CN103497236B - Specific heptapeptide targeting to WISP-1 protein and application thereof - Google Patents

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

The specific heptapeptide of targeting WISP-1 albumen and its application

Technical field

The invention belongs to biomedicine technical field, and in particular to target the specific heptapeptide of WISP-1 albumen and its answer With.

Background technology

The secreted protein of Wnt-1 inductions（WISP-1）Gene is earliest in mammary gland of mouse epithelial cell line（C57MG）In It was found that, the gene is located at human chromosome 8q24.1-8q24.3, and coded product is the secreted protein of 367 amino acid compositions, Containing a segment signal peptide（SP）And IGFBP, VWFC, TSP and CTCK4 domain（Fig. 5）.WISP-1 albumen belongs to connection group Knit growth factor（CCN）Family member, sequence analysis shows that WISP-1 keeps the sequence similarity of height with CCN family members.

With another two CCN family member CTGF as Cyr-61, WISP-1 is subject to the signals-modulating of integrin receptor.It is whole Close plain acceptor to play a role in cell transfer and in breeding, and the invasion and attack and growth of cancer cell can be affected.WISP-1 is at some Mitosis effect, the ability with sertoli cell system long term growth can be caused in clone to be embodied in：Participate in DNA to close Into, change cellular morphology, increase cell saturation degree, accelerated cell growth etc..WISP-1 can not only accelerate cell to breed, moreover it is possible to drive Make conversion of the normal cell to tumour cell.At present, discovery WISP-1 genes have been studied in some tumor cell lines and neoplastic disease Amplification, the WISP-1 protein-specifics overexpression in the tumours such as breast cancer, intestinal cancer, endometrioid carcinoma, chondroma in human body.

At present, the expression of WISP-1 is had proven in cell and animal level by the Wnt-1 in Wnt signal paths and its The promoter element transcription response Wnt-1 and beta-catenin white signal of the regulation of downstream beta-catenin, i.e. WISP-1 genes.β- Catenin is the important molecule of Wnt signal paths, just regulation and control of its expression by Wnt-1 signals.Under WISP-1 Wnt-1 Trip target, converts the high expression of WISP-1 in the C57MG cells of Wnt-1 genes.Send out in the research to WISP-1 gene promoters It is existing, cAMP response elements binding albumen（CREB）Rise during the transcription activating of beta-catenin mediation WISP-1 albumen in site Important function, and lymph enhancer/T cell factor（TCF/LEF）Site has been small effect.

WISP-1 albumen also shows the effect for suppressing growth of tumour cell and transfer in some cell types.In height In the mouse MC of transfer, the mELML expression being equal to WISP-1 is lowered, and mELML is transfected and expressed in the cell When, cell transfer is reduced.In lung cancer cell line（H460）In, overexpression WISP-1 can suppress lung carcinoma cell to shift and external Cell invasion and activity.The activation of Akt is highly important for Cytoskeleton.Research finds, in WISP-1 excess Akt activity is suppressed in the H460 cells of expression.When the blocking antibody of integrin is acted on, the Akt in the cell is swashed again It is living.

It is swollen that WISP-1 promotes the characteristic of cell propagation and conversion, inhibited apoptosis and Nasopharyngeal neoplasms to become The promising target of knurl biological therapy.The expression of WISP-1 can be blocked with antibody in vivo or in vitro, so that relevant cell It is restored with the function of tissue.

The acellular organism treatment of tumour includes antibody, polypeptide（Or protein）Vaccine, gene vaccine, vivo gene therapy Deng.Wherein, antibody and polypeptide therapeutic are presently the most two active fields.At present all WISP-1 for scientific research resist Body is all non-clinical applications type, and antibody prepares expensive and there is murine antibody antiantibody is also easy to produce in human body Etc. drawback.

