CN103497236A - Specific heptapeptide targeting to WISP-1 protein and application thereof - Google Patents
Specific heptapeptide targeting to WISP-1 protein and application thereof Download PDFInfo
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Abstract
The invention discloses a specific heptapeptide targeting to WISP-1 protein and application therefore. The amino acid sequence of the specific heptapeptide is as shown in any one of SEQ ID No.1 to 114. According to the invention, 114 antagonist peptides capable of specifically targeting to WISP-1 protein are screened out through a phage display technology; WISP-1 protein peptide antagonists can be prepared from the antagonist peptides; the WISP-1 protein peptide antagonists are capable of obviously inhibiting the activity of the WISP-1 protein, high in efficiency and low in toxicity, and also low in production cost, low in dosage and easy to synthesize.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to specificity seven peptides and the application thereof of target WISP-1 albumen.
Background technology
The secreted protein that Wnt-1 induces (WISP-1) gene is found the earliest in mammary gland of mouse epithelial cell line (C57MG), this gene is positioned at human chromosome 8q24.1-8q24.3, coded product is the secreted protein that 367 amino acid form, and contains a segment signal peptide (SP) and IGFBP, VWFC, a TSP and CTCK4 structural domain (Fig. 5).WISP-1 albumen belongs to conjunctive tissue somatomedin (CCN) family member, and sequential analysis shows that WISP-1 and CCN family member keep the sequence similarity of height.
The same with Cyr-61 with another two CCN family member CTGF, WISP-1 is subject to the signals-modulating of integrin receptor.Integrin receptor plays a role in cell shifts and breeds, and can affect invasion and attack and the growth of cancer cells.WISP-1 can cause the mitotic division effect in some clones, and having sustenticular cell is the ability of long term growth, is embodied in: participate in DNA and synthesize, change cellular form, increase cell saturation ratio, accelerated cell growth etc.WISP-1 can not only accelerate cell proliferation, can also order about the conversion of normal cell to tumour cell.At present, studied and found that the WISP-1 gene increases in some tumor cell lines and neoplastic disease human body, WISP-1 protein-specific overexpression in the tumours such as breast cancer, intestinal cancer, endometrioid carcinoma, chondroma.
At present, all confirmed Wnt-1 and the adjusting of downstream beta-catenin, i.e. the promoter element transcription response Wnt-1 of WISP-1 gene and the beta-catenin white signal during the expression of WISP-1 is subject to the Wnt signal path in cell and animal level.Beta-catenin is the important molecule of Wnt signal path, and its expression is subject to the just regulation and control of Wnt-1 signal.WISP-1 is the downstream targets of Wnt-1, WISP-1 high expression level in the C57MG cell of conversion Wnt-1 gene.In the research to the WISP-1 gene promoter, find, cAMP response element binding albumen (CREB) site plays an important role in the transcription activating process of beta-catenin mediation WISP-1 albumen, and lymph enhanser/T cytokine (TCF/LEF) site has been small effect.
The effect that WISP-1 albumen also shows the inhibition tumor cell growth and shifts in some cell types.In the mouse melanoma cell highly shifted, with the mELML down-regulated expression that WISP-1 is equal to, when this transit cell dyes and expresses mELML, cell shifts and reduces.In lung cancer cell line (H460), overexpression WISP-1 can suppress lung carcinoma cell transfer and cell in vitro invasion and attack and reactivity.The activation of Akt is very important for Cytoskeleton.The research discovery, in the H460 of WISP-1 overexpression cell, the Akt activity is suppressed.Do the used time when the blocking antibody of integrin, the Akt in this cell is reactivated.
The characteristic that WISP-1 promotes cell proliferation and conversion, inhibited apoptosis and tumour cell to shift makes it become the desirable target spot of tumor biotherapy.In vivo or external use antibody can both block the expression of WISP-1, thereby the function of relevant cell and tissue is restored.
The non cellular organism treatment of tumour comprises antibody, polypeptide (or protein) vaccine, gene vaccine, vivo gene treatment etc.Wherein, antibody and polypeptide treatment are two the most active at present fields.The WISP-1 antibody of the scientific research that is useful at present is all non-clinical application type, and the preparation of antibody is expensive and exist mouse antibody easily to produce the drawbacks such as anti-antibody in human body.
