, add and contain the resuspended precipitation of TBS 200 μ L that concentration is the sodium azide of 0.2g/L, centrifugal again 1 minute, collect supernatant liquor, the specificity bonded phage that must increase is measured titre;
Repeat above-mentioned steps and carry out the three-wheel elutriation altogether, enrichment can with FGFR3 specificity bonded phage, the phage that the third round elutriation goes out is no longer increased; Each phage rate of recovery of taking turns elutriation sees Table 1, and as shown in Table 1, after the biological elutriation of three-wheel, the yield of specificity bonded phage increases gradually, shows that the phage of institute's elutriation has obtained selective enrichment.
Table 1, respectively take turns the rate of recovery of elutriation process pnagus medius
DNA extraction of b, phage and order-checking
The ER2738 bacterium is seeded in the LB substratum, 37 ℃ of concuss of temperature are cultured to mid-log phase, branch is got 1mL and is put in the culture tube, the phage mono-clonal of picking at random on the titer determination flat board of the phage that adding goes out from the third round elutriation, 37 ℃ of concuss of temperature were cultivated 4.5 hours, centrifugal, get and contain phage supernatant liquor 500 μ L, add PEG/NaCl 200 μ L, room temperature was placed 10 minutes, centrifugal 10 minutes, abandon supernatant liquor, of short duration centrifugal, the remaining supernatant liquor of careful sucking-off, precipitation is resuspended among the iodide damping fluid 100 μ L, add ethanol 250 μ L, incubated at room made single stranded phage DNA precipitation in 10 minutes and most of phage albumen is retained in the solution, centrifugal 10 minutes, abandoned supernatant liquor, precipitation is the washing with alcohol of 700mL/L with concentration, (by concentration is 0.01M to be resuspended in TE after the of short duration vacuum-drying, the pH value is that the EDTA that 8.0 Tris-HCl and concentration are 1mM forms) among the 30 μ L, get this solution 5 μ L, entrust Shanghai to give birth to worker's biotechnology company limited and check order;
Present embodiment 45 phage mono-clonals of picking at random carries out determined dna sequence, derives the aminoacid sequence of the allogenic polypeptide of showing on the phage according to measured dna sequence dna; Found that the polypeptide that 23 phage mono-clonals are showed has same aminoacid sequence P1:Val-Ser-Pro-Pro-Leu-Thr-Leu-Gly-Gln-Leu-Leu-Ser (SEQ ID No.1); Also there is high homology between peptide sequence that all the other 22 phage mono-clonals are showed and the P1, comprising P2:Val-Thr-Ser-Pro-Thr-Thr-Leu-Pro-Gln-Leu-Met-Ser (SEQ ID No.2).
Two, artificial synthetic polypeptide
According to the aminoacid sequence of gained allogenic polypeptide, entrust Hua Datian source, Shanghai biotech company to adopt standard Fmoc scheme solid-phase synthetic peptide, gained polypeptide purity all is higher than 95%, puts temperature-70 ℃ preservation.
Three, the ELISA method detects combining of polypeptide and FGFR3
Experiment purpose: polypeptide and keying action between the FGFR3 and bonding strength that checking filters out.