With monoclonal antibody medicine phases ratio, less polypeptide is almost without immunogenicity；Chemically synthesize, product purity is high, It is quality controllable, a kind of new method can be provided for the biological therapy of tumour.In recent years, with the continuous development of biotechnology, It was found that many tumor-related genes and tumour produce regulatory factor, screening and the polypeptide of the special antagonism of these target spots, it is likely to become The breach of cancer drug development.

In polypeptide drugs exploitation, using various sides such as extraction, chemical synthesis, display technique of bacteriophage and proteasome degradation Formula is obtained a variety of peptide storehouses, and active peptides can be filtered out from these peptide storehouses, and identifies its structure.Phage peptide library It is a new technology of the rise nineties in 20th century, its principle is：With bacteriophage as carrier, by the gene piece of encoding exogenous polypeptide Duan Dingxiang is inserted into the coat protein gene area of bacteriophage, allogenic polypeptide is expressed simultaneously by merging with bacteriophage coat protein Phage surface is showed in, the polypeptide being demonstrated can keep relatively independent space structure and biologically active, and then by affine The bacteriophage of the methods such as enrichment screening expression specific polypeptides.

The content of the invention

The invention provides the specific heptapeptide of targeting WISP-1 albumen, the specific heptapeptide is using phage peptide library skill What art was screened, can specifically target the albumen of Wnt signal path downstream target genes WISP-1 codings.

A kind of specific heptapeptide of targeting WISP-1 albumen, amino acid sequence is as shown in SEQ ID No.1～114 are arbitrary.

Present invention also offers the gene of the coding specific heptapeptide, and the expression unit or weight containing the gene Group carrier.

Present invention also offers the fusion protein containing the specific heptapeptide.

Present invention also offers surface display has the bacteriophage of the specific heptapeptide.

Specific heptapeptide of the present invention is screened from phage random heptapeptide storehouse and obtained, and concrete steps include：

（1）The prokaryotic expression and purifying of target protein WISP-1；

Using the directional cloning method of protein coding sequence, using pFN19A (HaloTag7) T7SP6Flexi carrier structures The expression vector of target protein WISP-1 is built, HaloTag albumen is located at the N-terminal of WISP-1 fusion proteins；HaloTag is used as one kind Novel protein label, can strengthen the expression productivity and solubility of recombinant protein.

Using one-step method（KRX）Competent cell carries out effective conversion of carrier, by rhamnose promoter（rhaBAD）Drive Dynamic t7 rna polymerase is expressed, and the expression by T7 promoters to WISP-1 fusion proteins is flexibly controlled.

The WISP-1 fusion proteins of abduction delivering are covalently fixed on HaloLink resins, using TEV protease from WISP-1 albumen is cut on HaloLink resins, is reused HisLink resins specificity and is removed TEV enzymes, thus obtained The WISP-1 albumen that yield is big and purity is high.

（2）The screening of bacteriophage antagonistic peptide；

By target protein WISP-1 direct coateds on solid support, bacteriophage heptapeptide storehouse is added（New England Biolabs companies produce）, carry out " affine absorption → wash-out → recovery → amplification " and take turns affine screening enrichment more, wash away unadsorbed Or non-specific binding（Reference protein GUS is screened）Bacteriophage, screen from bacteriophage heptapeptide storehouse and target protein specificity With reference to, expression have antagonistic peptide（Specific heptapeptide）Bacteriophage.

3）Separate and expand single phage clone；

WISP-1 specific bond bacteriophages to screening are enriched with, dilute after pave plate, select monoclonal phage, And the culture amplification in 96 well culture plates；To screen antagonistic peptide as much as possible, 4608 clones of total coamplification（Put down in 24 piece of 96 hole Plate）, including some positive and negative control clones.

（4）The identification of antagonistic peptide；

ELISA identifications are carried out to WISP-1 specific bonds bacteriophage：First, by target protein WISP-1 coated elisa plates, so After be incubated each dilution monoclonal phage, finally with antiphagin ML3 antibody test positive monoclonal bacteriophages；It is positive single Cloned phage extracts phage single-chain DNA and is sequenced Jing after amplification；The polypeptide sequence that height repeats is found by sequence analysis Row, determine the specific heptapeptide motif of WISP-1 albumen.

