CN114134221B - Method for screening tumor specific TCR - Google Patents

Method for screening tumor specific TCR Download PDF

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CN114134221B
CN114134221B CN202210103753.XA CN202210103753A CN114134221B CN 114134221 B CN114134221 B CN 114134221B CN 202210103753 A CN202210103753 A CN 202210103753A CN 114134221 B CN114134221 B CN 114134221B
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张超亭
陆哲明
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Beijing Cancer Hospital
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Abstract

The invention relates to a method for screening tumor specific TCR, belonging to the technical field of tumor immunity. The invention is characterized in that after tumor cells of a tumor patient and tumor infiltrating T cells (TILs) corresponding to autologous tumor cells are incubated together in vitro, the cells are subjected to single cell transcriptome and TCR group sequencing to obtain a TCR sequence of each TILs cell, for all TILs cells expressing the same TCR, the average value of expression values of 10T cell markers of the TILs cells is taken as the activation score of the TCR marker, and the TCR with the higher score is screened as the TCR corresponding to the tumor specific T cell according to the activation score.

Description

Method for screening tumor specific TCR
Technical Field
The invention relates to the technical field of tumor immunity, in particular to a method for screening tumor specific TCR.
Background
T cells recognize the corresponding antigen through their cell surface T Cell Receptor (TCR), a receptor molecule on the surface of T cells that specifically recognizes the antigen peptide-MHC complex on antigen presenting cells, and thereby elicits a T cell immune response. Since most TCRs on the surface of T cells cannot recognize tumor cells, T cells cannot effectively kill tumor cells, resulting in rapid expansion of tumor cells. If a T cell for specifically recognizing the tumor cell is found, the corresponding TCR is cloned, and the TCR is introduced into the T cell through gene editing (such as lentivirus), a T cell receptor gene modified T cell (TCR-T) is generated, after the TCR specific to the tumor antigen is transferred into a common T cell, the T cell can be endowed with the capacity of recognizing the tumor antigen, the TCR-T has the capacity of specifically recognizing and killing the tumor cell, and the TCR-T can be activated and proliferated in vitro and then is input into a patient body, so that the anti-tumor efficacy can be exerted, and the effective anti-tumor activity can be shown in various cancers, therefore, the screening of the TCR specific to the tumor is the key for successful treatment of the TCR-T, and is a hotspot for research in tumor immunotherapy.
T cells are activated by tumor antigens and express T cell markers, so earlier studies stimulated T cells by tumor antigens, further screened T cells expressing T cell markers and completed cloning of corresponding TCRs, thereby obtaining tumor-specific TCRs, where the markers are mainly IFN- γ (corresponding to the gene name IFNG) or 41BB (corresponding to the gene name TNFRSF 9). However, different types of T cells (e.g., naive T cells, effector T cells, and memory T cells) and different time periods after antigen stimulation all affect the expression of T cell markers, i.e., the expression levels of different markers are very different, so that screening for tumor-specific T cells using a single marker may miss some tumor-specific T cells that do not express a certain marker, thereby causing false negatives. To make the screening results more accurate, it is necessary to use more comprehensive scores of T cell markers to screen for tumor-specific T cells and thus tumor-specific TCRs. However, in the process of selecting a plurality of T cell markers for screening tumor-specific T cells, the inventors found that the screening effect corresponding to the evaluation of expression values of all the markers known in the art to have the strongest correlation with the immune response of stimulated T cells is the best, and indeed, the inventors have found through continuous research that the average expression value of a specific T cell marker combination is used for screening tumor-specific TCR, and the effect is more accurate and the process is more efficient.
Disclosure of Invention
The invention aims to provide a method for efficiently and accurately screening tumor TCR.
During the research process, the inventors selected a plurality of commonly recognized T Cell markers, and performed single Cell transcriptome sequencing on activated TILs obtained by incubating TILs with corresponding tumor cells, so as to obtain the expression level of each T Cell marker (gene), i.e. the mRNA content corresponding to each gene, and further utilized the software program (Seurat AddModuleCore) (refer to and use description in the literature: Cell. 2021 Nov 11;184(23):5838. Cell 177, 1888. 1902 e1821 (2019) and https:// rrr. io/cran/Seurat/man/AddModuleCore. html), and the activation score of each TILs Cell was calculated according to the default parameters of the software, and the general calculation process is as follows: inputting the normalized expression value of each T cell marker into the software to obtain the activation score of the TILs cells, wherein the activation score is the average expression value obtained by subtracting the background value from the normalized expression value of the marker, so that each TILs cell can calculate the activation score of one TILs cell, and then averaging the activation scores of the TILs cells of all TILs cells with the same TCR to obtain the value of the TCR activation score.
Through continuous research and exploration, the invention determines that the screening of the tumor specific TCR by adopting the comprehensive score of the expression quantity of 10T cell markers has the highest sensitivity and specificity, namely the false positive rate and the false negative rate are the lowest, and the genes corresponding to the 10 markers are respectively as follows: IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF 1. The invention incubates tumor cells of tumor patients and corresponding TILs in vitro for 4-24 h, then carries out single cell transcriptome and TCR group sequencing on the cells, and screens the TCRs with the highest scores of the first few tumor specific T cells according to the comprehensive expression scores of the 10 markers.
Specifically, the present invention provides a method for screening tumor specific TCR, comprising the steps of:
(1) activating tumor infiltrating T cells (TILs) corresponding to autologous tumor cells of a tumor patient;
it will be understood by those skilled in the art that the methods for activating TILs are all methods commonly used in the art, such as co-incubating tumor cells of a tumor patient with corresponding TILs in vitro, or because tumor antigen-loaded presenting cells, such as dendritic cells or B cells, etc., can be used to replace tumor cells of a tumor patient and co-incubate TILs of a patient in vitro, the purpose of this is to activate TILs with tumor antigens, and then to screen TCR of tumor-specific T cells. In one embodiment of the present invention, the activation of TILs can be achieved by in vitro co-incubation of tumor cells of a tumor patient with corresponding TILs for 4-24 hours.
(2) Sequencing the cells by a single cell transcriptome and a TCR set to obtain a TCR sequence of each TILs cell and an expression value of each gene;
(3) for all TILs expressing the same TCR, taking the average value of the expression values of the 10 markers as the activation score of the TCR, and screening the TCR with higher score as the TCR corresponding to the tumor specific T cell according to the TCR activation score;
the 10 markers are respectively: IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF 1.
The method for calculating the TCR activation score of each TILs in the step (3) comprises the following steps: inputting the normalized expression values of 10 markers of each TILs into Seurat addModuleCore, calculating the activation score of the TILs according to the default parameters of the software, wherein the activation score is the average expression value of the normalized expression values of the 10T cell markers minus the background value, each TILs can calculate an activation score, and then averaging the activation scores of all TILs with the same TCR, namely the value of the activation score of the TCR.
References to the Seurat AddModuleCore software program calculation methods and the instructions for use are described above.
In the method provided by the present invention, the step (3) further comprises screening the first three TCRs with higher activation score values as the tumor-specific TCRs of the tumor patient. After a large number of experiments, the inventor finds that the first three TCRs with higher activation scores can be proved to be tumor-specific TCRs.
The invention provides application of the method for screening the tumor specific TCR in preparing TCR-T cells, tumor immunization and/or tumor treatment.
The invention also provides application of the TCR obtained by screening the tumor specific TCR method in preparation of TCR-T cells. It will be appreciated by those skilled in the art that the above tumor-specific TCR may be used in the preparation of TCR-T cells, in tumor immunization and/or in tumor therapy, and that such use is within the scope of the present invention.
In another aspect, the present invention provides a marker combination for screening tumor-specific TCR, consisting of the following 10 genes or corresponding proteins: IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF 1.
The invention provides applications of the marker combination in vitro screening of tumor specific TCR and/or in improving screening efficiency and accuracy of tumor specific TCR.
The invention also provides application of the marker combination in preparing TCR-T cells.
It will be appreciated by those skilled in the art that the use of the combination of reagents for detecting the expression level of each of the above-described marker combinations in screening for tumor-specific TCRs and in preparing TCR-T cells is within the scope of the present invention.
The invention utilizes a single cell sequencing technology and establishes a set of method for efficiently screening tumor specific TCR according to the comprehensive score of 10T cell markers, can efficiently screen various tumor specific TCR for each tumor patient, is superior to the method for screening tumor specific TCR according to a single marker (IFN-gamma or 41 BB), and is more superior to the method for screening tumor specific TCR by the combined score of other multiple markers.
Drawings
FIG. 1 is a graph comparing the concentration of secreted IFN-. gamma.after in vitro co-incubation of control T cells (Un-transfected) untransfected TCR and T cells transfected with each TCR (P1-IFN-TCR 1, P1-IFN-TCR2, P1-IFN-TCR3, P1-41BB-TCR2, P1-41BB-TCR 3) with the corresponding Autologous Tumor Cells (ATC) for 12 h.
FIG. 2 is a graph comparing the concentration of secreted IFN-. gamma.after in vitro co-incubation of control T cells (Un-transfected) untransfected TCR and T cells transfected with each TCR (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3) with the corresponding Autologous Tumor Cells (ATC) for 12 h.
FIG. 3 is a graph comparing the concentration of secreted IFN-. gamma.after in vitro co-incubation of control T cells (Un-transfected) untransfected with TCR and T cells transfected with P1-score2-TCR2 with corresponding Autologous Tumor Cells (ATC) for 12 h.
FIG. 4 is a graph comparing the concentration of secreted IFN-. gamma.after in vitro co-incubation of control T cells (Un-transfected) untransfected with TCR and T cells transfected with P1-score5-TCR3 with corresponding Autologous Tumor Cells (ATC) for 12 h.
FIG. 5 is the ability of control T cells untransfected with TCR (Un-tranded) and T cells transfected with each TCR (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3) to kill Autologous Tumor Cells (ATC) at different potency-to-target ratios.
