CN107446021A

CN107446021A - Folacin receptor alpha specific binding peptide 5 and its application

Info

Publication number: CN107446021A
Application number: CN201710649315.2A
Authority: CN
Inventors: 王旻; 邱郑; 徐祎凤; 邢黎军; 王红
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2017-07-26
Filing date: 2017-07-26
Publication date: 2017-12-08
Anticipated expiration: 2037-07-26
Also published as: CN107446021B

Abstract

The small peptide combined the present invention relates to a kind of folacin receptor alpha specific and its application, belong to biological technical field.The peptide sequence of the present invention is MNPYPRTPWPHV.This polypeptide is obtained by being screened from phage display library, can be with expression folacin receptor α tumour cell specific bond, and targeted therapy and tumor imaging for tumour provide a kind of new targeted molecular, the potential value with new drug development.

Description

Folacin receptor alpha specific binding peptide 5 and its application

Technical field：

The invention belongs to biological technical field, is related to a kind of small peptide of folacin receptor alpha specific combination and its in tumor target Application in.

Background technology：

Display technique of bacteriophage is that allogenic polypeptide or certain of albumen and bacteriophage capsid protein are carried out into amalgamation and expression, is made Foreign protein can be illustrated in surfaces of viral particles, while the DNA of encoding foreign proteins is located in the virion.Random 12 Peptide phage display library is that random dodecapeptides are fused on M13 phage minor coat proteins (pIII), for a combination Library.N-terminal of the dodecapeptide expression shown in pIII.This is just between substantial amounts of rondom polypeptide and its DNA encoding sequence Direct bridge is established, then target is obtained to carry out in-vitro screening with various target molecules (antibody, enzyme, cell surface receptor etc.) Molecule binding peptide.External option program is exactly that phage library is incubated altogether with solid phase target molecule in simple terms, and washing removes uncombined Bacteriophage, then afford the bacteriophage that can be specifically bound with target molecule.The bacteriophage being eluted also needs to be expanded, and enters Combination/amplification cycles of row next round, to be enriched with specific binding bacteriophage., can by DNA sequencing after 3~4 wheels " elutriation " To obtain the peptide sequence of each specific binding.

Folacin receptor (folate receptor, FR) is to combine and transport folic acid and its derivative into the important of cell Transporter, mainly exist in vivo with three kinds of hypotypes：FR α, β and γ.Folacin receptor α (folate receptor α, FR α) is one Kind is anchored to the glycoprotein of cell membrane surface by glycolsyl-phosphatidylinositol (GPI).Existing document report, oophoroma, lung cancer, liver FR α express in high in the tissue such as cancer, breast cancer, and restricted expression in the normal tissue, therefore are considered as great potential Ovary carcinoma marker or tumor associated antigen (TAA)；The high degree of specificity that FR α have to oophoroma, treatment can be used as related swollen The target spot of knurl；Part research also indicates that early diagnosis of the FR α for oophoroma has important value.Based on this, if can obtain with FR α have the short peptide sequence of higher affinity, will provide new approaches for the magnetic target therapy of cancer and diagnosis.

The content of the invention

Goal of the invention

It is an object of the invention to using display technique of bacteriophage obtain it is a kind of can be combined with folacin receptor alpha specific it is more Peptide.The polypeptide can be used for the positive tumour cell of targeting folacin receptor alpha expression.

Technical scheme

To achieve these goals, the technical solution adopted in the present invention is as follows：

1) the affine screening of phage display peptide library：Using folacin receptor α recombinant proteins as target, with phage display ten Dipeptides storehouse carries out four-wheel biological screening.The selective mechanisms rate of recovery and polyclonal ELISA are often taken turns, to judge whether screening is effective；It is logical Cross each clone of monoclonal ELISA detections and the binding ability of folacin receptor α recombinant proteins；

2) bacteriophage positive monoclonal DNA preparation and sequencing identification：According to above-mentioned ELISA results, select positive value high Bacteriophage monoclonal, after being expanded to it carry out sequencer module fast purifying surveyed with producing sufficiently pure template Sequence, it is sent to company from -96gIII sequencing primers and is sequenced, analysis positive bacteriophage accordingly shows peptide sequence；

3) cell ELISA detection phage clone and native cell surface folacin receptor α combination：Choose FR alpha expressions sun Property Cell line SKOV3, and using FR alpha expressions negative cells strain HepG2 as control, pass through ELISA and detect phage clone and cell The combination of strain；

4) phage clone and FR alpha expression positive cell strains SKOV3 combination are further confirmed that by flow cytometry.

