CN111087448A - Tumor-associated gene JAK2 mutation-associated antigen short peptide and application thereof - Google Patents
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Abstract
The invention discloses JAK2 mutation-associated antigen short peptide and application thereof. The sequence of the JAK2 mutation-associated antigen short peptide is shown in any one of SEQ ID NO 1-19. The short peptide has high affinity with MHC I molecules on DC cells, can effectively stimulate and induce to generate specific Cytotoxic T Lymphocytes (CTLs), can be used for immune elimination of JAK2 gene mutant cells, and further prevents diseases related to JAK2 gene mutation, particularly prevents tumor diseases related to JAK2 gene mutation; therefore, the JAK2 antigen short peptide has good potential of polypeptide vaccine and DC vaccine, and has good clinical transformation and disease prevention potential. Moreover, the JAK2 antigen short peptide has short length and small chemical synthesis difficulty, can be directly synthesized to obtain a high-purity product, greatly reduces the application cost, has clear effect and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to tumor-associated gene JAK2 mutation-associated antigen short peptide and application thereof.
Background
The Janus kinases (JAKs) protein family is a non-receptor tyrosine kinase, 4 members of JAK1, JAK2, JAK3 and TYK2 are found so far respectively, wherein a JAK2 gene is located in a 4-zone of a short arm 2 region of chromosome 9, JAK2 consists of 7 JAK homology domains (JH), JH1-JH7 is located at a carboxyl end from a carboxyl end to an amino end, JH2 is a main catalytic domain with tyrosine kinase activity, JH2 is a pseudo-kinase domain and plays a role in regulating the kinase activity of JH1, 2 mediates the intracellular signal transduction of I-type cytokines through a JAK-signal pathway, including erythropoietin (erythropoetin), thrombopoietin (thrombopoietin, TPO), granulocyte colony stimulating factor (granulocyte-macrophage stimulating factor), IFN-macrophage stimulating factor (EPO), platelet stimulating factor (TNF-macrophage stimulating factor), IFN-macrophage growth factor (TNF-macrophage growth factor), blood growth factor, blood protein.
Tumor antigen peptide specific CTL is induced and established by using tumor associated antigen short peptide, thereby obtaining CTL clone with specific killing tumor cells, and the activated CTL can kill corresponding target cells and play a role in immune monitoring. The key premise of the technology is to obtain related antigen short peptides which have good immunogenicity and specificity and can induce and generate specific cytotoxic T lymphocytes.
Disclosure of Invention
The invention aims to develop JAK2 gene mutation-related antigen short peptide aiming at tumor-related gene JAK2, the JAK2 gene mutation-related antigen short peptide has high affinity with MHC I molecules on DC cells, can effectively stimulate and induce generation of specific Cytotoxic T Lymphocytes (CTLs), can be used for immune elimination of JAK2 gene mutant cells, and has good potential of polypeptide vaccine and DC vaccine.
The invention aims to provide a short peptide of tumor-associated gene JAK2 mutation-associated antigen.
The invention also aims to provide application of the short peptide in preparing a DC vaccine for JAK2 mutation.
It is a further object of the present invention to provide a DC vaccine for JAK2 mutation.
The above purpose of the invention is realized by the following technical scheme:
the inventor discovers that mutant polypeptides of COSM12600, COS M29300 and COSM29302 can be combined with MHC-I molecules (the mutant sites of JAK2 are detected in cancers in blood systems) through bioinformatics prediction by online analysis of a T cell epitope prediction comprehensive platform NetCTL database (http:// www.cbs.dtu.dk/services/NetCTL). The site is an important target for immune elimination of JAK2 gene mutant cells. The immunological research proves that the principle that CD8 positive T lymphocyte CTL plays cellular immunity is as follows: CTL cells are activated by recognizing antigen peptides bound to MHC-I molecules, and the activated CTL can kill corresponding target cells to exert an immune surveillance effect. Therefore, the binding capacity of the JAK2 mutant sequence with a T lymphocyte receptor (TCR) and an MHC I molecule is predicted through a bioinformatics technology, the expression of the JAK2 mutant sequence is analyzed and positioned outside a cell membrane, the JAK2 mutant related antigen short peptide aiming at a tumor related gene is designed and optimized, the polypeptide sequence of the JAK2 antigen short peptide obtained through screening is preferably shown in any one of SEQ ID NO:1-19, the obtained JAK2 antigen short peptide has high affinity with the MHC I molecule on a DC cell, can effectively stimulate and induce generation of specific Cytotoxic T Lymphocytes (CTLs), inhibits growth of the tumor cell, and has good potential of a polypeptide vaccine and a DC vaccine, and has good clinical transformation and disease prevention prospects.