FIG. 6 is a statistical analysis of tumor volume following reinfusion of transplanted tumor mice with control T cells untransfected with TCR (Un-tranded) and T cells transfected with each TCR (P1-score-TCR1, P1-score-TCR2, P1-score-TCR 3).
Represents P <0.05, represents P <0.001, and is not labeled to indicate that no statistical difference exists.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Lentiviral packaging plasmids (pMDL, VSV-G, REV), pCDH-EF1-Luc2-T2A-tdTomato plasmids were purchased from Wuhan vast Ling Biotech, Inc. XbaI and SalI endonucleases were purchased from New England Biolabs (Beijing) LTD. PEI was purchased from Sigma. X-VIVO15 medium was purchased from Lonza. IL-2, IL-7, IL-15 cytokines were purchased from Peprotech, Inc. OKT3 was purchased from acrobaiiosystems, inc. The CD28 antibody was purchased from Hoodia. 293ft cells were purchased from ATCC. Flow antibodies, such as CD3, CFSE, PI, were purchased from BD, and flow antibodies against the murine TCR β chain constant region were purchased from eBioscience. IFN-gamma detection kits were purchased from Eikesai Biotechnology, Inc. TILs and transplantable tumor models of lung cancer patients are all constructed in the experiment. NOD/SCID immunodeficient mice were purchased from Experimental animals technology, Inc. of Weitongliwa, Beijing.
Example 1 sequencing of TILs Single cell transcriptome and TCR set
Culturing of TILs
After tumor tissue of a tumor patient is surgically excised, the tumor tissue is minced into 1-2 mm pieces, each piece of tumor tissue is placed in one well of a 24-well cell culture plate, and then a T cell culture medium is added. T-cell culture media comprise X-VIVO15 serum-free medium (Lonza, USA) and IL2 (50U/mL; Peprotech, USA), IL-7 (10 ng/mL; Peprotech, USA), IL-15(10 ng/mL, Peprotech, USA), O3 antibody KT (50ng/mL; ACRO, USA) and anti-CD28 antibody (1ug/mL; T & L Biotechnology, China). The small tumor tissue is cultured in a cell culture incubator until final TILs are obtained.
Co-incubation with corresponding tumor cells
TILs and corresponding autologous tumor cells were incubated in X-VIVO15 serum-free medium for 12h, and then washed with PBS, according to the requirements of 10Xgenomic, cell size: less than or equal to 30 mu m, and the cell survival rate is more than or equal to 85 percent; the agglomeration rate and the fragment rate are less than 5 percent (bright field detection); cell concentration and total amount: the concentration is 700-. The establishment of single-cell transcriptome and TCR library and the completion of on-machine sequencing were accomplished by Annuodda Gene technology (Beijing) Ltd.
Single cell transcriptome and TCR panel sequencing
According to the basic quality control requirements of the single cell transcriptome and the TCR set, the following quality control standards are simultaneously required to be met. Single cell transcriptome quality control standard: the number of genes per cell is 800-10000, the ratio of mitochondrial genes is less than 20%, and the UMI average value of CD3D, CD3E and CD3G is more than 0. Single cell TCR panel quality control standard: the UMI of both TCR α and β chains is greater than 0. And simultaneously, the T cells meeting the quality control standards of the single Cell transcriptome and the TCR set are brought into final analysis, the expression value of each TILs Cell gene normalization is calculated by using a 'LogNormal' method of Seurat, and Cell range vdj pipeline is used for assembling and identifying TCR alpha and beta chain sequences to obtain 6144 TILs cells meeting the requirements.
Theoretically, if a certain TCR can specifically recognize a corresponding tumor cell, all the TILs expressing the TCR should be activated by the corresponding tumor cell, and considering that the TILs having the same TCR cannot necessarily be contacted with the corresponding tumor cell or the heterogeneity of the tumor cell, which may cause a part of T cells not to be specifically activated by the corresponding tumor cell, but most of the TILs expressing the same tumor-specific TCR as a whole can be specifically activated, the present invention uniformly analyzes the TILs expressing the same TCR, i.e., the average value of the expression values of all the TILs expressing the same TCR is taken as the expression value of the TCR marker.
The marker normalized expression value of each TILs is input into Seurat AddModuleCore (the software program calculation method is referred to the reference and the use description: Cell. 2021 Nov 11;184(23):5838. Cell 177, 1888-one 1821 (2019): https:// rdrr. io/cran/Seurat/man/AddModuleCore. html), TILs activation scores are calculated according to the default parameters of the software, the activation scores are the average expression value of the marker normalized expression values minus the background value, each TILs calculates an activation score, and then the activation scores of all TILs with the same TCR are averaged to obtain the value of the TCR activation score.
The following test groups were conducted on 6144 TILs cells that met the above requirements. It was found that there were 447 different TCRs in 6144 TILs, of which the highest frequency TCR had 1116 TILs with the TCR, the lowest frequency TCR had only one TILs with the TCR, and about 14 TILs on average had one identical TCR. The activation scores of all the TILs cells in the following test groups are directly calculated by using the software, and then the activation scores of all the TILs cells expressing the same TCR are averaged to obtain the activation score of the TCR. The only difference in the calculation of different activation scores is the difference in the types of markers initially included, as is the calculation method described later. The invention proves that 3 TCRs (P1-score-TCR1, P1-score-TCR2 and P1-score-TCR3) which are screened and determined by the test group (3) and have the functions of specifically recognizing and killing corresponding tumor cells in 6144 TILs (P1-score-TCR1, P1-score-TCR2 and P1-score-TCR3) respectively have the TCRs. The grouping test was as follows:
test group (1) a marker IFNG
The test panel was ranked according to the expression values of IFNG, and the TILs cell activation scores were calculated according to the above method and 3 TCRs with the highest expression values were selected (P1-IFN-TCR 1, P1-IFN-TCR2, P1-IFN-TCR3, with the sequence shown in SEQ ID Nos. 1-3).
Test group (2) a marker TNFRSF9
The test group was ranked according to the expression values of TNFRSF9 (41 BB), the TILs cell activation score was calculated according to the above method and 3 TCRs with the highest expression values were selected (P1-41BB-TCR1, P1-41BB-TCR2, P1-41BB-TCR3, the sequence is shown in SEQ ID NO. 4-6).
Test group (3) 10 marker combinations
The test groups were ranked according to the mean of the expression values (designated as activation integration score, score) of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK and PRF1 (total of 10 markers), and the 3 TCRs with the highest mean were selected (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3, sequences as shown in SEQ ID Nos. 7-9). Wherein the P1-IFN-TCR1, P1-41BB-TCR1 and P1-score-TCR3 sequences are identical, i.e. the SEQ ID NO.1, 4, 9 sequences are identical and point to the same TCR.
Test group (4) 19 marker combinations
The test group was ranked by the mean value (designated as activation integration score2, score 2) of the expression values of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, PRF1, LAMP1, LTA, CD40LG, GZMM, HLA-DRA, TNFRSF4, GZMH, GNLY and FASLG (19 markers in total), and 3 TCRs (P1-score 2-TCR1, P1-score2-TCR2, P1-score2-TCR 3) with the highest mean value were selected, the sequence is as set forth in SEQ ID NO.10-12, wherein P1-score2-TCR1 has the same sequence as P1-score-TCR2 of trial group (3) (i.e., SEQ ID NO.10 is identical to the sequence of SEQ ID NO.8 and points to the same TCR), and P1-score2-TCR3 has the same sequence as P1-score-TCR1 of trial group (3) (i.e., SEQ ID NO.12 is identical to the sequence of SEQ ID NO. 7).
Test group (5) 15 marker combinations
The test group was ranked according to the average of the expression values (designated as activation integration score3, score 3) of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, PRF1, LAMP1, LTA, TNFRSF4, GZMH and GNLY (15 markers in total) and the highest 3 TCRs (P1-score 3-TCR1, P1-score3-TCR2, P1-score3-TCR 3) were selected as in SEQ ID NO.13-15, wherein P1-score3-TCR1 (SEQ ID NO. 13) has the same sequence as that of test group (3) P1-score-TCR2 (SEQ ID NO. 8) and P1-score 1-TCR 1 (SEQ ID NO. 14) has the same sequence as that of test group (SEQ ID NO. 3) and P1-score 1-TCR 1 (SEQ ID NO. 1).
Test group (6) additional 10 marker combinations
The highest 3 TCRs (P1-score 4-TCR1, P1-score4-TCR2, P1-score4-TCR 3) were selected in the test group by ranking the mean (activation integration score 4) of the expression values of (IFNG, IL2, TNF, TNFRSF9, GZMB, LAMP1, LTA, TNFRSF4, GZMH, GNLY) and having the sequence shown in SEQ ID NO.16-18, wherein P1-score4-TCR1 (SEQ ID NO. 16) has the same sequence as that of test group (3) P1-score-TCR3 (SEQ ID NO. 9), P1-score4-TCR3 (SEQ ID NO. 18) has the same sequence as that of test group (3) P1-score-TCR2 (SEQ ID NO. 8).
Test group (7) 5 marker combinations
The average of the expression values (activation composite score 5) of the 5 genes (IFNG, IL2, TNF, TNFRSF9, GZMB) was selected for ranking in this panel, and the highest 3 TCRs (P1-score 5-TCR1, P1-score5-TCR2, P1-score5-TCR 3) were selected as SEQ ID NO.19-21, where P1-score5-TCR1 (SEQ ID NO. 19) has the same sequence as P1-score-TCR3 (SEQ ID NO. 9) of the (3) panel.
Meanwhile, the four sequences of P1-score2-TCR2 (SEQ ID NO. 11), P1-score3-TCR3 (SEQ ID NO. 15), P1-score4-TCR2 (SEQ ID NO. 17) and P1-score5-TCR2 (SEQ ID NO. 20) are the same.