Beneficial effect

FR alpha bindings provided by the invention, FR α recombinant proteins can be specifically bound, while pass through cell ELISA and streaming Cytometry experiment is analyzed, and the phage display small peptide for finding to filter out can be combined with the natural FR alpha specifics of cell surface, is had latent Medical science and pharmacy value.

Brief description of the drawings：

The polyclonal ELISA of Fig. 1；

Fig. 2 positive monoclonals and the research of folacin receptor α recombinant protein binding abilities.Fig. 2A monoclonal phages ELISA screening positive clones (clone irised out is the displaying of folacin receptor alpha specific binding peptide 5 clone)；Fig. 2 B. positive monoclonals The further checking combined with folacin receptor α recombinant proteins；

The combination of positive monoclonal and FR alpha expression positive cells SKOV3 that the detection of Fig. 3 cell ELISAs screens, with M13 And FR alpha expression negative cells HepG2 is control；

Fig. 4 flow cytometry tests analyze positive monoclonal and SKOV3 specific binding situation, in control group such as figure Shown in dotted line, the SKOV3 cells of anti-M13 antibody and FITC fluorescence antibodies are only added.

Embodiment：

In order to more clearly illustrate the present invention, the description below provides more specific embodiment, the technology of this area Personnel are it should be understood that the present invention is not merely defined in following embodiments.

The affine screening of the phage display peptide library of embodiment 1

Random dodecapeptides phage display library is purchased from NEB companies, 100 μ l, and titre is 1.5 × 10¹³pfu/ml.It is stored in In TBS buffer solutions (50mM Tris-HCl, 150mM NaCl [PH7.5]) containing 50% glycerine, storage capacity 2.7 × 10⁹Individual conversion Son.Escherichia coil ER2738 are the Host Strains in this peptide storehouse.

(1) preparation

FR α recombinant proteins are diluted to final concentration 50 μ g/ml with coating buffer solution (carbonate buffer solution [PH9.6]), it is dilute Solution after releasing is rotated until surface moistens completely repeatedly with every μ l coated elisa plates of hole 100.4 DEG C were incubated in wet box Night.

(2) affine screening

Second day coating buffer outwelled in ELISA Plate, with TBST buffer solutions (TBS+ volume ratios 0.1% [v/v] Tween-20) Board-washing, incline buffer solution, claps and is got rid of to remove remaining liquid on clean blotting paper.200 μ l envelopes are added in every hole afterwards Blocking solution (0.1M NaHCO₃, 5mg/ml BSA, 0.02%NaN₃[PH8.6]), put 4 DEG C of overnight incubations or 37 DEG C of incubations in wet box At least 1 hour.Discard and blockade liquid, TBST buffer solutions are filled it up with per hole, board-washing 6 times, washing 5 minutes, are placed on decolorization swinging table every time Carry out.Incline buffer solution again, claps and is got rid of to remove remaining liquid on clean blotting paper, performs quick during this experimental procedure To avoid orifice plate from drying.Primary libraries are diluted with TBST buffer solutions, take the μ l of library liquid 100 after dilution to be added to coating FR in advance In the ELISA Plate micropore of α recombinant proteins, the bacteriophage quantity of addition is about 5 × 10¹².In wet box 4 DEG C be incubated overnight or 37 DEG C It is incubated 2 hours, discards liquid in hole, removing residual solution is got rid of into ELISA Plate inversion bat, and with TBST buffer solutions board-washing 10 times, grasps Make the same (washing away uncombined bacteriophage).After last time washing, liquid in hole is clapped and dried only, is added into ELISA Plate micropore It is bound to elute to enter 100 μ l non-specificity buffer solution such as 0.2M glycine-HCIs Glycine-HCl buffer solutions [PH2.2] Bacteriophage, it is gentle on decolorization swinging table to shake 10 minutes fully to elute under room temperature condition, then with 15 μ l 1M Tris-HCl [PH9.1] neutralizes above-mentioned eluent, is transferred in a clean EP pipe.Routinely M13 methods take 5 μ l eluents to be used for determining The titre of eluate, remaining eluent are added to 20ml and are in progress first round bacteriophage in the ER2738 strains of logarithm early stage The amplification of eluate, in 37 DEG C, 220rpm shaking table cultures 4.5 hours, amplified production is transferred in clean centrifuge tube, through 4 DEG C, After 10, the 000rpm operations of centrifugation 10 minutes, about 80% bacteriophage supernatant is taken, is transferred in another clean centrifuge tube, adds 1/6 The PEG/NaCl (20% [w/v] PEG-800,2.5M NaCl) of volume, 4 DEG C are placed at least 1 hour or handled overnight, are bitten Bacterial sediment.4 DEG C, 10,000rpm centrifugations 10 minutes, remaining supernatant is discarded with micropipettor, it is thorough that of short duration centrifugation can be carried out again Bottom, which is inhaled, abandons supernatant.Sediment is resuspended in 200 μ l TBS buffer solutions, is taken supernatant after centrifuging again, is transferred to clean centrifuge tube In, this is the eluate after expanding.5 μ l eluates are taken to carry out the measure of phage titre, remainder is used for the second wheel parent And screening.Repeat above step and carry out four-wheel screening altogether.The washing times of TBST in increase washing step are screened by wheel.

