CN103980349A

CN103980349A - Polypeptide capable of regulating activity of FGFR2 (Fibroblast Growth Factor Receptor 2)

Info

Publication number: CN103980349A
Application number: CN201410244482.5A
Authority: CN
Inventors: 陈林; 谭乔燕; 金旻; 杜晓兰; 王晓凤; 王权; 徐伟
Original assignee: Third Affiliated Hospital of TMMU
Current assignee: Third Military Medical University TMMU; Third Affiliated Hospital of TMMU
Priority date: 2014-06-04
Filing date: 2014-06-04
Publication date: 2014-08-13
Anticipated expiration: 2034-06-04
Also published as: CN103980349B

Abstract

The invention discloses a polypeptide capable of regulating the activity of FGFR2 (Fibroblast Growth Factor Receptor 2). The amino acid sequence of the polypeptide is Asn-Leu-Gly-Gln-Glu-Leu-Ala-Phe-Arg-Val-Pro-Asn. The polypeptide disclosed by the invention can be specifically combined with the FGFR2, plays an obviously regulating role on the activity of the FGFR2, can outstanding inhibit the activity of p-ERK (Phospho-Extracellular Signal-Regulated Kinase) contained in the main downstream signal channel MAPK (Mitogen Activated Protein Kinase) of the FGFR2, has the advantages of low molecular weight, easiness for preparation, controllability in quality, low immunogenicity and the like and has good potential application value in the field of medicines.

Description

Regulate the polypeptide of FGFR2 activity

Technical field

The invention belongs to biological technical field, relate to a peptide species.

Background technology

FGFR (fibroblast growth factor acceptor) is the cross-film tyrosine kinase receptor that a class has autophosphorylation activity, with its respective ligand FGF (fibroblast growth factor) combination, in histoorgan growth and injury repairing process, play a significant role.

4 kinds of FGFR are found at present, i.e. FGFR1, FGFR2, FGFR3 and FGFR4.Wherein, FGFR2 has critical function in bone and skull growth.Previously studies show that, two kinds of increased functionality types of FGFR2 point mutation (FGFR2-Ser252Trp, FGFR2-Pro253Arg) causes modal mankind craniosynostosis-Apert syndrome, shows as that coronal suture early closes, brachycephalism, also finger that the bad companion of Craniofacial growth is serious; In conjunction with the expression of RNAi and Cre-loxP system regulation FGFR2 gene, tamoxifen (tamoxifen) induction significantly reduces the lethality rate of Apert mouse after processing; In death 10.5 days embryonic stages, there are the multiple dysorganoplasias including four limbs in FGFR2 knock out mice.Mitogen-activated protein kinase signal path MAPK is one of topmost signal path in FGFR2 downstream, and the activation situation of MAPK directly or indirectly affects propagation, differentiation, conversion and the apoptosis etc. of cell.At mammalian cell, find to exist 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, between parallel MAPK path, both can mutually distinguish by complicated mechanism, can mutually regulate again.Studies have found that, blocker SB203580, the PD98059 of p38 and Erk1/2 processes respectively the shin bone of FGFR2-Ser252Trp mouse, and vitro culture is significantly alleviated bone forming hysteresis phenomenon for 7 days afterwards.Therefore, launch target biology treatment for FGFR2 and be undoubtedly important research direction from now on.

Regulate at present measure and the means of FGFR2 activity more, can activate FGFR2 by FGFs, plasmid, the virus etc. of expressing FGFs, and check FGFR2 activity with blocker, neutralizing antibody, RNAi etc.But there is the shortcoming of poor stability and induction of immunity reaction in the former, neutralizing antibody, RNAi may cause that other non-specific FGFRs is as the activity change of FGFR1, FGFR3 etc., and various FGFRs are not quite identical to the effect of bone, even contrary, as activate FGFR3 and may prevent and treat osteoarthritis, activate FGFR1 and but promote osteoarthritis to occur.In addition, FGFs/FGFRs tool is urged tumor vessel formation effect, has certain risk, and this just needs us to find other molecules that can specificity regulate FGFR2 activity.

Can regulate the molecular species of acceptor more, common are micromolecular compound, antibody and polypeptide.Wherein to have space structure simple for polypeptide, and molecular weight is little, and specificity is high, seepage force is strong, easily reach histoorgan, and be difficult for being identified by enzyme and degrading, immunogenicity is low, the features such as the less immunne response that causes anti-polypeptide, and polypeptide can synthetic, and purification process is easy, thereby more and more comes into one's own in new drug development, existing multiple polypeptides medicine is successfully applied to clinical, as alpha-thymulin etc.Exploitation yet there are no the polypeptide of specific combination FGFR2, therefore can and regulate its active polypeptide to have good application prospect with FGFR2 specific combination.

