CN109593125A - Antigen epitope polypeptide CD44-P1 and its application based on prostate cancer stem cells marker CD44 - Google Patents
Antigen epitope polypeptide CD44-P1 and its application based on prostate cancer stem cells marker CD44 Download PDFInfo
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Abstract
The present invention provides a kind of antigen epitope polypeptide CD44-P1 based on prostate cancer stem cells marker CD44 and its applications, it is related to bio-pharmaceutical engineer technology domain, a kind of antigen epitope polypeptide and its application based on prostate cancer stem cells marker CD44 provided by the invention, the antigen epitope polypeptide based on prostate cancer stem cells surface marker CD44 has been designed and synthesized by bioinformatics means, specific activation cytotoxic T lymphocyte, with good fragmentation effect, it can be used for developing the therapeutic peptide vaccine of targeting prostate cancer stem cells, new technical solution is provided for the accurate immunization therapy of malignant prostate cancer, and can selectively targeted prostate cancer stem cells fundamentally reduce prostate cancer recurrence.
Description
Technical field
The present invention relates to bio-pharmaceutical engineer technology domains, are based on prostate cancer stem cells marker more particularly, to one kind
The antigen epitope polypeptide CD44-P1 of CD44 and its application.
Background technique
Prostate-cancer incidence, the death rate have become the head for seriously threatening human health in trend is risen year by year at present
Number killer.Although the operative treatment of prostate cancer, chemotherapy, radiotherapy and molecular targeted therapy constantly make further progress, preceding
Effective treatment of column gland cancer is still critical issue urgently to be resolved.
In recent years, as people are to the molecule mechanism of prostate cancer and the continuous announcement of immunologic mechanism, prostate cancer specific
Immunization therapy be paid more and more attention, and obtaining ideal tumour antigen is the key that immunotherapy of tumors.With Immunology
The rapid development of method and molecular biology method can largely induce the discovery of the tumour antigen of immune response, be swollen
Tumor immunization therapy opens new era.
" tumor stem cell " theory thinks to have self-renewal capacity there are a group in tumor tissues and can generate heterogeneous
The cell of property tumor cell group, proliferation, transfer, recurrence and insensitive in close relations, the tomour specific to chemicotherapy with tumour
The promising target of property immunization therapy.In recent years, with CD133+/CD44+For phenotype prostate cancer stem cells be found after, it
Feature with stem cell function is confirmed in many aspects.
In recent years, with going deep into immune response Study on Molecular Mechanism, the immunocyte of body is gradually recognized
It is not the overall molecule corresponding to various pathogen or native antigen, but for the anti-of various antigen molecule
Former epitope, i.e. proteantigen are that its immunologic specificity is embodied by its epitope.Studies have shown that CD8+T cell was identified
Tumour antigen needs first to handle through antigen presenting cell, is presented in the form of " Antigenic Peptide-MHC-I class molecule " compound later anti-
Former presenting cells or target cell surface, the Antigenic Peptide in conjunction with MHC-I class molecule is CTL epitope accordingly.It is existing anti-swollen
Tumor treatment technology specificity is low, fragmentation effect is limited, and is easy tumor recurrence.
In view of this, the present invention is specifically proposed.
Summary of the invention
The present invention in order to solve the problems in the prior art, provides a kind of based on prostate cancer stem cells marker CD44's
Antigen epitope polypeptide CD44-P1 and its application.
To solve the above-mentioned problems, the technical solution adopted by the present invention is as described below:
The present invention provides a kind of isolated antigen epitope polypeptide CD44-P1, the antigen epitope polypeptide CD44-P1 is selected from
As follows (a) or (b):
(a) polypeptide that the amino acid sequence shown in SEQ ID NO:1 forms；
(b) amino acid sequence shown in SEQ ID NO:1 by the substitution of one or several amino acid residues and/or is lacked
It loses and/or adds, and the polypeptide as derived from (a) with prostate cancer stem cells marker CD44 antigen epitope function.
Further, the molecular weight of the antigen epitope polypeptide CD44-P1 is 1000-1300.
Further, the antigen epitope polypeptide CD44-P1 is obtained by prokaryotic cell or eukaryotic cell expression purifying；
Or,
The antigen epitope polypeptide CD44-P1 passes through artificial synthesized.