With the monoclonal antibody medicine, compare, less polypeptide does not almost have immunogenicity; But chemosynthesis, product purity is high, quality controllable, and a kind of new method can be provided for the biotherapy of tumour.In recent years, along with the development of biotechnology, found that a lot of tumor-related genes and tumour produce regulatory factor, the polypeptide of screening and the special antagonism of these target spots, may become the breach of cancer drug development.
In the polypeptide drugs exploitation, utilize the various ways such as extraction, chemosynthesis, display technique of bacteriophage and proteasome degradation can obtain various peptide storehouse, can filter out active polypeptide from these peptide storehouses, and identify its structure.Phage peptide library is a new technology of rising the nineties in 20th century, its principle is: take phage as carrier, the gene fragment orientation of encoding exogenous polypeptide is inserted into to the coat protein gene district of phage, make allogenic polypeptide by with bacteriophage coat protein, merging and express and be showed in phage surface, the polypeptide be demonstrated can keep relatively independent space structure and biological activity, and then the phage of by the screening of the methods such as affine enrichment, expressing specific polypeptides.
Summary of the invention
The invention provides specificity seven peptides of target WISP-1 albumen, these specificity seven peptides utilize phage peptide library to screen, specifically the albumen of target Wnt signal path downstream target gene WISP-1 coding.
A kind of specificity seven peptides of target WISP-1 albumen, as shown in aminoacid sequence is as arbitrary as SEQ ID No.1～114.
The present invention also provides the gene of described specificity seven peptides of encoding, and the expression unit or the recombinant vectors that contain described gene.
The present invention also provides the fusion rotein that contains described specificity seven peptides.
The present invention also provides surface display that the phage of described specificity seven peptides is arranged.
Specificity seven peptides of the present invention screen acquisition from phage random seven peptide storehouses, and concrete steps comprise:
(1) prokaryotic expression of target protein WISP-1 and purifying;
Adopt the directed cloning method of protein coding sequence, use the expression vector of pFN19A (HaloTag7) T7SP6Flexi vector construction target protein WISP-1, HaloTag albumen is positioned at the N-terminal of WISP-1 fusion rotein; HaloTag, as a kind of novel protein label, can strengthen expression productive rate and the solubility of recombinant protein.
Adopt single stage method (KRX) competent cell to carry out effective conversion of carrier, by rhamnose promoter (rhaBAD), drive t7 rna polymerase to express, and by the T7 promotor, the expression of WISP-1 fusion rotein is controlled flexibly.
The WISP-1 fusion rotein covalency of abduction delivering is fixed on the HaloLink resin, use TEV proteolytic enzyme from the HaloLink resin, WISP-1 albumen to be cut, re-use HisLink resin specificity and remove the TEV enzyme, so just obtained the WISP-1 albumen that output is large and purity is high.
(2) screening of phage antagonistic peptide;
By target protein WISP-1 direct coated on solid support, add phage seven peptide storehouses (production of New England Biolabs company), carry out the affine screening enrichment of " affine absorption → wash-out → recovery → amplification " many wheels, wash away absorption or phage non-specific binding (reference protein GUS screening), screen with the target protein specific binding from phage seven peptide storehouses, express the phage that antagonistic peptide (specificity seven peptides) are arranged.
3) separate and the single phage clone that increases;
The WISP-1 specific combination phage screened is carried out paving plate after enrichment, dilution, select the mono-clonal phage, and cultivate amplification in 96 well culture plates; For screening antagonistic peptide as much as possible, total 4608 clones of coamplification (24 96 hole flat boards), comprising some positives and negative control clone.
(4) evaluation of antagonistic peptide;
WISP-1 specific combination phage is carried out to the ELISA evaluation: at first, by target protein WISP-1 coated elisa plate, then hatch each dilution mono-clonal phage, finally use phage-resistance ML3 antibody test positive monoclonal phage; The positive monoclonal phage, after amplification, is extracted phage single-chain DNA and is checked order; Find the peptide sequence highly repeated by sequential analysis, determine specificity seven peptide motifs of WISP-1 albumen.
