CN104650186A - Active peptide capable of being specifically combined with TfR1 and application of active peptide - Google Patents

Abstract

The invention discloses an active peptide capable of being specifically combined with TfR1. The active peptide has an amino acid sequence as shown in SEQ ID NO:1. The in-vitro experiment shows that the active peptide can be combined with cells capable of high-expressing transferrin receptors, and coupled fluorescein isothiocyanate (FITC) can be transferred into cells specifically. Therefore, the combined peptide disclosed by the invention can be applied to a medicine targeted carrier which takes the transferrin receptors as target points, and has significant meanings for development of tumor target medicines, target treatment on brain-derived diseases, prediction on the transferrin receptors as targets, and tumor happening and development diagnosis.

Description

A kind of can with the bioactive peptide of TfR1 specific binding and application thereof

Technical field

The invention belongs to a kind of biomedicine field, be specifically related to a kind of can with the bioactive peptide of TfR1 specific binding and application thereof.

Background technology

Transferrins,iron complexes has another name called transferrin transferrin, TRF, siderophilin) be iron-protein main in blood plasma, be responsible for delivering the iron absorbed by alimentary canal and the iron of being degraded release by red corpuscle.With TRF-Fe ³⁺composite form enter in marrow, for the generation of mature erythrocyte.TfR (Transferrinreceptor, TfR) is the key protein molecule of body mediation iron metabolism, in the transport of iron, conversion and utilization, play keying action.Further study rear discovery along with to TfR in recent years, Transferrins,iron complexes and acceptor thereof are except the function of transport iron, also relevant with the metabolism of Growth of Cells and propagation and tumour cell thereof.Known two kinds of dissimilar TfRs and TfR 1 (TfR1) and transferrin receptor 2 (TfR2) at present.

TfR1 expresses in many cells, as red blood cell, liver cell, monocyte and hemato encephalic barrier.TfR1 is a kind of II type transmembrane glycoprotein, is to be formed by two disulfide bond crosslinkings by two subunits of homodimer (180kDa).Each monomer is containing 760 amino acid, molecular weight is 90 ~ 95kDa, comprise the outer C end regions (671 amino acid) of large born of the same parents, a single membrane span region (28 amino acid) and a short N end regions (61 amino acid).C end regions is an ectodomain, and it comprises the binding site of Transferrins,iron complexes (Tf), and 3 N connect glycosylation site and an O connection glycosylation site, and the glycosylation in these sites is that TfR1 function is necessary.TfR1 can environmentally pH change and change conformation, and conformation result of variations is converted to the change to Transferrins,iron complexes bonding force power.

TfR2 is a kind of II type transmembrane glycoprotein, it is the TfR1 homologous gene that Kawa-bata clones from Tf cell and HL-60 cell cDNA library, the identical sequence of 45% is had with TfR1, there is the similarity of 66% extracellular regions with TfR1, but the affinity of TfR2 and Transferrins,iron complexes is lower, lower than TfR1 25 times.TfR2 is mainly at liver expression, and its mRNA encodes and non-coding region lacks regulation and control ferric ionic component, and this just shows that the expression of TfR2 is not regulated and controled by iron ion response protein Feedback mechanism.

The Several Kinds of Malignancy cells such as research display nasopharyngeal carcinoma, cancer of the stomach, liver cancer, hematological system tumor are compared with their healthy tissues, and TfR presents high expression level situation, and the expression of TfR is usually relevant with cell proliferation activity.Serum Obtained From Advance Gastric Cancer TfR level comparatively benign stomach disease patient and normal people obviously increases, and the TfR of III, IV phase tumour patient is also apparently higher than I, II phase tumour patient simultaneously.Illustrate that the expression increase of TfR makes tumour cell obtain abnormal proliferation activity.In addition, Transferrins,iron complexes/TfR system improves physiologic barrier if intestinal mucosa, hemato encephalic barrier are to the permeability of medicine by endocytosis.The TfR of hemato encephalic barrier is considered to almost completely saturated by Transferrins,iron complexes, and Transferrins,iron complexes is restricted as medicine Brain targeting carrier.But, because Transferrin Receptor Antibody is different with the binding site of Transferrins,iron complexes and TfR from the binding site of TfR, and non-interference, the antibody of TfR thus can be adopted to carry out drug transport.Coupling has the monoclonal antibody (OX26) of Chinese People's Anti-Japanese Military and Political College's mouse TfR of medicine by Mediated by Transferrin Receptor endocytosis, can to stride across hemato encephalic barrier and transport, and OX26 demonstrates the stronger ability entering central nervous system.