The present invention also provides a kind of nucleic acid, the above-mentioned antigen epitope polypeptide CD44-P1 of the nucleic acid encode.
Further, the nucleic acid is selected from any one of following (A)-(C):
(A) nucleotide sequence as shown in SEQ ID NO:2；
(B) polynucleotides of polypeptide shown in SEQ ID NO:1 are encoded；
(C) with SEQ ID NO:2 shown in nucleotide sequence homology 80% or more and coding prostate cancer stem cells
The nucleotide of the polypeptide of marker CD44 antigen epitope function.
The present invention also provides a kind of carrier, the carrier includes above-mentioned nucleic acid.
The present invention also provides a kind of host cell, the host cell includes above-mentioned nucleic acid or carrier.
In addition, the present invention also provides above-mentioned antigen epitope polypeptide CD44-P1 in preparation prevention and/or treatment prostate
Application in the product of cancer.
Further, the product is vaccine or drug.
Further, before the antigen epitope polypeptide CD44-P1 is by the prevention of killing LNCAP- many cells ball and/or treatment
Column gland cancer；And/or
The antigen epitope polypeptide CD44-P1 passes through the prevention of killing VCaP- many cells ball and/or treatment prostate cancer.
Isolated antigen epitope polypeptide CD44-P1 provided by the invention, for based on prostate cancer stem cells marker CD44
Antigen epitope polypeptide.The present invention has been designed and synthesized by bioinformatics means based on prostate cancer stem cells surface markers
The antigen epitope polypeptide of object CD44, antigen epitope polypeptide CD44-P1 have good immunogenicity, can specific activation it is thin
Cytotoxic T Lymphocytes have good fragmentation effect to prostate cancer stem cells, are prostate cancer stem cells surface marker
The research of epitope is had laid a good foundation.
Antigen epitope polypeptide CD44-P1 provided by the invention based on prostate cancer stem cells marker CD44 can be stimulated
Body generates the immune response of anti-prostate cancer stem cell protection, so that the inhibition and removing of prostate cancer stem cells are imitated in enhancing
Fruit is based on this, and epitope antibody provided by the invention can be used for developing the therapeutic peptide epidemic disease of targeting prostate cancer stem cells
Seedling provides new technical solution for the accurate immunization therapy of malignant prostate cancer, and being capable of selectively targeted prostate cancer stem cells
Fundamentally reduce prostate cancer recurrence.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the schematic diagram for the CD44 Protein Epitopes analysis that the embodiment of the present invention 1 provides；
Fig. 2 is the mass spectral analysis figure for the antigen epitope polypeptide CD44-P1 that the embodiment of the present invention 1 provides；
Fig. 3 is the efficient liquid phase chromatographic analysis figure for the antigen epitope polypeptide CD44-P1 that the embodiment of the present invention 1 provides；
Flow cytometry analysis result schematic diagram when Fig. 4 A is the 0th day in the embodiment of the present invention 2；
Flow cytometry analysis result schematic diagram when Fig. 4 B is the 14th day in the embodiment of the present invention 2；
Fig. 5 A is that the result that antigen epitope polypeptide CD44-P1 acts on LNCAP- many cells ball in the embodiment of the present invention 2 is shown
It is intended to；
Fig. 5 B is the killing effect that antigen epitope polypeptide CD44-P1 acts on LNCAP- many cells ball in the embodiment of the present invention 2
Fruit schematic diagram；
Flow cytometry analysis result schematic diagram when Fig. 6 A is the 0th day in the embodiment of the present invention 3；
Flow cytometry analysis result schematic diagram when Fig. 6 B is the 14th day in the embodiment of the present invention 3；
Fig. 7 A is the result signal that antigen epitope polypeptide CD44-P1 acts on VCaP- many cells ball in the embodiment of the present invention 3
Figure；
Fig. 7 B is the fragmentation effect that antigen epitope polypeptide CD44-P1 acts on VCaP- many cells ball in the embodiment of the present invention 3
Schematic diagram.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
It should be understood that
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method
It can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with
Intercombination forms new technical solution.
