CN107236025B - Polypeptide and its application in conjunction with CD56 molecular specificity - Google Patents
Polypeptide and its application in conjunction with CD56 molecular specificity Download PDFInfo
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- CN107236025B CN107236025B CN201710206120.0A CN201710206120A CN107236025B CN 107236025 B CN107236025 B CN 107236025B CN 201710206120 A CN201710206120 A CN 201710206120A CN 107236025 B CN107236025 B CN 107236025B
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- C07—ORGANIC CHEMISTRY
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Abstract
The present invention provides polypeptide with CD56 molecular specificity in conjunction with and its application, the polypeptide selected from least one of No. 7 polypeptides, No. 8 polypeptides, No. 7 polypeptides, No. 8 polypeptides amino acid sequence respectively as shown in sequence 1 in sequence table, sequence 2.Polypeptide provided by the invention can manually synthesize in conjunction with CD56 molecular specificity or the method for genetic engineering produces.Compared with antibody, polypeptide provided by the invention has easily prepared, and molecular weight is small, the features such as being not likely to produce immunological rejection；And toxic side effect is small, and the combination of CD56+ cell, to target cell without the effect of significantly killing and Inhibit proliferaton.Polypeptide provided by the invention is used as the marker of CD56 positive expression cell, also can be used as the sub etc. of immunofluorescence CD56 antibody.
Description
Technical field
The present invention relates to biomedicine field, more particularly in conjunction with CD56 molecular specificity polypeptide and its application.
Background technique
CD56 molecule is called N-CAM (neural cell adhesion molecule, NCAM), is
One member of immunoglobulin superfamily.CD56 is the glycoprotein containing polypeptide chain, the extracellular portion packet of CD56 molecule
Include 5 Ig samples structural domain and 2 III type fibronectin spline structure domains.Due to the different montage modes of its mRNA, 3 are ultimately produced
The different molecular isoform of kind: 1.120kDa hypotype is anchored on cell by GPI (glycosyl-phosphatidylinositol)
On film, it is mainly expressed in the tissue of normal tissue and well differentiated, such as NK cell, nerve fiber, neuro-muscular junction, mind
Through endocrine tissue, endocrine tissue etc..2.140kDa and 180kDa hypotype includes a transmembrane domain, is mainly expressed in
In indifferent tissue or malignant cell, such as tumour (such as retinoblastoma, astrocyte of neuroectodermal origin
Tumor, neuroblastoma etc.), endocrine tumors, Small Cell Lung Cancer, NK/T cell lymphoma/leukaemia, Huppert's disease and
Acute myeloid leukemia etc..Therefore, CD56 has been used for the marker of NK cell and above-mentioned tumour cell, can be used for cell mark
Note, sorting and tumor cells diagnosis.
CD56 molecule not only plays mechanical one important receptor of anchoring or cell surface in cell adherence,
Adjust intracellular downstream signaling pathway.Report that the ligand having found includes: CD56 molecule (combination of the same race), L1- in document
CAM, FGFR, GNDF, GFR α, extracellular matrix components such as CSPG, SHPG, collagen, heparin etc..The study found that CD56 exists
Play a significant role in the atomization of nervous system and the plasticity of cynapse, and can be inhibited using the signal transduction in downstream
The proliferation of astroglia and nerve protrusion elongation precursor, it is even more important that CD56 downstream signal can influence to dislike
Property tumour cell bioactivity, and CD56 lose expression it is closely related with some Malignant tumor of bonal metastasis potential and poor prognosis.Cause
This, the ligand (such as antibody) of CD56 molecule can be used as a kind of potential inhibitor to inhibit tumour growth.
Currently, using CD56 molecule to be directed to CD56 antibody, previous source of mouse in the research of the oncotherapy of target
Property antibody can generate in clinical treatment human anti-mouse antibody reaction (human anti-mouse antibodies, HAMA), cause
Action time shortens therapeutic antibodies in vivo, weakens its antineoplastic action, and other pairs that may cause on body are made
With.There is researcher using the method for genetic engineering murine antibody is transformed, to weaken HAMA reaction.But simple anti-CD56
The therapeutic effect of antibody is not very ideal, and then has molecule of the anti-CD56 antibody of research and utilization as a targeting transport, connection
Closing radioactive isotope, cytotoxic drug, cell factor, double potency antibodies (can be in combination with the tumour of tumor cell surface
The activating molecules of related antigen and immunocyte surface) etc., enhance the lethal effect to tumour cell.Therefore, in tumour immunity
Key in the clinical application for the treatment of is the high degree of specificity antibody found in conjunction with CD56 molecule.