Because the specific heptapeptide can specifically bind WISP-1 albumen, therefore present invention also offers the specificity Application of the heptapeptide in WISP-1 polypeptide antagonists are prepared.The WISP-1 polypeptides antagonist can be used to block The expression of WISP-1 albumen, so that the function of relevant cell and tissue is restored.

Applicant's research finds that WISP-1 is the esophageal cancer cell of Radioresistance（KYSE-150R）Epithelial-mesenchymal is thin Dysuria with lower abdominal colic（EMT）Key factor.In KYSE-150R cells, the RNA and protein expression of WISP-1 are significantly raised. Using the expression in antibody with WISP-1, the Radioresistance for finding KYSE-150R cells is obviously reduced therewith applicant, in nude mice Also similar result is obtained in model.Therefore, WISP-1 is the extremely valuable therapy target for overcoming Radioresistance.

Therefore, WISP-1 polypeptides antagonist of the present invention can be radiotherapeutic sensitizer.The radiotherapeutic sensitizer can For weakening the Radioresistance of esophageal cancer cell, be conducive to carrying out radiotherapy to patient with esophageal carcinoma.

The present invention utilizes phage peptide library, and the albumen with Wnt signal path downstream target genes WISP-1 codings is as target Point, screens with the bacteriophage for substantially suppressing WISP-1 protein actives, Jing ELISA identifications and sequence from bacteriophage heptapeptide storehouse Analysis obtains the sequence of antagonistic peptide.

Compared with prior art, beneficial effects of the present invention are：

The present invention utilizes phage peptide library, and screening can specifically target 114 specificity of WISP-1 albumen Heptapeptide, prepare WISP-1 polypeptides antagonist using these specific heptapeptides and the radiotherapeutic sensitizer in esophageal carcinoma therapy not It is only capable of substantially suppressing the activity of WISP-1 albumen, high-efficiency low-toxicity, and low production cost, consumption is little, be easily-synthesized.

Description of the drawings

Fig. 1 is the screening process figure of the specific heptapeptide of present invention targeting WISP-1 albumen；

Fig. 2 is the abduction delivering result electrophoretogram of WISP-1 recombinant proteins；Wherein, M is protein marker；

Fig. 3 is the Western blot qualification figures of WISP-1 recombinant proteins；

Fig. 4 is the ELISA qualification figures of the bacteriophage positive colony of affine WISP-1 albumen；

Fig. 5 is the structure chart of WISP-1 albumen.

Specific embodiment

The screening technique of the specific heptapeptide of targeting WISP-1 albumen, its screening process is shown in Fig. 1, specifically includes：

（1）The structure of recombinant plasmid pFN19A Halo-WISP-1

With pFN2K-WISP-1 as template, the Specific PCR primers of WISP-1 genes are designed and synthesized, enter performing PCR amplification, PCR primer sequence is as follows：

Upstream primer：5'-CCCGGCGATCGCCATGAGGTGGTTCCTGCCCTG-3'；

Downstream primer：5'-CTTGGTTTAAACGTTGGCAATTTCTGAGAAGTC-3'；

Specific restriction restriction endonuclease is used after PCR primer is purified（Sgf I&Pme I）Digestion is carried out, by pFN19A (HaloTag7) T7SP6Flexi carriers identical restriction enzyme carries out digestion, and by corresponding cDNA fragments and carrier Connection, connection product conversion Escherichia coli JML09, PCR identification positive colonies are simultaneously sequenced, by the correct positive colony of sequence verification It is named as pFN19A Halo-WISP-1.