In the present invention, unless otherwise indicated, it is each reaction or operating procedure can sequentially carry out, can also in sequence into
Row.Preferably, reaction method herein is that sequence carries out.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
According to an aspect of the invention, there is provided a kind of isolated antigen epitope polypeptide CD44-P1, the epitope
Peptide C D44-P1 is selected from following (a) or (b):
(a) polypeptide that the amino acid sequence shown in SEQ ID NO:1 forms；
(b) amino acid sequence shown in SEQ ID NO:1 by the substitution of one or several amino acid residues and/or is lacked
It loses and/or adds, and the polypeptide as derived from (a) with prostate cancer stem cells marker CD44 antigen epitope function.
Wherein, SEQ ID NO:1 are as follows:
Ala-Gly-Val-Phe-His-Val-Glu-Lys-Asn-Gly-Arg-Tyr。
In this application, term " antigen epitope polypeptide " refers to any polypeptide piece that can be identified by the antibody of specificity
Section.
Isolated antigen epitope polypeptide CD44-P1 provided by the invention, for based on prostate cancer stem cells marker CD44
Antigen epitope polypeptide.The present invention has been designed and synthesized by bioinformatics means based on prostate cancer stem cells surface markers
The antigen epitope polypeptide of object CD44, antigen epitope polypeptide CD44-P1 have good immunogenicity, can specific activation it is thin
Cytotoxic T Lymphocytes have good fragmentation effect to prostate cancer stem cells, are prostate cancer stem cells surface marker
The research of epitope is had laid a good foundation.
Mankind's CD44 gene is located at No. 11 the short arm of a chromosome, overall length about 50kb, altogether by 20 highly conserved exon groups
At each exon length 70-210bp is differed, intermediate to be separated by introne different in size.CD44 is as a kind of cell surface
Molecule functions effect in cell adherence and signal transduction, by the tumor markers as prostate cancer stem cells, has
Very potent epitope, the antigen epitope polypeptide provided by the invention based on prostate cancer stem cells marker CD44 is to preceding
Column gland cancer stem cell has stronger immunogenicity and lethal effect.
The substitution and/or deletion and/or addition of one or several amino acid residues can be 1-10 amino acid residue
Substitution and/or deletion and/or addition；The substitution and/or deletion and/or addition of one or several amino acid residues can be sent out
Life is except above structure domain.
In some preferred embodiments, the molecular weight of the antigen epitope polypeptide CD44-P1 is 1000-1300, example
It such as can be, but be not limited to 1202.35,1208.26 or 1084.18, preferably 1202.35.
In some preferred embodiments, the antigen epitope polypeptide CD44-P1 passes through prokaryotic cell or eukaryocyte
Expression and purification obtains；Or,
The antigen epitope polypeptide CD44-P1 passes through artificial synthesized.
In some embodiments, the present invention also provides a kind of nucleic acid, can encode above-mentioned antigen epitope polypeptide
CD44-P1。
Herein, nucleic acid sequence includes the variant (such as displacement of degenerate codon) and complementation sequence of its conservative substitution
Column.Term " nucleic acid " and " polynucleotides " be it is synonymous, include gene, cDNA molecule, mRNA molecule and their segment example
Such as oligonucleotides.
In some preferred embodiments, the nucleic acid is selected from any one of following (A)-(C):
(A) nucleotide sequence as shown in SEQ ID NO:2；
(B) polynucleotides of polypeptide shown in SEQ ID NO:1 are encoded；
(C) with SEQ ID NO:2 shown in nucleotide sequence homology 80% or more and coding prostate cancer stem cells
The nucleotide of the polypeptide of marker CD44 antigen epitope function.
SEQ ID NO:2 in sequence table is gcaggtgtattccacgtggagaaaaatggtcgctac.
In some embodiments, the present invention also provides the nucleic acid sequences containing coding antigen epitope polypeptide CD44-P1
The expression vector of column.
In the present invention, carrier can refer to it is comprising nucleic acid of the invention or its segment, hereditary information can be carried and
The molecule or reagent that hereditary information can be delivered in cell.Typical carrier include plasmid, virus, bacteriophage, sticking grain and
Minichromosome.Carrier can be cloning vector, and (carrier i.e. for being transferred to hereditary information in cell can be bred described
Cell and can choose the cell presence or absence of the hereditary information) or expression vector (i.e. comprising necessary something lost
Pass carrier of the element to allow the hereditary information of the carrier to express in cell).Therefore, cloning vector may include selection
Label, and the replication orgin to match with cell type specified by the cloning vector, and expression vector then include for
Influence the necessary regulating element of expression in specified target cell.
