CN112442113A - Target specific polypeptide and application thereof - Google Patents
Target specific polypeptide and application thereof Download PDFInfo
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- CN112442113A CN112442113A CN201910817056.9A CN201910817056A CN112442113A CN 112442113 A CN112442113 A CN 112442113A CN 201910817056 A CN201910817056 A CN 201910817056A CN 112442113 A CN112442113 A CN 112442113A
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- iron oxide
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/06—Ferric oxide [Fe2O3]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention provides a targeted specific polypeptide, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1, said polypeptide can specifically bind to cells highly expressing the chemokine receptor CXCR 4. The polypeptide P12 has high affinity to CXCR4, and can be specifically bound to CXCR 4; meanwhile, P12 can be used as a targeting probe to modify the iron oxide nanoparticles and guide the iron oxide nanoparticles to be combined with cells with high expression of CXCR 4; the polypeptide P12 can be used as a carrier for cell screening, targeted therapy and molecular imaging, and provides a feasible method for improving the specificity and sensitivity of tumor targeted therapy and molecular imaging.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a targeted specific polypeptide and application thereof.
Background
The tumor cell has great difference from normal cell in the aspects of gene expression, gene transcription and the like, and certain protein over-expressed on the surface of the tumor cell can be used as a tumor-related target for tumor targeted therapy and diagnosis.
The chemokine receptor CXCR4 is highly expressed in lymphocytes, tumor cells, epithelial cells and mesenchymal tissues and can respond to the activation of a ligand CXCL12, so that the interaction of CXCR4/CXCL12 biological axes plays an important role in the proliferation of tumor cells, the metastasis to organs (such as liver, lung, lymph nodes, bone marrow and the like) with the high expression of CXCL12, the homing of lymphocytes and the chemotaxis.
Compared with antibodies, synthetic polypeptides are generally low in molecular weight, pose a lower risk of causing immune reactions, and are simple in preparation process and low in cost. CXCR4 antagonists are currently mainly classified into three categories: small molecule antagonists, polypeptide antagonists and CXCR4 antibodies. The small molecule antagonist is mainly AMD3100, and a plurality of studies report that the AMD3100 can effectively inhibit the activity of CXCR4, inhibit the migration of tumor cells caused by CXCL12 and inhibit the activation of downstream signal paths in-vitro and in-vivo tumor models; another class of polypeptide antagonists, such as LY-2510924, significantly inhibits tumor cell migration by CXCL12 and inhibits CXCL12/CXCR4 mediated intracellular signaling; in addition, using CXCR4 antibodies to specifically bind CXCR4, HIV infection and tumor cell migration can also be significantly inhibited. Existing studies indicate that CXCR4 is likely to be a target for new tumor therapies and targeted molecular imaging. The antagonists have antagonism on the function of the cells with high expression of CXCR4, and no polypeptide which is specially used as a CXCR4 targeting probe and does not influence the function of the cells exists. The development of polypeptides that target CXCR4 without interfering with cellular function could provide novel probe molecules for tumor-targeted therapy, related cell tracking and molecular imaging.
Disclosure of Invention
Therefore, the present invention aims to overcome the defects in the prior art and provide a target specific polypeptide and application thereof.
Before the technical solution of the present invention is explained, the terms used herein are defined as follows:
the term "FITC" refers to: fluorescein isothiocyanate.
To achieve the above object, the first aspect of the present invention provides a target specific polypeptide capable of specifically binding to a cell highly expressing CXCR4, the amino acid sequence of the polypeptide is SEQ ID NO: 1, and the polypeptide can specifically bind to cells highly expressing the chemokine receptor CXCR 4.
The polypeptide according to the first aspect of the present invention, wherein the N-terminus of the polypeptide is modified by Biotin or FITC;
preferably, the biotin is linked to the N-terminus of the polypeptide by GG, and/or
And the FITC is connected with the N end of the polypeptide through aminocaproic acid.
In a second aspect, the present invention provides a target-specific nanoparticle, wherein the nanoparticle is the polypeptide-modified iron oxide nanoparticle of the first aspect;
preferably, the particle size of the iron oxide nanoparticles is 3nm to 100nm, preferably 3nm to 20 nm.