（2）Abduction deliverings of the recombinant plasmid pFN19A Halo-WISP-1 in KRX Escherichia coli

PFN19A Halo-WISP-1 recombinant plasmid transformed competence colibacillus are expressed into bacterium KRX, is spread and be inverted on LB agar plates Culture, picking colony, the positive clone of PCR checkings is inoculated in the LB culture mediums containing ampicillin, and shaken cultivation is entered in thalline When entering Exponential growth stage, rhamnose abduction delivering, collects thalline, with Halo protein purification liquid are added（50mM HEPES, 150mM NaCl, pH7.5）Resuspended, SDS-PAGE analyses determine optimum expression condition.

（3）The purifying of fusion protein

According to the expression condition of optimization（37 DEG C, 3.5h）A large amount of abduction deliverings are carried out, centrifugation goes supernatant to collect bacterium solution, precipitation Preserve in -80 DEG C or put standby on ice；HaloLink resin suspensions 1mL is in advance with the Halo protein purification liquid of 2.5 times of volumes 4 DEG C balance.

With the resuspended step of 15mL Halo protein purification liquid（2）The thalline of collection, adds 100 μ L lysozymes（100mg/mL） With 100 μ L nucleases（5mg/mL）, ultrasonication being carried out on ice and is clarified to bacterium solution, supernatant is collected in centrifugation；Take 12mL supernatants Liquid（S）The HaloLink resin suspension 1mL of pre-balance are added, 2h is incubated at room temperature, purification column is crossed and is abandoned filtrate（FT）, use Halo eggs White refined solution washing purification column, is eventually adding the Halo protein purification liquid 3mL containing 8U/mLTEV enzymes in purification column, mixes rear chamber Temperature reaction 1h；Cross post and collect filtrate, add Halo protein purifications liquid washing pillar, be repeated once, collect filtrate（E1）, add 10 μ L HisLink resins are collected by centrifugation supernatant in 3mL E1 after incubation at room temperature 0.5h（E2）.Inhale respectively from S, FT, E1 and E2 Sample is taken, 10%SDS-PAGE analyses are carried out, electrophoresis result is shown in Fig. 2." S-TEV " sample is referred to and takes 10 μ L containing 8U/mL in Fig. 2 The solution that the S example reactions of the Halo protein purifications liquid of TEV enzymes and 50 μ L are obtained.

（4）The identification of recombinant protein

The recombinant protein plus 5 μ L5 × protein electrophoresis sample-loading buffer of 10 μ g purifying are taken, in 95 DEG C 5min is heated, carry out 10% SDS-PAGE electrophoresis；Electrophoresis carries out Coomassie light blue dyeing after terminating, or with half-dried electrotransfer system by protein delivery to nitric acid Cellulose membrane 17min（15V）, skim milk closing 2h, add rabbit-anti WISP-1 antibody in 4 DEG C of overnight incubations, wash number with PBS It is secondary, each 10min；Again with goat antirabbit-HRP antibody incubation 1h, detected with ECL after washing three times with PBS, exposed in darkroom To on X-ray（See Fig. 3）.

（5）Phage random heptapeptide storehouse targets the affine screening of WISP-1 albumen

1）Wash pipe：0.1M NaHCO₃（pH8.6）Solution cleaning coating immunity pipe；

2）Coating：[WISP-1 albumen is dissolved in 0.1M NaHCO to prepare 100 μ g/mL molecule solutions₃（pH8.6）In], room During immune pipe is coated under temperature；

3）Closing：Coating buffer is poured out, immune pipe is upside down in be clapped on clean paper handkerchief and gets rid of to remove residual solution, and often pipe is filled it up with Confining liquid, 4 DEG C of jog closings；

4）It is affine：Confining liquid is abandoned, with TBST（TBS+0.1%Tween-20）Washing；With the TBSB of 4mL（TBS+0.5%BSA） Buffer solution dilution 2 × 10¹¹Pfu unit bacteriophages（10μL）, in being then added to immune pipe, 4 DEG C of jog incubations；