Summary of the invention
The present invention be make up the deficiencies in the prior art, provide it is a kind of can with polypeptide CD56 molecular specificity ins conjunction with, tool
There is the features such as easily prepared, molecular weight is small, and application security is high.
The present invention is reaches its purpose, the technical solution adopted is as follows: the polypeptide in conjunction with CD56 molecular specificity is provided,
The polypeptide includes at least one of No. 7 polypeptides, No. 8 polypeptides, No. 7 polypeptides, No. 8 polypeptides amino acid sequence respectively such as
Sequence 1 in sequence table, shown in sequence 2.
Polypeptide provided by the invention can manually synthesize or the method for genetic engineering in conjunction with CD56 molecular specificity
Production.The polypeptide is also referred to as a kind of non-antibody binding proteins (non-antibody binding protein), non-antibody
Binding protein is the also referred to as bracket binding protein (scaffolds for the feature of antibody/antigen specific binding
binding protein).The bracket that this albuminoid is formed in three-dimensional structure by its surface amino groups acid can be in conjunction with specific point
Son has molecular weight small compared with antibody, and penetration is strong, and heat-resisting, stability is good, easy to operate and suitable large-scale production etc.
Feature.
The present invention also provides a kind of marker of CD56 positive expression cell, which includes as described above more
Peptide.
Polypeptide provided by the invention can be applied to label, identification, enrichment, sorting or purifying CD56 positive cell, or application
In preparing the preparation for marking, identifying, be enriched with, sort or purifying CD56 positive cell.The cell includes neuron, star
Cell, Schwann cell, NK cell and NKT cell, also comprising neuroectodermal origin tumour (such as retinoblastoma,
Astrocytoma, neuroblastoma etc.), endocrine tumors, NK/T cell lymphoma and Small Cell Lung Cancer tumour cell.
Another aspect of the present invention provide it is a kind of for sorting the preparation of CD56 positive cell, including as described above more
Peptide is coupled the polypeptide bead complexes to be formed including the polypeptide and magnetic bead and (or is coated with the polypeptide that magnetic bead is formed for polypeptide
Bead complexes).
Polypeptide provided by the invention can also be used in the diagnosis or parting or treatment of disease, such as tumour, viral infection disease
Disease, parasitic disease, leukaemia etc. can also be applied to diagnosis or parting or the preparation for the treatment of of the preparation for disease.The tumour
Tumour, endocrine tumors, NK/T cell lymphoma and Small Cell Lung Cancer including neuroectodermal origin etc., the outer embryo of nerve
The tumour in layer source includes but is not limited to retinoblastoma, astrocytoma, neuroblastoma etc..
Polypeptide of the invention applies also for the preparation of drug targeting preparation.
The present invention also provides a kind of pharmaceutical preparations, including polypeptide described above.The further drug can also wrap
Include the auxiliary material or carrier pharmaceutically allowed.These carriers or auxiliary material, those skilled in the art can be according to the drug agent of required preparation
Type etc. is required and is specifically chosen, such as carrier can be diluent, excipient, adhesive, wetting agent, disintegrating agent, absorption enhancement
One of agent, surfactant, absorption carrier, lubricant are a variety of；Auxiliary material for example may include savory dose, sweetener
Deng.Pharmaceutical dosage form for example can be capsule, soft capsule, tablet, oral solution, dispersible tablet, freeze-dried powder, injection or dripping pill etc..
The existing corresponding reagent in this field can be selected in these carriers or auxiliary material.
Present inventor by T7 display technique of bacteriophage screened from the liver cancer cDNA library of people it is a plurality of can
The albumen or polypeptide of specific binding CD56 molecule finally pick out two and CD56 molecule knot in conjunction with elisa technique and PCR
Stronger polypeptide with joint efforts, respectively No. 7 and No. 8 polypeptides, and artificial synthesized polypeptide carries out subsequent experimental study.Inventor
It is connected to FITC fluorescein in the end of polypeptide, is tested by cellular immunofluorescence, it is found that polypeptide of the invention can be with CD56
Antibody is combined jointly in CD56+ cell surface, and not with the cell combination of CD56-, it was demonstrated that polypeptide of the invention is in cell table
Face can be in conjunction with CD56, and has stronger specificity.Further, inventor has found that polypeptide of the invention does not have cell
Toxicity, and the proliferation of cell is not influenced, the expression rate of cell CD56 is nor affected on, illustrates that polypeptide of the invention does not have cell
Stimulation, does not influence the survival condition and function of cell, this just gives subsequent cell sorting to provide feasibility, ensure that more
Safety of the peptide to required sorting cell.Polypeptide can be detained up to 3.5h, explanation in Mice Body after being injected into Mice Body
Mouse is more slow to the catabolic process of polypeptide, has the potentiality as target medicine carrier, can greatly prolong drug
Retention time in vivo, will not the quick catabolism with blood circulation, thus improve the therapeutic effect of drug, extend
Dosing interval.