A third aspect of the present invention provides a method for preparing the nanoparticle of the second aspect, the method comprising the steps of:
(1) activating carboxyl on the surface of the iron oxide nanoparticle MNP by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS);
(2) reacting MNP with FITC-labeled Avidin (Avidin-FITC) to obtain FITC and Avidin-labeled iron oxide nanoparticle MNP (MNP-Avidin-FITC); and
(3) adding biotin-labeled polypeptide (biotin-P12), and incubating with FITC and Avidin-labeled iron oxide nanoparticles (MNP-Avidin-FITC) to obtain FITC-P12 polypeptide-modified iron oxide nanoparticles (FITC-P12-MNP).
In a fourth aspect, the invention provides a targeting probe comprising a polypeptide according to the first aspect and/or a nanoparticle according to the second aspect.
In a fifth aspect, the present invention provides a vector for cell screening, targeted therapy and/or molecular imaging, the vector comprising a polypeptide according to the first aspect.
According to a sixth aspect of the invention there is provided the use of a polypeptide of the first aspect in the manufacture of a medicament for the detection and/or treatment of a disease in which there is high expression of the chemokine receptor CXCR 4.
The use according to the sixth aspect of the invention, wherein the chemokine receptor CXCR4 high expression disease is a solid tumor, leukemia and/or a lymphatic disease; preferably, the solid tumor is breast cancer.
The seventh aspect of the invention provides the use of the nanoparticle of the second aspect or the targeting probe of the fourth aspect in the preparation of a medicament for detecting and/or treating a disease with high expression of chemokine receptor CXCR 4.
The use according to a seventh aspect of the invention wherein the disease in which the chemokine receptor CXCR4 is overexpressed is a lymphoid disease.
In order to overcome the defects of the prior art, the invention aims to provide a targeting polypeptide aiming at a cell with high CXC R4 expression and application thereof, wherein the polypeptide can be specifically combined with CXCR4 on the surface of the cell and has higher affinity.
The invention relates to a polypeptide, in particular to a polypeptide with tumor targeting specificity and application thereof, wherein the polypeptide can be specifically combined with a chemokine receptor CXCR4 and is applied to the fields of targeted molecular imaging, targeted therapy and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a polypeptide P12, wherein the amino acid sequence of the polypeptide P12 is as follows: QGCRRRNTVDDWISRRRAL, and derivatives thereof, biotin-labeled polypeptide P12: Biotin-GG-QGCRRRNTVDDWISRRRAL, and FITC-labeled polypeptide P12 FITC-Acp-QGCRRRNTVDDWISRRRAL.
The polypeptide P12 can be specifically combined with cells highly expressing CXCR 4. The cells are CXCR4 high-expression solid tumor cells, leukemia cells and lymphocytes, and more preferably CXCR4 high-expression solid tumor cells are breast cancer cells.
The polypeptide P12 has no obvious influence on the activity of the cell with high expression of CXCR 4.
Preferably, the cells are CXCR4 high expressing solid tumor cells, leukemia cells and lymphocytes, more preferably CXCR4 high expressing solid tumor cells are breast cancer cells.
The polypeptide P12 can be used for modifying iron oxide nanoparticles and can be used as a targeting probe for tumor detection.
Preferably, the polypeptide P12 is used for modifying iron oxide nanoparticles, and the iron oxide nanoparticles modified by P12 can be specifically combined with cells highly expressing CXCR 4. Preferably, the cell is a lymphocyte highly expressing CXCR 4.
In one embodiment of the invention, the binding of P12-modified iron oxide nanoparticles to lymphocytes was observed using an iron staining method.
The polypeptide provided by the invention can be specifically combined with a chemokine receptor CXCR4 with high expression of tumor cells, has higher affinity, and has important significance in tumor targeted therapy and targeted molecular imaging.