5）Wash-out：Topple over the unconjugated bacteriophage of removing, be inverted immune pipe and clap that to get rid of removing remnants molten on clean paper handkerchief Liquid, pipe is washed for several times with TBST buffer solutions, and jog is washed every time；Last time is abandoned after cleaning solution, and often pipe adds the 0.2M of 500 μ L Glycine-HCl（pH2.2）, jog rocks；

6）Neutralization：Eluent is sucked in a clean 1.5mL microcentrifugal tubes, 1M Tris-HCl are used（pH9.1）In neutralization State eluent；

7）50 μ L bacteriophage elutions things are left and taken for phage titre measure：Routinely μ L of program determination 1 in ML3 methods Bacteriophage elution thing is 10^-1-10^-4Titre under different dilution factors（Referring to step（6））；

8）Remaining eluate amplification：Bacteriophage elution thing is added in ER2738 cultures, 37 DEG C of culture 4.5hr；

9）Culture is proceeded in a 50mL centrifuge tubes, is centrifuged, supernatant is proceeded in another centrifuge tube, then is centrifuged；

10）The top of supernatant 80% is proceeded in a fresh tube, the PEG/NaCl of 1/6 volume, 4 DEG C of precipitations of bacteriophage are added Overnight；

11）Centrifugation PEG sediments, abandon supernatant, then of short duration centrifugation, suck residual supernatant；Precipitation is resuspended molten with 1mL TBS Solution, centrifugation, precipitates residual cells, then takes supernatant and proceeds to another fresh microcentrifugal tube, adds the PEG/NaCl of 1/6 volume It is incubated 60min on ice to precipitate again；Centrifugation, abandons supernatant, is precipitated and dissolved in 200 μ L TBS（Containing 0.02%NaN₃）In, as expand Eluent；

12）Use reference protein GUS（Lay in laboratory）Coating immunity pipe, according to above-mentioned 2）-11）The step of carry out it is " affine Absorption → wash-out → reclaim → amplification " screening, removes Non-specific phage；

13）Three-wheel screening is carried out again by above-mentioned steps, and second takes turns and with the later several rounds respectively with the amplification of last round of eluent Bacteriophage, the input phagocytosis scale of construction is each about 2 × 10¹¹pfu。

（6）The titer determination in the bacteriophage heptapeptide storehouse that screening is obtained

1）In LB culture mediums, shaking table culture is to mid-log phase for inoculation ER2738 single bacterium colonies；

2）Prepare autoclaved Top Agar culture mediums and LB/IPTG/Xgal flat boards；

3）Prepare 10 times of bacteriophages being serially diluted with LB nutrient solutions；

4）When ER2738 thalli growths to mid-log phase, by culture equal portions in microcentrifugal tube, each bacteriophage is dilute Degree of releasing one pipe ER2738 cultures of correspondence；

5）Add the different dilution bacteriophages of 10 μ L one by one respectively in every pipe ER2738 cultures, quick concussion is mixed It is even, incubation at room temperature a few minutes, carry out phage-infect；

6）Infection thalline is added in the Top Agar agar culture tubes of pre-temperature, every time a pipe, it is quick to mix, pour into immediately On LB/IPTG/Xgal flat boards, appropriate tilt flat plate uniformly spreads out top-layer agar；

7）After flat board cooling, 37 DEG C of lucifuge overnight incubations are inverted in；

8）Flat board is checked, the plaque number on flat board is counted, plaque forming unit (pfu) is calculated.