Due to described specificity seven Toplink specific binding WISP-1 albumen, so the present invention also provides the application of described specificity seven peptides in preparation WISP-1 protein polypeptide antagonist.Described WISP-1 protein polypeptide antagonist can be used for blocking the expression of WISP-1 albumen, thereby the function of relevant cell and tissue is restored.
The applicant studies discovery, and WISP-1 is the key factor of esophageal cancer cell (KYSE-150R) Epithelial-mesenchymaltransition (EMT) of Radioresistance.In the KYSE-150R cell, the RNA of WISP-1 and protein expression all significantly raise.The applicant uses in antibody and the expression of WISP-1, finds that the Radioresistance of KYSE-150R cell significantly weakens thereupon, also obtains similar result in nude mice model.Therefore, WISP-1 is the extremely valuable treatment target spot that overcomes Radioresistance.
Therefore, WISP-1 protein polypeptide antagonist of the present invention can be radiotherapeutic sensitizer.Described radiotherapeutic sensitizer can be used for weakening the Radioresistance of esophageal cancer cell, is conducive to patient with esophageal carcinoma is carried out to radiotherapy.
The present invention utilizes phage peptide library, the albumen of the Wnt signal path downstream target gene WISP-1 of take coding is target spot, screen the phage with obvious inhibition WISP-1 protein-active from phage seven peptide storehouses, through the sequence of ELISA evaluation and sequential analysis acquisition antagonistic peptide.
Compared with prior art, beneficial effect of the present invention is:
The present invention utilizes phage peptide library, screen 114 specificity seven peptides of target WISP-1 albumen specifically, utilize these specificity seven peptides to prepare the activity that radiotherapeutic sensitizer in WISP-1 protein polypeptide antagonist and esophageal carcinoma therapy can not only obviously suppress WISP-1 albumen, high-efficiency low-toxicity, and production cost is low, consumption is little, easily synthetic.
The accompanying drawing explanation
The screening process figure of specificity seven peptides that Fig. 1 is target WISP-1 albumen of the present invention;
The abduction delivering that Fig. 2 is the WISP-1 recombinant protein is electrophorogram as a result; Wherein, M is protein marker;
The Western blot evaluation figure that Fig. 3 is the WISP-1 recombinant protein;
The ELISA evaluation figure of the phage positive colony that Fig. 4 is affine WISP-1 albumen;
The structure iron that Fig. 5 is WISP-1 albumen.
Embodiment
The screening method of specificity seven peptides of target WISP-1 albumen, its screening process is shown in Fig. 1, specifically comprises:
(1) structure of recombinant plasmid pFN19A Halo-WISP-1
Take pFN2K-WISP-1 as template, design and synthesize the specific PCR primer of WISP-1 gene, carry out pcr amplification, the PCR primer sequence is as follows:
Upstream primer: 5'-CCCGGCGATCGCCATGAGGTGGTTCCTGCCCTG-3';
Downstream primer: 5'-CTTGGTTTAAACGTTGGCAATTTCTGAGAAGTC-3';
The PCR product is purified rear with specificity restriction enzyme (Sgf I& Pme I) carrying out enzyme cuts, pFN19A (HaloTag7) T7SP6Flexi carrier is carried out to enzyme with identical restriction enzyme to be cut, and corresponding cDNA fragment is connected with carrier, connect product and transform intestinal bacteria JML09, PCR identifies positive colony order-checking, the correct positive colony called after pFN19A Halo-WISP-1 by sequence verification.
(2) abduction delivering of recombinant plasmid pFN19A Halo-WISP-1 in the KRX intestinal bacteria
PFN19A Halo-WISP-1 recombinant plasmid transformed competence colibacillus is expressed to bacterium KRX, spread on the LB agar plate and be inverted and cultivate, picking colony, PCR verifies that positive clone is inoculated in the LB substratum containing penbritin, shaking culture, when thalline enters Exponential growth stage, add the rhamnosyl abduction delivering, collect thalline, with Halo protein purification liquid (50mM HEPES, 150mM NaCl, pH7.5) resuspended, the expression condition of SDS-PAGE Analysis deterrmination optimum.
(3) purifying of fusion rotein
According to the expression condition of optimizing (37 ℃, 3.5h) carry out a large amount of abduction deliverings, the centrifugal supernatant that goes is collected bacterium liquid, is deposited in-80 ℃ and preserves or put standby on ice; HaloLink resin suspension 1mL uses the Halo protein purification liquid of 2.5 times of volumes 4 ℃ of balances in advance.