Polypeptide is coated with onto nanometer magnetic bead by inventor, carries out cell sorting using magnetic field environment, as a result, it has been found that polypeptide point
The positive rate of cell is selected close to the reagent of commercialization, shows that polypeptide of the present invention has feasibility in cell sorting.The polypeptide can
The anti-CD56 antibody of the mouse of substitute goods has important face in sorting natural killer cells (NK cell), NKT cell
Bed application value.
The coded sequence of polypeptide of the invention derives from the library of people, can react, prolong to avoid HAMA in clinical treatment
The action time of long polypeptide in vivo enhances the antineoplastic action of its drug delivery, and weakens and may cause on body
Other pairs are made.Moreover, the polypeptide that the present invention screens has molecular weight small, synthesis is convenient, many advantages, such as saving is easy, in clinic
There is higher application value.
Detailed description of the invention
OD value of Fig. 1 .CD56 bacteriophage in conjunction with CD56 recombinant protein
Fig. 2 CD56 non-antibody binding proteins bacteriophage PCR result
Fig. 3 CD56-nABP and CD56 antibody mediated immunity fluorescence is imaged altogether
The non-specific binding research of Fig. 4 CD56-nABP and CD56 antibody
The Study of cytotoxicity of Fig. 5 CD56-nABP
Fig. 6 CD56-nABP cel l proliferation
Fluorescence imaging analysis is metabolized in Fig. 7 CD56-nABP body
Fig. 8 CD56-nABP is metabolized Fluirescence observation in mice organs
The research of Fig. 9 CD56-nABP magnetic cell sorting
Specific embodiment
Technical scheme is described further with reference to the accompanying drawing:
It is mass percent if the percentage in text is not specified；If reagent used herein is not specified,
For the reagent obtained can be bought from market.It is this field if experimental implementation conducted in Examples below is not specified
The customary technical operation that technical staff is grasped.
The polypeptide combined hereinafter with regard to CD56 molecular specificity can be expressed as CD56-nABP or nABP-CD56.
One T7 bacteriophage liver cancer cDNA library of embodiment screens CD56 non-antibody binding proteins
Using T7 display technique of bacteriophage, screening can be specifically bound more with CD56 from the liver cancer cDNA library of people
Peptide picks out the stronger polypeptide of binding force, and the DNA sequence dna of sequencing analysis insertion bacteriophage by ELISA, artificial synthesized more
Peptide.
The preparation of 1.1 culture mediums
(1) preparation (1L volume) of LB liquid medium:
| Ingredient | Weight |
| Bacto typtone | 10g |
| Yeast extract | 5g |
| NaCl | 10g |
It is dissolved with second level water, and adjusts pH to 7.5, high pressure steam sterilization 20min with 1N NaOH.
(2) preparation (1L volume) of LB solid medium:
| Ingredient | Weight |
| Bacto typtone | 10g |
| Yeast extract | 5g |
| NaCl | 10g |
| Agar | 15g |
It is dissolved with second level water, and adjusts pH to 7.5, high pressure steam sterilization 20min with 1N NaOH.
(3) Top agarose prepares (1L volume):
It is dissolved with second level water, and adjusts pH to 7.5, high pressure steam sterilization 20min with 1N NaOH.
(4) preparation of M9LB culture medium:
| Ingredient | Volume |
| 20×M9salts | 5ml |
| 20%glucose | 2ml |
| 1M MgSO4 | 0.1ml |
| LB | 100ml |
The preparation (1L volume) of (5) 20 × M9salts:
| Ingredient | Weight |
| NH4Cl | 20g |
| KH2PO4 | 60g |
| Na2HPO4·7H2O | 120g |
It is dissolved with second level water, high pressure steam sterilization 20min.
1.2 96 orifice plates of coating:
(1) plate is washed with deionized water several times, is patted dry on paper.
(2) CD56 recombinant protein is diluted to 5 with deionized water or TBS (10mM Tris-HCl, pH8.0,150mM NaCl)
μg/ml。
(3) albumen that 100 μ l have diluted is added, 4 DEG C of overnight incubations are wrapped with sealed membrane.
(4) with 300 μ 1 × TBS of l board-washing 3 times.