Example 2 construction of TCR-T Lentiviral vectors
(1) In example 1, 21 TCRs were found in total, but since some TCRs of TCR1-3 obtained from different experimental groups were identical, the present application synthesized the above 9 non-repetitive TCRs separately, added XbaI and SalI cleavage sites at both ends of each TCR nucleotide sequence, and cloned into pUC57 vector;
(2) using XbaI and SalI to double-enzyme cut pUC57 vector containing target gene, cutting gel and recovering target gene fragment;
(3) the original vector pCDH-EF1-Luc2-T2A-tdTomato is subjected to double enzyme digestion by XbaI and SalI, and a vector fragment of about 6.5kb is recovered by gel cutting;
(4) and connecting the recovered target gene and the vector fragment by using DNA ligase to obtain the recombinant lentiviral vector carrying each TCR.
Example 3 preparation of TCR Lentivirus
The 9 recombinant lentiviral vectors of example 2 were transfected into 293ft cells by transfection reagent (PEI) to produce lentiviruses. The specific method comprises the following steps: the plasmid mixture (pMDL: VSV-G: REV =5:3:2, mass ratio) and TCR1 lentiviral vector were added to 500. mu.L of serum-free medium Opti-MEM at a mass ratio of 1:1, vortexed to mix well. 32g PEI was added to 500. mu.L serum free medium Opti-MEM, vortexed to mix well. Then 500ul of plasmid mixture and 500ul of PEI, and its added to the confluence of about 90% 293ft cells, after 48 hours to collect the virus supernatant, after ultracentrifugation, the virus concentration 100 times, and then obtain the concentrated virus.
EXAMPLE 4 preparation of TCR-T cells
Peripheral blood T cells were sorted, cultured in T cell media, and activated T cells were infected with the virus after concentration in example 3, MOI =10:1 (OKT 3 and CD28 antibodies in the T cell media components activated T cells). 72 hours after infection, the expression of each TCR was detected by flow antibody against the murine TCR β chain constant region.
Example 5 in vitro and in vivo functional characterization of TCR-T cells
Each 10 kinds of6TCR-T cells and 105The supernatant IFN- γ concentration was measured after 12 hours of co-incubation of individual autologous lung cancer cells. The results showed that only one TCR (P1-IFN-TCR 1 and P1-41BB-TCR1 with the same sequence) gene-modified T cells among the three TCRs selected by IFNG or TNFRSF9 single indexes can be specifically activated to secrete IFN-gamma (FIG. 1), and all the 3 TCRs (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3) selected by the 10 marker activation scores of the test group (3) can specifically recognize the corresponding tumor cells to secrete IFN-gamma (FIG. 2).
The test group (4) was 3 TCRs screened based on the activation composite score2 (P1-score 2-TCR1, P1-score2-TCR2, P1-score2-TCR 3), in which P1-score2-TCR1 and P1-score-TCR2 have the same sequence, and P1-score2-TCR3 and P1-score-TCR1 have the same sequence, so that P1-score2-TCR1 and P1-score2-TCR3 gene modified T cells have the ability to specifically recognize tumor cells, but P1-score2-TCR2 gene modified T cells cannot be specifically activated to secrete IFN-. gamma. (FIG. 3).
The test group (5) showed that 3 TCRs (P1-score 3-TCR1, P1-score3-TCR2 and P1-score3-TCR 3) screened based on the activation integration score3, wherein P1-score3-TCR1 has the same sequence as P1-score-TCR2 and P1-score3-TCR2 has the same sequence as P1-score-TCR1, so that P1-score3-TCR1 and P1-score3-TCR2 genetically modified T cells have the ability to specifically recognize tumor cells, but P1-score3-TCR3 and P1-score2-TCR2 have the same sequence, and thus P1-score3-TCR3 genetically modified T cells also cannot specifically recognize tumor cells.
The test group (6) showed that 3 TCRs (P1-score 4-TCR1, P1-score4-TCR2 and P1-score4-TCR 3) screened according to the activation integration score of 4, wherein the P1-score4-TCR1 and the P1-score-TCR3 have the same sequence, and the P1-score4-TCR3 and the P1-score-TCR2 have the same sequence, so that the P1-score4-TCR1 and the P1-score4-TCR3 gene modified T cells have the ability of specifically recognizing tumor cells, but the P1-score4-TCR2 and the P1-score2-TCR2 have the same sequence, and therefore the P1-score4-TCR2 gene modified T cells cannot specifically recognize tumor cells.
The test group (7) was 3 TCRs (P1-score 5-TCR1, P1-score5-TCR2, P1-score5-TCR 3) screened based on the activation composite score of 5, in which P1-score5-TCR1 and P1-score-TCR3 had the same sequence, so that P1-score5-TCR1 gene-modified T cells were able to specifically recognize and kill tumor cells, but P1-score5-TCR2 and P1-score2-TCR2 had the same sequence, so that P1-score5-TCR2 gene-modified T cells could not specifically recognize tumor cells, and P1-score5-TCR3 gene-modified T cells could not specifically recognize tumor cells (FIG. 4).
All of the 3 TCRs selected based on the T cell activation scores (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3) can specifically recognize the corresponding tumor cells, and all of the other groups of tumor-specific TCRs belong to the above three TCRs, so that the three TCRs in total (P1-score-TCR1, P1-score-TCR2, P1-score-TCR3) can specifically recognize the corresponding tumor cells. Further, this example evaluates the ability of these 3 TCRs to specifically kill the corresponding tumor cells by in vitro killing experiments, each TCR-T cell was incubated with CFSE pre-labeled autologous lung cancer tumor cells at an effective-to-target ratio of 30:1,10:1,2:1, respectively, and the cell death ratio (PI) in CFSE-labeled target cells was identified by flow-based assay+ Percentage of target cells). The results showed that the three TCR-T cells specifically killed the corresponding tumor cells in vitro (FIG. 5), and that each of the three TCR-T cells (6X 10T)6) The transplanted tumor mouse model constructed by corresponding lung cancer tumor cells was back-transfused, and the results showed that the three TCR gene modified T cells can kill the mouse transplanted tumor model in vivo compared with the non-gene modified T cells (fig. 6).
Example 6 other 4 patients with Lung cancer validated the strategy for screening TCRs described above
This example also included 4 lung cancer patients, who obtained TILs and tumor cells from the above-described pattern, were subjected to sequencing of single-cell transcriptome and TCR panel according to the method described in example 1 to obtain TCR sequences of each T cell and expression values of each marker, and then tumor-specific TCRs were screened in the above-described test panels (1) to (7), respectively, and finally found that 3 TCRs having the highest scores were able to specifically recognize and kill the corresponding tumor cells, sorted according to the mean value (activation integrated score) of the expression values of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, gzmkmkmkmkmkm, and PRF1 of the test panel (3 TCRs) having the highest scores, and 3 TCRs screened with the single IFNG or TNFRSF9 scores and each activation integrated score (activation integrated score 2/3/4/5) calculated from the multiple T cell marker scores of the test panels (4) to (7), only 1-2 TCRs can specifically recognize corresponding tumor cells at most;
therefore, the screening method of the tumor specific TCR provided by the invention has better screening effect than the screening method of single T cell markers, and the combined screening of a plurality of T cell markers in other combined modes, even more than 10T cell markers.