The thalline of collecting by the resuspended step of 15mL Halo protein purification liquid (2), add 100 μ L N,O-Diacetylmuramidases (100mg/mL) and 100 μ L nucleases (5mg/mL), carries out ultrasonication on ice to the clarification of bacterium liquid, centrifugal, collects supernatant liquor; Get the HaloLink resin suspension 1mL that 12mL supernatant liquor (S) adds pre-balance, incubated at room 2h, cross purification column and abandon filtrate (FT), with Halo protein purification liquid washing purification column, finally add Halo protein purification liquid 3mL containing the 8U/mLTEV enzyme in purification column, mix rear room temperature reaction 1h; Cross post and collect filtrate, add Halo protein purification liquid washing pillar, repeat once, collect filtrate (E1), add 10 μ L HisLink resins in 3mL E1, centrifugal collection supernatant (E2) after incubated at room 0.5h.Draw respectively sample from S, FT, E1 and E2, carry out the 10%SDS-PAGE analysis, electrophoresis result is shown in Fig. 2.In Fig. 2, " S-TEV " sample refers to and gets the solution that 10 μ L obtain containing the S example reaction of the Halo protein purification liquid of 8U/mL TEV enzyme and 50 μ L.
(4) evaluation of recombinant protein
The recombinant protein of getting 10 μ g purifying adds 5 μ L5 * protein electrophoresis sample-loading buffer, in 95 ℃ of heating 5min, carries out the 10%SDS-PAGE electrophoresis; Electrophoresis is examined the dyeing of Ma Shi light blue after finishing, or by half-dried electrotransfer system, protein delivery is arrived to nitrocellulose filter 17min(15V), skimmed milk sealing 2h, add the anti-WISP-1 antibody of rabbit in 4 ℃ of overnight incubation, with the PBS washing for several times, each 10min; Again with goat antirabbit-HRP antibody incubation 1h, with after PBS washing three times, with ECL, detect, be exposed to (see figure 3) on X-ray in darkroom.
(5) the affine screening of phage random seven peptide storehouse target WISP-1 albumen
1) wash pipe: 0.1M NaHCO
3(pH8.6) solution cleans coated immunity pipe;
2) coated: [WISP-1 albumen is dissolved in 0.1M NaHCO to prepare 100 μ g/mL target molecule solution
3(pH8.6) in], under room temperature, be coated in immune pipe;
3) sealing: pour out coating buffer, immune pipe is upside down in clean paper handkerchief arsis and gets rid of to remove residual solution, and every pipe is filled it up with confining liquid, 4 ℃ of jog sealings;
4) affine: as to abandon confining liquid, with TBST(TBS+0.1%Tween-20) washing; TBSB(TBS+0.5%BSA with 4mL) the damping fluid dilution 2 * 10
11pfu unit phage (10 μ L), then be added in immune pipe, and 4 ℃ of jogs are hatched;
5) wash-out: topple over and remove unconjugated phage, be inverted immune pipe and get rid of and remove residual solution at clean paper handkerchief arsis, with the TBST damping fluid, wash pipe for several times, wash jog at every turn; After abandoning washings for the last time, every pipe adds the 0.2M Glycine-HCl(pH2.2 of 500 μ L), jog rocks;
6) neutralization: elutriant is sucked in a clean 1.5mL Eppendorf tube, use 1M Tris-HCl(pH9.1) neutralize above-mentioned elutriant;
7) leaving and taking 50 μ L phage eluates measures for phage titre: the program determination 1 μ L phage eluate of ML3 method is 10 routinely
-1-10
-4titre (referring to step (6)) under different extent of dilution;
8) residue eluate amplification: the phage eluate is joined in the ER2738 culture, cultivate 4.5hr for 37 ℃;
9) culture is proceeded in a 50mL centrifuge tube, centrifugal, supernatant liquor proceeds in another centrifuge tube, more centrifugal;
10) top of supernatant 80% is proceeded in a fresh tube, add the PEG/NaCl of 1/6 volume, 4 ℃ of precipitations of phage are spent the night;
11) centrifugal PEG throw out, abandon supernatant, ofer short duration centrifugal, sucks residual supernatant liquor; 1mL TBS resuspended dissolving for precipitation, centrifugal, make the residual cells precipitation, then get supernatant and proceed to another fresh Eppendorf tube, add the PEG/NaCl of 1/6 volume to hatch 60min on ice and again precipitate; Centrifugal, abandon supernatant, be precipitated and dissolved in 200 μ L TBS(containing 0.02%NaN
3) in, be the elutriant of amplification;
12) with reference protein GUS(laboratory deposit) coated immunity pipe, according to above-mentioned 2)-11) step carry out " affine absorption → wash-out → recoverys → amplification " and screen, remove non-specific phage;
13) carry out the three-wheel screening by above-mentioned steps, second takes turns and uses respectively with the later several rounds amplification phage of last round of elutriant again, drops into the phagocytosis scale of construction and all is about 2 * 10
11pfu.