(5) 5% skim milk is prepared with water, 200 μ l are added in every hole.
(6) it is incubated at room temperature 60min or 4 DEG C overnight.
(7) it is washed with deionized water plate 5 times, then 200 μ l water are added in every hole, seal plate and are stored in 4 DEG C.
1.3 phage selection steps (carry out) referring to specification:
(1) suitable 5403 host strain (50 μ l) and antibiotic are inoculated with to the M9LB culture medium of 50ml, shaking bacterium to OD is
0.5-0.6。
(2) prepare 1 × TBST (10mM Tris-HCl, pH 8.0,150mM NaCl, 0.1%-20)。
(3) bacteriophage needed for being added, if volume is supplied less than 100 μ l with 1 × TBST.
(4) it is incubated at room temperature 30min.
(5) with 1 × TBST board-washing 5 times, every hole is greater than 200 μ l.
(6) 200 μ l T7 eluents are added, are incubated at room temperature 10-20min.
(7) bacteriophage of transfer elution takes 10 μ l to calculate phage titre to sterile EP tube.
(8) bacteriophage of elution is added to 5403 host strains of ready 6ml at the beginning, 37 DEG C are shaken bacterium until splitting
Solution.
(9) the second wheel, third round phage selection process are carried out again, and step is screened essentially identical with the first round.
The phagocytosis scale of construction of the phagocytosis scale of construction and elution that each round is added is as shown in table 1, by the phage selection of three-wheel, energy
Enough bacteriophages in conjunction with CD56 recombinant protein are progressively enriched with, and the low bacteriophage of binding force is removed.
The result of 1 CD56 bacteriophage four-wheel of table screening enrichment
The binding force of 1.4 ELISA method detection bacteriophage monoclonals and CD56 recombinant protein:
The CD56 recombinant protein that 30 μ l concentration are 5 μ g/ml is added in every hole on (1) 96 orifice plate, and 4 DEG C of coatings are overnight.
(2) unbonded recombinant protein is washed away, then closes 1h with 3% skimmed milk power room temperature.
(3) bacteriophage after screening third round is cultivated in LB solid medium, 45 bacteriophage monoclonals of random picking
And expand, phage lysate is added in hole, 37 DEG C of incubation 1h.
(4) be added after board-washing the 100 diluted T7Tail-Fiber Monoclonal Antibody of μ l confining liquid (1:
2500) (Merck Millipore) is incubated at room temperature 1h.
(5) be added after board-washing the diluted Anti-Mouse IgG-HRP (1:5000) of 100 μ l confining liquids (Abcam,
Cambridge, UK), it is incubated at room temperature 1h.
(6) 100 μ l TMB chromogenic substrates are added after board-washing, room temperature, which is protected from light, is incubated for 15-60min.
(7) the 250mM HCl that 100 μ l are added terminates reaction.
(8) absorbance (OD) in each hole is detected at 450nm wavelength.
45 plaques are selected at random from the bacteriophage after third round screening, carry out ELISA after expanding in 5403 bacterium
Detection, as a result, it has been found that No. 1, No. 7, No. 11, No. 14, No. 15, No. 17, No. 18, No. 22, No. 24, No. 25, No. 27, No. 29, No. 32,
No. 35, No. 37, No. 38, No. 40, No. 44, No. 45 OD values are higher, as shown in Figure 1.
1.5 CD56 positive bacteriophage gene sequencing, CD56-nABP sequence is determining and synthesizes
The higher bacteriophage of OD value is chosen from ELISA result to carry out PCR detection and be sequenced, and is passed through using DNASTAR software
It crosses and is compared with T7 bacteriophage own sequence, analyze the DNA fragmentation of each bacteriophage insertion, and carry out BLAST on the website NCBI,
Finally determine the amino acid sequence of CD56-nABP simultaneously, and artificial synthetic polypeptide (Shanghai Qiangyao Biotechnology Co., Ltd.).According to
It needs in the fluorescence labels albumen such as N-terminal addition FITC (green fluorescence) of a part of CD56-nABP, purity > 95%, preparation
CD56-nABP fluorescent reagent.