The polypeptides of the invention may have, but are not limited to, the following beneficial effects:
(1) the polypeptide P12 has high affinity to CXCR4, and can be specifically bound to CXCR 4; meanwhile, P12 can be used as a targeting probe to modify the iron oxide nanoparticles and guide the iron oxide nanoparticles to be combined with cells with high expression of CXCR 4;
(2) the polypeptide P12 can be used as a carrier for cell screening, targeted therapy and molecular imaging, and provides a feasible method for improving the specificity and sensitivity of tumor targeted therapy and molecular imaging.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the expression of CXCR4 protein in solid tumor cell lines (MDA-MB-231 cells), leukemia cell lines (K562 cells) and lymphocyte cell lines (Jurkat cells) preferred in example 2 of the present invention.
FIG. 2 shows the experimental results of the effect of the biotin-labeled polypeptide P12 on the activity of leukemia cell line (K562 cells) in example 3 of the present invention.
FIG. 3 shows the experimental results of the effect of the biotin-labeled polypeptide P12 on the activity of human breast cancer cell line (MDA-MB-231 cells) in example 4 of the present invention.
FIG. 4 shows the experimental results of the effect of the biotin-labeled polypeptide P12 on the activity of the lymphocyte cell line (Jurkat cells) in example 5 of the present invention.
FIG. 5 is a graph showing the experimental results of the affinity of FITC-labeled polypeptide P12 for leukemia cell line (K562 cells) in example 6 of the present invention.
FIG. 6 shows the experimental results of the affinity of FITC-labeled polypeptide P12 for human breast cancer cell line (MDA-MB-231 cells) in example 7 of the present invention.
FIG. 7 shows the experimental results of the affinity of FITC-labeled polypeptide P12 for the lymphocyte cell line (Jurkat cells) in example 8 of the present invention.
FIG. 8 shows the experimental results of the affinity of FITC-labeled polypeptide P12-modified iron oxide nanoparticles to lymphocyte cell lines (Jurkat cells) in example 9 of the present invention.
FIG. 9 shows the results of experiments on binding of FITC-labeled polypeptide P12 modified iron oxide nanoparticles to cells observed by iron staining after co-incubation with lymphocyte cell line (Jurkat cells) in example 10 of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
Unless otherwise indicated, the human chronic myelogenous leukemia cell line K562, the human acute T-cell leukemia cell line Jurkat, and the breast cancer MDA-MB-231 cell lines used in the following examples were purchased from the cell resource center of the institute of basic medicine, national academy of medicine, China.
The solvents of the aqueous solutions used in the examples below were sterile ultrapure aqueous solutions unless otherwise specified.
Unless otherwise indicated, all reagents used in the following examples were analytical reagents.
Unless otherwise specified, all PBS solutions used in the following examples are 1 × PBS solutions.
The reagents used in the following examples were purchased from the following sources and instrument models, respectively:
(1) reagent purchase source:
PBS buffer, DMEM medium, 1640 medium, fetal bovine serum, and double antibody were purchased from Thermo Fisher Scientific;
the 4% paraformaldehyde solution was obtained from Beijing Solaibao Tech Co., Ltd;
CXCR4 antibodies were purchased from Biolegend;
CCK8 reagent test kit was purchased from Sigma.
(2) The instrument model is as follows:
a water purifier (Merck Millipore, Germany, model Milli-Q Integral 3);
centrifuge (Beijing Rebo centrifuge, Inc., model LD 5-2A);
flow cytometry (BD biosciences, model BD accuri C6);
multifunctional microplate readers (Molecular Devices, model spectra Max i3, USA);
example 1: preparation of polypeptide P12
The amino acid sequence of polypeptide P12 is SEQ ID NO: 1;
the amino acid sequence of the polypeptide P12 marked by the biotin is as follows: Biotin-GG-QGCRRRNTVDDWISRRRAL; the amino acid sequence of the FITC-labeled polypeptide P12 is FITC-Acp-QGCRRRNTVDDWISRRRAL.
The polypeptide (synthesized by Shanghai peptide Biotech Co., Ltd., purity 98%) was synthesized according to the designed sequence, and a mother solution of an appropriate concentration was prepared before the experiment.
Polypeptide dissolution: dissolving the polypeptide powder with ultrapure water to obtain a mother solution with a concentration of 1mM to ensure sufficient dissolution of the polypeptide, and storing at-20 deg.C for use.
