CN105524141B - A kind of preparation and application of FAPa activation type diagnosing tumor polypeptide bead complexes - Google Patents
A kind of preparation and application of FAPa activation type diagnosing tumor polypeptide bead complexes Download PDFInfo
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Abstract
The invention discloses a kind of novel FAP α activation type diagnosing tumor polypeptide bead complexes FAP-GP-NWs.The compound is by the FAP alpha specific digestion peptide substrate with FAM and Biotin label with amino magnetic bead (NWs) by passing through flexible linker SM (PEG)2Built-up, the sequence of peptide substrate is Biotin-DGlu‑G‑DVal‑DAsn‑DAsp‑DAsn‑DGlu‑DGlu‑G‑DPhe‑DPhe‑DSer‑DAla‑DArg-Lys (5FAM)-GPGPNQC, wherein D represented amino acid type is D type；SM(PEG)2Represent NHS- (PEG)2- Maleimide connexon reduces interfering with each other between substrate and magnetic bead.The advantage of the invention is that for the first time diagnosing tumor will be used for after FAP alpha specific substrate and amino magnetic bead coupling, the polypeptide of nano material coupling can recycle the longer time in vivo, to be transported to diseased tissue by blood vessel, amplify biomarker concentration using the activity of the fibroblast activation protein FAP α of unconventionality expression in tumour, make signaling molecule from be discharged into blood in urine facilitate extraction identify.Meanwhile FAP-GP-NWs to mouse without overt toxicity, established solid foundation for further clinical research and application.
Description
Technical field
The invention belongs to biology and pharmaceutical technology fields, and in particular to a kind of polypeptide magnetic bead with diagnosing tumor meaning is multiple
Close the preparation and application of object FAM-GP-NWs.
Background technique
Research in relation to finding biomarker is to be able to identify reliable index to comment disease progress risk
Estimate, early diagnose, design monitors to the therapeutic scheme of patient and effectively recurrent disease.So far, widely
Biomolecule such as products of cellular metabolism, polypeptide, protein, cell-free nucleic acid, foreign molecules etc. have developed into various forms of
Biomarker.However, being difficult dissolution in body fluid and in body since concentration of the biomarker in body circulation is very low
It is interior and can all degrade rapidly in vitro, detection technique and biology are received to diagnose the illness with naturally occurring biomarker
Limitation.Another endogenous biological marker is to give exogenous drugs by system come the biological shape of the body detected
State.These methods prompt us to can use the physiology behavior of body or interfere the molecular process of disease specific to study spy
A possibility that determining Substitute Indexes of the drug as medical diagnosis on disease.
We devise a kind of artificial synthesized nanoscale reagent, they in body circulation can by histoorgan or
The blood vessel gap of person's disease specific gathers illing tissue, and after reaching disease settings, they are by the protease of abnormal activity
Hydrolysis, releases biological marker of the peptide substrate with fluorescent molecule connected on surface into host's urine, as synthesis
Object is identified and is detected by ELISA or mass spectrum.Due to overexpression protease and many disorders such as cancers, thrombus, inflammation,
Atherosclerosis etc. is in close relations, so be to judge morbid state and provide to the multi-path monitoring of these imbalance protease by stages
It may.By the inspiration of glomerulus selectively filtration mode, we devise a kind of protease-sensitive nanometer segment we
Referred to as artificial synthesized biomarker FAM-GP- magnetic bead (FAM-GP-NWs), the egg that nanometer magnetic bead is marked with fluorescence group
White zymolyte is modified, and by the hydrolysis for the Protease F APa that tumour associated fibroblast cell is overexpressed, signal is released
It is then focused in host's urine into blood circulation and receives detection, we utilize FAPa positive tumor models, detect urine
Whether middle signal strength, inquiring into this artificial synthesized urine biomarker can be as a kind of effective progress cancer getting up early diagnosis
Method.