The length for finding most of segments by PCR detection shows Partial Fragment between 200bp-700bp in Fig. 2
PCR result.After bacteriophage is sequenced, sequencing result DNASTAR software alignment and assembbly goes out the sequence of each insertion bacteriophage,
And BLSAT is carried out on NCBI, last artificial synthesized two non-antibody binding proteins SEQ7 and SEQ8, amino acid sequence is respectively such as
Shown in lower:
The amino acid sequence of No. 7 polypeptides: Ser Ser Val Leu Thr Lys Trp Tyr Leu Val Cys Trp
Ser Leu Gly Glu Ser Ile Ser Asn Glu Arg Arg Lys Asn Phe His Leu Val Gly His
Trp Gly Cys Leu Arg Lys Leu Thr Phe Val Val Glu(SSVLTKWYLVCWSLGESISNERRKNFHL
VGHWGCLRKLTFVVE)。
The amino acid sequence of No. 8 polypeptides: Ser Ser Val Tyr Cys Lys Gln Thr Asn Lys Lys Lys
Arg Lys Glu Arg Lys Arg Asp Thr Gly Met Lys Gly Asp Ile Ile Gly Thr Gly Val
Asn Asp Leu Ala Pro Leu Arg Gly Pro Ile Lys Ala Ser Thr Met Val Ala Val Ala
Gln Asp Met Ala Gly Thr Gly Ala Asp Phe Leu Phe Leu(SSVYCKQTNKKKRKERKRDTGMKG
DIIGTGVNDLAPLRGPIKASTMVAVAQDMAGTG ADFLFL)。
The special Journal of Sex Research of embodiment two, CD56-nABP polypeptide and cell combination
The special Journal of Sex Research of 2.1 CD56-nABP polypeptides and U2 OS cell, CTSC-2 cell combination
(1) it is placed in 6 orifice plates after coverslip disinfects in alcohol, is coated with lid glass with 0.1% rat-tail Type I collagen at room temperature
Piece 1h.
(2) it after being washed once with PBS, seeds cells on coverslip, it is adherent to cell for 24 hours in 37 DEG C of incubator cultures
Afterwards, No. 7 and No. 8 polypeptide hatching combination 30min are added.
(3) it is washed once with PBS again, the fixed 15min of 4%PFA is added.
(4) 1h is incubated for confining liquid (5%BSA+10% lowlenthal serum) after washing 3 times with PBS.
(5) the CD56 antibody (1:100) after diluting is added to be incubated overnight at 4 DEG C.
(6) it is then washed 3 times with PBS, is added Anti-Mouse IgG fluorescence secondary antibody (1:1000), is protected from light incubation at room temperature
1h。
(7) DAPI is added after washing 3 times with PBS and is protected from light dye core 5min, after PBS washes 1 time, is sealed with anti-fluorescent quenching mountant
Piece.
(8) it observes and takes pictures under laser scanning co-focusing microscope.
The special Journal of Sex Research of the cell combinations such as 2.2 CD56-nABP and NK92, Jurkat, PBMC:
(1) polypeptide (No. 7 or No. 8 polypeptides) of 10 μ g/ml is added in cell after being resuspended, and mixes and is incubated in 37 DEG C of incubators
30min。
(2) it is washed once with PBS again, the fixed 15min of 4%PFA is added.
(3) 1h is incubated for confining liquid (5%BSA+10% lowlenthal serum) after washing 3 times with PBS.
(4) the CD56 antibody (1:100) after diluting is added to be incubated overnight at 4 DEG C.
(5) after washing 3 times with PBS, addition Anti-Mouse IgG fluorescence secondary antibody (1:1000) (Abcam, Cambridge,
UK), it is protected from light at room temperature and is incubated for 1h.
(6) DAPI reagent is added after washing 3 times with PBS and is protected from light dye core 5min, after PBS washes 1 time, with anti-fluorescent quenching mountant
Mounting.
(7) it observes and takes pictures under laser scanning co-focusing microscope.
Experimental result: U2 OS is applied, the fluorescent marker experiment display of NK92 and Jurkat cell pearl is with FITC fluorescence
CD56-nABP and CD56 antibody can mark these CD56+ cells jointly, and Fig. 3 shows CD56-nABP and CD56 antibody fluorescence mark
The confocal microscopy image of note, the CD56-nABP with FITC fluorescence is together with CD56 overlapping antibody, explanation
CD56-nABP can be in conjunction with the CD56 molecule of cell surface.And CTSC-2 cell is the cell of CD56-, cell surface without
CD56 antibody, also without polypeptide fluorescence, as shown in figure 4, showing that CD56-nABP polypeptide with CTSC-2 cell combination, does not illustrate CD56-
NABP polypeptide combines specificity with higher.
The cytotoxicity experiment of embodiment three, CD56-nABP polypeptide
Cell count after U2OS cell dissociation is resuspended, every hole is inoculated with 3000 cells on 96 orifice plates, with 100 μ l (DF+10%
FBS) culture medium culture cell, in CO2For 24 hours, polypeptide (the 0.1 μ g/ of various concentration is added in incubator culture after cell is adherent
Ml, 0.5 μ g/ml, 1 μ g/ml, 5 μ g/ml, 10 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml), it is detected after 72h with CCK8
Cell activity.