Sequence listing
<110> Beijing tumor Hospital (Beijing university tumor Hospital)
<120> a method for screening tumor specific TCR
<130> KHP211122054.8YS
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgggcacta gcctgctgtg ctggatggcc ctgtgcctgc tcggcgccga tcacgccgac 60
accggcgtga gccaggaccc taggcacaag attacaaaga gggggcagaa cgtgacattc 120
cggtgcgatc ccattagcga gcataacagg ctctactggt accggcagac cctcggccag 180
ggccctgagt tcctgaccta ctttcagaac gaggcccagc tggagaagag caggctgctg 240
tccgaccggt ttagcgccga gaggcctaag ggcagcttct ccaccctgga gatccagcgc 300
acagagcagg gcgatagcgc catgtacctg tgcgccagca gctccaccag cggggggccc 360
atccaggaga cccagtactt cggccccggc acccggctgc tggtgctgga ggatctgcgg 420
aacgtgaccc cccctaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 480
cagaaggcca cactggtgtg cctggccagg gggttctttc ccgatcacgt ggagctgtcc 540
tggtgggtga acggcaagga ggtccacagc ggggtgtgca cagaccccca ggcctacaag 600
gagtccaact acagctactg cctcagcagc aggctgcggg tctccgccac attttggcac 660
aaccccagga accacttccg gtgccaggtg cagtttcacg gcctgagcga ggaggataag 720
tggcctgagg ggagccctaa gcccgtgacc cagaacatct ccgccgaggc ctggggcaga 780
gccgattgcg ggattactag cgcctcctac cagcagggcg tgctgagcgc caccattctc 840
tacgagatcc tgctcggcaa ggccaccctc tacgccgtgc tggtgtccac actggtcgtg 900
atggccatgg tgaagcggaa gaacagccgg gccaagcgga gcgggagcgg ggccaccaac 960
ttctccctgc tgaagcaggc cggcgacgtc gaggagaacc ccggccccat ggagactctg 1020
ctgggcctgc tgatcctgtg gctccagctg cagtgggtga gcagcaagca ggaggtgaca 1080
cagatccccg ccgccctgtc cgtgcccgag ggggagaacc tggtgctgaa ctgcagcttc 1140
acagatagcg ccatctacaa cctgcagtgg ttccggcagg accccggcaa gggcctgaca 1200
tccctgctcc tgatccagag cagccagcgg gagcagacta gcggcaggct caacgccagc 1260
ctggacaaga gcagcgggag gtccaccctg tacatcgccg ccagccagcc aggggatagc 1320
gccacttacc tgtgcgccgg ccacggctac aaccaggggg gcaagctgat tttcgggcag 1380
gggacagagc tgagcgtgaa gccaaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 2
<211> 1803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gcttgggcac tgaagctttc 360
tttggacaag gcaccagact cacagttgta gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgatgaaat ccttgagagt tttactagtg 1020
atcctgtggc ttcagttgag ctgggtttgg agccaacaga aggaggtgga gcagaattct 1080
ggacccctca gtgttccaga gggagccatt gcctctctca actgcactta cagtgaccga 1140
ggttcccagt ccttcttctg gtacagacaa tattctggga aaagccctga gttgataatg 1200
ttcatatact ccaatggtga caaagaagat ggaaggttta cagcacagct caataaagcc 1260
agccagtatg tttctctgct catcagagac tcccagccca gtgattcagc cacctacctc 1320
tgtgccgcct gggggagcaa ctatcagtta atctggggcg ctgggaccaa gctaattata 1380
aagccaaata ttcagaatcc ggagcctgcc gtgtaccagc tgaaggaccc acggagccag 1440
gattccaccc tgtgcctgtt cacagacttt gattcccaga tcaacgtgcc caagaccatg 1500
gagtctggca cctttatcac agacaagtgc gtgctggaca tgaaggccat ggacagcaag 1560
tccaacggcg ccatcgcctg gtccaatcag acctctttca catgccagga tatctttaag 1620
gagacaaacg ccacatatcc atcctctgac gtgccctgtg atgccaccct gacagagaag 1680
agcttcgaga cagacatgaa cctgaatttt cagaacctgc tggtcatcgt gctgcggatc 1740
ctgctgctga aggtggctgg gtttaatctg ctgatgactc tgagactgtg gtcctcctga 1800
taa 1803
<210> 3
<211> 1815
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgagcatcg ggctcctgtg ctgtgtggcc ttttctctcc tgtgggcaag tccagtgaat 60
gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120
cagtgtgccc aggatatgaa ccataactcc atgtactggt atcgacaaga cccaggcatg 180
ggactgaggc tgatttatta ctcagcttct gagggtacca ctgacaaagg agaagtcccc 240
aatggctaca atgtctccag attaaacaaa cgggagttct cgctcaggct ggagtcggct 300
gctccctccc agacatctgt gtacttctgt gccagcaggc ctcggacagg gggcgcttac 360
aatgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gcgcaacgtc 420
accccaccaa aggtcagttt gtttgagcca tcaaaggcgg agatcgccaa caaacagaaa 480
gctacgctcg tgtgtttggc tcggggcttc ttcccagacc acgtagaact ttcctggtgg 540
gtcaatggaa aggaggttca ttcaggagtg tgcactgatc cccaagcgta caaggaatcc 600
aactatagct actgtctctc atctcggctc cgggtgagtg cgacattctg gcataatcct 660
cggaaccact ttcgatgcca agtgcagttt catgggttga gcgaggaaga caagtggccc 720
gagggcagtc ctaaaccagt cactcaaaac ataagcgccg aggcatgggg tagagccgat 780
tgtgggatta ctagcgcttc ataccaacaa ggggtattga gcgctacaat tctttacgaa 840
attctcctcg gcaaggcgac gctctacgcc gtactggtgt ctactctcgt ggttatggca 900
atggtgaaac ggaaaaacag cagagccaaa agaagtggtt ctggcgcgac gaattttagt 960
ttgcttaagc aagccggaga tgtggaggaa aatcctggac cgatgctgac tgccagcctg 1020
ttgagggcag tcatagcctc catctgtgtt gtatccagca tggctcagaa ggtaactcaa 1080
gcgcagactg aaatttctgt ggtggagaag gaggatgtga ccttggactg tgtgtatgaa 1140
acccgtgata ctacttatta cttattctgg tacaagcaac caccaagtgg agaattggtt 1200
ttccttattc gtcggaactc ttttgatgag caaaatgaaa taagtggtcg gtattcttgg 1260
aacttccaga aatccaccag ttccttcaac ttcaccatca cagcctcaca agtcgtggac 1320
tcagcagtat acttctgtgc tctgaggatt gatgacaaga tcatctttgg aaaagggaca 1380
cgacttcata ttctccccaa tattcagaat ccggagcctg ccgtgtacca gctgaaggac 1440
ccacggagcc aggattccac cctgtgcctg ttcacagact ttgattccca gatcaacgtg 1500
cccaagacca tggagtctgg cacctttatc acagacaagt gcgtgctgga catgaaggcc 1560
atggacagca agtccaacgg cgccatcgcc tggtccaatc agacctcttt cacatgccag 1620
gatatcttta aggagacaaa cgccacatat ccatcctctg acgtgccctg tgatgccacc 1680
ctgacagaga agagcttcga gacagacatg aacctgaatt ttcagaacct gctggtcatc 1740
gtgctgcgga tcctgctgct gaaggtggct gggtttaatc tgctgatgac tctgagactg 1800
tggtcctcct gataa 1815
<210> 4
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgggcacta gcctgctgtg ctggatggcc ctgtgcctgc tcggcgccga tcacgccgac 60
accggcgtga gccaggaccc taggcacaag attacaaaga gggggcagaa cgtgacattc 120
cggtgcgatc ccattagcga gcataacagg ctctactggt accggcagac cctcggccag 180
ggccctgagt tcctgaccta ctttcagaac gaggcccagc tggagaagag caggctgctg 240
tccgaccggt ttagcgccga gaggcctaag ggcagcttct ccaccctgga gatccagcgc 300
acagagcagg gcgatagcgc catgtacctg tgcgccagca gctccaccag cggggggccc 360
atccaggaga cccagtactt cggccccggc acccggctgc tggtgctgga ggatctgcgg 420
aacgtgaccc cccctaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 480
cagaaggcca cactggtgtg cctggccagg gggttctttc ccgatcacgt ggagctgtcc 540
tggtgggtga acggcaagga ggtccacagc ggggtgtgca cagaccccca ggcctacaag 600
gagtccaact acagctactg cctcagcagc aggctgcggg tctccgccac attttggcac 660
aaccccagga accacttccg gtgccaggtg cagtttcacg gcctgagcga ggaggataag 720
tggcctgagg ggagccctaa gcccgtgacc cagaacatct ccgccgaggc ctggggcaga 780
gccgattgcg ggattactag cgcctcctac cagcagggcg tgctgagcgc caccattctc 840
tacgagatcc tgctcggcaa ggccaccctc tacgccgtgc tggtgtccac actggtcgtg 900
atggccatgg tgaagcggaa gaacagccgg gccaagcgga gcgggagcgg ggccaccaac 960
ttctccctgc tgaagcaggc cggcgacgtc gaggagaacc ccggccccat ggagactctg 1020
ctgggcctgc tgatcctgtg gctccagctg cagtgggtga gcagcaagca ggaggtgaca 1080
cagatccccg ccgccctgtc cgtgcccgag ggggagaacc tggtgctgaa ctgcagcttc 1140
acagatagcg ccatctacaa cctgcagtgg ttccggcagg accccggcaa gggcctgaca 1200
tccctgctcc tgatccagag cagccagcgg gagcagacta gcggcaggct caacgccagc 1260
ctggacaaga gcagcgggag gtccaccctg tacatcgccg ccagccagcc aggggatagc 1320
gccacttacc tgtgcgccgg ccacggctac aaccaggggg gcaagctgat tttcgggcag 1380
gggacagagc tgagcgtgaa gccaaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 5
<211> 1824
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgagcctcg ggctcctgtg ctgtggggcc ttttctctcc tgtgggcagg tccagtgaat 60
gctggtgtca ctcagacccc aaaattccgg gtcctgaaga caggacagag catgacactg 120
ctgtgtgccc aggatatgaa ccatgaatac atgtactggt atcgacaaga cccaggcatg 180
gggctgaggc tgattcatta ctcagttggt gagggtacaa ctgccaaagg agaggtccct 240
gatggctaca atgtctccag attaaaaaaa cagaatttcc tgctggggtt ggagtcggct 300