The ability simply penetrating tumor tissues due to micromolecule polypeptide structure is strong, and not easily repel by immunity system, therefore finding can be that target spot develops new tumor-targeting drug with TfR with the active small peptide of TfR1 specific binding, carry out the targeted therapy of brain-borne disease, and to TfR be target tumor development prediction, diagnose significant.

Summary of the invention

Primary and foremost purpose of the present invention is to overcome the shortcoming of prior art with not enough, provide a kind of can with the bioactive peptide of TfR1 specific binding and application thereof.

The concrete technical scheme of the present invention is as follows:

Can with a bioactive peptide CPU-312-01 for TfR1 specific binding, aminoacid sequence is NRAHSLH(SEQ ID NO:1), molecular weight is 1193.5Da.

Prior art field ordinary method can be adopted to modify bioactive peptide of the present invention.Polypeptide drug commonly uses that modifying method comprises the modification of main chain terminal, the modification of intermediary residues, cyclisation, amino acid are replaced, glycosylation modified and PEG modifies.

The main chain terminal modifying method that such as polypeptide drug is commonly used is the acetylizing of N end and the amidation of C end, protects respectively, make polypeptide can not soon by corresponding polypeptide protein enzyme liberating to peptide chain two Amino End Group and carboxyl.This technology has been widely used in the chemosynthesis of polypeptide at present.N holds acetylize normally after the whole peptide chain combination of solid-phase synthetic peptide reaction, adds diacetyl oxide and makes its acetylize.The amidation of C end is then by selecting split product to be the resin of acid amides or to select different fragmentation pattern.Main chain terminal connects the lipid acid of different lengths, main chain C holds or the PEG of N end modifies and glycosylation modified, its ultimate principle is all increase the relative molecular weight of peptide molecule and sterically hindered, improve its stability to polypeptide hydrolyzes enzyme, reduce the filtration of renal glomerulus.Certain the several amino acid replaced in peptide chain is the mode that another kind of postponement enzyme liberating makes the Increased Plasma Half-life of polypeptide drugs, replaces the amino acid that object is generally the easy enzymolysis in peptide chain.In addition, L-type amino acid being replaced with D type alpha-non-natural amino acid is also a kind of ordinary method that amino acid is replaced.

Present invention also offers the modified peptides of above-mentioned bioactive peptide, bioactive peptide modified, hold at the N of bioactive peptide, C end or intermediary residues on connect chemical group, amino acid, polypeptide, protein or PEG.

Present invention also offers the modified peptides of above-mentioned bioactive peptide, one or more amino acid in described bioactive peptides sequence are replaced to corresponding amino acid derivative or special acid.

Present invention also offers gene that the is of the present invention and bioactive peptide of TfR1 specific binding of encoding, there is the nucleotide sequence as shown in SEQ ID NO:2.

Bioactive peptide with TfR1 specific binding of the present invention or its modified peptides may be used for preparing target tumor medicine.Further, described tumour is liver cancer or mammary cancer.

Bioactive peptide with TfR1 specific binding of the present invention or its modified peptides may be used for preparing blood-brain barrier drug.

Bioactive peptide with TfR1 specific binding of the present invention or its modified peptides can be used in the detection of TfR.

TfR specific binding peptides of the present invention is obtained by display technique of bacteriophage.Display technique of bacteriophage is created in 1985 by Smith, to change the phage of structure for carrier, gene fragment orientation to be selected is inserted bacteriophage coat protein plasmagene district, make allogenic polypeptide or protein expression and be showed in phage surface, and then being expressed the phage having special peptide or protein by affine concentration method.It is set up based on 3 points: (1) inserts foreign gene at the N end of p III and p VIII capsid protein, the expressing fusion protein formed is on the surface of phage particle, do not affect and disturb the life cycle of phage, keep foreign gene native conformation simultaneously, also can by corresponding antibody or acceptor identify; (2) utilize target molecule that is fixing and solid support, adopt suitable elutriation method, wash away the phage of non-specific binding, filter out object phage; (3) allogenic polypeptide or protein expression are on the surface of phage, and its encoding gene is derived as the single stranded DNA order-checking of the part in viral genome by secretor type phage.This technology achieves the conversion of genotype and phenotype, more and more widely for Characterization of antigenic epitopes, protein-protein interaction Locus Analysis in Shoots, zymolyte analysis, find there is the simulating peptide of the albumen of biological function, the discovery of lead compound, Isolation and ldentification disease-specific antigens simulating peptide, screening cell and organ specificity binding peptide, Study on Protein and nucleic acid binding properties, signal for locating transduction pathway etc.