As a result as shown in figure 5, figure A, B are respectively No. 7, No. 8 polypeptides are to the toxic effect of U2 OS cell.Statistical analysis
The results show that U2 OS cell is cultivated in the culture medium that the polypeptide of various concentration gradient is added and the culture medium that polypeptide is not added,
Its cell viability does not have difference, illustrates No. 7 and No. 8 polypeptides do not have toxic effect to U2 OS cell.
The influence of example IV, CD56-nABP polypeptide cell proliferation
U2 OS and CTSC-2 cell culture are in 24 orifice plates, with 500 μ l (DF+10%FBS) culture medium culture cells, in CO2
Incubator culture after cell is adherent, is separately added into No. 7 polypeptides and No. 8 polypeptides for 24 hours, makes its final concentration of 10 μ g/mL, and set up
One is not added the negative control group of polypeptide, adds a polypeptide every 48h, and count cell daily, continuous counter 7 days, draws
Cell Proliferation curve.
Experimental result: see Fig. 6, figure A, B are the experimental result of U2 OS, CTSC-2 cell respectively, with negative control group ratio
Compared with there was no significant difference (Fig. 6) for the proliferative capacity of group of cells, and CD56-nABP cell proliferation ability does not make significant difference.
Embodiment five, CD56-nABP are tested in Mice Body intracellular metabolite
Female mice weight 18.4g enters No. 8 polypeptides of 400ul CD56-nABP, concentration 1mg/ from mouse tail vein injection
ml.Start to image after yellow Jackets (concentration 20mg/ml) anesthetized mice of 60ul is injected intraperitoneally.Every 15min shooting
Fluorescence images (Fig. 7) display, and CD56-nABP polypeptide blood loop distribution also has fluorescence to mouse systemic and mouse lip；1h
Afterwards, polypeptide fluorescence is gradually weak, and is progressively enriched with Mouse Liver, kidney and intestines, and polypeptide only resides in liver and enteric cavity, table after 3.5h
It is bright main by liver metabolism.
Mouse Liver, spleen, lung and kidney are dissected, is observed under frozen section Laser Scanning Confocal Microscope, it is as a result as shown in figure 8, each
Internal organs have a small amount of polypeptide residual, illustrate that polypeptide enters each internal organs in Mice Body after blood circulation, but
After 3.5 hours fluorescence is taken pictures, each remaining polypeptide of internal organs is less, may most of catabolism in vivo.
Embodiment six, the sorting research of CD56-nABP polypeptide magnetic cell
The preparation of 6.1 CD56-nABP polypeptides-bead complexes
(1) the 300nm diameter magnetic bead for taking 40 μ l, with 500 μ l buffer2, (PBS of MES containing 0.1M, 0.5M NaCl are molten
Liquid, pH5.0) cleaning magnetic bead 3 times.
(2) No. 8 polypeptides of 100 μ l are added after magnetic bead being resuspended with 500 μ l buffer2,1mg EDC, 1.5mg NHSS shakes
Bed is incubated for 2h.
(3) 5mgBSA and 5mg Gly is added, continues shaking table and is incubated for 4h.
(4) it is cleaned magnetic bead 3 times with 1ml PBS.
(5) liquid is saved with 1ml again (contain 0.1%BSA, 0.01%Tween, 0.05%NaN3PBS, pH7.4) cleaning magnetic
Pearl 3 times, 500 μ l save liquid and magnetic bead, 4 DEG C of preservations are resuspended.
The research of 6.2 CD56-nABP polypeptide cells magnetic sortings
(1) by U2 OS cell and CTSC-2 cell dissociation at single cell suspension, supernatant is abandoned in 300g centrifugation.
(2) 100 μ l polypeptides-bead complexes (experimental group) and 100 μ l magnetic beads (negative control), another set are separately added into
20 μ lCD56 MicroBeads (U.S. day Ni, positive control) is added.
(3) 4 DEG C of incubation 15min after mixing.
(4) it is separately added into 1ml buffer1 cleaning, then 300g is centrifuged 5min, and 500 μ l buffer1 are resuspended.
(5) magnetic liquid separates cell, collects positive and negative cells group, and machine testing on CD56 streaming antibody is added.