(3) measure of phage titre

ER2738 strains are inoculated with 10ml LB culture mediums, 37 DEG C of shaking table cultures to mid-log phase (OD₆₀₀It is left 0.5 It is right).Period dissolves by heating top-layer agar in micro-wave oven, is dispensed into 5ml sterilizing EP pipes, often pipe 3ml, pipe are several according to phagocytosis Depending on body dilution gradient, each pipe of dilution gradient one.It is standby that the EP pipes of packing are placed in 45 DEG C of water-baths；Prepare LB/ simultaneously IPTG/Xgal culture plates, it is standby to be placed in 37 DEG C of incubators.10 times of gradients are carried out with LB culture mediums to the bacteriophage supernatant of collection Dilute (the bacteriophage dilution range typically expanded：10⁸~10¹¹).By the ER2738 bacterium solutions in mid-log phase after the completion of dilution It is dispensed into several 1.5ml sterilizing EP pipes, often the μ l of pipe 200.10 μ l are taken to add immediately the dilution of each gradient diluted Into the EP pipes containing bacterium solution, vibration mixes to be incubated 5 minutes after 37 DEG C.Then the top-layer agar of 45 DEG C of placements is taken out, Guan Zhong Content is transferred completely into top-layer agar immediately, and the LB/IPTG/Xgal cultures that pre-temperature is crossed are poured over after quick reverse mixing On flat board, gently rocking flat board is uniformly distributed top-layer agar.After solidification a period of time to be cooled, 37 DEG C of inversion overnight incubations. Flat board of the plaque sum at 100 or so is chosen, the plaque number to be grown on flat board is counted and calculates bacteriophage effect Valency (pfu).According to the phagocytosis scale of construction (input titre Input) of every wheel input screening and the phagocytosis scale of construction afforded, (output is dripped Spend Output), the output/input ratio often taken turns can be calculated, reflects the enrichment degree (rate of recovery Recovery) of specific bacteriophage. Screen and find by four-wheel, having obtained effective enrichment with the bacteriophage of the high-affinity of FR α recombinant proteins specific binding (is shown in Table 1)。

Table 1 often takes turns the situation of input titre, output titre and the rate of recovery