gctccctccc aaacatctgt gtacttctgt gccagcagtt ggtccaaggg ggaggggttt 360
agctcctaca atgagcagtt cttcgggcca gggacacggc tcaccgtgct agaggacctg 420
cgcaacgtca ccccaccaaa ggtcagtttg tttgagccat caaaggcgga gatcgccaac 480
aaacagaaag ctacgctcgt gtgtttggct cggggcttct tcccagacca cgtagaactt 540
tcctggtggg tcaatggaaa ggaggttcat tcaggagtgt gcactgatcc ccaagcgtac 600
aaggaatcca actatagcta ctgtctctca tctcggctcc gggtgagtgc gacattctgg 660
cataatcctc ggaaccactt tcgatgccaa gtgcagtttc atgggttgag cgaggaagac 720
aagtggcccg agggcagtcc taaaccagtc actcaaaaca taagcgccga ggcatggggt 780
agagccgatt gtgggattac tagcgcttca taccaacaag gggtattgag cgctacaatt 840
ctttacgaaa ttctcctcgg caaggcgacg ctctacgccg tactggtgtc tactctcgtg 900
gttatggcaa tggtgaaacg gaaaaacagc agagccaaaa gaagtggttc tggcgcgacg 960
aattttagtt tgcttaagca agccggagat gtggaggaaa atcctggacc gatgctcctg 1020
ctgctcgtcc cagcgttcca ggtgattttt accctgggag gaaccagagc ccagtctgtg 1080
acccagcttg acagccaagt ccctgtcttt gaagaagccc ctgtggagct gaggtgcaac 1140
tactcatcgt ctgtttcagt gtatctcttc tggtatgtgc aataccccaa ccaaggactc 1200
cagcttctcc tgaagtattt atcaggatcc accctggtta aaggcatcaa cggttttgag 1260
gctgaattta acaagagtca aacttccttc cacttgagga aaccctcagt ccatataagc 1320
gacacggctg agtacttctg tgctgtgacc gtaccctatg gtcagaattt tgtctttggt 1380
cccggaacca gattgtccgt gctgcccaat attcagaatc cggagcctgc cgtgtaccag 1440
ctgaaggacc cacggagcca ggattccacc ctgtgcctgt tcacagactt tgattcccag 1500
atcaacgtgc ccaagaccat ggagtctggc acctttatca cagacaagtg cgtgctggac 1560
atgaaggcca tggacagcaa gtccaacggc gccatcgcct ggtccaatca gacctctttc 1620
acatgccagg atatctttaa ggagacaaac gccacatatc catcctctga cgtgccctgt 1680
gatgccaccc tgacagagaa gagcttcgag acagacatga acctgaattt tcagaacctg 1740
ctggtcatcg tgctgcggat cctgctgctg aaggtggctg ggtttaatct gctgatgact 1800
ctgagactgt ggtcctcctg ataa 1824
<210> 6
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60
gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120
agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180
ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240
ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300
gagctggggg actcggccct ttatctttgc gccagcagcc tagggacctc tgagcagtac 360
ttcgggccgg gcaccaggct cacggtcaca gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgtcacttt ctagcctgct gaaggtggtc 1020
acagcttcac tgtggctagg acctggcatt gcccagaaga taactcaaac ccaaccagga 1080
atgttcgtgc aggaaaagga ggctgtgact ctggactgca catatgacac cagtgatcaa 1140
agttatggtc tattctggta caagcagccc agcagtgggg aaatgatttt tcttatttat 1200
caggggtctt atgacgagca aaatgcaaca gaaggtcgct actcattgaa tttccagaag 1260
gcaagaaaat ccgccaacct tgtcatctcc gcttcacaac tgggggactc agcaatgtat 1320
ttctgtgcaa tgacccacaa gaggggggaa tatggaaaca agctggtctt tggcgcagga 1380
accattctga gagtcaagtc caatattcag aatccggagc ctgccgtgta ccagctgaag 1440
gacccacgga gccaggattc caccctgtgc ctgttcacag actttgattc ccagatcaac 1500
gtgcccaaga ccatggagtc tggcaccttt atcacagaca agtgcgtgct ggacatgaag 1560
gccatggaca gcaagtccaa cggcgccatc gcctggtcca atcagacctc tttcacatgc 1620
caggatatct ttaaggagac aaacgccaca tatccatcct ctgacgtgcc ctgtgatgcc 1680
accctgacag agaagagctt cgagacagac atgaacctga attttcagaa cctgctggtc 1740
atcgtgctgc ggatcctgct gctgaaggtg gctgggttta atctgctgat gactctgaga 1800
ctgtggtcct cctgataa 1818
<210> 7
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gctcgactag cgggggacct 360
atccaagaga cccagtactt cgggccaggc acgcggctcc tggtgctcga ggacctgcgc 420
aacgtcaccc caccaaaggt cagtttgttt gagccatcaa aggcggagat cgccaacaaa 480
cagaaagcta cgctcgtgtg tttggctcgg ggcttcttcc cagaccacgt agaactttcc 540
tggtgggtca atggaaagga ggttcattca ggagtgtgca ctgatcccca agcgtacaag 600
gaatccaact atagctactg tctctcatct cggctccggg tgagtgcgac attctggcat 660
aatcctcgga accactttcg atgccaagtg cagtttcatg ggttgagcga ggaagacaag 720
tggcccgagg gcagtcctaa accagtcact caaaacataa gcgccgaggc atggggtaga 780
gccgattgtg ggattactag cgcttcatac caacaagggg tattgagcgc tacaattctt 840
tacgaaattc tcctcggcaa ggcgacgctc tacgccgtac tggtgtctac tctcgtggtt 900
atggcaatgg tgaaacggaa aaacagcaga gccaaaagaa gtggttctgg cgcgacgaat 960
tttagtttgc ttaagcaagc cggagatgtg gaggaaaatc ctggaccgat ggagaccctc 1020
ttgggcctgc ttatcctttg gctgcagctg caatgggtga gcagcaaaca ggaggtgacg 1080
cagattcctg cagctctgag tgtcccagaa ggagaaaact tggttctcaa ctgcagtttc 1140
actgatagcg ctatttacaa cctccagtgg tttaggcagg accctgggaa aggtctcaca 1200
tctctgttgc ttattcagtc aagtcagaga gagcaaacaa gtggaagact taatgcctcg 1260
ctggataaat catcaggacg tagtacttta tacattgcag cttctcagcc tggtgactca 1320
gccacctacc tctgtgctgg acacggttat aaccagggag gaaagcttat cttcggacag 1380
ggaacggagt tatctgtgaa acccaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 8
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggt ccccatggaa 60
acgggagtta cgcagacacc aagacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatctggg tcataacgct atgtattggt acaagcaaag tgctaagaag 180
ccactggagc tcatgtttgt ctacagtctt gaagaacggg ttgaaaacaa cagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctcacttat tccttcacct acacaccctg 300
cagccagaag actcggccct gtatctctgc gccagcagcc caggagtggg ggaatataat 360
tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gcgcaacgtc 420
accccaccaa aggtcagttt gtttgagcca tcaaaggcgg agatcgccaa caaacagaaa 480
gctacgctcg tgtgtttggc tcggggcttc ttcccagacc acgtagaact ttcctggtgg 540
gtcaatggaa aggaggttca ttcaggagtg tgcactgatc cccaagcgta caaggaatcc 600
aactatagct actgtctctc atctcggctc cgggtgagtg cgacattctg gcataatcct 660
cggaaccact ttcgatgcca agtgcagttt catgggttga gcgaggaaga caagtggccc 720
gagggcagtc ctaaaccagt cactcaaaac ataagcgccg aggcatgggg tagagccgat 780
tgtgggatta ctagcgcttc ataccaacaa ggggtattga gcgctacaat tctttacgaa 840
attctcctcg gcaaggcgac gctctacgcc gtactggtgt ctactctcgt ggttatggca 900
atggtgaaac ggaaaaacag cagagccaaa agaagtggtt ctggcgcgac gaattttagt 960
ttgcttaagc aagccggaga tgtggaggaa aatcctggac cgatgtgggg agttttcctt 1020
ctttatgttt ccatgaagat gggaggcact acaggacaaa acattgacca gcccactgag 1080
atgacagcta cggaaggtgc cattgtccag atcaactgca cgtaccagac atctgggttc 1140
aacgggctgt tctggtacca gcaacatgct ggcgaagcac ccacatttct gtcttacaat 1200
gttctggatg gtttggagga gaaaggtcgt ttttcttcat tccttagtcg gtctaaaggg 1260
tacagttacc tccttttgaa ggagctccag atgaaagact ctgcctctta cctctgtgct 1320
gtgagagatc aagaggaata ctctggggct gggagttacc aactcacttt cgggaagggg 1380
accaaactct cggtcatacc aaatattcag aatccggagc ctgccgtgta ccagctgaag 1440
gacccacgga gccaggattc caccctgtgc ctgttcacag actttgattc ccagatcaac 1500
gtgcccaaga ccatggagtc tggcaccttt atcacagaca agtgcgtgct ggacatgaag 1560
gccatggaca gcaagtccaa cggcgccatc gcctggtcca atcagacctc tttcacatgc 1620
caggatatct ttaaggagac aaacgccaca tatccatcct ctgacgtgcc ctgtgatgcc 1680
accctgacag agaagagctt cgagacagac atgaacctga attttcagaa cctgctggtc 1740
atcgtgctgc ggatcctgct gctgaaggtg gctgggttta atctgctgat gactctgaga 1800
ctgtggtcct cctgataa 1818
<210> 9
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgggcacta gcctgctgtg ctggatggcc ctgtgcctgc tcggcgccga tcacgccgac 60
accggcgtga gccaggaccc taggcacaag attacaaaga gggggcagaa cgtgacattc 120
cggtgcgatc ccattagcga gcataacagg ctctactggt accggcagac cctcggccag 180
ggccctgagt tcctgaccta ctttcagaac gaggcccagc tggagaagag caggctgctg 240
tccgaccggt ttagcgccga gaggcctaag ggcagcttct ccaccctgga gatccagcgc 300
acagagcagg gcgatagcgc catgtacctg tgcgccagca gctccaccag cggggggccc 360
atccaggaga cccagtactt cggccccggc acccggctgc tggtgctgga ggatctgcgg 420
aacgtgaccc cccctaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 480
cagaaggcca cactggtgtg cctggccagg gggttctttc ccgatcacgt ggagctgtcc 540