Nucleic acid of the invention or its segment are inserted into suitable carrier and carry nucleic acid fragment of the present invention to be formed
Cloning vector or expression vector.This new support is also a part of the invention.The carrier may include plasmid, bacteriophage,
Sticking grain, minichromosome or virus also include the naked DNA of the only transient expression in specific cells.Cloning vector and table of the present invention
Duplication that can be spontaneous up to carrier, thus can for for rear clone high level expression or high level duplication purpose height is provided
Copy number.Expression vector may include the promoter for driving nucleic acid fragment of the invention to express, and optional coding makes described
The nucleic acid sequence of the secretion of peptide expression product or the signal peptide being integrated on film, nucleic acid fragment of the invention, and optional coding
The nucleic acid sequence of terminator.When operating expression vector in producing bacterial strain or cell line, when carrier is introduced into host cell
It can be integrated into the genome of host cell, can also cannot be integrated into host cell gene group.Carrier usually carries
Replication site, and the flag sequence of Phenotypic Selection can be provided in transformed cells.
Expression vector of the invention is for converting host cell.This transformed cells are also a part of the invention, can be with
It is the culture cell or cell for being proliferated nucleic acid fragment and carrier of the invention or for being prepared by recombinant polypeptide of the invention
System.Transformed cells of the invention include microorganism such as bacterium (such as Escherichia coli, bud pole bacterium).Host cell also includes coming from
Multicellular organism such as fungi, insect cell, plant cell or mammalian cell.
In addition, the present invention also provides above-mentioned antigen epitope polypeptide CD44-P1 in preparation prevention and/or treatment prostate
Application in the product of cancer.
Antigen epitope polypeptide provided by the invention based on prostate cancer stem cells marker CD44 can stimulate body to produce
Raw anti-prostate cancer stem cell protection immune response, thus enhance the inhibition to prostate cancer stem cells and understand effect, base
In this, epitope antibody provided by the invention can be used for developing the therapeutic peptide vaccine of targeting prostate cancer stem cells, be
The accurate immunization therapy of malignant prostate cancer provides new technical solution, and can selectively targeted prostate cancer stem cells from basic
Upper reduction prostate cancer recurrence.
In some preferred embodiments, the product is vaccine or drug.
In some embodiments, vaccine further includes adjuvant, and drug further includes pharmaceutically acceptable carrier.
In some preferred embodiments, the antigen epitope polypeptide CD44-P1 passes through killing LNCAP- many cells ball
Prevention and/or treatment prostate cancer；And/or
The antigen epitope polypeptide CD44-P1 passes through the prevention of killing VCaP- many cells ball and/or treatment prostate cancer.
In order to facilitate it is clearer understand the contents of the present invention, be described in detail as follows now in conjunction with specific embodiment.
Unless otherwise instructed, experimental animal, drug used in the embodiment of the present invention and the equal source of reagent are regular and easy
Purchase channel:
A.LNCaP cell and VCaP cell are purchased from American Type Culture collection warehousing (ATCC)；
B. erythrocyte cracked liquid, IFN-γ, IL1 α and AntiCD3 McAb and AntiCD28 McAb, IL-2, AIM-V culture solution, IL4,
TNF β, the GM-CSF factor are purchased from Beijing source Tong Lihai Biotechnology Co., Ltd；
C. flow cytometer uses the LSRFortessa Cell Analyzer of BD bioscience company；
D. Tissue Culture Dish, culture bottle are purchased from U.S. Corning company.
In following example D C cell cultivation process, if any jaundice, then change liquid, add IL-4, TNF β and GM-CSF because
Son.