Summary of the invention

In view of this, the object of the present invention is to provide a peptide species, can and regulate its activity with FGFR2 specific binding, also have molecular weight little, prepare the advantages such as simple, quality controllable, immunogenicity is low.

After deliberation, the invention provides following technical scheme:

The polypeptide that regulates FGFR2 activity, aminoacid sequence is Asn-Leu-Gly-Gln-Glu-Leu-Ala-Phe-Arg-Val-Pro-Asn (SEQ ID No.1).

Beneficial effect of the present invention is: polypeptide provided by the invention can with FGFR2 specific binding, and its activity is had to obvious regulating effect, can significantly suppress the activity of p-ERK in the main downstream signal path of FGFR2 MAPK, also have molecular weight little, prepare the advantages such as simple, quality controllable, immunogenicity is low, in field of medicaments, have good potential using value.

Brief description of the drawings

In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe:

Fig. 1 is the combination situation of ELISA method checking P3 peptide and FGFR2.

Fig. 2 is that luciferase reporter gene method detects 10 impacts of μ M P3 peptide on FGFR2 downstream signal p-ERK level, and * * represents and blank group (blank) ratio, P<0.01; * * represents and blank group (blank) ratio, P<0.001.

Fig. 3 is the impact that mtt assay detects different concns P3 peptide processing C3H10T1/2 cell on cell proliferation after 24 hours, and * represents and blank group (blank) ratio, P<0.05.

Fig. 4 is that ALP staining detects 10 μ M P3 peptide impacts to osteoblast differentiation on C3H10T1/2 cell.

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.

The FGFR2 using in preferred embodiment, mouse-anti FGFR2 antibody is all purchased from Sigma company of the U.S., phage 12 peptide library selection test kits (comprising ER2738 bacterial classification) are purchased from New England Lab company of the U.S., the rabbit anti-mouse igg of horseradish peroxidase (HRP) mark is purchased from biological reagent company of Zhong Shan Golden Bridge, pcDNA3.1-Myc empty carrier is preserved by this seminar, Myc-FGFR2 plasmid is so kind as to give by David professor Ornitz of Washington, DC university, efficient eucaryon transfection reagent VigoFect and luciferase reporter gene detection kit are purchased from prestige lattice Lars biotechnology (Beijing) company limited.

One, from phage 12 peptide storehouses elutriation can with the polypeptide of FGFR2 specific binding

1, the elutriation in phage 12 peptide storehouses

Be that the FGFR2 solution of 20 μ g/mL is (taking concentration as 0.1mol/L by concentration, pH is that 8.6 sodium hydrogen carbonate solution is solvent) 150 μ L coated elisa plates, 4 DEG C of coated spending the night, discard coating buffer, it is that the BSA solution of 5mg/mL is (taking concentration as 0.1mol/L that full hole adds concentration, pH is that 8.6 sodium hydrogen carbonate solution is solvent), 37 DEG C of sealing 1h, discard confining liquid, (contain the TBS damping fluid of Tween-20 with TBST, when first round elutriation, the concentration of Tween-20 is 1mL/L, the concentration of the second Tween-20 while taking turns with third round elutriation is increased to 5mL/L) washing 6 times, drop into phage and (when first round elutriation, drop into the mixed solution of phage 12 peptide storehouse stostes and TBST, second drops into the phage of the specific binding that previous round elutriation goes out while taking turns with third round elutriation), under room temperature, 1h is hatched in gentle concussion, discard the phage of non-specific binding, with TBST washing 10 times, adding concentration is 0.2mol/L, pH is glycine-hydrochloride buffer of 2.2, the phage of gentle concussion 8min wash-out specific binding under room temperature, draw elutriant, adding concentration is 1mol/L, pH is 9.1 Tris-HCl damping fluid 150 μ L neutralizations, measure titre, for subsequent use,

ER2738 bacterium is seeded in LB substratum 20mL, 37 DEG C of concuss are cultured to mid-log phase (OD600 value approximately 0.5), add aforementioned with in Tris-HCl damping fluid and after phage elutriant 100 μ L, 4.5h is cultivated in 37 DEG C of concussions, 4 DEG C, the centrifugal 10min of rotating speed 10000rpm, the supernatant liquor that absorption contains phage, add and be equivalent to the PEG/NaCl solution of supernatant liquor 1/6 volume (NaCl that the PEG8000 that is 200g/L by concentration and concentration are 2.5mol/L forms, lower same), 4 DEG C of hold over night precipitation phages, 4 DEG C, the centrifugal 15min of rotating speed 10000rpm, abandon supernatant liquor, by the resuspended phage precipitation of TBS damping fluid, centrifuging and taking supernatant liquor again, add the PEG/NaCl solution that is equivalent to supernatant liquor 1/6 volume, ice bath precipitation phage 90min, the centrifugal supernatant liquor of abandoning, with containing the resuspended phage precipitation of TBS damping fluid 1mL that concentration is the sodium azide of 0.2g/L, centrifuging and taking supernatant liquor again, add the PEG/NaCl solution that is equivalent to supernatant liquor 1/6 volume, ice bath precipitation phage 90min, the centrifugal supernatant liquor of abandoning, with containing the resuspended phage precipitation of TBS damping fluid 200 μ L that concentration is the sodium azide of 0.2g/L, centrifuging and taking supernatant liquor again, measure titre, as the input phage of next round elutriation,