The polyclonal ELISA identifications of the bacteriophage of embodiment 2

FR α recombinant proteins are diluted to the μ g/ml of final concentration 10 with carbonate buffer solution [PH9.6], the μ l of coating 100 per hole, 4 DEG C coating overnight.Molecule solution is discarded every other day, and is clapped on clean blotting paper and gets rid of removal residual liquid, and button is dry, is delayed with TBS After fliud flushing washed once, 200 μ l are added per hole and blockade liquid, 37 DEG C are placed 1~2 hour.Throw away and blockade liquid, TBS buffer solutions washing 3 It is secondary, 5 minutes every time, firmly patted on clean blotting paper, get rid of net cleaning solution.Wash and 100 μ l dilutions are added in every hole Each round screening amplification after eluent (i.e. bacteriophage supernatant), 37 DEG C of incubations discard liquid in hole after 2 hours, and use TBST Buffer solution and TBS buffer solutions wash 3 times successively, 5 minutes every time, clap and add the anti-M13 of mouse that HRP is marked after getting rid of removing cleaning solution As secondary antibody (being diluted to working concentration with liquid is blockaded), 37 DEG C are incubated 2 hours antibody, discard liquid in hole, washing operation is same as above One operation.Add 100 μ l tmb substrate liquid per hole to be developed the color, room temperature lucifuge is incubated 10 minutes, and liquid should be by without discoloration in hole For blueness；50 μ l 1M H are added per hole₂SO₄Terminate liquid, color development stopping reaction, detects OD on ELIASA₄₅₀Value.

As a result show, as number of screening round increases, the affinity that eluate is combined with FR α recombinant proteins is stepped up, but In fourth round, affinity has declined (see Fig. 1).

The ELISA identifications and sequencing identification of the bacteriophage monoclonal of embodiment 3

(1) ELISA detects binding ability of the monoclonal phage displaying small peptide to target molecule

The acquisition of bacteriophage to be measured：ER2738 strains need to be inoculated with advance, 37 DEG C of cultures to logarithm early stage, by this logarithm early stage Culture is transferred in 2ml deep-well plates, per the μ l of hole 600.It is used to detecting the flat board of titre that (total amount is 100 in selection four-wheel screening The flat board of individual or so and following plaque), with the multiple blue plaques of the random picking of pipette tips into deep-well plates, 37 DEG C, 220rpm shaking table cultures 4.5 hours.4 DEG C, 10,000rpm centrifugations 10 minutes, precipitation is discarded, takes 100 μ l supernatants to be used for per hole ELISA is detected, and remaining supernatant collects temporary 4 DEG C of preservations.

ELISA is detected：Target molecule FR α recombinant proteins are dissolved in by carbonate buffer solution [PH9.6] with the μ g/ml of final concentration 10 In, 100 μ l are coated with per hole, 4 DEG C of coatings are stayed overnight in the wet box of sealing.Unnecessary molecule solution is thrown away, and in clean paper handkerchief After the unnecessary liquid of removing is got rid of in upper bat, 200 μ l confining liquids are added per hole, 37 DEG C are closed 1~2 hour.Throw away and blockade liquid, TBST washings 6 times, clap after drying net ELISA Plate, the 100 μ l supernatants obtained in deep-well plates are sequentially added in order, room temperature reaction 1~2 is small When, then 6 times (operation is same as above) are washed with TBST, with the anti-M13 antibody blockaded liquid and marked by 1: 10000 dilution proportion HRP, by every In the μ l adding holes of hole 100, react at room temperature 1~2 hour, TBST fully washs 6 times (step is same as above), and 100 μ l TMB are added per hole (now with the current) colour developing of substrate solution, lucifuge add 50 μ l 1M H in every hole after acting on 10 minutes₂SO₄Terminate liquid, color development stopping are anti- Should, OD is detected on ELIASA₄₅₀Value.

As a result Fig. 2 is seen.By Preliminary detection result, (clone that Fig. 2A is irised out is folacin receptor alpha specific binding peptide 5 to Fig. 2A Displaying clone), independent picking clone, by its binding ability (figure with FR α recombinant proteins of further ELISA experimental verifications 2B), wherein PBS is as blank control, and there were significant differences compared with PBS control group for experimental group (P ＜ 0.05).

(2) bacteriophage positive monoclonal DNA preparation and sequencing identification

According to above-mentioned ELISA results, choose after positive bacteriophage monoclonal is expanded and carry out the quick of sequencer module again Purifying is sequenced with producing sufficiently pure template：ER2738 overnight cultures are inoculated in LB culture mediums according to 1: 100 and shaken After shaking logarithmic phase, 1ml is into culture tube for packing.10 μ l bacteriophages positive colonies are taken into above-mentioned 1ml culture tubes.37 DEG C of shaking tables Culture 4.5~5 hours.Culture is transferred in microcentrifugal tube, is centrifuged 30 seconds.Supernatant is transferred to a clean centrifuge tube, and this is Bacteriophage reservoir is expanded, can temporary 4 DEG C of storages if not carrying out subsequent experiment immediately.