tggtgggtga acggcaagga ggtccacagc ggggtgtgca cagaccccca ggcctacaag 600
gagtccaact acagctactg cctcagcagc aggctgcggg tctccgccac attttggcac 660
aaccccagga accacttccg gtgccaggtg cagtttcacg gcctgagcga ggaggataag 720
tggcctgagg ggagccctaa gcccgtgacc cagaacatct ccgccgaggc ctggggcaga 780
gccgattgcg ggattactag cgcctcctac cagcagggcg tgctgagcgc caccattctc 840
tacgagatcc tgctcggcaa ggccaccctc tacgccgtgc tggtgtccac actggtcgtg 900
atggccatgg tgaagcggaa gaacagccgg gccaagcgga gcgggagcgg ggccaccaac 960
ttctccctgc tgaagcaggc cggcgacgtc gaggagaacc ccggccccat ggagactctg 1020
ctgggcctgc tgatcctgtg gctccagctg cagtgggtga gcagcaagca ggaggtgaca 1080
cagatccccg ccgccctgtc cgtgcccgag ggggagaacc tggtgctgaa ctgcagcttc 1140
acagatagcg ccatctacaa cctgcagtgg ttccggcagg accccggcaa gggcctgaca 1200
tccctgctcc tgatccagag cagccagcgg gagcagacta gcggcaggct caacgccagc 1260
ctggacaaga gcagcgggag gtccaccctg tacatcgccg ccagccagcc aggggatagc 1320
gccacttacc tgtgcgccgg ccacggctac aaccaggggg gcaagctgat tttcgggcag 1380
gggacagagc tgagcgtgaa gccaaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 10
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggt ccccatggaa 60
acgggagtta cgcagacacc aagacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatctggg tcataacgct atgtattggt acaagcaaag tgctaagaag 180
ccactggagc tcatgtttgt ctacagtctt gaagaacggg ttgaaaacaa cagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctcacttat tccttcacct acacaccctg 300
cagccagaag actcggccct gtatctctgc gccagcagcc caggagtggg ggaatataat 360
tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gcgcaacgtc 420
accccaccaa aggtcagttt gtttgagcca tcaaaggcgg agatcgccaa caaacagaaa 480
gctacgctcg tgtgtttggc tcggggcttc ttcccagacc acgtagaact ttcctggtgg 540
gtcaatggaa aggaggttca ttcaggagtg tgcactgatc cccaagcgta caaggaatcc 600
aactatagct actgtctctc atctcggctc cgggtgagtg cgacattctg gcataatcct 660
cggaaccact ttcgatgcca agtgcagttt catgggttga gcgaggaaga caagtggccc 720
gagggcagtc ctaaaccagt cactcaaaac ataagcgccg aggcatgggg tagagccgat 780
tgtgggatta ctagcgcttc ataccaacaa ggggtattga gcgctacaat tctttacgaa 840
attctcctcg gcaaggcgac gctctacgcc gtactggtgt ctactctcgt ggttatggca 900
atggtgaaac ggaaaaacag cagagccaaa agaagtggtt ctggcgcgac gaattttagt 960
ttgcttaagc aagccggaga tgtggaggaa aatcctggac cgatgtgggg agttttcctt 1020
ctttatgttt ccatgaagat gggaggcact acaggacaaa acattgacca gcccactgag 1080
atgacagcta cggaaggtgc cattgtccag atcaactgca cgtaccagac atctgggttc 1140
aacgggctgt tctggtacca gcaacatgct ggcgaagcac ccacatttct gtcttacaat 1200
gttctggatg gtttggagga gaaaggtcgt ttttcttcat tccttagtcg gtctaaaggg 1260
tacagttacc tccttttgaa ggagctccag atgaaagact ctgcctctta cctctgtgct 1320
gtgagagatc aagaggaata ctctggggct gggagttacc aactcacttt cgggaagggg 1380
accaaactct cggtcatacc aaatattcag aatccggagc ctgccgtgta ccagctgaag 1440
gacccacgga gccaggattc caccctgtgc ctgttcacag actttgattc ccagatcaac 1500
gtgcccaaga ccatggagtc tggcaccttt atcacagaca agtgcgtgct ggacatgaag 1560
gccatggaca gcaagtccaa cggcgccatc gcctggtcca atcagacctc tttcacatgc 1620
caggatatct ttaaggagac aaacgccaca tatccatcct ctgacgtgcc ctgtgatgcc 1680
accctgacag agaagagctt cgagacagac atgaacctga attttcagaa cctgctggtc 1740
atcgtgctgc ggatcctgct gctgaaggtg gctgggttta atctgctgat gactctgaga 1800
ctgtggtcct cctgataa 1818
<210> 11
<211> 1803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gcttgggcac tgaagctttc 360
tttggacaag gcaccagact cacagttgta gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgatgaaat ccttgagagt tttactagtg 1020
atcctgtggc ttcagttgag ctgggtttgg agccaacaga aggaggtgga gcagaattct 1080
ggacccctca gtgttccaga gggagccatt gcctctctca actgcactta cagtgaccga 1140
ggttcccagt ccttcttctg gtacagacaa tattctggga aaagccctga gttgataatg 1200
ttcatatact ccaatggtga caaagaagat ggaaggttta cagcacagct caataaagcc 1260
agccagtatg tttctctgct catcagagac tcccagccca gtgattcagc cacctacctc 1320
tgtgccgcct gggggagcaa ctatcagtta atctggggcg ctgggaccaa gctaattata 1380
aagccaaata ttcagaatcc ggagcctgcc gtgtaccagc tgaaggaccc acggagccag 1440
gattccaccc tgtgcctgtt cacagacttt gattcccaga tcaacgtgcc caagaccatg 1500
gagtctggca cctttatcac agacaagtgc gtgctggaca tgaaggccat ggacagcaag 1560
tccaacggcg ccatcgcctg gtccaatcag acctctttca catgccagga tatctttaag 1620
gagacaaacg ccacatatcc atcctctgac gtgccctgtg atgccaccct gacagagaag 1680
agcttcgaga cagacatgaa cctgaatttt cagaacctgc tggtcatcgt gctgcggatc 1740
ctgctgctga aggtggctgg gtttaatctg ctgatgactc tgagactgtg gtcctcctga 1800
taa 1803
<210> 12
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gctcgactag cgggggacct 360
atccaagaga cccagtactt cgggccaggc acgcggctcc tggtgctcga ggacctgcgc 420
aacgtcaccc caccaaaggt cagtttgttt gagccatcaa aggcggagat cgccaacaaa 480
cagaaagcta cgctcgtgtg tttggctcgg ggcttcttcc cagaccacgt agaactttcc 540
tggtgggtca atggaaagga ggttcattca ggagtgtgca ctgatcccca agcgtacaag 600
gaatccaact atagctactg tctctcatct cggctccggg tgagtgcgac attctggcat 660
aatcctcgga accactttcg atgccaagtg cagtttcatg ggttgagcga ggaagacaag 720
tggcccgagg gcagtcctaa accagtcact caaaacataa gcgccgaggc atggggtaga 780
gccgattgtg ggattactag cgcttcatac caacaagggg tattgagcgc tacaattctt 840
tacgaaattc tcctcggcaa ggcgacgctc tacgccgtac tggtgtctac tctcgtggtt 900
atggcaatgg tgaaacggaa aaacagcaga gccaaaagaa gtggttctgg cgcgacgaat 960
tttagtttgc ttaagcaagc cggagatgtg gaggaaaatc ctggaccgat ggagaccctc 1020
ttgggcctgc ttatcctttg gctgcagctg caatgggtga gcagcaaaca ggaggtgacg 1080
cagattcctg cagctctgag tgtcccagaa ggagaaaact tggttctcaa ctgcagtttc 1140
actgatagcg ctatttacaa cctccagtgg tttaggcagg accctgggaa aggtctcaca 1200
tctctgttgc ttattcagtc aagtcagaga gagcaaacaa gtggaagact taatgcctcg 1260
ctggataaat catcaggacg tagtacttta tacattgcag cttctcagcc tggtgactca 1320
gccacctacc tctgtgctgg acacggttat aaccagggag gaaagcttat cttcggacag 1380
ggaacggagt tatctgtgaa acccaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 13
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggt ccccatggaa 60
acgggagtta cgcagacacc aagacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatctggg tcataacgct atgtattggt acaagcaaag tgctaagaag 180
ccactggagc tcatgtttgt ctacagtctt gaagaacggg ttgaaaacaa cagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctcacttat tccttcacct acacaccctg 300
cagccagaag actcggccct gtatctctgc gccagcagcc caggagtggg ggaatataat 360
tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gcgcaacgtc 420
accccaccaa aggtcagttt gtttgagcca tcaaaggcgg agatcgccaa caaacagaaa 480
gctacgctcg tgtgtttggc tcggggcttc ttcccagacc acgtagaact ttcctggtgg 540
gtcaatggaa aggaggttca ttcaggagtg tgcactgatc cccaagcgta caaggaatcc 600
aactatagct actgtctctc atctcggctc cgggtgagtg cgacattctg gcataatcct 660
cggaaccact ttcgatgcca agtgcagttt catgggttga gcgaggaaga caagtggccc 720
gagggcagtc ctaaaccagt cactcaaaac ataagcgccg aggcatgggg tagagccgat 780
tgtgggatta ctagcgcttc ataccaacaa ggggtattga gcgctacaat tctttacgaa 840
attctcctcg gcaaggcgac gctctacgcc gtactggtgt ctactctcgt ggttatggca 900
atggtgaaac ggaaaaacag cagagccaaa agaagtggtt ctggcgcgac gaattttagt 960
ttgcttaagc aagccggaga tgtggaggaa aatcctggac cgatgtgggg agttttcctt 1020
ctttatgttt ccatgaagat gggaggcact acaggacaaa acattgacca gcccactgag 1080
atgacagcta cggaaggtgc cattgtccag atcaactgca cgtaccagac atctgggttc 1140
aacgggctgt tctggtacca gcaacatgct ggcgaagcac ccacatttct gtcttacaat 1200
gttctggatg gtttggagga gaaaggtcgt ttttcttcat tccttagtcg gtctaaaggg 1260
tacagttacc tccttttgaa ggagctccag atgaaagact ctgcctctta cctctgtgct 1320
gtgagagatc aagaggaata ctctggggct gggagttacc aactcacttt cgggaagggg 1380
accaaactct cggtcatacc aaatattcag aatccggagc ctgccgtgta ccagctgaag 1440
