CN106117339A - The CTL of a kind of specific tumor antigen NSE identifies epitope peptide and application thereof - Google Patents
The CTL of a kind of specific tumor antigen NSE identifies epitope peptide and application thereof Download PDFInfo
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Abstract
The invention discloses the antitumor CTL epitope peptide in a kind of NSE source, for nonapeptide, its aminoacid sequence is Asn Ser Glu Ala Leu Glu Leu Val Lys.The invention also discloses above-mentioned epitope peptide and there is in preparation the application of the tumor therapeutic polypeptide vaccine that NSE expresses.The invention also discloses and prepared corresponding DC cell and the method for DC CIK cell by above-mentioned epitope peptide.Specific DC CIK cell prepared by the present invention demonstrates certain killing rate to lung carcinoma cell NCI H446, can be used for preparing the preparation of pulmonary carcinoma specific autoimmune cell.
Description
Technical field
The present invention relates to epitope peptide technical field, the CTL particularly relating to a kind of specific tumor antigen NSE identifies epitope peptide
And application, further relate to the preparation method and applications of a kind of tumor antigen NSE specificity DC cell, and a kind of tumor antigen
The preparation method and applications of NSE specificity DC-CIK cell.
Background technology
NSE is a kind of isozyme of Enolase.NSE is the tumor markers of small cell lung cancer (SCLC), the diagnosis positive
Rate is 91%.Contribute to small cell lung cancer and the Differential Diagnosis of nonsmall-cell lung cancer (NSCLC).To small cell lung cancer (SCLC)
Observation of curative effect and recurrence monitoring also have important value.Neuroblastoma, the serum NSE concentration of APUDoma can be bright
Aobvious rising.Due to NSE unconventionality expression in tumor tissues so that it is become a kind of potential tumor marker thing.At present
Have been used for diagnosis and the biologic treatment of tumor.
In recent years, research shows that in cancerous lung tissue, the expression of NSE can show the biological characteristics of small cell lung cancer
And occur with it, shift, prognosis etc. closely related, so NSE becomes an important target spot of therapy for small cell lung cancer.
The most clinically in ideas of cancer therapy, the treatment of the local such as operation and radiotherapy has preferable curative effect to local tumor,
And the treatment to general, transitivity and minimal disease relies primarily on chemotherapy and immunization therapy.Current research shows, tumor exists
The early stage occurred just has Partial tumors cell to transfer to its hetero-organization or organ, temporarily hides, and this is probably a lot of tumor
After the treatment, recurrence and the main cause of transfer occur.Therefore the treatment of tumor needs to treat as systemic disease, no
The tumor of local lesion to be removed, the Preventive of tumor to be prevented and the invasion and attack to internal organs, be only possible to the most effectively
Improve cure rate and the life cycle of tumour patient.Although but traditional chemotherapeutics obtains one in terms of the treatment of some tumor
Fixed progress, but from the point of view of the 5 of tumor patient years survival rates, it is not obviously improved, and obtaining easily occurs in chemotherapeutics simultaneously
Obtain the many drug resistances of property and toxic and side effects, limit its Clinical practice, it is therefore desirable to the preferably means of whole body therapeutic.
Immune cell therapy is that recent two decades grows up, and including CIK and DC-CIK etc., DC-CIK shows preferably
Targeting and specificity, to the treatment of tumor more effectively, have no side effect, to improving quality of life, extending life cycle and have aobvious
Write ground effect, be one of the best means of current tumor whole body therapeutic, there is huge clinical potentials.
But, still having much room for improvement at the antigen inducing special DC-CIK cell at present, shortage can efficiently stimulate tumor special
The CTL epitope polypeptide antigen of opposite sex DC-CIK.
Summary of the invention
The technical problem existed based on background technology, present invention aim at providing a kind of specific tumor antigen NSE's
CTL identifies epitope peptide.
The object of the invention also resides in the application providing the CTL of above-mentioned specific tumor antigen NSE to identify epitope peptide.
The object of the invention also resides in the preparation method providing a kind of tumor antigen NSE specificity DC cell.
The object of the invention also resides in the preparation method providing a kind of tumor antigen NSE specificity DC-CIK cell.
The object of the invention also resides in the above-mentioned tumor antigen NSE specificity DC cell of offer and the application of DC-CIK cell.
For achieving the above object, the present invention adopts the following technical scheme that:
The CTL of a kind of specific tumor antigen NSE that the present invention proposes identifies epitope peptide, and described CTL identifies that epitope peptide is
Nonapeptide, its aminoacid sequence is as follows: Asn-Ser-Glu-Ala-Leu-Glu-Leu-Val-Lys.