Summary of the invention
The object of the present invention is to provide a kind of tumour associated fibroblast cell-stimulating albumen (FAPa) activation type diagnosing tumors
The preparation and its application of polypeptide bead complexes.
The technical solution adopted by the present invention is that: a kind of enzyme activition formula diagnosing tumor polypeptide bead complexes are provided, are tied
Structure formula is such as shown in (I): X-SM (PEG)2- Y (I), in which: X represent it is a kind of by biotin (Biotin) mark with FAM group
FAPa digestion substrate polypeptide, structural formula Biotin-DGlu-G-DVal-DAsn-DAsp-DAsn-DGlu-DGlu-G-DPhe-DPhe-DSer-DAla-DArg-Lys (5FAM)-GPGPNQC, wherein D represented amino acid type is D type；Y represents average grain
The amino magnetic bead of diameter 100nm, SM (PEG)2MHS- (PEG) 2-Maleimide connexon is represented, is reduced mutual dry between X and Y
It disturbs.
As a further improvement of the present invention, the GPGP amino acid of the motif N-terminal of X is the digestion substrate GP sequence of FAP α
Sub-series, FAP α can hydrolyze P amino acid N end amido bond.
Using FAP α as target, successful design has simultaneously synthesized and is marked by Biotin and carry the FAP α of FAM group the present invention
Activation type peptide substrate, and using amino nanometer magnetic bead as carrier, with SM (PEG)2For connexon, FAP α activation type is successfully synthesized
Diagnosing tumor polypeptide bead complexes to realize the diagnosis to tumour, and do not generate injury to body, to similar diagnosis
The design studies of reagent have reference significance.
The preparation method of polypeptide of the present invention uses solid phase polypeptide synthesis.
The beneficial effects of the present invention are: experiment in vivo proves, which can
By FAP alpha specific digestion, discharges fluorescent polypeptide signal and to urine and be detected, with realizing the mesh of the diagnosis to tumour, and
Normal tissue is nontoxic.
The preparation method that the present invention studies simultaneously is convenient and easy, has good field of medicaments application and popularization value.
Detailed description of the invention
Fig. 1 is FAM-GP-NWs structure and action principle；
Fig. 2 is MS and the HPLC analysis chart of the substrate polypeptide containing Biotin and FAM group；
Fig. 3 is FAM-GP-NWs structure, synthesis and identification；
Fig. 4 is the Fluorescence Identification and external digestion Efficiency testing of FAM-GP-NWs
Fig. 5 is small animal imaging system detection FAM-GP-NWs in the intracorporal fluorescence distribution of mouse and cyclic process.
Specific embodiment
Peptide substrate sequence used in following experiments is as follows:
Biotin-DGlu-G-DVal-DAsn-DAsp-DAsn-DGlu-DGlu-G-DPhe-DPhe-DSer-DAla-DArg-
Lys(5FAM) -GPGPNQC；
Data analysis: count results applied statistics software SPSS16.0 is analyzed, and is counted using Levene method to each group
As a result homogeneity test of variance is carried out.If each group homogeneity of variance (when P > 0.05, homogeneity of variance), then examined using independent sample t
It tests, whether there were significant differences between analysis group (when P < 0.05, there were significant differences)
The synthesis of case study on implementation 1:FAPa specific substrate
1. 2-Chlorotrityl Chloride Resin resin is put into reaction tube, add DCM (15ml/g), vibrates
30min；
2. leaching out solvent by sand core, Fmoc-L-Cys (Trt)-OH of 3 times of molar excess is added, adds 10 times and rubs