gacccacgga gccaggattc caccctgtgc ctgttcacag actttgattc ccagatcaac 1500
gtgcccaaga ccatggagtc tggcaccttt atcacagaca agtgcgtgct ggacatgaag 1560
gccatggaca gcaagtccaa cggcgccatc gcctggtcca atcagacctc tttcacatgc 1620
caggatatct ttaaggagac aaacgccaca tatccatcct ctgacgtgcc ctgtgatgcc 1680
accctgacag agaagagctt cgagacagac atgaacctga attttcagaa cctgctggtc 1740
atcgtgctgc ggatcctgct gctgaaggtg gctgggttta atctgctgat gactctgaga 1800
ctgtggtcct cctgataa 1818
<210> 14
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gctcgactag cgggggacct 360
atccaagaga cccagtactt cgggccaggc acgcggctcc tggtgctcga ggacctgcgc 420
aacgtcaccc caccaaaggt cagtttgttt gagccatcaa aggcggagat cgccaacaaa 480
cagaaagcta cgctcgtgtg tttggctcgg ggcttcttcc cagaccacgt agaactttcc 540
tggtgggtca atggaaagga ggttcattca ggagtgtgca ctgatcccca agcgtacaag 600
gaatccaact atagctactg tctctcatct cggctccggg tgagtgcgac attctggcat 660
aatcctcgga accactttcg atgccaagtg cagtttcatg ggttgagcga ggaagacaag 720
tggcccgagg gcagtcctaa accagtcact caaaacataa gcgccgaggc atggggtaga 780
gccgattgtg ggattactag cgcttcatac caacaagggg tattgagcgc tacaattctt 840
tacgaaattc tcctcggcaa ggcgacgctc tacgccgtac tggtgtctac tctcgtggtt 900
atggcaatgg tgaaacggaa aaacagcaga gccaaaagaa gtggttctgg cgcgacgaat 960
tttagtttgc ttaagcaagc cggagatgtg gaggaaaatc ctggaccgat ggagaccctc 1020
ttgggcctgc ttatcctttg gctgcagctg caatgggtga gcagcaaaca ggaggtgacg 1080
cagattcctg cagctctgag tgtcccagaa ggagaaaact tggttctcaa ctgcagtttc 1140
actgatagcg ctatttacaa cctccagtgg tttaggcagg accctgggaa aggtctcaca 1200
tctctgttgc ttattcagtc aagtcagaga gagcaaacaa gtggaagact taatgcctcg 1260
ctggataaat catcaggacg tagtacttta tacattgcag cttctcagcc tggtgactca 1320
gccacctacc tctgtgctgg acacggttat aaccagggag gaaagcttat cttcggacag 1380
ggaacggagt tatctgtgaa acccaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 15
<211> 1803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gcttgggcac tgaagctttc 360
tttggacaag gcaccagact cacagttgta gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgatgaaat ccttgagagt tttactagtg 1020
atcctgtggc ttcagttgag ctgggtttgg agccaacaga aggaggtgga gcagaattct 1080
ggacccctca gtgttccaga gggagccatt gcctctctca actgcactta cagtgaccga 1140
ggttcccagt ccttcttctg gtacagacaa tattctggga aaagccctga gttgataatg 1200
ttcatatact ccaatggtga caaagaagat ggaaggttta cagcacagct caataaagcc 1260
agccagtatg tttctctgct catcagagac tcccagccca gtgattcagc cacctacctc 1320
tgtgccgcct gggggagcaa ctatcagtta atctggggcg ctgggaccaa gctaattata 1380
aagccaaata ttcagaatcc ggagcctgcc gtgtaccagc tgaaggaccc acggagccag 1440
gattccaccc tgtgcctgtt cacagacttt gattcccaga tcaacgtgcc caagaccatg 1500
gagtctggca cctttatcac agacaagtgc gtgctggaca tgaaggccat ggacagcaag 1560
tccaacggcg ccatcgcctg gtccaatcag acctctttca catgccagga tatctttaag 1620
gagacaaacg ccacatatcc atcctctgac gtgccctgtg atgccaccct gacagagaag 1680
agcttcgaga cagacatgaa cctgaatttt cagaacctgc tggtcatcgt gctgcggatc 1740
ctgctgctga aggtggctgg gtttaatctg ctgatgactc tgagactgtg gtcctcctga 1800
taa 1803
<210> 16
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgggcacta gcctgctgtg ctggatggcc ctgtgcctgc tcggcgccga tcacgccgac 60
accggcgtga gccaggaccc taggcacaag attacaaaga gggggcagaa cgtgacattc 120
cggtgcgatc ccattagcga gcataacagg ctctactggt accggcagac cctcggccag 180
ggccctgagt tcctgaccta ctttcagaac gaggcccagc tggagaagag caggctgctg 240
tccgaccggt ttagcgccga gaggcctaag ggcagcttct ccaccctgga gatccagcgc 300
acagagcagg gcgatagcgc catgtacctg tgcgccagca gctccaccag cggggggccc 360
atccaggaga cccagtactt cggccccggc acccggctgc tggtgctgga ggatctgcgg 420
aacgtgaccc cccctaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 480
cagaaggcca cactggtgtg cctggccagg gggttctttc ccgatcacgt ggagctgtcc 540
tggtgggtga acggcaagga ggtccacagc ggggtgtgca cagaccccca ggcctacaag 600
gagtccaact acagctactg cctcagcagc aggctgcggg tctccgccac attttggcac 660
aaccccagga accacttccg gtgccaggtg cagtttcacg gcctgagcga ggaggataag 720
tggcctgagg ggagccctaa gcccgtgacc cagaacatct ccgccgaggc ctggggcaga 780
gccgattgcg ggattactag cgcctcctac cagcagggcg tgctgagcgc caccattctc 840
tacgagatcc tgctcggcaa ggccaccctc tacgccgtgc tggtgtccac actggtcgtg 900
atggccatgg tgaagcggaa gaacagccgg gccaagcgga gcgggagcgg ggccaccaac 960
ttctccctgc tgaagcaggc cggcgacgtc gaggagaacc ccggccccat ggagactctg 1020
ctgggcctgc tgatcctgtg gctccagctg cagtgggtga gcagcaagca ggaggtgaca 1080
cagatccccg ccgccctgtc cgtgcccgag ggggagaacc tggtgctgaa ctgcagcttc 1140
acagatagcg ccatctacaa cctgcagtgg ttccggcagg accccggcaa gggcctgaca 1200
tccctgctcc tgatccagag cagccagcgg gagcagacta gcggcaggct caacgccagc 1260
ctggacaaga gcagcgggag gtccaccctg tacatcgccg ccagccagcc aggggatagc 1320
gccacttacc tgtgcgccgg ccacggctac aaccaggggg gcaagctgat tttcgggcag 1380
gggacagagc tgagcgtgaa gccaaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 17
<211> 1803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gcttgggcac tgaagctttc 360
tttggacaag gcaccagact cacagttgta gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgatgaaat ccttgagagt tttactagtg 1020
atcctgtggc ttcagttgag ctgggtttgg agccaacaga aggaggtgga gcagaattct 1080
ggacccctca gtgttccaga gggagccatt gcctctctca actgcactta cagtgaccga 1140
ggttcccagt ccttcttctg gtacagacaa tattctggga aaagccctga gttgataatg 1200
ttcatatact ccaatggtga caaagaagat ggaaggttta cagcacagct caataaagcc 1260
agccagtatg tttctctgct catcagagac tcccagccca gtgattcagc cacctacctc 1320
tgtgccgcct gggggagcaa ctatcagtta atctggggcg ctgggaccaa gctaattata 1380
aagccaaata ttcagaatcc ggagcctgcc gtgtaccagc tgaaggaccc acggagccag 1440
gattccaccc tgtgcctgtt cacagacttt gattcccaga tcaacgtgcc caagaccatg 1500
gagtctggca cctttatcac agacaagtgc gtgctggaca tgaaggccat ggacagcaag 1560
tccaacggcg ccatcgcctg gtccaatcag acctctttca catgccagga tatctttaag 1620
gagacaaacg ccacatatcc atcctctgac gtgccctgtg atgccaccct gacagagaag 1680
agcttcgaga cagacatgaa cctgaatttt cagaacctgc tggtcatcgt gctgcggatc 1740
ctgctgctga aggtggctgg gtttaatctg ctgatgactc tgagactgtg gtcctcctga 1800
taa 1803
<210> 18
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggt ccccatggaa 60
acgggagtta cgcagacacc aagacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatctggg tcataacgct atgtattggt acaagcaaag tgctaagaag 180
ccactggagc tcatgtttgt ctacagtctt gaagaacggg ttgaaaacaa cagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctcacttat tccttcacct acacaccctg 300
cagccagaag actcggccct gtatctctgc gccagcagcc caggagtggg ggaatataat 360
tcacccctcc actttgggaa cgggaccagg ctcactgtga cagaggacct gcgcaacgtc 420
accccaccaa aggtcagttt gtttgagcca tcaaaggcgg agatcgccaa caaacagaaa 480
gctacgctcg tgtgtttggc tcggggcttc ttcccagacc acgtagaact ttcctggtgg 540
gtcaatggaa aggaggttca ttcaggagtg tgcactgatc cccaagcgta caaggaatcc 600
aactatagct actgtctctc atctcggctc cgggtgagtg cgacattctg gcataatcct 660
cggaaccact ttcgatgcca agtgcagttt catgggttga gcgaggaaga caagtggccc 720
gagggcagtc ctaaaccagt cactcaaaac ataagcgccg aggcatgggg tagagccgat 780
tgtgggatta ctagcgcttc ataccaacaa ggggtattga gcgctacaat tctttacgaa 840
attctcctcg gcaaggcgac gctctacgcc gtactggtgt ctactctcgt ggttatggca 900
atggtgaaac ggaaaaacag cagagccaaa agaagtggtt ctggcgcgac gaattttagt 960
ttgcttaagc aagccggaga tgtggaggaa aatcctggac cgatgtgggg agttttcctt 1020
ctttatgttt ccatgaagat gggaggcact acaggacaaa acattgacca gcccactgag 1080
atgacagcta cggaaggtgc cattgtccag atcaactgca cgtaccagac atctgggttc 1140
aacgggctgt tctggtacca gcaacatgct ggcgaagcac ccacatttct gtcttacaat 1200
gttctggatg gtttggagga gaaaggtcgt ttttcttcat tccttagtcg gtctaaaggg 1260