(6) titer determination in the phage seven peptide storehouses that screening obtains
1) the mono-bacterium colony of inoculation ER2738 is in the LB substratum, and shaking table is cultured to mid-log phase;
2) prepare autoclaved Top Agar substratum and LB/IPTG/Xgal flat board;
3) prepare the phage of 10 times of serial dilutions with the LB nutrient solution;
4) when the ER2738 thalli growth to mid-log phase, by the culture equal portions in Eppendorf tube, the corresponding pipe ER2738 culture of each phage extent of dilution;
5) add one by one respectively the different dilution phages of 10 μ L in every pipe ER2738 culture, concussion mixes fast, and room temperature incubation several minutes, carry out phage-infect;
6) will infect in the Top Agar agar culture tube that thalline adds pre-temperature, each pipe, mix fast, is poured into immediately on the LB/IPTG/Xgal flat board, and suitably tilt flat plate evenly spreads out top-layer agar;
7) after flat board is cooling, be inverted in 37 ℃ of lucifuge overnight incubation;
8) check flat board, the plaque number on the counting flat board, calculate plaque forming unit (pfu).
(7) the plaque mono-clonal of target WISP-1 amplification
1) the ER2738 overnight culture is inoculated in to the LB-Tet substratum, constant-temperature table shakes to logarithmic phase;
2) by step (5), last takes turns the Phage Infection Host Strains ER2738 of the target WISP-1 under the screening wash-out, and bed board spends the night;
3) choose respectively 16-48 blue plaque to above-mentioned steps 1) in the ER2738 culture that increases in logarithmic phase, 37 ℃ of shaking tables are cultivated 4.5-5h;
4) culture is proceeded in a 1.5mL centrifuge tube, centrifugal, collect supernatant, this is amplification phage storage liquid, 4 ℃ of storages;
5) top of supernatant 80% is proceeded in a fresh tube, add the PEG/NaCl of 1/6 volume, allow 4 ℃ of precipitations of phage spend the night;
6) centrifugal PEG throw out, abandon supernatant, ofer short duration centrifugal, sucks residual supernatant liquor; The resuspended dissolving of 1mL TBS for precipitation, centrifugal, make the residual cells precipitation, then get supernatant and proceed to another fresh Eppendorf tube, add the PEG/NaCl of 1/6 volume, hatch 60min on ice, centrifugal, abandon supernatant, be precipitated and dissolved in 200 μ L TBS(containing 0.02%NaN
3) in, be the elutriant of amplification.
(8) ELISA identifies the phage positive colony of affine WISP-1
1) with the target molecule solution of 100 μ g/mL, [WISP-1 albumen is dissolved in 0.1M NaHCO
3(pH8.6) in] coated elisa plate, every hole 100 μ L, slightly vibration in wet box;
2) throw away unnecessary target molecule solution, and be inverted flat board and get rid of and remove raffinate at the paper handkerchief arsis, every hole adds the sealing fluid-tight and closes;
3) throw away confining liquid, with 0.1%PBST, wash plate for several times;
4) every hole adds the mono-clonal phage after step (7) increases, and 4 ℃ of shaken overnight are hatched;
5) wash plate for several times with 0.1%PBST, then every hole adds the anti-ML3 antibody of 100 μ L HRP marks, room temperature concussion effect 1h;
6) wash plate for several times with 0.1%PBST, every hole adds 100 μ L tmb substrate chromophoric solutions, and after room temperature effect to blueness obviously manifests, every hole adds the 1M hydrochloric acid termination reaction of 50 μ L, measures the light absorption value at 450nm place.