The fast purifying of sequencing template：(the volume about 1ml) containing bacteriophage supernatant of above-mentioned collection is taken, adds 400 μ l PEG/ NaCl, overturn and mix, 12,000rpm is centrifuged 10 minutes after room temperature is placed 10 minutes, is abandoned supernatant, can be carried out centrifugally operated again, thorough Bottom, which is inhaled, abandons remaining supernatant.200 μ l iodide buffer solutions (10mM Tris-HCl, 1mM EDTA, 4M NaI is added in sediment [PH8.0]) and sediment is thoroughly resuspended, 500 μ l ethanol are added, are incubated at room temperature 10 minutes (precipitating DNA)；12,000rpm Centrifugation 10 minutes, abandons supernatant.12,000rpm is centrifuged 10 minutes after cleaning precipitation once with 70% ethanol of 1ml precoolings, abandons supernatant After uncap and air-dry overnight, ethanol is fully volatilized.Finally precipitation is resuspended in 30 μ l distilled waters, this is sequencing template, takes 5 μ l are detected with 1% agarose gel electrophoresis, and the DNA selection -96gIII sequencing primers after detection is qualified are sent to company and are sequenced, Analyze corresponding 12 peptide sequence of bacteriophage.

By comparing, applicant obtains a small peptide SEQ ID NO.1：MNPYPRTPWPHV.

The cell ELISA of embodiment 4 detects the combination of phage display small peptide and cell

To verify the combination situation of FR α under the native state of the polypeptide screened and cell surface expression, high table is chosen Up to FR α ovarian cancer cell SKOV3, and unrelated hepatocellular carcinoma H22 is set to carry out cell ELISA for control.SKOV3 is thin Born of the same parents and HepG2 cell culture are to cell density up to more than 80%, and observation cellular morphology is good under the microscope, through 0.25% pancreatin Add corresponding fresh culture after digestion to be resuspended and carry out viable count, adjustment cell density to 2 × 10⁵Individual/ml, by every The μ l of hole 100 spread 96 porocyte culture plates, 37 DEG C of overnight incubations.Every other day inhale abandon supernatant, with sterile PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄, 2mM KH₂PO₄[PH7.4]) softly wash twice, 5 minutes every time, at room temperature with more than 10% Polyformaldehyde fixes 15 minutes, and fixer is abandoned in suction, and with the soft board-washing of PBS 3 times, 5 minutes every time, with 5% skim milk (PBS dilutions) is closed 2 hours for 37 DEG C as confining liquid.Confining liquid is abandoned, after PBS can do simple rinsing, corresponding bacteriophage is fitted Added after dilution in Tissue Culture Plate, one group of negative control (wild type M13) is set, per the μ l of hole 100,4 DEG C of overnight incubations.Every Supernatant is abandoned in its suction, and washs 3 times successively with PBST (PBS+ volume ratios 0.1% [v/v] Tween-20), PBS, and every time 5 Minute.The anti-M13 antibody for being diluted to the HRP of working concentration (1: 10000) with confining liquid and being marked is added, per the μ l of hole 100,37 DEG C It is incubated 2 hours, after ibid carrying out washing operation, chromogenic reaction is with terminating reaction with Phage-ELISA step.

As a result such as Fig. 3.Compared with wild type M13, experimental group polypeptide and SKOV3 have a preferable combination, and compared to HepG2 combination, there is obvious specific (P ＜ 0.05), illustrate FR alpha bindings and tumor cell surface table that screening obtains The natural FR α reached can be specifically bound.

The combination situation of the Flow cytometry phage display small peptide of embodiment 5 and cell surface FR α

Experiment purpose is tested with cell ELISA.The positive bacteriophage monoclonal screened using Flow cytometry Combination situation to SKOV3 tumour cells.