The CTL of above-mentioned specific tumor antigen NSE identifies that epitope peptide uses solid-phase synthesis to synthesize.Basic procedure is such as
Under: first an amino is connected on insoluble solid phase carrier Wang resin by the aminoacid of Fmoc radical protection, then takes off
Falling the protection group of amino, first aminoacid is i.e. connected on solid phase carrier；Secondly by second amino by Fmoc radical protection
Amino acid whose carboxyl condensing agent activation, second aminoacid carboxyl after activation again with first ammonia being connected on solid phase carrier
The amino reaction of base acid forms peptide bond, has now been generated as a dipeptides with protection group on solid phase carrier.Repeat above-mentioned
Peptide bond formed reaction, make peptide chain from C end to N end grow, until it reaches required peptide chain length, finally cutting obtain purpose
Peptide, then through HPLC purification, its purity is more than 90%.
The CTL of the above-mentioned specific tumor antigen NSE that the present invention also proposes identifies that epitope peptide has what NSE expressed in preparation
The application of tumor therapeutic polypeptide vaccine.
Preferably, the CTL of above-mentioned specific tumor antigen NSE identifies that epitope peptide is in preparing lung cancer therapy polypeptide vaccine
Application, especially lung carcinoma cell NCI-H446 is had lethality.
The preparation method of a kind of tumor antigen NSE specificity DC cell that the present invention also proposes, uses above-mentioned specificity to swell
The CTL of tumor antigen NSE identifies that epitope peptide addition DC cell carries out cultivation and obtains.
The specificity DC cell that the preparation method of the above-mentioned tumor antigen NSE specificity DC cell that the present invention also proposes obtains
Application in preparation treatment lung-cancer medicament.
The preparation method of a kind of tumor antigen NSE specificity DC-CIK cell that the present invention also proposes, uses above-mentioned special
Property tumor antigen NSE CTL identify that epitope peptide carries out induction and obtains.
The specificity that the preparation method of the above-mentioned tumor antigen NSE specificity DC-CIK cell that the present invention also proposes obtains
The application in preparation treatment lung-cancer medicament of the DC-CIK cell.
The present invention is ingenious utilizes NSE as the stronger tumor associated antigen of a species specificity, presents high expressed in pulmonary carcinoma,
And not express in the normal tissue, thus the theoretical method screening combined with experiment is used to obtain the table of tumor associated antigen
Position peptide, gained epitope peptide has no that document report, for the tumor vaccine of based on antigen NSE of development or tumor-specific CTL cell
Preparation is provided fundamental basis, and the antigenic peptides vaccine construction for follow-up multivalence lays the foundation.
Accompanying drawing explanation
Fig. 1 is the mass spectral analysis figure of the CTL identification epitope peptide of a kind of specific tumor antigen NSE that the present invention proposes.
Fig. 2 is the embodiment of the present invention 1 gained P1, P2, P3 ... ... the Specific CTL Cells secretion that each self-induction of P20 obtains
The ability comparison diagram of INF-γ.
Fig. 3 is that the Specific CTL Cells that obtains of the embodiment of the present invention 1 each self-induction of gained P5, P7, P11, P19 is to pulmonary carcinoma
Cell NCI-H446 kills ability comparison diagram.
Fig. 4 is that the CTL of a kind of specific tumor antigen NSE proposed by the present invention identifies the preparation-obtained spy of epitope peptide
The statistical analysis figure of the flow cytomery data of opposite sex CTL cellular immunization cell mass.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1: synthesis epitope peptide
Use the method integrated theory with practice, according to the primary structure of antigen, integrated use Immunoinformatics means,
SYFPEITHI, BIMAS, NetCTL, WAPP and EpiJen antigens c TL epi-position comprehensively to NSE is used to be predicted analyzing, choosing
The peptide sequence taking scoring front 20 carries out experiment sieving, the most named P1, P2, P3 ... P20.
Basic procedure is as follows: first by the aminoacid of Fmoc radical protection, one amino is connected to insoluble solid phase carrier
On Wang resin, then taking off the protection group of amino, first aminoacid is i.e. connected on solid phase carrier；Secondly by second ammonia
Base is activated by the amino acid whose carboxyl condensing agent of Fmoc radical protection, and second aminoacid carboxyl after activation is consolidated with being connected on again
First amino acid whose amino reaction of phase carrier forms peptide bond, has now been generated as one on solid phase carrier with protection group
Dipeptides.Repeat above-mentioned peptide bond and form reaction, make peptide chain grow to N end from C end, until it reaches required peptide chain length,
Finally cutting obtains purpose epitope peptide crude product.Through HPLC purification, purpose epitope peptide crude product is obtained purpose epitope peptide fine peptide, and it is pure
Degree is more than 90%, and mass spectral analysis confirms its molecular weight coincidence theory value.