The design and synthesis of embodiment 1CD44 epitope small peptide
1. sharing the full amino acid sequence (NP_ for searching people CD44 albumen in gene pool NCBI Genbank from international openness
000601.3) totally 742 amino acid.Its amino acid sequence is as follows:
MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTLPTMAQMEKA
LSIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITIT
IVNRDGTRYVQKGEYRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTDRIPATT
LMSTSATATETATKRQETWDWFSWLFLPSESKNHLHTTTQMAGTSSNTISAGWEPNEENEDERDRHLSFSGSGIDDD
EDFISSTISTTPRAFDHTKQNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEE
TPHSTSTIQATPSSTTEETATQKEQWFGNRWHEGYRQTPKEDSHSTTGTAAASAHTSHPMQGRTTPSPEDSSWTDFF
NPISHPMGRGHQAGRRMDMDSSHSITLQPTANPNTGLVEDLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTST
LTSSNRNDVTGGRRDPNHSEGSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQDT
FHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLALALILAVCIAVNSRRRCGQKKKLVIN
SGNGAVEDRKPSGLNGEASKSQEMVHLVNKESSETPDQFMTADETRNLQNVDMKIGV
2. the present embodiment by DNA Star (Protean) software according to the hydrophily of amino acid sequence, antigenic index with
And surface accessibility comprehensive analysis predicts the position of its antigen small peptide.
3. as shown in Figure 1, in conjunction with Immune Epitope Database (IEDB, http://www.iedb.org/) number
The antigen epitope polypeptide comprising CD44 Protein Epitopes is devised according to library, and is named as CD44-P1.
As shown in Fig. 2, the molecular weight of antigen epitope polypeptide CD44-P1 are as follows: 1202.35, desolventizing temperature are as follows: 350 DEG C, take off
Its flow velocity of solvent 350L/h.
As shown in figure 3, the efficient liquid phase chromatographic analysis flow velocity of antigen epitope polypeptide CD44-P1 are as follows: 1.0ml/min, wavelength:
220nm, applied sample amount: 10 μ l, concrete outcome are as shown in table 1:
The efficient liquid phase chromatographic analysis result of 1 antigen epitope polypeptide CD44-P1 of table
| Sequentially | Time | Sample size | Peak area | Peak height |
| 1 | 9.111 | 0.0555 | 4687 | 946 |
| 2 | 9.532 | 0.0693 | 5858 | 1164 |
| 3 | 9.795 | 99.5406 | 8414181 | 402756 |
| 4 | 10.333 | 0.3346 | 28285 | 9802 |
| It amounts to | NA | 100 | 8453011 | 414668 |
Embodiment 2
The antigen epitope polypeptide CD44-P1 of the CD44 Protein Epitopes provided using the embodiment of the present invention 1 studies CD44
Lethal effect of the effector T cell of induction to Human Prostate Cancer Cells LNCAP- many cells ball.Specifically comprise the following steps:
1. prepared by the separation of peripheral blood mononuclear cells: HLA-A2 positive healthy volunteer's peripheral blood 10mL of acquisition is received
Collect in 2 centrifuge tubes, 2000r/min is centrifuged 5min, abandons supernatant, sedimentation cell is mixed, adds physiological saline to 25mL, makes
Sedimentation cell sufficiently suspends, and forms blood cell suspension.1 centrifuge tube is separately taken, lymphocyte separation medium 20mL is added, it will with dropper
Blood cell suspension is slowly transferred to the surface of lymphocyte separation medium, makes to form clearly interface therebetween.
2. dividing from tube bottom to liquid level after the above-mentioned effective 2000r/min centrifugation 20min of the centrifugation for forming clearly interface
It is 4 layers, is followed successively by red blood cell and granulocyte layer, lymphocyte separation medium layer, peripheral blood mononuclear cells layer i.e. PBMC layers, blood plasma
Layer；Peripheral blood mononuclear cells layer is sucked out with suction pipe, is transferred in another centrifuge tube, it is spare.
3. adding physiological saline to 40mL, 1500r/min centrifugation into the centrifuge tube equipped with peripheral blood mononuclear cells layer
5min, after abandon supernatant, physiological saline is added, sedimentation cell suspends to 40mL, after mixing well, 1500r/min centrifugation
5min；Centrifuge washing 3 times altogether.
4. after last time is centrifuged, abandoning supernatant, by sedimentation cell and being transferred in culture bottle, be added 40mL's
AIM-V culture solution culture, and it is labeled as A.
5. taking out the cell of culture after culture 2h, the lymphocyte to suspend in A is transferred in another 1 culture bottle, is marked
For B.