By the OD with negative target protein PRDX
450value compares (the part comparative result is shown in Fig. 4), acquisition can with 114 positive colony phage (OD of WISP-1 albumen specific combination
4500.4).
(9) fast purifying of sequencing template
1) the ER2738 overnight culture is pressed to the 1:100 dilution, get 1mL mycelium dilution thing, add the positive colony phage 10 μ L of step (8) screening, the 4.5h that increased, after centrifugal, get 500 μ L supernatants and proceed to a fresh centrifuge tube;
2) add 200 μ L PEG/NaCl in centrifuge tube, put upside down and mix, room temperature is placed 10-20min, centrifugal, abandons supernatant, more centrifugal, sucks remaining supernatant;
3) throw out thoroughly is resuspended in 100 μ L iodide damping fluids, adds 250 μ L ethanol, and room temperature incubation 10-20min is centrifugal, abandons supernatant, with 70% ethanol, washes precipitation, centrifugal, abandons supernatant, of short duration placement drying;
4) precipitation is resuspended in 30 μ L containing the H of RNA enzyme
2o or TE[10mM Tris-HCI (pH8.0), 1mMEDTA] in, obtain template solution;
5) get the above-mentioned template solution of 5 μ L, checked order, order-checking obtains the nucleotide sequence of 114 seven peptides of coding.
Claims (9)
1. specificity seven peptides of target WISP-1 albumen, is characterized in that, as shown in aminoacid sequence is as arbitrary as SEQ ID No.1～114.
2. the gene of specificity seven peptides as claimed in claim 1 of encoding.
3. contain the expression unit of gene as claimed in claim 2.
4. contain the recombinant vectors of gene as claimed in claim 2.
5. contain the fusion rotein of specificity seven peptides as claimed in claim 1.
6. surface display has the phage of specificity seven peptides as claimed in claim 1.
7. the application of specificity seven peptides in preparation WISP-1 protein polypeptide antagonist as claimed in claim 1.
8. application as claimed in claim 7, is characterized in that, described WISP-1 protein polypeptide antagonist is radiotherapeutic sensitizer.
9. application as claimed in claim 8, is characterized in that, described radiotherapeutic sensitizer is for weakening the Radioresistance of esophageal cancer cell.
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Cited By (2)
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| CN105111283A (en) * | 2015-08-21 | 2015-12-02 | 天津医科大学 | Small peptide and compositions formed by small peptide connection |
| CN112125954A (en) * | 2020-09-28 | 2020-12-25 | 宁夏大学 | Heptapeptide specifically combined with BCG vaccine, coding gene, preparation method and application |
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| WO2002020822A2 (en) * | 2000-09-08 | 2002-03-14 | Board Of Regents, The University Of Texas System | Biopanning and rapid analysis of selective interactive ligands (brasil) |
| WO2006054262A2 (en) * | 2004-11-18 | 2006-05-26 | Universita Degli Studi Di Roma 'tor Vergata' | Use of phage display technique for identifying peptides capable of binding progenitor/stem cells, peptides thereby obtained and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002020822A2 (en) * | 2000-09-08 | 2002-03-14 | Board Of Regents, The University Of Texas System | Biopanning and rapid analysis of selective interactive ligands (brasil) |
| WO2006054262A2 (en) * | 2004-11-18 | 2006-05-26 | Universita Degli Studi Di Roma 'tor Vergata' | Use of phage display technique for identifying peptides capable of binding progenitor/stem cells, peptides thereby obtained and uses thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111283A (en) * | 2015-08-21 | 2015-12-02 | 天津医科大学 | Small peptide and compositions formed by small peptide connection |
| CN112125954A (en) * | 2020-09-28 | 2020-12-25 | 宁夏大学 | Heptapeptide specifically combined with BCG vaccine, coding gene, preparation method and application |
| CN112125954B (en) * | 2020-09-28 | 2023-02-28 | 宁夏医科大学总医院 | Heptapeptide specifically combined with BCG (bacillus calmette guerin), coding gene, preparation method and application |
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