（7）The plaque monoclonal amplification of targeting WISP-1

1）ER2738 overnight cultures are inoculated in into LB-Tet culture mediums, constant-temperature table is shaken to logarithmic phase；

2）By step（5）The Phage Infection Host Strains ER2738 of the targeting WISP-1 under last wheel screening wash-out, paving Plate is overnight；

3）16-48 blue plaque is chosen respectively to above-mentioned steps 1）In the ER2738 cultures increased in logarithmic phase, 37 DEG C of shaking table cultures 4.5-5h；

4）Culture is proceeded in a 1.5mL centrifuge tubes, is centrifuged, collect supernatant, this is and expands bacteriophage reservoir, 4 DEG C Storage；

5）The top of supernatant 80% is proceeded in a fresh tube, the PEG/NaCl of 1/6 volume is added, allows 4 DEG C of bacteriophage to precipitate Overnight；

6）Centrifugation PEG sediments, abandon supernatant, then of short duration centrifugation, suck residual supernatant；Precipitation is resuspended molten with 1mL TBS Solution, centrifugation, precipitates residual cells, then takes supernatant and proceeds to another fresh microcentrifugal tube, adds the PEG/ of 1/6 volume NaCl, is incubated on ice 60min, centrifugation, abandons supernatant, is precipitated and dissolved in 200 μ L TBS（Containing 0.02%NaN₃）In, as expand Eluent.

（8）ELISA identifies the bacteriophage positive colony of affine WISP-1

1）With the molecule solution of 100 μ g/mL, [WISP-1 albumen is dissolved in 0.1M NaHCO₃（pH8.6）In] coating ELISA Plate, per the μ L of hole 100, the slight oscillatory in wet box；

2）Unnecessary molecule solution is thrown away, and is inverted flat board and is clapped on paper handkerchief and get rid of removing raffinate, add closing fluid-tight to close per hole；

3）Confining liquid is thrown away, with 0.1%PBST board-washings for several times；

4）Step is added per hole（7）Monoclonal phage after amplification, 4 DEG C of shaken overnight incubations；

5）With 0.1%PBST board-washings for several times, the anti-ML3 antibody of 100 μ L HRP marks, room temperature concussion is then added to make per hole Use 1h；

6）With 0.1%PBST board-washings for several times, 100 μ L tmb substrate chromophoric solutions, room temperature are added to act on to blue and substantially showing per hole After now, the 1M hydrochloric acid terminating reactions of 50 μ L are added per hole, determine the light absorption value at 450nm.

By the OD with negative target protein PRDX₄₅₀Value is compared（Part comparative result is shown in Fig. 4）, obtain energy and WISP- 114 positive colony bacteriophages of 1 albumen specific bond（OD₄₅₀>0.4）.

（9）The fast purifying of sequencing template

1）ER2738 overnight cultures are pressed into 1:100 dilutions, take 1mL mycelium dilution things, add step（8）The positive of screening The μ L of cloned phage 10, carry out amplification 4.5h, after centrifugation, take 500 μ L of supernatant and proceed to a fresh centrifuge tube；

2）200 μ L PEG/NaCl are added in centrifuge tube, are overturned and is mixed, room temperature places 10-20min, supernatant is abandoned in centrifugation, It is centrifuged again, sucks remaining supernatant；

3）Sediment is thoroughly resuspended in 100 μ L iodide buffer solutions, 250 μ L ethanol of addition, incubation at room temperature 10-20min, Centrifugation, abandons supernatant, and with 70% ethanol precipitation is washed, and is centrifuged, and abandons supernatant, of short duration placement drying；

4）Precipitation is resuspended in Hs of the 30 μ L without RNase₂In O or TE [10mM Tris-HCI (pH8.0), 1mMEDTA], Obtain template solution；

5）The above-mentioned template solution of 5 μ L is taken, is sequenced, sequencing obtains the nucleotide sequence of 114 heptapeptides of coding.

Claims (6)

1. the specific heptapeptide of WISP-1 albumen is targetted, it is characterised in that amino acid sequence is as shown in SEQ ID No.97.

2. the gene of specificity heptapeptide as claimed in claim 1 is encoded.

3. the expression unit of gene as claimed in claim 2 is contained.

4. the recombinant vector of gene as claimed in claim 2 is contained.

5. containing the fusion protein of specificity heptapeptide as claimed in claim 1.

6. surface display has the bacteriophage of specificity heptapeptide as claimed in claim 1.