Experimental technique: adopt indirect elisa method, in 96 well culture plates, polypeptide P1 that every hole adding concentration is 1mmol/ μ L or P2 solution 20 μ L and coating buffer (are got yellow soda ash 0.3g, sodium bicarbonate 0.58g and sodium azide 0.04g, be dissolved in water, regulate pH to 9.6, thin up is to 200mL, i.e.) 80 μ L, 4 ℃ of bags of temperature are spent the night, discard coating buffer, full hole adds the calf serum solution that concentration is 50g/L (pH is that 7.4 PBS is a solvent), 37 ℃ of sealings of temperature 40 minutes, discard confining liquid, (contain the PBS that concentration is the tween 20 of 0.5mL/L with PBST, pH is 7.4) wash 3 times, add concentration and be the FGFR3 solution 30 μ L of 100 μ g/mL and pH and be 7.4 PBS 70 μ L, temperature was hatched 1 hour for 37 ℃, with PBST washing 3 times, add mouse-anti FGFR3 antibody (U.S. Sigma company by dilution in 1: 500, mass percentage concentration is that 1% BSA solution is diluting solvent) 100 μ L, temperature was hatched 1 hour for 37 ℃, discard an anti-solution, with PBST washing 5 times, add rabbit anti-mouse igg (middle China fir Golden Bridge biological reagent company) the 100 μ L of horseradish peroxidase-labeled, temperature was hatched 30 minutes for 37 ℃, discarded two anti-solution, with PBST washing 5 times, add tetramethyl biphenyl diamines-hydrogen peroxide urea solution 100 μ L, 37 ℃ of lucifuge colour developings of temperature 10 minutes, adding concentration is the sulphuric acid soln termination reaction of 2M, measure the OD450 value at wavelength 450nm place, the result is with the equal value representation in 3 multiple holes; Negative control group (add polypeptide P1 coated elisa plate, but all reagent all being 7.4 PBS replacement with pH in the subsequent step), P1 blank group and P2 blank group (do not add polypeptide P1 or P2 coated elisa plate, all the other operations are constant) are set simultaneously.
Experimental result: see Table 2, polypeptide P1 and P2 have higher combination rate with FGFR3.
Table 2, ELISA detect the combining of polypeptide and FGFR3 (X ± SD)
Four, Western blot method detects the activation situation of polypeptide to the main downstream signal path of FGFR3 MAPK
Research at present is clear and definite, and mitogen-activated protein kinase signal path MAPK is one of topmost signal path in FGFR3 downstream, and the activation situation of MAPK directly or indirectly influences propagation, differentiation, conversion and the apoptosis etc. of cell.At cells of mamma animals, found to exist following three parallel MAPK signal paths at present, be respectively ERK signal path, p38MAPK path and JNK/SAPK path.Same stimulus can activate many MAPK paths simultaneously, can activate ERK, p38MAPK and three paths of JNK/SAPK simultaneously as stress stimulation, and EGF can activate ERK and two paths of JNK/SAPK simultaneously.Mechanism by complexity between the parallel MAPK path not only can distinguish mutually, but also can regulate mutually.
Experiment purpose: the polypeptide that detection filters out is to the activation situation of signaling molecule in the main downstream signal path of the FGFR3 MAPK path, with the mechanism of clear and definite polypeptides for modulating cell function.
Experimental technique: in 12 well culture plates, every hole inoculation 1.2 * 10
5Individual precartilage cell ATDC5, with containing concentration is that the DMEM/F12 substratum of the foetal calf serum of 50g/L is 37 ℃ in temperature, the CO2 gas concentration is to cultivate 24 hours under the condition of 50mL/L, after treating cell attachment, absorb substratum, wash cell 2 times with serum-free DMEM/F12 substratum, add serum-free DMEM/F12 substratum 1mL, cultivated 24 hours, absorb substratum, adding and containing the final concentration that DMEM/F12 substratum of foetal calf serum that concentration is 50g/L and polypeptide P1 that concentration is 10mM or P2 solution make polypeptide P1 or P2 is 2 μ M, blank group (not adding polypeptide P1 or P2) is set simultaneously, handle 0.5 respectively, 1.5,2.5 hour, collecting cell is blown and beaten lysing cell with cell pyrolysis liquid, in collecting cell homogenate on ice, adopt BCA protein quantification kit measurement protein concentration; The protein sample of having surveyed concentration adds equal-volume 2 * sample-loading buffer, boiled 5 minutes, (adopting concentration is the concentrated glue of 50g/L to carry out SDS-PAGE after the room temperature cooling, concentration is the separation gel of 90g/L, the electrophoresis parameter is 80V 20 minutes, 120V 80 minutes), after electrophoresis finishes, with albumen electricity transfer printing (constant current 300mA 60 minutes) to PVDF membrane, adding concentration is the skim-milk solution (TBST is a solvent) of 50g/L, room temperature sealing 1 hour, add ERK respectively, P-ERK (phosphorylation ERK), P38, one of P-P38 (phosphorylation P38) or β-actin resists, 4 ℃ of overnight incubation of temperature are washed film 5 times with TBST, add horseradish peroxidase-labeled two and resist, temperature was hatched 1 hour for 37 ℃, wash film 5 times with TBST, colour developing, exposure, photographic fixing, detect the variation of ERK or P38 phosphorylation level, and do contrast with the amount of total ERK or P38 molecule, β-actin is an internal reference.