You are excessive DIEA, is eventually adding DMF dissolution, vibrates 30min, methanol end socket, 30min；
3. removing solvent, add 20% piperidines/DMF solution (15ml/g), 5min, removes and add 20% piperidines/DMF solution again
(15ml/g), 15min；
4. taking out piperidine solution, more than ten grainy resins are taken, is washed three times with ethyl alcohol, ninhydrin, KCN, phenol solution each one is added
Drop, 105 DEG C of -110 DEG C of heating 5min, deepening blue is positive reaction；
Twice, twice, DMF (10ml/g) is twice for methanol (10ml/g) by 5.DMF (10ml/g)；
6. it is excessive to protect amino acid Fmoc-L-Asp (OtBu)-OH three times, HBTU three times are excessive, use and lack DMF dissolution as far as possible,
Reaction tube is added, is added immediately ten times of excess of DIEA, reacts 30min；
7. taking out solution, more than ten grainy resins are taken, are washed three times with ethyl alcohol, ninhydrin, KCN is added, phenol solution each one is dripped,
105 DEG C of -110 DEG C of heating 5min, colourless is negative reaction, illustrates fully reacting；
Once, twice, DMF (10ml/g) is twice for methanol (10ml/g) by 8.DMF (10ml/g)；
9. repeating the operation of three to eight steps, it is sequentially connected remaining amino acid in sequence, until the last one, removes the last one
The Fmoc protecting group of amino acid；
10.DMF (10ml/g) twice, methanol (10ml/g) three times, DCM (10ml/g) three times, dry adsorbent；
11. preparing cutting liquid (10/g) TFA 94.5%；Water 1%；EDT 2.5%；TIS 2.5%, clipping time:
120min；
12. lysate is dried up as far as possible with nitrogen, washed six times with ether, then room temperature volatilizes；
13. using HPLC purified polypeptide, solution after purification is lyophilized, finished product is both obtained；
14. taking a small amount of finished product polypeptide respectively, the molecular weight identification of MS and the Purity (Fig. 2) of HPLC analysis are done；
15. sealed package, -20 degree save by the polypeptide of white powder；
The same above method of the synthesis of remaining polypeptide.
The synthesis of FAM-GP-NWs
(1) according to SM (PEG)2It is mixed in 10ml EP with the nanometer magnetic bead amino number molar ratio about 50: 1 after activation
(pH7.4,5mM EDTA), room temperature combines overnight in shaking table under nitrogen protection, and product is cleaned with PBS, and magnetic frame separates standby
With；
(2) peptide substrate is dissolved in PBS and first step product normal-temperature reaction stays overnight (molar ratio 1: 1, pH 7.4,5mM
EDTA, nitrogen protection), it is separated with magnetic frame with magnetic product, dissolves product after being cleaned with appropriate PBS, -20 DEG C of guarantors
It deposits.
The diagnosing tumor effect disquisition of case study on implementation 2:FAM-GP-NWs
The verifying of Vitro Tumor diagnosis effect:
According to mesh polypeptide can directly react FAP- to the inhibiting effect of the cell growth of expression different molecular marker
The Vitro Tumor of GP-NWs diagnoses activity.Therefore, we pass through the expression quantity for detecting the molecular marker of different cell strains first
Suitable cell strain is selected, the diagnosing tumor meaning of ELISA experiment detection FAP-GP-NWs is then passed through.Concrete operations are such as
Under:
The selection of cell strain:
We have selected the L929 cell strain of FAPa+ and the 4T1 cell strain of FAPa-, and are had detected with Western-bolt
The expression quantity of FAPa.