Experimental result: separation results are as shown in figure 9, curve 1,2,3 respectively represents negative control, experimental group and the positive in figure
The experimental result of control group.As seen from Figure 9, the positive rate of the U2 OS cell sorted out by No. 8 polypeptide-bead complexes
Reach 84.6%, is 97.9% with the U2 OS cell positive rate that commercialized U.S. day Ni sorting reagent sorts out, illustrates with more
Peptide-bead complexes sorting U2 OS cell is feasible, and already close to the separating effect of U.S. day Ni.In negative control group,
I.e. individual magnetic bead sorting U2 OS cell reaches 47.9%, illustrates that magnetic bead has certain non-specific adsorption to act in cell, but
It is also to need to optimize experiment condition in follow-up study, reduce the non-specificity of magnetic bead far below polypeptide-magnetic bead separating effect
Suction-operated.And in the CTSC-2 cell processes for sorting CD56- with No. 8 polypeptides-bead complexes, negative control group, experiment
Group and the sorting indexes of positive controls are 2.74%, 1.6%, 54.9% respectively, it is seen then that polypeptide of the present invention not with CTSC-2 cell
In conjunction with non-specific adsorption is less.For No. 7 polypeptides, cell sorting experimental result is similar to No. 8 polypeptides, repeats no more.
To sum up, the present invention by T7 display technique of bacteriophage screened from the liver cancer cDNA library of people it is a plurality of can be special
The opposite sex combines the albumen or polypeptide of CD56 molecule finally to pick out two in conjunction with CD56 molecule in conjunction with elisa technique and PCR
The stronger polypeptide of power, respectively No. 7 and No. 8 polypeptides, and artificial synthesized polypeptide carries out subsequent experimental study.Inventor exists
The end of polypeptide is connected to FITC fluorescein, is tested by cellular immunofluorescence, it is found that polypeptide of the present invention can be with CD56 antibody
It is common to combine in CD56+ cell surface, and not with the cell combination of CD56-, it was demonstrated that polypeptide of the invention is in cell surface energy
Enough in conjunction with CD56, and there is stronger specificity.Further, inventor has found that polypeptide does not have toxicity to cell, and not
The proliferation for influencing cell, nor affects on the expression rate of cell CD56, illustrates that polypeptide of the present invention does not have stimulation to cell, not shadow
The survival condition and function of cell are rung, this just gives subsequent cell sorting to provide feasibility, ensure that polypeptide to required sorting
The safety of cell.It can be detained in Mice Body after polypeptide of the present invention is injected into Mice Body up to 3.5h, illustrate mouse
More slow to the catabolic process of polypeptide, polypeptide of the present invention has the potentiality as target medicine carrier, can prolong significantly
The retention time of long drug in vivo will not be imitated quickly in the catabolism with blood circulation, the treatment for thus improving drug
Fruit extends dosing interval.
Inventor also passes through EDC and NHSS crosslinking agent in polypeptide coating to nanometer magnetic bead, is carried out using magnetic field environment thin
Born of the same parents' sorting, as a result, it has been found that the positive rate of polypeptide of the present invention sorting cell shows polypeptide in cell sorting close to the reagent of commercialization
In have feasibility.The anti-CD56 antibody of the mouse of the alternative commercialization of the polypeptide, sorting natural killer cells (NK cell),
There is important clinical value in NKT cell.NK cell is the important immunocyte of one kind of human body, participates in the anti-of body
Tumour, viral infection resisting, also the IIth type hypersensitivity of participation and graft-versus-host reaction.NK cell is primarily present in peripheral blood
In, the 5-10% of PBMC is accounted for, also there is NK cell in lymph node and marrow.NKT cell is a subgroup of T cell, cell
The existing T cell receptor TCR in surface, and have NK cell receptor, it plays a role in the inherent immunity of human body, mainly plays immunological regulation
And cytotoxicity.
Therefore, using CD56 molecule combination polypeptide of the invention, the NK and NKT that can be isolated from human peripheral blood are thin
Born of the same parents group carries out amplification cultivation in vitro, and feeds back to the immunity for enhancing body in patient body, improves to tumour cell, disease
The lethality of poison and helminth.It, in clinical treatment can be to avoid HAMA since the coded sequence of polypeptide derives from the library of people
Reaction extends the action time of polypeptide in vivo, enhances the antineoplastic action of its drug delivery, and weaken and may cause body
Other side effects on body, and the polypeptide that we screen has molecular weight small, synthesis is convenient, it is easy many advantages, such as saving,
There is higher application value in clinic.