Experimental result: see Fig. 1～4, polypeptide P1 has promoter action to the phosphorylation level of ERK molecule in the MAPK signal path, and the phosphorylation level of P38 molecule is had restraining effect, polypeptide P2 also has promoter action to the phosphorylation level of ERK molecule, and prompting polypeptide P1 and P2 regulate the cells physiological process by the activity of regulating the ERK molecule.
Five, mtt assay detects the situation that influences of different concns polypeptide on cell proliferation
Experiment purpose: detect the cell proliferation situation of FGFR3 express cell after the effect of different concns polypeptide.
Experimental technique: in 96 well culture plates, 8000 precartilage cell ATDC5 of every hole inoculation or mesenchymal cell C3H10T1/2 are that the DMEM/F12 substratum of the foetal calf serum of 50g/L is 37 ℃, CO in temperature with containing concentration
2Gas concentration is to cultivate 24 hours under the condition of 50mL/L, after treating cell attachment, absorb substratum, wash cell 2 times with serum-free DMEM/F12 substratum, add serum-free DMEM/F12 substratum 1mL, cultivated 24 hours, absorb substratum, add and to contain the DMEM/F12 substratum of foetal calf serum that concentration is 50g/L and polypeptide P1 or the P2 solution that concentration is 10mM makes the final concentration of polypeptide P1 or P2 be respectively 1,5,10,50,100 μ M, blank group (not adding polypeptide P1 or P2) is set simultaneously, handling 24 hours, and added cell proliferation detection reagent 20 μ L, is 37 ℃ in temperature, CO
2Gas concentration is that lucifuge was hatched 2～6 hours under the condition of 50mL/L, treats that liquid becomes pink colour by purple in the hole, measures OD570 value at 570nm wavelength place, and the result is with 3 equal value representations of answering holes.
Experimental result: see Table 3～4, polypeptide P1 all has promoter action to the propagation of ATDC3 and two kinds of cells of C3H10T1/2 under low concentration, does not have obviously effect (seeing Fig. 5～6) under higher concentration; And polypeptide P2 also has promoter action to the propagation of two kinds of cells, but there is not obvious dependency with peptide concentration.
Table 3, mtt assay detect the situation that influences of polypeptide P1 on cell proliferation
Table 4, mtt assay detect the situation that influences of polypeptide P2 on cell proliferation
Six, cell counting detects the long lasting effect situation of finite concentration polypeptide on cell proliferation
Experiment purpose: detect the cell proliferation situation of FGFR3 express cell behind finite concentration polypeptide continuous action.
Experimental technique: in 96 well culture plates, inoculation 3000 precartilage cell ATDC5 in every hole are that the DMEM/F12 substratum of the foetal calf serum of 50g/L is 37 ℃, CO in temperature with containing concentration
2Gas concentration is to cultivate 24 hours under the condition of 50mL/L, after treating cell attachment, absorb substratum, wash cell 2 times with serum-free DMEM/F12 substratum, add serum-free DMEM/F12 substratum 1mL, cultivated 24 hours, absorb substratum, adding and containing the final concentration that DMEM/F12 substratum of foetal calf serum that concentration is 50g/L and polypeptide P1 that concentration is 10mM or P2 solution make polypeptide P1 or P2 is 2 μ M, blank group (not adding polypeptide P1 or P2) is set simultaneously, handles 1 respectively, 2,3,4,5 days, absorb substratum, adding concentration is the pancreatin 100 μ L of 2.5g/L, digested 5 minutes, and added and contain the DMEM/F12 substratum 200 μ L termination digestion that concentration is the foetal calf serum of 50g/L, dispel cell, obtained cell suspension 10 μ L carry out cell counting, and the result is with the equal value representation in 3 multiple holes.