Advantage of the present invention

The present invention has following advantage and effect relative to prior art: TfR specific binding peptides of the present invention is obtained by display technique of bacteriophage, is brand-new sequence.Build easily through biological and chemical process synthesis.Bioinformatics technique shows that this is combined with TfR in conjunction with Toplink, experiment in vitro shows this Cell binding in conjunction with Toplink and high expression level TfR, and can coupling fluorescein isothiocyanate (FITC) be proceeded in born of the same parents, can be used as the detection of pharmaceutical carrier and TfR.Therefore binding peptide of the present invention can be used for the exploitation of the tumor-targeting drug taking TfR as target spot, the targeted therapy of brain-borne disease, and to TfR be target tumor development prediction, diagnose significant.

Accompanying drawing explanation

Fig. 1 is that fluorescence microplate reader detects the HepG2 cell picked-up experimental result to coupling FITC binding peptide and FITC.

Fig. 2 is that fluorescence microplate reader detects HepG2 cell and LO2 cell absorbs experimental result to coupling FITC binding peptide.

Fig. 3 is that fluorescence microscope HepG2 cell engulfs experimental result to coupling FITC binding peptide.

Fig. 4 is that fluorescence microscope HepG2 cell engulfs experimental result to FITC.

Embodiment

Concrete steps of the present invention are described by the following examples, but do not limit by embodiment.

Term used in the present invention, except as otherwise noted, generally has the implication that those of ordinary skill in the art understand usually.

Below in conjunction with specific embodiment and comparable data describes in further detail the present invention.Should be understood that these embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.

In the examples below, the various process do not described in detail and method are ordinary methods as known in the art.

Following examples material therefor and plant and instrument are:

Implement material: intestinal bacteria ER2738 (New England Biolabs company of the U.S.), phage display random 7 peptide library (New England Biolabs company of the U.S.), TfR (German CalBiochem company), HRP-M13 antibody (GE Healthcare company), RPMI-1640 (Gibco company), calf serum (Gibco company), trypsin Gibco company).

Embodiment 1

By the method for phage display, obtain TfR specific binding peptides CPU-312-01:

(1) by the screening of phage random peptide library, the nucleotide sequence of TfR specific binding peptides CPU-312-01 is obtained:

A. the Biopanning of phage display random 7 peptide library: with coating buffer (0 .1 M NaHCO3, pH 8 .6) TfR is diluted to 100 μ g/ml, add 150ul in a hole of enzyme plate, repeatedly rotate until surface is completely moistening, keep moisture state, 4 DEG C are spent the night.Outwell coating buffer, clappers removing raffinate, fills it up with confining liquid (0 .1 M NaHCO3, pH 8 .6,5 mg/ml BSA), and 4 DEG C at least act on one hour.Pour out confining liquid, wash 6 times with 0. 1%TBST [TBS+0 .1% (v/v) Tween-20], have the final say at every turn.Dilute l0 μ l naive phage peptide storehouse with 100 μ l TBST, add in the hole with TfR bag quilt, incubation at room temperature 1 hour, topples over unconjugated phage, rinses 10 times, have the final say at every turn with 0. 1%TBST.Then add 100 μ l elutriants [0. 2M Glycine-HC1 (ph2. 2), lmg/mLBSA] room temperature and slightly shake 15min, sucking-off elutriant, then in the 1M Tris-HCL using 15 μ l and above-mentioned elutriant.Draw the elutriant mensuration titre that 1 μ l has neutralized, all the other liquid add 20mlLB substratum (the bacterium liquid containing 20 μ l tsiklomitsin+200 μ l incubated overnight), and 37 DEG C, 225rpm cultivates 5 hours; Centrifugal amplification phage, 4 DEG C, centrifugal 10 minutes of 12000g, proceeds to 80% of supernatant in a new centrifuge tube, adds the PEG8000/NaCl of 1/6 volume, allows phage 4 DEG C of precipitates overnight; Centrifugal phage, 12000g, 15 minutes, 4 DEG C.Outwell supernatant, draw residual solution.Add the resuspended precipitation of 1ml TBS, proceed in Eppendorf tube.4 DEG C, 14000rpm makes residual cells precipitate in centrifugal 5 minutes.Supernatant proceeds in another Eppendorf tube, adds the PEG8000/Nacl of 1/6 volume, ice bath 1h.4 DEG C, centrifugal 10 minutes of 14000rpm, abandons supernatant, precipitates resuspended with 200 μ lTBS.Centrifugal 1min precipitates any undissolvable material, and supernatant proceeds in a new Eppendorf tube, this phage of namely increasing.Undertaken the 2nd by above step, 3 take turns screening, often screen one and take turns and reduce the bag of TfR by concentration, change 50 μ g/ml and 30 μ g/ml into, in conjunction with time add 2 × 10 ¹¹last round of wash-out amplified material, in conjunction with phage with free TfR TfR molecule (100g μ g/ml is dissolved in TBS) competitive elution, use 5%TBST instead and wash during wash-out, improve screening specificity.

B. phage titre measures: with LB substratum dilution phage, do not increase phage dilution range: 10 ¹-10 ⁴, phage dilution range after amplification: 10 ⁸-10 ¹¹.Get the phage of the above-mentioned dilution of l0 μ l, add in 200 μ l mid-log phase ER2738, act on 2 minutes, the infection thalline obtained is added in 3ml liquid top-layer agar and mixes, uniform spreading is on the IPTG/Xgal/Tet flat board of preheating, be inverted lucifuge for 37 DEG C to spend the night, calculate the blue plaque quantity on flat board, extension rate is multiplied by plaque quantity and is phage titre.

C. the amplification of plaque: measure the phage titre that third round does not increase, 300 μ l overnight culture, 30 μ l Tet, 30ml

Be divided in 30 test tubes after the mixing of LB liquid nutrient medium, each test tube 1ml, each test tube picking locus coeruleus adds, 37 DEG C of shaking tables cultivate 5 hours, and culture proceeds in Eppendorf tube, centrifugal 30 seconds, supernatant proceeds in new centrifuge tube centrifugal again, proceeded to by 80% supernatant in new centrifuge tube, this is the phage storage liquid of amplification, 4 DEG C of storages.

D. ELISA identifies positive phage clones: the phage storage liquid getting gained in 5 μ l step C, add 20mlLB substratum (containing 20 μ l tsiklomitsins, the bacterium liquid of 200 μ l incubated overnight) in, cultivate 4. 5 hours for 37 DEG C, centrifugal amplification phage, centrifugal 10 minutes of 4 DEG C of 12000g, proceed in a new centrifuge tube by 80% of supernatant, add the PEG8000/ NaCl of 1/6 volume, allow phage 4 DEG C of precipitates overnight; Centrifugal phage, 12000g, 15min, 4 DEG C.Outwell supernatant, draw residual solution.Add the resuspended precipitation of 1ml TBS, proceed in Eppendorf tube.Centrifugal, 4 DEG C, 14000rpm, 5min make residual cells precipitate.Supernatant proceeds in another Eppendorf tube, adds the PEG8000/NaCl of 1/6 volume, ice bath 1h.Centrifugal, 4 DEG C, 14000rpm, 10min, abandon supernatant, and precipitation is resuspended in 50 μ l TBS, and this is the phage storage liquid of amplification, measures titre, prepares to carry out ELISA qualification.