Repeat above-mentioned steps carry out altogether three-wheel elutriation, enrichment can with the phage of FGFR2 specific binding; The phage that third round elutriation goes out is no longer increased with ER2738 bacterium.

The phage rate of recovery of each wheel elutriation is in table 1.As shown in Table 1, after three-wheel elutriation, increase gradually with the rate of recovery of the phage of FGFR2 specific binding, show to have compared with the phage of high-affinity by effectively enrichment with FGFR2.

The rate of recovery of the each wheel of table 1. elutriation process pnagus medius

Round	Drop into phage (pfu)	Reclaim phage (pfu)	The rate of recovery (%)
				1	1×10 ¹¹	1×10 ³	1×10 ^-8
2	9×10 ¹¹	1.93×10 ⁵	2.14×10 ^-7
				3	8×10 ¹¹	2.9×10 ⁶	3.62×10 ^-6

2, the DNA extraction of phage and order-checking

ER2738 bacterium is seeded in LB substratum, 37 DEG C of concuss are cultured to mid-log phase, point get 1mL and put in culture tube, add the phage mono-clonal of random picking the titer determination flat board of the phage going out from third round elutriation, 37 DEG C of concuss are cultivated 4.5h, centrifugal, get the supernatant liquor 500 μ L that contain phage, add PEG/NaCl solution 200 μ L, precipitation at room temperature phage 10min, the centrifugal supernatant liquor of abandoning, add the resuspended phage precipitation of iodide damping fluid 100 μ L, add again ethanol 250 μ L, incubated at room 10min makes phage DNA precipitation and most of phage albumen is retained in solution, the centrifugal supernatant liquor of abandoning, the washing with alcohol that DNA precipitation is 700mL/L by concentration, after vacuum-drying, be resuspended in TE damping fluid 30 μ L.Get this solution 5 μ L, entrust Jia Gen bio tech ltd, Shanghai to check order.

The phage that the present embodiment goes out from third round elutriation as stated above, random 29 phage mono-clonals of picking carry out DNA extraction and order-checking, derive the aminoacid sequence of the exogenous polypeptid of showing on phage according to the DNA sequence dna recording, found that P3 peptide (aminoacid sequence is as shown in SEQ ID No.1) can specific binding FGFR2.

Two, synthetic can with the polypeptide of FGFR2 specific binding

According to the aminoacid sequence of P3 peptide, entrust Shanghai Qiangyao Biotechnology Co., Ltd. to adopt standard Fmoc scheme solid phase synthesis P3 peptide.After measured, synthesize polypeptide purity higher than 95%, temperature-70 DEG C preservation.

Three, the combination situation of the synthetic polypeptide of ELISA method checking and FGFR2

In 96 hole enzyme plates, it is that P3 peptide solution 20 μ L and the coated damping fluid of 1mmol/ μ L (got sodium carbonate 0.3g that every hole adds concentration, sodium bicarbonate 0.58g and sodium azide 0.04g, be dissolved in water, regulate pH to 9.6, be diluted with water to again 200mL, obtain) 80 μ L, blank group (not adding P3 peptide) is set simultaneously, 4 DEG C of coated spending the night, discard solution, it is the calf serum solution (PBS taking pH as 7.4 is as solvent) of 50g/L that full hole adds concentration, 37 DEG C of sealing 60min, discard solution, (contain the PBS that concentration is the Tween-20 of 0.5mL/L with PBST, pH is 7.4) wash 3 times, adding concentration is the FGFR2 solution 30 μ L of 100 μ g/mL and the PBS70 μ L that pH is 7.4, hatch 1h for 37 DEG C, with PBST washing 3 times, add mouse-anti FGFR2 antibody (the BSA solution taking mass percentage concentration as 1% is as diluting solvent) the 100 μ L of 1:500 dilution, hatch 1h for 37 DEG C, discard solution, with PBST washing 5 times, add the rabbit anti-mouse igg 100 μ L of HRP mark, hatch 30min for 37 DEG C, discard solution, with PBST washing 5 times, add tetramethyl biphenyl diamines-hydrogen peroxide urea solution 100 μ L, 37 DEG C of lucifuge colour developing 10min, adding concentration is the sulphuric acid soln termination reaction of 2mol/L, measure OD450 value at wavelength 450nm place, result is with the equal value representation in 3 multiple holes.As shown in Figure 1, P3 peptide and FGFR2 have higher combination rate to result.