Therefore, the following products and applications should be considered within the scope of the present invention:
the sequence of the tumor-associated gene JAK2 mutation-associated antigen short peptide is shown in any one of SEQ ID NO 1-19. JAK2 mutant short peptide products can be prepared by taking the short peptide as an active antigen component and an auxiliary agent.
The application of the short peptide in preparing products capable of inducing the generation of specific cytotoxic T lymphocytes. Specifically, the induction method of the specific cytotoxic T lymphocyte comprises the following steps: at least one of JAK2 mutant short peptides of SEQ ID NO. 1-19 is co-cultured with CD8+ T cells through antigen presenting cells, so that JAK2 mutant specific cytotoxic T cells can be obtained through induction.
The short peptide is applied to preparing a human body immunological activity regulator, and particularly is used as an active antigen component for regulating the human body immunological activity. The short peptide antigen is processed in cells to form specific fragments, namely, the antigen peptide is completely matched with MHC 1 molecules and then presented on the cell surface to be recognized by a corresponding T Cell Receptor (TCR) to form an antigen peptide-MHC-TCR complex, so that Cytotoxic T Lymphocytes (CTL) are activated.
The short peptide is applied to the preparation of products for preventing JAK2 gene mutation and related diseases, in particular to JAK2 gene mutation related tumors. The product comprises vaccines, such as DC vaccines or monocyte vaccines and the like.
Antigenic peptide-MHC-TCR complex, activating cytotoxic T lymphocytes, the activated specific T lymphocytes being able to kill abnormal cells carrying the JAK2 mutation. Induction method of specific cytotoxic T lymphocytes: at least one of JAK2 mutant short peptides shown in SEQ ID NO. 1-19 is used to react with CD8 by an antigen presenting cell+TCells were co-cultured and JAK2 mutation-specific cytotoxic T cells were induced.
Based on the above, the invention also provides a DC vaccine or monocyte vaccine for preventing JAK2 gene mutation and related diseases, which is prepared by loading the short peptide shown in any one of SEQ ID NO. 1-19 and dendritic cells or monocytes.
The JAK2 short peptide shown in any one of SEQ ID NO 1-19 and Dendritic Cells (DC) are loaded and infused back, and can be used as a DC vaccine for disease prevention and for stimulating an organism to generate polypeptide specific anti-cytotoxic T cells, so that the prevention of diseases related to JAK2 gene mutation is realized, and particularly the prevention of tumors related to JAK2 gene mutation is realized.
In particular, the vaccine is an intravenous infusion vaccine.
In the preparation method of the DC vaccine for preventing JAK2 gene mutation and related diseases, a vitrogen factor is selected as an adjuvant to increase the activity of DC cells, and specific antigen peptide is used for sensitizing dendritic cells in vitro to obtain an autologous dendritic cell preparation which is used as a vaccine for preventing and treating chronic diseases related to chlamydia pneumoniae of venous return transfusion type. The preparation method comprises the following steps: the maturation promoting factor and the Vitrogen factor are used as adjuvants to promote the maturation of DC cells; and then adding the short peptide of the invention into a DC cell culture system for inducing maturation, collecting DC cells loaded with the short peptide fragments, washing with physiological saline, and then resuspending with the physiological saline to obtain the DC vaccine.
More specifically, as an alternative, the DC vaccine is prepared by the following steps:
s1, extracting and inducing DC cells:
s11, obtaining immature DC cells
Collecting peripheral blood of healthy donor, separating mononuclear cells by lymphocyte separation, culturing at 37 deg.C in culture medium with 5% CO2After culturing for 3 hours under the conventional condition, the adherent cells are immature DC cells;
s12, amplification culture of immature DC cells
37℃、5％CO2Culturing for 5 days under the condition, and changing the culture solution every other day to complete the amplification culture of immature DC cells (imDC cells);
S13.Induction of DC cells
Adding a maturation promoting factor, and simultaneously taking a Vitrogen factor as an adjuvant to promote the maturation of the DC cells;
s2, loading of polypeptide:
after inducing DC cells to mature for 5 days, adding the short peptide into a culture system;
s3, preparing a DC vaccine:
and (3) centrifuging to collect the DC cells loaded with the short peptide fragments, washing the cells for 3 times by using physiological saline, and finally, resuspending the DC cells loaded with the short peptide fragments by using the physiological saline to obtain the DC vaccine.