The TfR molecule preparing 100 μ g/ml (is dissolved in 0.1M NaHCO ₃, PH8.6).4 phage gradients are arranged, each gradient 3 holes for 30 phages to be identified, and arranges that not wrap by blank well be negative control, wrap and placed 4 DEG C by good enzyme plate and spend the night.The unnecessary molecule solution of sucking-off, adds liquid of blockading, and 4 DEG C act on 1 hour, and negative control is closed equally.Throw away liquid of blockading, wash plate 6 times with 0.5%TBST, get the phage of increasing in step C, dilute four quantity gradients and (be respectively 10 ⁹, 10 ¹⁰, 10 ¹¹, 10 ¹²pfu), bag is added by good hole, room temperature effect 1 hour.Wash plate 6 times with 0. 5%TBST, add 1:5000 and dilute HRP-M13 antibody, act on 1 hour.Wash plate 6 times with 0.5%TBST, add OPD nitrite ion, develop the color and add 2M H after 30 minutes ₂sO ₄termination reaction.Select 490nm wavelength, enzyme-linked immunosorbent assay instrument measures each hole absorption value, record result.Using OD value higher than negative control 2.1 doubly as positive colony.

E. the extraction of positive bacteriophage single stranded DNA and order-checking: get 500 μ l above-mentioned phage storage liquid, add 200 μ lPEG8000/NaCl

Place 10 minutes, centrifugal 10 minutes of 14000rpm, abandons supernatant, and precipitation is resuspended in 100 μ l iodide damping fluids, and add 250 μ l ethanol, room temperature effect 10 minutes, centrifugal 10 minutes of 14000rpm, abandons supernatant, wash precipitation with 70% ethanol, of short duration vacuum-drying.Precipitation is resuspended in 30 μ l TE(10mMTris-HCl, 1mMEDTA) in, get the order-checking of above-mentioned solution.This order-checking is completed by Jin Wei intelligence bio tech ltd.

(2) the sequencing result translation will obtained, choose the positive value of Elisa high by (0.834,0.717) and the consistent sequence of nucleotide sequence obtains binding peptide CPU-312-01, and deliver to the biochemical company limited synthesis of Ningbo health shellfish and mark fluorescent probe, its nucleotides sequence is classified as SEQ ID NO:2, and aminoacid sequence is SEQ ID NO:1.

Embodiment 2 fluorescence microplate reader detects the picked-up of cell to coupling FITC binding peptide and FITC and tests:

The HepG2 cell (liver cancer cell) of taking the logarithm vegetative period, with appropriate 0. 25% trypsin solution digestion, jog makes Digestive system stepless action in cell, when the contracting of cell in blocks circle, in individual cells state time, add rapidly the appropriate RPMI-1640 substratum containing 10% calf serum, blow and beat gently with suction pipe and make into single cell suspension; Every hole adds 8 × 10 ⁴individual cell in 24 orifice plates, and adds 500 μ l containing the RPMI-1640 substratum of 10% serum, in 37 DEG C, containing 5 % CO ₂constant incubator in cultivate 24 little of monolayer cell state; Before detecting, 1h, 2h, 3h, 4h add the RPMI-1640 substratum of 500 μ l containing 10% calf serum, the coupling FITC binding peptide of 50um respectively, and the multiple hole of each time point 3, is placed in 37 DEG C, 5% C0 ₂cultivate in constant incubator; Simultaneously using FITC and LO2 cell (normal liver cell) as negative control, detect fluorescence (λ ex=485 nm, λ em=528 nm) with fluorescence microplate reader.During detection fluorescence, the above-mentioned substratum of sucking-off, washs 2 times with PBS.Experimental result as depicted in figs. 1 and 2, time (h)-fluorescence intensity level (AU) curve that in Fig. 1, CPU-312-01-FITC series records for the coupling FITC binding peptide of HepG2 cellular uptake, time (h)-fluorescence intensity level (AU) curve that FITC series records for HepG2 cellular uptake FITC.As can be seen from Figure 1 the former fluorescence intensity is higher than the latter, illustrate FITC and polypeptide coupling after, by the endocytosis of Mediated by Transferrin Receptor cell to CPU-312-01-FITC, with cell to compared with nonspecific endocytosis of FITC, intake improves greatly.Time (h)-fluorescence intensity level (AU) curve that in Fig. 2, HepG2 series records for the coupling FITC binding peptide of HepG2 cellular uptake, time (h)-fluorescence intensity level (AU) curve that LO2 series records for the coupling FITC binding peptide of LO2 cellular uptake, after 2h, the HepG2 cell fluorescence intensity value of high expression level TfR is apparently higher than the LO2 cell fluorescence intensity value of low expression TfR, Expression of Transferrin Receptor level improves can improve the picked-up of cell to binding peptide, illustrate that this small peptide has the target bonding force with high expression level TfR tumour cell, and can by fluorescein isothiocyanate (FITC) targeted cells.