IFN-γ, IL1 α and AntiCD3 McAb and AntiCD28 McAb are added into the B bottle containing lymphocyte, and at second day
IL-2, Fiber differentiation CIK cell is added；The AIM-V culture solution and IL4 of addition 40mL into the A bottle containing attached cell, and
TNF β is added in third day, is added within the 5th day, the GM-CSF factor, Fiber differentiation DC cell.
In 6.DC cell cultivation process, if any jaundice, then liquid is changed, adds IL-4, TNF β and the GM-CSF factor.
In 7.CIK cell cultivation process, such as finds culture solution jaundice, then change liquid, add IL-2 after changing liquid.As discovery has carefully
Born of the same parents' agglomerate becomes bad cotton-shaped, then standing sedimentation 5min or so after being collected with centrifuge tube, after floccule precipitating, carefully by supernatant
Cell suspension is refunded in culture bottle.
It is added CD44 antigen epitope polypeptide CD44-P1 when 8.DC cell culture was to the 7th day into A culture bottle, the 8th day when shovels
Lower DC cell is mixed with the CIK cell in corresponding B after collecting and is co-cultured, and DC Tissue Culture Flask is discarded.
If total cell concentration is excessively huge, assigns in A and B bottles and continue after DC cell and CIK cell can also being mixed
Culture.By induced t cell at cytotoxic T cell (CTL), i.e. effector T cell.
9. the DC-CIK cell co-cultured did flow cytometry analysis CD3, CD56, CD4 and CD8 positive cell in the 14th day
Ratio simultaneously carries out cell killing experiment.
10. the preparation of target cell: recovery prostate gland cancer cell LNCAP is normally cultivated, is passed on.Phosphoric acid after being digested by pancreatin
Salt buffer cleaning is resuspended；After marking target cell, cleaning to be resuspended with the Calcein-AM of the concentration of 10mM according to effector cell with
The effect target ratio of tumor stem cell 5:1,10:1,37 DEG C of co-cultivation 4h；Culture solution is collected, l000rpm is centrifuged 5min, takes 100 μ L
Supernatant measures average fluorescent strength and adds detergent as most using simple target cell using simple target cell as spontaneous release
Big burst size.
As shown in Figure 4 A and 4 B shown in FIG., by flow cytometer comparative analysis, pass through CD44 antigen epitope polypeptide as the result is shown
CIK cell ratio after activation significantly increases；At the 0th day (Fig. 4 A), flow cytometry analysis is the result shows that CD3 positive T is thin
It is 41.90%, CD8 positive T cell ratio is 26.90% that born of the same parents' ratio, which is 69.10%, CD4 positive T cell ratio, CIK cell
(CD3/CD56 is bis- positive) ratio is 2.70%；At the 14th day (Fig. 4 B), flow cytometry analysis is the result shows that CD3 positive T
It is 14.90%, CD8 positive cell ratio is 84.30% that cell proportion, which is 97.50%, CD4 positive cell ratio, CIK cell ratio
Example is 32.20%.
As fig. 5 a and fig. 5b, it is analyzed by CD44 antigen epitope polypeptide prostate cancer stem cells fragmentation effect, as a result
Show that the CIK cell of CD44 antigen epitope polypeptide activation has significant prostate cancer stem cells lethal effect.CD44 antigen table
Position peptide C D44-P1 is capable of the effect of specific inducing cell killing, kills cell proportion by 2.70% and is promoted to 32.20%；
And have a significant lethal effect to the LNCAP- tumour many cells ball of Human Prostate Cancer Cells stem cell enrichment, fragmentation effect by
The 7.90% of background is promoted to 31.21%, and wherein lethal effect is indicated with killing rate (%).
Embodiment 3
The antigen epitope polypeptide CD44-P1 of the CD44 Protein Epitopes provided using the embodiment of the present invention 1 studies CD44
Lethal effect of the effector T cell of induction to Human Prostate Cancer Cells VCaP- many cells ball.
1. the method for separating and preparing of peripheral blood mononuclear cells is the same as embodiment 2.
The cultural method of 2.DC-CIK cell is the same as embodiment 2.
3. the preparation of target cell VCaP- many cells glomus cell and fragmentation effect detection method are the same as embodiment 2.