The invention selects specific epitope polypeptide, sensitizes autologous DC cells in vitro, prepares DC cell preparation, can carry out venous return transfusion on patients, rebuilds the whole body immune balance of organisms, starts immune system and carries out specific treatment on gene mutation tumor cells. The DC technology is adopted, and a plurality of specific antigen peptides are used for jointly activating dendritic cells, have extremely strong specificity, induce dendritic cells with higher activity to carry a plurality of antigen information, can stimulate the immunity of an organism after being infused back into a human body, and can achieve the aim of inducing the human body to generate specific antibodies and specific CTL cells, thereby effectively preventing JAK2 gene mutation and the occurrence and development of related diseases.
The invention has the following beneficial effects:
the JAK2 antigen short peptide has high affinity with MHC I molecules on DC cells, and can effectively stimulate and induce the generation of specific Cytotoxic T Lymphocytes (CTLs); the generated specific cytotoxic T lymphocyte can be used for immune elimination of JAK2 gene mutation cells, so that diseases related to JAK2 gene mutation are prevented, and particularly, tumor diseases are prevented.
The JAK2 antigen short peptide can be used for preparing polypeptide vaccines and DC vaccines and has good clinical transformation and disease prevention prospects.
Moreover, the JAK2 antigen short peptide has short length, incomparable application advantages, small chemical synthesis difficulty, capability of directly synthesizing to obtain a high-purity product, greatly reduced application cost, definite effect and good application potential.
Drawings
FIG. 1 shows the result of the JAK2 mutation-associated antigen short peptide-specific CTL IFN-gamma release experiment.
Figure 2 is a comparison of the killing activity of JAK2 mutation-associated antigen short peptides on target cells.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1T cell epitope prediction of JAK2 Gene mutant peptide and JAK2 short peptide design
1. The peptide sequence with high affinity to T cell epitope receptor (TCR) and MHC class I molecules is analyzed and predicted by a T cell epitope prediction data comprehensive platform (http:// www.cbs.dtu.dk/services/NetCTL) and a bioinformatics technology. The research obtains a series of JAK2 mutation-associated antigen short peptides.
2. From the above polypeptides, 50 polypeptide fragments with high predictive score and less predictive toxicity were selected and further studied. The specific experiment is as follows:
the establishment method of JAK2 mutant short peptide specific CTL clone is as follows:
sorting healthy donors by flow 105An individual CD8+T cells, adding JAK2 mutant short peptide-loaded Mo-DCs104In vitro Peripheral Blood Mononuclear Cells (PBMC)10 loaded with JAK2 short peptide treated with mitomycin C and stimulated 2 times at 1 week intervals5Each was stimulated 1 time, thereby obtaining CTL cells.
The method for establishing JAK2 gene mutation short peptide specific CTL cells by adopting in vitro induction is adopted, cell culture supernatant is sucked, and the IFN-gamma content in the supernatant is detected.
The results showed that 20 polypeptides shown in Table 1 have specific immune response effect, can activate T lymphocytes, and induce secretion of IFN-gamma (shown in FIG. 1). Meanwhile, the results also show that the corresponding relation between the 20 polypeptides and the predicted prediction scores of the 50 polypeptides is not obviously related, and the reference of the prediction results is poor.