Embodiment 4 fluorescence microscope cell engulfs experiment to coupling FITC binding peptide and FITC's:

The HepG2 cell (liver cancer cell) of taking the logarithm vegetative period, with appropriate 0. 25% trypsin solution digestion, jog makes Digestive system stepless action in cell, when the contracting of cell in blocks circle, in individual cells state time, add rapidly the appropriate RPMI-1640 substratum containing 10% calf serum, blow and beat gently with suction pipe and make into single cell suspension, every hole adds 8 × 10 ⁴individual cell in 24 orifice plates, and adds 500 μ l containing the RPMI-1640 substratum of 10% serum, in 37 DEG C, containing 5 % CO ₂constant incubator in cultivate 24 little of monolayer cell state, every hole adds the RPMI-1640 substratum of 500 μ l containing 10% calf serum, the coupling FITC binding peptide of 50um, and 3 multiple holes, are placed in 37 DEG C, 5% C0 ₂cultivate in constant incubator; Simultaneously using FITC and LO2 cell (normal liver cell) as negative control.After 4h, the above-mentioned substratum of sucking-off, washs 2 times with PBS, takes pictures with fluorescence microscope.Experimental result as shown in Figure 3, Figure 4, Fig. 3 is that HepG2 cell engulfs situation to coupling FITC binding peptide, Fig. 4 is that HepG2 cell engulfs situation to FITC, illustrate when same time same concentrations, coupling FITC binding peptide is more easily by HepG2 cellular uptake, and this interacts in conjunction with Toplink and TfR and make cytophagic FITC more.

Above experimental result shows, TfR specific binding peptides CPU-312-01 of the present invention, can with the Cell binding of TfR and high expression level TfR, and by FITC targeted cells, the ability of transhipment material targeted cells can be had.To the exploitation of tumor-targeting drug taking TfR as target spot, the targeted therapy of brain-borne disease and with TfR be target tumor development prediction, diagnose significant.

Sequence table

Organization Applicant

----------------------

Street: Tong Jia No. 24, lane

City: Nanjing

State: Jiangsu Province

Country: China

PostalCode : 210009

PhoneNumber :

FaxNumber :

EmailAddress :

<110> OrganizationName: China Medicine University

 

Application Project

<120> Title: a kind of can with the bioactive peptide of TfR1 specific binding and application thereof

<130> AppFileReference :

<140> CurrentAppNumber :

<141> CurrentFilingDate :

<160>1

 

Sequence

 

<210> 1

<211> 7

<212> PRT

<213> artificial sequence

NRAHSLH

 

<210> 2

<211> 21

<212> DNA

<213> artificial sequence

AAT CGC GCT CAT TCT CTT CAT

 

Claims (8)

1. can with a bioactive peptide for TfR1 specific binding, there is the aminoacid sequence as shown in SEQ ID NO:1.

2. a modified peptides for bioactive peptide as claimed in claim 1, is characterized in that modifying bioactive peptide, hold at the N of bioactive peptide, C end or intermediary residues on connect chemical group, amino acid, polypeptide, protein or PEG.

3. a modified peptides for bioactive peptide as claimed in claim 1, is characterized in that one or more amino acid in described bioactive peptides sequence to replace to corresponding amino acid derivative or special acid.

4. encode described in claim 1 with the gene of the bioactive peptide of TfR1 specific binding, there is the nucleotide sequence as shown in SEQ ID NO:2.

5. preparing the application in target tumor medicine with the modified peptides of the bioactive peptide described in one of bioactive peptide or the claim 1-3 item of TfR1 specific binding described in claim 1.

6. apply as claimed in claim 5, it is characterized in that described tumour is liver cancer or mammary cancer.

7. preparing the application in saturating blood-brain barrier drug with the modified peptides of the bioactive peptide described in one of bioactive peptide or the claim 1-3 item of TfR1 specific binding as claimed in claim 1.

8. as claimed in claim 1 with the application of modified peptides in TfR detects of the bioactive peptide described in one of bioactive peptide or the claim 1-3 item of TfR1 specific binding.