The CTL of a kind of specific tumor antigen NSE that the present invention proposes identifies that epitope peptide uses Fmoc solid-phase synthesis to enter
Row synthesis, described CTL identifies that epitope peptide is nonapeptide, and numbered P11, its aminoacid sequence is as follows: Asn-Ser-Glu-Ala-
Leu-Glu-Leu-Val-Lys.P11 is carried out mass spectral analysis, and its mass spectrometry results is as shown in Figure 1, it can be verified that its molecule
Amount is 1002.1197g/mol, coincidence theory value.
Embodiment 2: polypeptide Function detection
Above-mentioned gained P11 and remaining 19 epitope peptide, can be used for preparing the tumor therapeutic polypeptide epidemic disease with NSE expression
Seedling.
1, above-mentioned CTL identifies that epitope peptide inducing specific CTL cell, IFN-γ secretion and the killing to tumor target cell are real
Test detection: the peripheral blood of extraction patient separates through density gradient centrifugation, it is thus achieved that PBMCs, add cytokine cultivate DC cell and
CTL cell, uses the NSE epitope peptide of the DC cell loading present invention further, co-cultures with CTL and stimulates special CTL amplification, enters
One step uses ELISA and LDH experiment to detect the secretion of special CTL INF-γ under specific antigen stimulates and to pulmonary carcinoma in vitro
The lethal effect of cell NCI-H446.
Concrete grammar is as follows:
(I), the separation of PBMC and induction:
1) peripheral blood 50mL anticoagulant processed, 2000rpm is centrifuged 10min；
2) collect upper plasma frozen, dilute remaining hemocyte with PBS (pH=7.4)；
3) hemocyte of dilution is joined on isopyknic lymph separation liquid liquid level；
4) 20 DEG C of centrifugal 20min, close centrifuge brake；
5) after centrifugal, it is divided into four layers, draws tunica albuginea layer (i.e. the second layer) with suction nozzle glass dropper；
6) the tunica albuginea layer PBS taken out washs twice；
7) by cell with 2～5 × 106/ mL is inoculated in 6 orifice plates, after 2h, reclaims the most adherent cell, with pre-coated
The culture plate of anti-CD3IgG and anti-CD28IgG carries out activating to be cultivated；
8) adherent cell adds GM-CSF with IL-4 stimulates cultivation to induce into DC cell in 5 days, within the 3rd day half, changes liquid；
9) the 5th day, collect DC cell, 10 μ g embodiment 1 gained purpose epitope peptide fine peptides are added in DC cell, after 1h,
The T cell of DC cell with activation is co-cultured, is simultaneously introduced IL2 and IL-15；
10), after continuing to cultivate 5 days, the CTL cell obtaining antigen-specific carries out cytokine secretion and to tumor cell
Killing experiments.
(II), IFN-γ cytokine secretion detection: Human IFN-gamma Platinum ELISA (IFN-γ
ELISA detection kit, eBioscience company) IFN-γ of detection CTL emiocytosis, step is as follows:
1), after CTL cell being removed cytokine cultivation 24h, it is inoculated in 96 orifice plates；
2) after in cell, addition stimulates corresponding polypeptide again to stimulate 24h, centrifugal segregation cell, collect cell conditioned medium；
3) using the expression of IFN-γ in ELISA detection supernatant, the CTL epitope peptide selecting IFN-γ secretion more kills
Wound experiment.
Result as in figure 2 it is shown, the DC cell of P11 and P5, P7, P19 load all can preferably to induce specific CTL thin
Born of the same parents, the IFN-γ of secretion higher amount.