Preparation of FITC-P12 polypeptide-modified iron oxide nanoparticles (FITC-P12-MNP): to a solution of iron oxide nanoparticles (MNP, available from Nanjing Donghana Biotechnology Co., Ltd./Xian supermagnetic NanoBiotechnology Co., Ltd., surface-coated carboxylated dextran) were added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, final concentration 2mM) and N-hydroxythiosuccinimide (Sulfo-NHS, final concentration 5mM), and reacted at room temperature for 15 minutes. Activated MNP was collected by centrifugation, resuspended, and added with FITC-labeled Avidin (Avidin-FITC, A2901, Sigma-Aldrich), and reacted at room temperature for two hours to obtain FITC and Avidin-labeled MNP (MNP-Avidin-FITC). Unreacted Avidin was removed by centrifugation, and then biotin-labeled polypeptide (biotin-P12) was added and incubated with MNP-Avidin-FITC for 30 minutes at 37 ℃. And (3) performing high-speed centrifugal separation, removing unbound polypeptides, and resuspending with PBS to obtain FITC-P12 polypeptide-modified iron oxide nanoparticles (FITC-P12-MNP).
Example 2: flow cytometry detection of CXCR4 expression in K562, MDA-MB-231 and Jurkat cells
K562, MDA-MB-231 and Jurkat cells were collected into a 1.5mL centrifuge tube, rinsed once with 1mL PBS, and then resuspended in 100. mu.L of 1% BSA in PBS, followed by addition of 1. mu.L of CXCR4 antibody stock solution and incubation at 4 ℃ for 1 h. After 1mL of PBS was rinsed three times, the cell positive rate was detected by flow cytometry. The results are shown in figure 1, the K562 cell positive rate is higher than 75%, and the MDA-MB-231 and Jurkat positive rates are higher than 95%, and the K562 cell positive rate and the MDA-MB-231 and Jurkat positive rates are both the cells with high expression of CXCR 4.
Example 3: effect of Biotin-labeled polypeptide P12 on K562 cell Activity
At a rate of 1X 10 per hole4Cells K562 cells were plated in 96-well plates and 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL and 1mg/mL Biotin-P12 polypeptide was added, respectively, and incubated with the cells at 37 ℃ for 24 h. Centrifuging for 5min in a centrifuge with the rotation speed of 1000rpm, sucking off the supernatant, adding 200 mu LPBS solution, blowing, uniformly mixing, rinsing, centrifuging again for 5min in the centrifuge with the rotation speed of 1000rpm, sucking off the supernatant, adding 10 mu LCCK8 detection reagent and 100 mu L culture medium into each hole, and incubating for 2h in an incubator at 37 ℃. And (4) sucking 100 mu L of the supernatant into a 96-well plate, detecting absorbance values at 450nm and 630nm, and calculating the cell survival rate. The P12 polypeptide had no significant effect on K562 cell viability over the tested concentration range, as shown in figure 2.
Example 4: effect of biotin-labeled polypeptide P12 on MDA-MB-231 cell Activity
At a rate of 1X 10 per hole4MDA-MB-231 cells were plated in 96-well plates, and 10ng/mL, 100ng/mL and 500ng/mL Biotin-P12 polypeptides were added, respectively, and incubated with the cells at 37 ℃ for 24 h. The supernatant was aspirated, rinsed once with 200. mu.L of PBS solution, and the supernatant was aspirated. Add 10. mu.L of LCCK8 assay reagent and 100. mu.L of medium to each well and incubate for 2h at 37 ℃ in an incubator. And (4) sucking 100 mu L of the supernatant into a 96-well plate, detecting absorbance values at 450nm and 630nm, and calculating the cell survival rate. P12 polypeptide was shown to be fine for MDA-MB-231 over the range of concentrations testedCell survival was not significantly affected, as shown in figure 3.
Example 5: effect of Biotin-labeled polypeptide P12 on lymphocyte Jurkat Activity
At a rate of 1X 10 per hole4Cells Jurkat cells were plated in 96-well plates and incubated with cells at 37 ℃ for 24h with the addition of 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, and 1mg/mL of the Biotin-P12 polypeptide. Centrifuging for 5min in a centrifuge with the rotation speed of 1000rpm, removing the supernatant, adding 200 mu LPBS solution, blowing, uniformly mixing, rinsing, centrifuging again for 5min in the centrifuge with the rotation speed of 1000rpm, sucking the supernatant, adding 10 mu LCCK8 detection reagent and 100 mu L culture medium into each hole, and incubating for 2h in an incubator at 37 ℃. And (4) sucking 100 mu L of the supernatant into a 96-well plate, detecting absorbance values at 450nm and 630nm, and calculating the cell survival rate. The P12 polypeptide had no significant effect on Jurkat cell viability over the tested concentration range, as shown in figure 4.