The above described is only a preferred embodiment of the present invention, limitation in any form not is done to the present invention, therefore
All contents without departing from technical solution of the present invention, it is made to the above embodiment according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
<110>Guangzhou generation Pharmaceutical Technology Co., Ltd
<120>polypeptide in conjunction with CD56 molecular specificity and its application
<160> 2
<170> Patentin version 3.3
<210> 1
<211> 43
<212> PRT
<213>artificial sequence
<400> 1
Ser Ser Val Leu Thr Lys Trp Tyr Leu Val Cys Trp Ser Leu Gly Glu Ser Ile Ser Asn Glu
1 5 10 15 20
Arg Arg Lys Asn Phe His Leu Val Gly His Trp Gly Cys Leu Arg Lys Leu Thr Phe Val Val Glu
25 30 35 40
(SSVLTKWYLVCWSLGESISNERRKNFHLVGHWGCLRKLTFVVE)
<210> 2
<211> 63
<212> PRT
<213>artificial sequence
<400> 2
Ser Ser Val Tyr Cys Lys Gln Thr Asn Lys Lys Lys Arg Lys Glu Arg Lys Arg Asp Thr Gly
1 5 10 15 20
Met Lys Gly Asp Ile Ile Gly Thr Gly Val Asn Asp Leu Ala Pro Leu Arg Gly Pro Ile Lys
25 30 35 40
Ala Ser Thr Met Val Ala Val Ala Gln Asp Met Ala Gly Thr Gly Ala Asp Phe Leu Phe Leu
45 50 55 60
(SSVYCKQTNKKKRKERKRDTGMKGDIIGTGVNDLAPLRGPIKASTMVAVAQDMAGTGADFLFL)
Claims (12)
1. the polypeptide in conjunction with CD56 molecular specificity, which is characterized in that the polypeptide include No. 7 polypeptides, in No. 8 polypeptides extremely
Few one kind, No. 7 polypeptides, No. 8 polypeptides amino acid sequence respectively as shown in sequence 1 in sequence table, sequence 2.
2. a kind of marker of CD56 positive expression cell, which is characterized in that including polypeptide as described in claim 1.
3. the application of polypeptide as described in claim 1, which is characterized in that be included in label, identification, enrichment, sorting or purifying
Nondiagnostic application in CD56 positive cell, or it is positive thin for marking, identifying, be enriched with, sort or purifying CD56 in preparation
Application in the preparation of born of the same parents.
4. application according to claim 3, which is characterized in that the cell is that CD56 expresses positive cell, the cell
Including neuron, astrocyte, Schwann cell, NK cell, NKT cell；And/or the cell includes neuroderm
The tumour in source, endocrine tumors, NK/T cell lymphoma and Small Cell Lung Cancer tumour cell.
5. application according to claim 4, which is characterized in that the tumour of the neuroectodermal origin includes retina mother
Cytoma, astrocytoma, neuroblastoma.
6. a kind of for sorting the preparation of CD56 positive cell, which is characterized in that including polypeptide as described in claim 1 or
The polypeptide bead complexes for being coated in magnetic bead including the polypeptide and being formed.
7. application of the polypeptide as described in claim 1 in preparation of the preparation for diagnosis or the treatment of disease, feature exist
In the disease includes tumour, virus infection, parasitic disease.
8. application according to claim 7, which is characterized in that the tumour includes the tumour, interior of neuroectodermal origin
Secreting tumor, NK/T cell lymphoma and Small Cell Lung Cancer.
9. application according to claim 8, which is characterized in that the tumour of the neuroectodermal origin includes retina mother
Cytoma, astrocytoma, neuroblastoma.
10. polypeptide as described in claim 1 is preparing the application in drug targeting preparation.
11. a kind of drug targeting preparation, which is characterized in that its target activity ingredient includes polypeptide as described in claim 1.
12. drug targeting preparation according to claim 11, which is characterized in that further include the auxiliary material pharmaceutically allowed or load
Body.
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| PCT/CN2017/099487 WO2018176732A1 (en) | 2017-03-30 | 2017-08-29 | Polypeptide specifically binding to cd56 molecule and use thereof |
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| CN112521479B (en) * | 2020-12-10 | 2022-04-15 | 中山大学附属口腔医院 | Polypeptide specifically bound with CD3 and application thereof |
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| CN105254717B (en) * | 2015-08-18 | 2018-08-24 | 中山大学 | The polypeptide combined with CD34 molecular specificities and its application |
| CN105254759A (en) * | 2015-10-26 | 2016-01-20 | 无锡傲锐东源生物科技有限公司 | Anti-CD56 protein monoclonal antibody hybridoma cell, anti-CD56 monoclonal antibody generated by same and application |
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