Protein immunoblot (Western Blot):
1, after cell is handled according to requirement of experiment, culture medium is discarded, is washed after 3 times with pre-cooling PBS and three decontamination cells are added are split
Liquid is solved, places 20~30min on ice.Cell pyrolysis liquid is collected, 12000rpm centrifugation 30min obtains supernatant；
2, albumen supernatant is after Bradford standard measure, with every 20~50 μ g total protein concentration loading of hole.Voltage 80V~120V
SDS-PAGE electrophoretic separation is carried out, then the pretreated pvdf membrane of methanol is gone to the condition electricity of 200mA, 90-120min；
3, after transferring film, pvdf membrane 5min is washed on shaking table with PBS/T, then closes liquid chamber with Western Blot
Temperature closing 2h, detected antibody are diluted with confining liquid by specification recommendation ratio, and 4 DEG C of incubation pvdf membranes are stayed overnight.Next day will
Pvdf membrane moves to 0.5~1h of room temperature rewarming, and PBS/T is washed 3 times, each 10min, and the secondary antibody (1: 5000- of HRP label is added
8000) it is incubated at room temperature 1-1.5h.ECL luminescent solution is added dropwise after washing 10min × 3 time in PBS/T, exposes, develops in darkroom, is fixed
Shadow；
4, the pvdf membrane after exposing is after PBS/T washing 10min × 3 time remove remaining luminescent solution, in 50 DEG C of water-baths
Middle stripping buffer impregnates oscillation incubation 30min to dissociate the antibody of combination.Then 10min × 3 are washed with PBS/T
Secondary, Western Blot confining liquid room temperature closes 2h, and with the diluted non-phosphorylating protein antibodies of certain proportion or internal reference antibody
It is incubated overnight in 4 DEG C, the washing of next day PBS/T is incubated for secondary antibody, and expose, develop, be fixed.
The results show that L923 cell is FAPa+ and 4T1 cell is FAPa negative (Fig. 4 C).
The Vitro Tumor diagnosis effect of FAP-GP-NWs is identified
Laser co-focusing experiment:
It in cell inoculation to culture dish and cultivates for 24 hours until 60% converge, culture medium is changed to containing 10%FBS and 1 μ
The polypeptide fresh culture of M FITC label.Then by cell culture 2h.By cells rinsed with PBS 3 times, then 4% poly first
Aldehyde fixes 10min.The nucleus of tumour cell is by 4,6- diamidino -2-phenylindone (DAPI) dyeing visualization.Cell is swashing
It is checked under light Laser Scanning Confocal Microscope.
The results show that the experimental results showed that, reaction product fluorescence intensity is very high, and can come into full contact with cell surface
It is possibly used for next step toy in-vivo imaging experiment (Fig. 4 A)
ELIS experiment:
Overnight by 4 DEG C of FAM antibody coatings after the dilution of 96 orifice plates, PBS is washed three times, then with the PBS containing 1%BSA
It is washed three times in 37 DEG C of closing 2h, PBST, 2h will be incubated in solution adding hole to be measured, PBST is washed three times, after dilution is added
Substrate TMB colour developing 1-5min is added in the horseradish peroxidase of HRP label, 37 DEG C of incubation 2h, and it is whole that sulfuric acid termination reaction solution is added
Only chromogenic reaction, microplate reader measure every suction luminosity at 492nm.
The results show that magnetic bead is easy to be gathered into bulk in static culture medium, FAPa may be will affect to substrate polypeptide
Digesting efficiency tested into one so digestion in vivo in next step need to be carried out but since vitro and vivo environment difference are larger
Step demonstrate,proves (Fig. 4 B, C), and cell culture supernatant fluorescent value highest when FAM antibody dilutes 2,000 times can choose this concentration and carry out down
Urine ELISA detection when one step experiment in vivo.
The in-vivo tumour diagnosis effect of FAP-GP-NWs is identified
The foundation of tumor model:
6-8 weeks big BALB/c-nu/nu nude mice obtains from Zhongshan University (Guangzhou, China) animal center, in pathogen-free domestic
Under the conditions of raise.All zooperies are according to the mechanism criterion progress for looking after and using experimental animal.2×106Eca-
109 cells are resuspended in 100 μ l physiological saline, inject (SC) in the left side oxter of mouse.Tumour growth and Avoirdupois monitoring every 2 days
1 time, the experiment of polypeptide magnetic bead internal injection and small animal living body imaging experiment are carried out after tumour growth to diameter 4-6mm.