Example 6: flow cytometry detectionFITC-labeledAffinity of polypeptide P12 for leukemia-associated cell K562 cell Force of
At a rate of 1X 10 per hole5K562 cells were seeded in 12-well plates, 20. mu.M FITC-labeled P12 polypeptide was added, and cells were incubated for 6h at 37 ℃. The cells were collected into 1.5mL centrifuge tubes and placed in a centrifuge at 1000rpm for 5 min. After removing the supernatant, the cells were rinsed twice with 1mL PBS, after the supernatant was aspirated, the cells were resuspended in 100. mu.L of cell suspension with PBS, the cell suspension was filtered through a 300-mesh screen, and the average fluorescence intensity of the cells was measured using a flow cytometer. The result is shown in fig. 5, the P12 polypeptide can mark K562 cells, the fluorescence intensity is obviously greater than that of the negative control group, and the P12 polypeptide can specifically recognize CXCR4 of the K562 cells.
Example 7: flow cytometry detectionFITC-labeledAffinity of polypeptide P12 for breast cancer-related cell MDA-MB-231 Force of mixing
At a rate of 1X 10 per hole5MDA-MB-231 cells were seeded into 12-well plates, 20. mu.M of FITC-labeled P12 polypeptide was added, and the cells were incubated for 6h at 37 ℃. Then collect the cells into a 1.5mL centrifuge tubePlacing in a centrifuge with the rotation speed of 1000rpm for 5min for centrifugation. After removing the supernatant, the cells were rinsed twice with 1mL PBS, after the supernatant was aspirated, the cells were resuspended in 100. mu.L of cell suspension with PBS, the cell suspension was filtered through a 300-mesh screen, and the average fluorescence intensity of the cells was measured with a flow cytometer. The result is shown in FIG. 6, the P12 polypeptide can mark MDA-MB-231 cells, the fluorescence intensity is significantly larger than that of a negative control group, and the P12 polypeptide can specifically recognize CXCR4 of MDA-MB-231 cells.
Example 8: flow cytometry detectionFITC-labeledAffinity of polypeptide P12 for lymphocyte Jurkat
At a rate of 1X 10 per hole5Jurkat cells were seeded in 12-well plates, 20. mu.M of FITC-labeled P12 polypeptide was added, and cells were incubated for 6h at 37 ℃. The cells were then collected into 1.5mL centrifuge tubes and placed in a centrifuge at 1000rpm for 5 min. After removing the supernatant, the cells were rinsed twice with 1mL PBS, after the supernatant was aspirated, the cells were resuspended in 100. mu.L of cell suspension with PBS, the cell suspension was filtered through a 300-mesh screen, and the average fluorescence intensity of the cells was measured with a flow cytometer. The results are shown in fig. 7, the P12 polypeptide can label Jurkat cells, the fluorescence intensity is significantly greater than that of the negative control group, and P12 can specifically recognize CXCR4 of Jurkat cells.
Example 9:FITC-labeledBinding of P12 polypeptide-modified iron oxide nanoparticles to lymphocyte Jurkat
At a rate of 1X 10 per hole5Jurkat cells were seeded in 12-well plates, and 40. mu.g/mL FITC-P12 polypeptide-modified iron oxide nanoparticles (FITC-P12-MNP) were added and incubated with the cells at 37 ℃ for 6 h. The cells were collected into 1.5mL centrifuge tubes and placed in a centrifuge at 1000rpm for 5 min. After removing the supernatant, the cells were rinsed twice with 1mL PBS, after the supernatant was aspirated, the cells were resuspended in 100. mu.L of cell suspension with PBS, the cell suspension was filtered through a 300-mesh screen, and the proportion of FITC-positive cells was determined by flow cytometry. The result is shown in fig. 8, the FITC-P12 polypeptide-modified iron oxide nanoparticles have strong binding force with Jurkat cells, and the positive rate reaches 97%, which indicates that the FITC-P12 polypeptide-modified iron oxide nanoparticles have strong binding force with Jurkat cells.