Cell processing：To cell density up to more than 80%, micro- Microscopic observation cellular morphology is good for SKOV3 cell culture, Digested through 0.25% pancreatin, add fresh culture and terminate digestion, 1,500rpm centrifugation 5 minutes, PBS resuspensions prepare slender Born of the same parents' suspension, carry out viable count, adjustment cell concentration to 1~2 × 10⁶Individual/ml, it is dispensed into 1.5ml EP pipes, often pipe 250 The μ l of μ l~350.After 3,000rpm centrifugations 5 minutes, the bacteriophage supernatant that 100 μ l of often pipe addition have diluted is as primary antibody (dilution For 2%FBS-PBS), select the bacteriophage supernatant corresponding with sequencing peptide sequence, after 4 DEG C are incubated 1 hour, take out 3, 000rpm is centrifuged 5 minutes, abandons supernatant, and often pipe plus 1ml PBS are washed 2~3 times, and supernatant is abandoned in suction.Add the mouse source that 100 μ l have diluted Anti- M13 secondary antibodies, 4 DEG C are placed 1 hour, are taken out ibid operation and are washed, are eventually adding the sheep anti mouse fluorescence two with FITC labels It is anti-, by 1: 500 thinner ratio (2%FBS-PBS dilutions), per the μ l of hole 100, notice that this step needs lucifuge to operate.4 DEG C of lucifuges are incubated 1 Hour, washing step is same as above, and last PBS for adding appropriate volume according to cell precipitation after having washed is resuspended, and up flow type is thin Born of the same parents' instrument is detected.

As a result show, see Fig. 4, the cell line that bacteriophage supernatant can be preferably with the high expression of FR α specifically binds, tied Conjunction rate is 41.8%.

Claims

1. a kind of polypeptide for targetting folacin receptor α, it is characterised in that amino acid sequence is SEQ ID NO.1：Met-Asn-Pro- Tyr-Pro-Arg-Thr-Pro-Trp-Pro-His-Val。

2. a kind of polypeptide for targetting folacin receptor α, it is characterised in that for the derivative of polypeptide described in claim 1, its derivative One is connected to including but not limited to acetylation modification thing, fatty acid chain trim, PEG trims, the motif both ends of polypeptide Individual Cys, polypeptide head and the tail acid amides ring.

3. a kind of fused polypeptide or albumen, it is characterised in that the N-terminal or C-terminal or tundish 1-2 containing claim of more peptide or proteins Described peptide sequence, its merge part including but not limited to human serum albumins (HSA), Fc fragments, antibody or antibody fragment, Go back to the nest molecule, targeted molecular, affinity ligand, cell penetrate peptide, in vivo escape molecule, subcellular fraction targeted molecular, core targeted molecular Or their conjugate and mixture.

4. encode the nucleotide sequence of polypeptide described in claim 1.

5. a kind of nucleotide sequence, it is characterised in that the both ends of nucleic acid molecules or tundish are containing the nucleotides sequence described in claim 4 Row.

6. a kind of pharmaceutical composition, it is characterised in that lead to comprising the peptide sequence described in claim 1-3 with active constituents of medicine Cross and be covalently or non-covalently coupled, or drug carrier is passed comprising the polypeptide described in claim 1-3.

7. a kind of molecular probe, it is characterised in that the probe includes the polypeptide described in claim 1-3.

8. a kind of pharmaceutical composition, it is characterised in that include the nucleotide sequence described in claim 4-5 and other nucleotides sequences Row, which pass through, to be covalently or non-covalently coupled, or passs drug carrier comprising the nucleotide sequence described in claim 4-5.

9. claim 1-3 peptide sequence, claim 4-5 nucleotide sequence or claim 6-8 pharmaceutical composition Application in medicine is prepared.

10. claim 1-3 peptide sequence, claim 4-5 nucleotide sequence or claim 6-8 pharmaceutical composition In the application of tumour diagnostic reagent.

11. according to claim 8-10 application, it is characterised in that the medicine is preferred for tumor-targeting drug.

12. according to claim 8-10 application, it is characterised in that the tumour, which includes folacin receptor α, what is expressed to a certain degree Benign or malignant tumour, such as oophoroma, adenocarcinoma of endometrium, non-small cell lung cancer, clear cell carcinoma of kidney, colon cancer and mammary gland Cancer.