2. 9 immunohistochemical stainings
It is routinely fixed after the perfusion of 4% paraformaldehyde, materials are placed in 20% sucrose solution and stay overnight for 4 DEG C.Production wax stone is cut
Piece, patch.After 0.01M PBS buffer solution cleans 5min × 3；The 0.3% hydrogen peroxide methanol solution (methanol prepared is added
80ml, 0.01M PBS buffer solution 100ml, 30% hydrogen peroxide) 30min is stood, to eliminate the shadow of endogenic peroxidase
It rings, 0.01M PBS buffer solution cleans 5min × 3；Be added prepare 0.3%Triton × 100 (30%Triton × 100,
0.01M PBS100ml) 30min is stood, to increase permeability of cell membrane, 0.01M PBS buffer solution cleans 5min × 3；It is added and uses
Primary antibody dilution (bovine serum albumin(BSA) 1.00g, 0.01M PBS 100ml, sodium azide 0.08g) diluted primary antibody, 4 DEG C of incubations
24h；Antibody is sucked, 0.01M PBS cleans 5min × 3；The diluted secondary antibody of 0.01M PBS is added, is incubated at room temperature 2h, 0.01M
PBS cleans 5min × 3；The antibody of ABC compound etc is added, is incubated at room temperature 2h, 0.01M PBS cleans 5min × 3；With distillation
Water flushes three times rapidly；Be added developing solution colour developing, be usually no more than 30min, when cell by coloring and background color it is thin when inhale
Developing solution is removed, 0.01M PBS is added after being flushed three times rapidly with distilled water and terminates reaction；After dehydration of alcohol, mounting is clapped
According to.
The results show that CAFs is full of in tumor stroma in Eca-109 tumor model, and high expression quantity is presented in FAPa
(Fig. 5 C-F).
Small animal living body imaging experiment:
After mouse tumor model is built up, 200 μ l polypeptide magnetic bead PBS re-suspension liquids are injected to mouse by tail vein, give mouse
After anaesthetizing shaving, respectively at 2h, 6h, 12h, anesthesia is taken pictures for 24 hours, observes fluorescent polypeptide in the intracorporal changes in distribution of mouse.
It, can be with but organize after taking out the results show that polypeptide magnetic bead cannot be detected in Mice Body by living imaging
Detect fluorescence, and fluorescence stable in vivo can remain above 12h, but fluorescence is gathered in liver, notification portion magnetic more
Pearl fails to hydrolyze (Fig. 5 G-J) by tumor tissues interior for 24 hours.
Collect and analyze urine
After mouse tail vein injection polypeptide magnetic bead, according to the result of study in relation to FAPa digestion activity in document, respectively at
2h, 6h, 12h carry out abdomen squash type and collect urine for 24 hours, to prevent polypeptide signal from degrading, are added in the urine that is collected into
1 to 10 times of urine proportional diluted being collected into is used for ELISA detection by EDTA final concentration 5mM., ELISA method is same as above.
The results show that urine detection is as the result is shown: compared with the control group, experimental mice urine light absorption value gradually rises,
Reach peak value when 8h, P=0.044 demonstrates the in-vivo diagnostic effect (Fig. 5 A, B) of FAM-GP- magnetic bead.
Claims (1)
1. a kind of FAP α activation type diagnosing tumor polypeptide bead complexes, it is characterised in that its structural formula is such as shown in (I): X-SM
(PEG)2- Y (I), in which: it is more that X represents a kind of FAP α digestion substrate with FAM group marked by biotin (Biotin)
Peptide, structural formula Biotin-DGlu-G-DVal-DAsn-DAsp-DAsn-DGlu-DGlu-G-DPhe-DPhe-DSer-DAla-DArg-Lys (5FAM)-GPGPNQC, wherein D represented amino acid type is D type；Y represents the amino magnetic bead of average grain diameter 100nm,
SM(PEG)2Represent NHS- (PEG)2- Maleimide connexon.
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| CN102212116A (en) * | 2010-04-02 | 2011-10-12 | 复旦大学 | Acetylcholine receptor mediated brain-targeted polypeptide and application thereof |
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