Four, synthetic polypeptide detects the regulating effect of FGFR2 activity

1, luciferase reporter gene method detects the activation situation of synthetic polypeptide to the main downstream signal path of FGFR2 MAPK

24h before transfection, 293T cell is seeded to 12 well culture plates, after growing to 80% fusion, with efficient eucaryon transfection reagent VigoFect transfection 0.1ng Myc-FGFR2 plasmid (comparing with pcDNA3.1-Myc plasmid) and ERK path reporter plasmid (50ng PFR-luc simultaneously, 50ng P-EIK-1 and 5ng internal reference PRL-TK), after 3h, change liquid, after 24h, add 10 μ M P3 peptide processing, blank group (not adding P3 peptide) and PD166866 control group (substituting P3 peptide with FGF inhibitor PD166866) are set simultaneously, after processing 24h, discard substratum, with 1 × PBS rinsing cell of precooling 2 times, every hole adds 300 μ L1 × Universal Lysis Buffer (ULB), put-80 DEG C of freezing cracking 1h of cryogenic refrigerator, room temperature vortex vibration 30min is to the complete cracking of cell again, measure uciferase activity, record Photinus pyralis LUC flat light emission (Rlus1) and renilla luciferase flat light emission (Rlus2), transcribe relative reactivity value using the relative ratio of Rlus1/Rlus2 as reporter gene, result represents with the means standard deviation in 3 multiple holes.As shown in Figure 2,10 μ M P3 peptides can significantly suppress the activity of FGFR2 downstream p-ERK to result, and prompting P3 peptide may be the physiological process that the activity by regulating p-ERK regulates cell.

2, mtt assay detects the impact of synthetic polypeptide on FGFR2 express cell propagation

In 96 well culture plates, 5000 mescenchymal stem cell C3H10T1/2 of every hole inoculation, are that the DMEM-H substratum of foetal calf serum of 50g/L is at 37 DEG C, 5%CO with containing concentration ₂condition under cultivate 24h, after cell attachment, discard substratum, rinse cell 2 times with serum-free DMEM-H substratum, add again serum-free DMEM-H substratum 1mL, cultivate 24h, discard substratum, add and contain DMEM-H substratum and the different final concentration (0.5 that concentration is the foetal calf serum of 50g/L, 1, 10, 25, 50, 100 μ M) P3 peptide, blank group (not adding P3 peptide) is set simultaneously, process 24h, add again cell proliferation detection reagent 20 μ L, lucifuge is hatched 4h, measure OD490 value at wavelength 490nm place, result is with the equal value representation in 6 multiple holes.Result as shown in Figure 3, process after C3H10T1/2 cell 24h by 10 μ M P3 peptides, and cell-proliferation activity significantly raises, and prompting P3 peptide can promote the propagation of C3H10T1/2 cell.

3, the synthetic polypeptide impact to osteoblast differentiation on FGFR2 express cell of ALP staining examine

C3H10T1/2 cell is seeded to 12 well culture plates, after growing to 80% fusion, with being added with 10% foetal calf serum, 10 ^-8α-MEM substratum of M dexamethasone, 50 μ g/mL vitamins Cs, 0.01M β-phospho-glycerol sodium and 10 μ M P3 peptides carries out osteoblast differentiation induction, single induction group (not adding P3 peptide) and blank group (not adding dexamethasone, vitamins C, β-phospho-glycerol sodium and P3 peptide) are set simultaneously, induce after 7 days, substratum is abandoned in suction, PBS rinsing 2 times, the fixing 15min of 4% 4 DEG C of paraformaldehydes, PBS rinsing 2 times, add ALP staining fluid, hatch 30min for 37 DEG C, observe C3H10T1/2 cell ALP secretion situation after Osteoinductive differentiation.As shown in Figure 4,10 μ M P3 more singly induction groups of inducing peptide treatment group and blank group ALP are active significantly to be strengthened result, and prompting P3 peptide can promote C3H10T1/2 cell to osteoblast differentiation.

Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.

Claims

1. the polypeptide that regulates FGFR2 activity, is characterized in that, aminoacid sequence is Asn-Leu-Gly-Gln-Glu-Leu-Ala-Phe-Arg-Val-Pro-Asn.