Example 10: iron staining observationFITC-labeledP12 polypeptide modified iron oxide nano-particles and lymphocytes Conjugation of Jurkat
At a rate of 1X 10 per hole5Jurkat cells were seeded in 12-well plates, and 40. mu.g/mL FITC-P12 polypeptide-modified iron oxide nanoparticles (FITC-P12-MNP) were added and incubated with the cells at 37 ℃ for 6 h. The cells were collected into 1.5mL centrifuge tubes and placed in a centrifuge at 1000rpm for 5 min. After removing the supernatant, the cells were rinsed twice with 1mL PBS, fixed for 10min with 4% paraformaldehyde at room temperature, and then dropped onto a slide to dry naturally. And dropwise adding an acidic potassium ferrocyanide solution onto the glass slide for iron dyeing, rinsing with PBS, and then re-dyeing with 0.1% nuclear solid red dye solution for 8 min. After PBS rinsing, the cells were covered with a cover slip and the staining of the cells was observed under a microscope. MNP appears blue after iron staining, cells appear red after nuclear fast red staining. As shown in FIG. 9, it was observed that the cell surface appeared noticeably blue in the FITC-P12-MNP group, but not in the control and MNP groups, indicating that MNP linked with P12 polypeptide had the ability to target receptor binding to cells.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Sequence listing
<110> national center for Nano science
INSTITUTE OF BASIC MEDICAL SCIENCES, CHINESE ACADEMY OF MEDICAL SCIENCES
<120> target specific polypeptide and application thereof
<130> YZDI-190042
<140> 2019108170569
<141> 2019-08-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Gly Cys Arg Arg Arg Asn Thr Val Asp Asp Trp Ile Ser Arg Arg
1 5 10 15
Arg Ala Leu
Claims (10)
1. A target specific polypeptide, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1, and the polypeptide can specifically bind to cells highly expressing the chemokine receptor CXCR 4.
2. The polypeptide of claim 1, wherein the N-terminus of the polypeptide is modified by Biotin or FITC;
preferably, the biotin is linked to the N-terminus of the polypeptide by GG, and/or
And the FITC is connected with the N end of the polypeptide through aminocaproic acid.
3. A target-specific nanoparticle, wherein the nanoparticle is the polypeptide-modified iron oxide nanoparticle of claim 1 or 2;
preferably, the particle size of the iron oxide nanoparticles is 3nm to 100nm, preferably 3nm to 20 nm.
4. A method for preparing nanoparticles as claimed in claim 3, characterized in that it comprises the following steps:
(1) activating carboxyl on the surface of the iron oxide nanoparticles by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide;
(2) reacting the ferric oxide nanoparticles with the FITC marked avidin to obtain FITC and avidin marked ferric oxide nanoparticles; and
(3) adding biotin-labeled polypeptide, and incubating with FITC and avidin-labeled iron oxide nanoparticles to obtain FITC-P12 polypeptide-modified iron oxide nanoparticles.
5. A targeting probe, characterized in that it comprises a polypeptide according to claim 1 or 2 and/or a nanoparticle according to claim 3.
6. A vector for cell screening, targeted therapy and/or molecular imaging, characterized in that the vector comprises a polypeptide according to claim 1 or 2.
7. Use of a polypeptide according to claim 1 or 2 for the manufacture of a medicament for the detection and/or treatment of a disease in which the chemokine receptor CXCR4 is highly expressed.
8. The use according to claim 7, wherein the high expression disease of the chemokine receptor CXCR4 is a solid tumor, leukemia and/or a lymphatic disease; preferably, the solid tumor is breast cancer.
9. Use of the nanoparticle of claim 3 or the targeting probe of claim 5 for the preparation of a medicament for the detection and/or treatment of a disease with a high expression of the chemokine receptor CXCR 4.
10. The use according to claim 9, wherein the disease of high expression of the chemokine receptor CXCR4 is a lymphatic disease.
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