As shown in Figure 6 A and 6 B, by flow cytometer comparative analysis, pass through CD44 antigen epitope polypeptide as the result is shown
CIK cell ratio after activation significantly increases；At the 0th day (Fig. 6 A), flow cytometry analysis is the result shows that CD3 positive T is thin
It is 35.50%, CD8 positive T cell ratio is 25.3% that born of the same parents' ratio, which is 65.20%, CD4 positive T cell ratio, CIK cell
(CD3/CD56 is bis- positive) ratio is 1.90%；At the 14th day (Fig. 6 B), flow cytometry analysis is the result shows that CD3 positive T
It is 22.60%, CD8 positive cell ratio is 57.50% that cell proportion, which is 95.30%, CD4 positive cell ratio, CIK cell
(CD3/CD56 is bis- positive) ratio is 26.60%.CD44 antigen epitope polypeptide CD44-P1, CD44-P1 and CD44-P3 can be special
The effect of anisotropic inducing cell killing, kills cell proportion by 1.90% and is promoted to 26.60%.
As shown in figures 7 a and 7b, the CIK cell after CD44 epitope inducing peptide is to Human Prostate Cancer Cells stem cell
The LNCAP- tumour many cells ball of enrichment has significant lethal effect, and fragmentation effect is promoted to 34.50% by the 6.60% of background.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Shenzhen, the People's Hospital, Longhua District
<120>antigen epitope polypeptide CD44-P1 and its application based on prostate cancer stem cells marker CD44
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213>artificial sequence
<400> 1
Ala Gly Val Phe His Val Glu Lys Asn Gly Arg Tyr
1 5 10
<210> 2
<211> 36
<212> DNA
<213>artificial sequence
<400> 2
gcaggtgtat tccacgtgga gaaaaatggt cgctac 36
<210> 3
<211> 742
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 3
Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro
1 5 10 15
Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly
20 25 30
Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu
35 40 45
Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala
50 55 60
Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly
65 70 75 80
Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser Ile
85 90 95
Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser
100 105 110
Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp
115 120 125
Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr
130 135 140
Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu
145 150 155 160
Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175
Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly
180 185 190
Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp
195 200 205
Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Thr Leu
210 215 220
Met Ser Thr Ser Ala Thr Ala Thr Glu Thr Ala Thr Lys Arg Gln Glu
225 230 235 240
Thr Trp Asp Trp Phe Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn
245 250 255
His Leu His Thr Thr Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile
260 265 270
Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg
275 280 285
His Leu Ser Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile
290 295 300
Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala Phe Asp His Thr Lys Gln
305 310 315 320
Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His Ser Asn Pro Glu Val
325 330 335
Leu Leu Gln Thr Thr Thr Arg Met Thr Asp Val Asp Arg Asn Gly Thr
340 345 350
Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala His Pro Pro Leu Ile
355 360 365
His His Glu His His Glu Glu Glu Glu Thr Pro His Ser Thr Ser Thr
370 375 380
Ile Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys
385 390 395 400
Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro
405 410 415
Lys Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser Ala His
420 425 430
Thr Ser His Pro Met Gln Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser
435 440 445
Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His Pro Met Gly Arg Gly
450 455 460
His Gln Ala Gly Arg Arg Met Asp Met Asp Ser Ser His Ser Ile Thr
465 470 475 480
Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly Leu Val Glu Asp Leu Asp
485 490 495
Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln Ser Asn Ser Gln Ser
500 505 510
Phe Ser Thr Ser His Glu Gly Leu Glu Glu Asp Lys Asp His Pro Thr
515 520 525
Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn Asp Val Thr Gly Gly Arg
530 535 540
Arg Asp Pro Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr
545 550 555 560
Thr Ser His Tyr Pro His Thr Lys Glu Ser Arg Thr Phe Ile Pro Val
565 570 575
Thr Ser Ala Lys Thr Gly Ser Phe Gly Val Thr Ala Val Thr Val Gly
580 585 590
Asp Ser Asn Ser Asn Val Asn Arg Ser Leu Ser Gly Asp Gln Asp Thr
595 600 605
Phe His Pro Ser Gly Gly Ser His Thr Thr His Gly Ser Glu Ser Asp
610 615 620
Gly His Ser His Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr Ser Gly
625 630 635 640
Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser
645 650 655
Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys Ile Ala Val Asn Ser
660 665 670
Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn Ser Gly Asn
675 680 685
Gly Ala Val Glu Asp Arg Lys Pro Ser Gly Leu Asn Gly Glu Ala Ser
690 695 700
Lys Ser Gln Glu Met Val His Leu Val Asn Lys Glu Ser Ser Glu Thr
705 710 715 720
Pro Asp Gln Phe Met Thr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val
725 730 735
Asp Met Lys Ile Gly Val
740
Claims (10)
1. isolated antigen epitope polypeptide CD44-P1, which is characterized in that the antigen epitope polypeptide CD44-P1 is selected from following (a)
Or (b):
(a) polypeptide that the amino acid sequence shown in SEQ ID NO:1 forms；
(b) amino acid sequence shown in SEQ ID NO:1 is passed through to the substitution and/or missing of one or several amino acid residues
And/or addition, and the polypeptide as derived from (a) with prostate cancer stem cells marker CD44 antigen epitope function.