TABLE 1
| Serial number | Pos | Sequence of | Serial number |
| 1 | 610 | VLNGVCFCGDENIL | SEQ ID NO:1 |
| 2 | 610 | VLNGVCFCGDENI | SEQ ID NO:2 |
| 3 | 610 | VLNGVCFCGDEN | SEQ ID NO:3 |
| 4 | 611 | LNGVCFCGDENIL | SEQ ID NO:4 |
| 5 | 612 | NGVCFCGDENIL | SEQ ID NO:5 |
| 6 | 613 | GVCFCGDENIL | SEQ ID NO:6 |
| 7 | 676 | AKNILLIGEEDRKTG | SEQ ID NO:7 |
| 8 | 676 | AKNILLIGEEDRKT | SEQ ID NO:8 |
| 9 | 676 | AKNILLIGEEDRK | SEQ ID NO:9 |
| 10 | 676 | AKNILLIGEEDR | SEQ ID NO:10 |
| 11 | 676 | AKNILLISEEDRKTG | SEQ ID NO:11 |
| 12 | 676 | AKNILLISEEDRKT | SEQ ID NO:12 |
| 13 | 677 | KNILLIGEEDRKTG | SEQ ID NO:13 |
| 14 | 678 | NILLIGEEDRKTG | SEQ ID NO:14 |
| 15 | 678 | LLISEEDRKTG | SEQ ID NO:15 |
| 16 | 679 | ILLIGEEDRKTG | SEQ ID NO:16 |
| 17 | 679 | NILLISEEDRKTG | SEQ ID NO:17 |
| 18 | 680 | LLIGEEDRKTG | SEQ ID NO:18 |
| 19 | 680 | ILLISEEDRKTG | SEQ ID NO:19 |
Example 2 inhibition of tumor growth assay
Example 120 JAK2 mutant short peptides with specific immune response effect and capable of activating T lymphocytes are obtained. From these 5 short peptides were randomly selected and the experiment was continued for further validation.
The cell culture supernatant induced by 5 short peptides was used to culture human myelogenous leukemia HL-60 cells. The control group is divided into two groups of short peptide loading-free and nonsense short peptide loading groups.
The results show that the 5 short peptides induced the supernatant can significantly inhibit tumor growth and significantly reduce tumor activity compared with the control group (as shown in fig. 2).
Experimental results show that the CTL epitope established by the invention is extremely effective. JAK2 mutant short peptide shown in any one of SEQ ID NO 1-19 is subjected to dendritic cell presentation and is co-cultured with cytotoxic T lymphocyte, so that JAK2 mutant specific cytotoxic T lymphocyte can be obtained through induced screening. The JAK2 mutant antigen specific cytotoxic T lymphocyte can be used for immune elimination of JAK2 gene mutant cells, so that diseases related to JAK2 gene mutation are prevented, and particularly, tumor diseases are prevented. Therefore, the JAK2 short peptide shown in any one of SEQ ID NO 1-19 and Dendritic Cells (DC) are loaded and infused back, and the JAK2 short peptide can be used as a DC vaccine for disease prevention and can stimulate an organism to generate polypeptide specific anti-cytotoxic T cells, so that the JAK2 gene mutation related disease prevention, particularly tumor prevention, is realized.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Wittaen (Guangzhou) pharmaceutical Co., Ltd
<120> tumor-associated gene JAK2 mutation-associated antigen short peptide and application thereof
<130>
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<170>PatentIn version 3.3
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Claims (10)
1. The tumor-associated gene JAK2 mutation-associated antigen short peptide is characterized in that the sequence is shown in any one of SEQ ID NO 1-19.
2. Use of a short peptide according to claim 1 for the preparation of a product inducing the production of specific cytotoxic T lymphocytes.
3. Use of the short peptide of claim 1 for the preparation of a modulator of human immune activity.
4. The use according to claim 3, wherein the short peptide is an active antigenic component for modulating the immune activity of humans.
5. The use of the short peptide of claim 1 in the preparation of a product for preventing mutation of JAK2 gene.
6. The use of the short peptide of claim 1 in the preparation of a product for preventing diseases associated with JAK2 gene mutation.
7. The use of the short peptide of claim 1 for the preparation of a DC vaccine or monocyte vaccine for the prevention of mutation in the JAK2 gene.
8. The use of the short peptide of claim 1 for the preparation of a DC vaccine or monocyte vaccine for the prevention of diseases associated with mutations in the JAK2 gene.
9. The use according to claim 6 or 8, wherein the disease associated with mutation of JAK2 gene is tumor associated with mutation of JAK2 gene.
10. A vaccine for preventing JAK2 gene mutation, which is prepared by loading the short peptide of claim 1 on dendritic cells or monocytes.
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2019
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| WO2007044894A2 (en) * | 2005-10-11 | 2007-04-19 | Chembridge Research Laboratories, Inc. | Cell-free protein expression systems and methods of use thereof |
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| Title |
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