(III), tumor cytotoxicity experiment: use lactic acid dehydrogenase (LDH) release detection method, use CytoTox
96Non-Radioactive Cytotoxicity Assay (citotoxicity detection kit, Promega company) carries out LDH examination
Test detection cellkilling capacity.Step is as follows:
1) detection culture plate (100 μ L/ hole) is set up
A. experimental group is set up: express positive lung carcinoma cell NCI-H446 as target cell with NSE, thin by effector lymphocyte and target
Born of the same parents add above-mentioned characteristic CTL cell than for 5:1,10:1,20:1
B. effector lymphocyte's spontaneous release group is set up
C. target cell spontaneous release group is set up
D. target cell maximum release group is set up
E. ground control group is set up
2) cell cracking and results supernatant
A.37 DEG C 5%CO2Co-culture 5h
B. adding lysate, after 45min, all of hole in target cell maximum release group, 250g/min is centrifugal collects supernatant
3) LDH detection
A. transferase 45 0 μ L of supernatant is to another 96 orifice plate
B. every hole adds the substrate mixture of 50 μ L dilutions, and room temperature lucifuge hatches 30min
C. 50 μ L stop buffers are added in every hole
D. under 490nm, detect light absorption value OD
Cell killing rate computing formula is as follows:
Killing rate (%)=[(ODExperimental group-ODEffector lymphocyte's spontaneous release group-ODTarget cell spontaneous release group)/(ODTarget cell maximum release group-ODTarget cell spontaneous release group)]
× 100%
Result is as it is shown on figure 3, Fig. 3 is the specificity that the embodiment of the present invention 1 each self-induction of gained P11, P5, P7, P19 obtains
CTL Cells on Lung Cancer cell NCI-H446 kills ability comparison diagram；From the figure 3, it may be seen that the CTL Cells on Lung Cancer cell of P11 induction
NCI-H446 fragmentation effect is best.
2, using the percentage ratio shared by various immunocytes in the special CTL of flow cytometer showed acquisition, result is as shown in Figure 4.
As shown in Figure 4, containing substantial amounts of CTL cell in the immunocyte group that the present invention is prepared by P11, also contain a definite proportion simultaneously
The NKT cell of example, i.e. this immunocyte group have preferable immunocompetence, have the energy of powerful killing specific tumors cell
Power.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and
Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.
Claims (7)
1. the CTL of a specific tumor antigen NSE identifies epitope peptide, it is characterised in that described CTL identifies that epitope peptide is nine
Peptide, its aminoacid sequence is Asn-Ser-Glu-Ala-Leu-Glu-Leu-Val-Lys.
2. the CTL of specific tumor antigen NSE described in claim 1 identifies that the tumor that epitope peptide has NSE expression in preparation is controlled
The application of the property treated polypeptide vaccine.
3. the CTL of specific tumor antigen NSE described in claim 1 identifies that epitope peptide is in preparing lung cancer therapy polypeptide vaccine
Application.
4. the preparation method of a tumor antigen NSE specificity DC cell, it is characterised in that use the most special
The CTL of opposite sex tumor antigen NSE identifies that epitope peptide addition DC cell carries out cultivation and obtains.
The specificity DC cell that the most according to claim 4, the preparation method of tumor antigen NSE specificity DC cell obtains exists
Application in preparation treatment lung-cancer medicament.
6. the preparation method of a tumor antigen NSE specificity DC-CIK cell, it is characterised in that use such as claim 1 institute
The CTL stating specific tumor antigen NSE identifies that epitope peptide carries out induction and obtains.
The specificity DC-that the most according to claim 6, the preparation method of tumor antigen NSE specificity DC-CIK cell obtains
CIK cell application in preparation treatment lung-cancer medicament.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111116728A (en) * | 2020-01-13 | 2020-05-08 | 郑州大学 | Tumor specific antigen CTL epitope peptide and application thereof |
| CN115785211A (en) * | 2022-06-10 | 2023-03-14 | 河北博海生物工程开发有限公司 | Lung cancer specific molecular target 04 and application thereof |
| CN115785208A (en) * | 2022-06-10 | 2023-03-14 | 河北博海生物工程开发有限公司 | Lung cancer specific molecular target 01 and application thereof |
Citations (1)
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| CN103784950A (en) * | 2014-01-22 | 2014-05-14 | 北京弘润源生物技术有限公司 | Preparation method of breast cancer-specific epitope polypeptide-loaded dendritic cell vaccine and kit thereof |
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2016
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| CN103784950A (en) * | 2014-01-22 | 2014-05-14 | 北京弘润源生物技术有限公司 | Preparation method of breast cancer-specific epitope polypeptide-loaded dendritic cell vaccine and kit thereof |
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| GREGORY B.: "Mapping of Antigenic Sites in Human Neuron-Specific Enolase by Expression Subcloning.", 《CLIN.CHEM.》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111116728A (en) * | 2020-01-13 | 2020-05-08 | 郑州大学 | Tumor specific antigen CTL epitope peptide and application thereof |
| CN111116728B (en) * | 2020-01-13 | 2021-04-27 | 郑州大学 | Tumor specific antigen CTL epitope peptide and application thereof |
| CN115785211A (en) * | 2022-06-10 | 2023-03-14 | 河北博海生物工程开发有限公司 | Lung cancer specific molecular target 04 and application thereof |
| CN115785208A (en) * | 2022-06-10 | 2023-03-14 | 河北博海生物工程开发有限公司 | Lung cancer specific molecular target 01 and application thereof |
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