Experimental result: see Table 5 and Fig. 7, polypeptide P1 has lasting short proliferation function to precartilage cell ATDC5.
Table 5, cell counting detect the long lasting effect situation of polypeptide P1 and P2 on cell proliferation
Based on above-mentioned experimental result, can draw as drawing a conclusion: polypeptide P1 of the present invention can combine with the FGFR3 specificity, and its activity there is obvious facilitation, can be used as activeconstituents, use separately or use with other compound and/or extract composition compound with pharmacologically active, make the medicine of the anti-FGFR3 hypofunction disease of various formulations with pharmaceutically acceptable carrier according to the conventional formulation method of pharmaceutical field again, be used for prevention and treatment FGFR3 hypofunction disease such as osteoarthritis, osteoporosis, fracture etc.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A. field operations Surgery Research Institute
＜120〉have the active polypeptide of fibroblast growth factor receptor3 of promotion and screening method and application
<160>2
<210>1
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉polypeptide P1
<400>1
Val?Ser?Pro?Pro?Leu?Thr?Leu?Gly?Gln?Leu?Leu?Ser
1 5 10
<210>2
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉polypeptide P2
<400>2
Val?Thr?Ser?Pro?Thr?Thr?Leu?Pro?Gln?Leu?Met?Ser
1 5 10
Claims (1)
1. have the active polypeptide of the fibroblast growth factor receptor3 of promotion, it is characterized in that: form by the aminoacid sequence shown in the Val-Ser-Pro-Pro-Leu-Thr-Leu-Gly-Gln-Leu-Leu-Ser.
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| CN102627686B (en) * | 2012-04-20 | 2013-10-02 | 中国人民解放军第三军医大学第三附属医院 | Polypeptide with effect of inhibiting activity of fibroblast growth factor receptor 3 and application thereof |
| CN103980349B (en) * | 2014-06-04 | 2016-03-23 | 中国人民解放军第三军医大学第三附属医院 | Regulate the polypeptide of FGFR2 activity |
| CN112546244B (en) * | 2017-06-22 | 2023-07-04 | 密执安州立大学董事会 | Fibroblast growth factor receptor 2 specific peptide reagents and methods |
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| CA2468125A1 (en) * | 2001-10-24 | 2003-05-01 | Dgi Biotechnologies, Inc. | Target specific screening and its use for identifying target binders |
| WO2003076467A1 (en) * | 2002-03-14 | 2003-09-18 | Yeda Research And Development Co. Ltd. | Compositions and methods for inducing and regulating bone formation |
| CN101027320A (en) * | 2004-06-18 | 2007-08-29 | 恩卡姆医药公司 | FGFR binding peptides |
| WO2007110079A8 (en) * | 2006-03-29 | 2007-12-13 | Enkam Pharmaceuticals As | Targeted delivery of fgfr ligands into the brain |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2468125A1 (en) * | 2001-10-24 | 2003-05-01 | Dgi Biotechnologies, Inc. | Target specific screening and its use for identifying target binders |
| WO2003076467A1 (en) * | 2002-03-14 | 2003-09-18 | Yeda Research And Development Co. Ltd. | Compositions and methods for inducing and regulating bone formation |
| CN101027320A (en) * | 2004-06-18 | 2007-08-29 | 恩卡姆医药公司 | FGFR binding peptides |
| WO2007110079A8 (en) * | 2006-03-29 | 2007-12-13 | Enkam Pharmaceuticals As | Targeted delivery of fgfr ligands into the brain |
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