2. antigen epitope polypeptide CD44-P1 according to claim 1, which is characterized in that the antigen epitope polypeptide CD44-
The molecular weight of P1 is 1000-1300.
3. antigen epitope polypeptide CD44-P1 according to claim 1, which is characterized in that the antigen epitope polypeptide CD44-
P1 is obtained by prokaryotic cell or eukaryotic cell expression purifying；Or,
The antigen epitope polypeptide CD44-P1 passes through artificial synthesized.
4. a kind of nucleic acid, which is characterized in that the described in any item antigen epitope polypeptides of the nucleic acid encode claim 1-3
CD44-P1。
5. nucleic acid according to claim 4, which is characterized in that the nucleic acid is selected from any one of following (A)-(C):
(A) nucleotide sequence as shown in SEQ ID NO:2；
(B) polynucleotides of polypeptide shown in SEQ ID NO:1 are encoded；
(C) with SEQ ID NO:2 shown in nucleotide sequence homology 80% or more and coding prostate cancer stem cells mark
The nucleotide of the polypeptide of object CD44 antigen epitope function.
6. a kind of carrier, which is characterized in that the carrier includes nucleic acid described in claim 4 or 5.
7. a kind of host cell, which is characterized in that the host cell includes that nucleic acid described in claim 4 or 5 or right are wanted
Carrier described in asking 6.
8. antigen epitope polypeptide CD44-P1 as described in any one of claims 1-3 is in preparation prevention and/or treatment prostate cancer
Product in application.
9. application according to claim 8, which is characterized in that the product is vaccine or drug.
10. application according to claim 8, which is characterized in that the antigen epitope polypeptide CD44-P1 passes through killing
The prevention of LNCAP- many cells ball and/or treatment prostate cancer；And/or
The antigen epitope polypeptide CD44-P1 passes through the prevention of killing VCaP- many cells ball and/or treatment prostate cancer.
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| CN201811521663.2A CN109593125A (en) | 2018-12-12 | 2018-12-12 | Antigen epitope polypeptide CD44-P1 and its application based on prostate cancer stem cells marker CD44 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811521663.2A CN109593125A (en) | 2018-12-12 | 2018-12-12 | Antigen epitope polypeptide CD44-P1 and its application based on prostate cancer stem cells marker CD44 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113699106A (en) * | 2021-08-24 | 2021-11-26 | 上海纳米技术及应用国家工程研究中心有限公司 | Separation and amplification method of killer cells induced by cytokines |
| CN114057874A (en) * | 2020-07-31 | 2022-02-18 | 北京市神经外科研究所 | anti-CD 44 single-chain antibody and application thereof in preparing medicine for treating tumor |
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| CN101687930A (en) * | 2007-05-30 | 2010-03-31 | 霍夫曼-拉罗奇有限公司 | The anti-CD44 antibody of chimeric and humanization of mediation cancer cell cytotoxicity |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057874A (en) * | 2020-07-31 | 2022-02-18 | 北京市神经外科研究所 | anti-CD 44 single-chain antibody and application thereof in preparing medicine for treating tumor |
| CN114057874B (en) * | 2020-07-31 | 2023-05-05 | 北京市神经外科研究所 | anti-CD 44 single-chain antibody and application thereof in preparation of medicines for treating tumors |
| CN113699106A (en) * | 2021-08-24 | 2021-11-26 | 上海纳米技术及应用国家工程研究中心有限公司 | Separation and amplification method of killer cells induced by cytokines |
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