tacagttacc tccttttgaa ggagctccag atgaaagact ctgcctctta cctctgtgct 1320
gtgagagatc aagaggaata ctctggggct gggagttacc aactcacttt cgggaagggg 1380
accaaactct cggtcatacc aaatattcag aatccggagc ctgccgtgta ccagctgaag 1440
gacccacgga gccaggattc caccctgtgc ctgttcacag actttgattc ccagatcaac 1500
gtgcccaaga ccatggagtc tggcaccttt atcacagaca agtgcgtgct ggacatgaag 1560
gccatggaca gcaagtccaa cggcgccatc gcctggtcca atcagacctc tttcacatgc 1620
caggatatct ttaaggagac aaacgccaca tatccatcct ctgacgtgcc ctgtgatgcc 1680
accctgacag agaagagctt cgagacagac atgaacctga attttcagaa cctgctggtc 1740
atcgtgctgc ggatcctgct gctgaaggtg gctgggttta atctgctgat gactctgaga 1800
ctgtggtcct cctgataa 1818
<210> 19
<211> 1821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atgggcacta gcctgctgtg ctggatggcc ctgtgcctgc tcggcgccga tcacgccgac 60
accggcgtga gccaggaccc taggcacaag attacaaaga gggggcagaa cgtgacattc 120
cggtgcgatc ccattagcga gcataacagg ctctactggt accggcagac cctcggccag 180
ggccctgagt tcctgaccta ctttcagaac gaggcccagc tggagaagag caggctgctg 240
tccgaccggt ttagcgccga gaggcctaag ggcagcttct ccaccctgga gatccagcgc 300
acagagcagg gcgatagcgc catgtacctg tgcgccagca gctccaccag cggggggccc 360
atccaggaga cccagtactt cggccccggc acccggctgc tggtgctgga ggatctgcgg 420
aacgtgaccc cccctaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 480
cagaaggcca cactggtgtg cctggccagg gggttctttc ccgatcacgt ggagctgtcc 540
tggtgggtga acggcaagga ggtccacagc ggggtgtgca cagaccccca ggcctacaag 600
gagtccaact acagctactg cctcagcagc aggctgcggg tctccgccac attttggcac 660
aaccccagga accacttccg gtgccaggtg cagtttcacg gcctgagcga ggaggataag 720
tggcctgagg ggagccctaa gcccgtgacc cagaacatct ccgccgaggc ctggggcaga 780
gccgattgcg ggattactag cgcctcctac cagcagggcg tgctgagcgc caccattctc 840
tacgagatcc tgctcggcaa ggccaccctc tacgccgtgc tggtgtccac actggtcgtg 900
atggccatgg tgaagcggaa gaacagccgg gccaagcgga gcgggagcgg ggccaccaac 960
ttctccctgc tgaagcaggc cggcgacgtc gaggagaacc ccggccccat ggagactctg 1020
ctgggcctgc tgatcctgtg gctccagctg cagtgggtga gcagcaagca ggaggtgaca 1080
cagatccccg ccgccctgtc cgtgcccgag ggggagaacc tggtgctgaa ctgcagcttc 1140
acagatagcg ccatctacaa cctgcagtgg ttccggcagg accccggcaa gggcctgaca 1200
tccctgctcc tgatccagag cagccagcgg gagcagacta gcggcaggct caacgccagc 1260
ctggacaaga gcagcgggag gtccaccctg tacatcgccg ccagccagcc aggggatagc 1320
gccacttacc tgtgcgccgg ccacggctac aaccaggggg gcaagctgat tttcgggcag 1380
gggacagagc tgagcgtgaa gccaaatatt cagaatccgg agcctgccgt gtaccagctg 1440
aaggacccac ggagccagga ttccaccctg tgcctgttca cagactttga ttcccagatc 1500
aacgtgccca agaccatgga gtctggcacc tttatcacag acaagtgcgt gctggacatg 1560
aaggccatgg acagcaagtc caacggcgcc atcgcctggt ccaatcagac ctctttcaca 1620
tgccaggata tctttaagga gacaaacgcc acatatccat cctctgacgt gccctgtgat 1680
gccaccctga cagagaagag cttcgagaca gacatgaacc tgaattttca gaacctgctg 1740
gtcatcgtgc tgcggatcct gctgctgaag gtggctgggt ttaatctgct gatgactctg 1800
agactgtggt cctcctgata a 1821
<210> 20
<211> 1803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60
actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120
aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180
ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240
agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300
acagagcagg gggactcggc catgtatctc tgtgccagca gcttgggcac tgaagctttc 360
tttggacaag gcaccagact cacagttgta gaggacctgc gcaacgtcac cccaccaaag 420
gtcagtttgt ttgagccatc aaaggcggag atcgccaaca aacagaaagc tacgctcgtg 480
tgtttggctc ggggcttctt cccagaccac gtagaacttt cctggtgggt caatggaaag 540
gaggttcatt caggagtgtg cactgatccc caagcgtaca aggaatccaa ctatagctac 600
tgtctctcat ctcggctccg ggtgagtgcg acattctggc ataatcctcg gaaccacttt 660
cgatgccaag tgcagtttca tgggttgagc gaggaagaca agtggcccga gggcagtcct 720
aaaccagtca ctcaaaacat aagcgccgag gcatggggta gagccgattg tgggattact 780
agcgcttcat accaacaagg ggtattgagc gctacaattc tttacgaaat tctcctcggc 840
aaggcgacgc tctacgccgt actggtgtct actctcgtgg ttatggcaat ggtgaaacgg 900
aaaaacagca gagccaaaag aagtggttct ggcgcgacga attttagttt gcttaagcaa 960
gccggagatg tggaggaaaa tcctggaccg atgatgaaat ccttgagagt tttactagtg 1020
atcctgtggc ttcagttgag ctgggtttgg agccaacaga aggaggtgga gcagaattct 1080
ggacccctca gtgttccaga gggagccatt gcctctctca actgcactta cagtgaccga 1140
ggttcccagt ccttcttctg gtacagacaa tattctggga aaagccctga gttgataatg 1200
ttcatatact ccaatggtga caaagaagat ggaaggttta cagcacagct caataaagcc 1260
agccagtatg tttctctgct catcagagac tcccagccca gtgattcagc cacctacctc 1320
tgtgccgcct gggggagcaa ctatcagtta atctggggcg ctgggaccaa gctaattata 1380
aagccaaata ttcagaatcc ggagcctgcc gtgtaccagc tgaaggaccc acggagccag 1440
gattccaccc tgtgcctgtt cacagacttt gattcccaga tcaacgtgcc caagaccatg 1500
gagtctggca cctttatcac agacaagtgc gtgctggaca tgaaggccat ggacagcaag 1560
tccaacggcg ccatcgcctg gtccaatcag acctctttca catgccagga tatctttaag 1620
gagacaaacg ccacatatcc atcctctgac gtgccctgtg atgccaccct gacagagaag 1680
agcttcgaga cagacatgaa cctgaatttt cagaacctgc tggtcatcgt gctgcggatc 1740
ctgctgctga aggtggctgg gtttaatctg ctgatgactc tgagactgtg gtcctcctga 1800
taa 1803
<210> 21
<211> 1809
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60
gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120
cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180
gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240
aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300
gctccctccc agacatctgt gtacttctgt gccagcagaa accgacagac ccaagagacc 360
cagtacttcg ggccaggcac gcggctcctg gtgctcgagg acctgcgcaa cgtcacccca 420
ccaaaggtca gtttgtttga gccatcaaag gcggagatcg ccaacaaaca gaaagctacg 480
ctcgtgtgtt tggctcgggg cttcttccca gaccacgtag aactttcctg gtgggtcaat 540
ggaaaggagg ttcattcagg agtgtgcact gatccccaag cgtacaagga atccaactat 600
agctactgtc tctcatctcg gctccgggtg agtgcgacat tctggcataa tcctcggaac 660
cactttcgat gccaagtgca gtttcatggg ttgagcgagg aagacaagtg gcccgagggc 720
agtcctaaac cagtcactca aaacataagc gccgaggcat ggggtagagc cgattgtggg 780
attactagcg cttcatacca acaaggggta ttgagcgcta caattcttta cgaaattctc 840
ctcggcaagg cgacgctcta cgccgtactg gtgtctactc tcgtggttat ggcaatggtg 900
aaacggaaaa acagcagagc caaaagaagt ggttctggcg cgacgaattt tagtttgctt 960
aagcaagccg gagatgtgga ggaaaatcct ggaccgatgc tcctgctgct cgtcccagtg 1020
ctcgaggtga tttttactct gggaggaacc agagcccagt cggtgaccca gcttgacagc 1080
cacgtctctg tctctgaagg aaccccggtg ctgctgaggt gcaactactc atcttcttat 1140
tcaccatctc tcttctggta tgtgcaacac cccaacaaag gactccagct tctcctgaag 1200
tacacatcag cggccaccct ggttaaaggc atcaacggtt ttgaggctga atttaagaag 1260
agtgaaacct ccttccacct gacgaaaccc tcagcccata tgagcgacgc ggctgagtac 1320
ttctgtgttg tggctgccca caataacaat gacatgcgct ttggagcagg gaccagactg 1380
acagtaaaac caaatattca gaatccggag cctgccgtgt accagctgaa ggacccacgg 1440
agccaggatt ccaccctgtg cctgttcaca gactttgatt cccagatcaa cgtgcccaag 1500
accatggagt ctggcacctt tatcacagac aagtgcgtgc tggacatgaa ggccatggac 1560
agcaagtcca acggcgccat cgcctggtcc aatcagacct ctttcacatg ccaggatatc 1620
tttaaggaga caaacgccac atatccatcc tctgacgtgc cctgtgatgc caccctgaca 1680
gagaagagct tcgagacaga catgaacctg aattttcaga acctgctggt catcgtgctg 1740
cggatcctgc tgctgaaggt ggctgggttt aatctgctga tgactctgag actgtggtcc 1800
tcctgataa 1809

Claims (7)

1. A method of screening for a tumor specific TCR, comprising the steps of:
(1) activating tumor infiltrating T cells TILs corresponding to autologous tumor cells of a tumor patient;
(2) sequencing a single cell transcriptome and a TCR set of the activated TILs to obtain a TCR sequence of each T cell;
(3) for all TILs expressing the same TCR, taking the average value of the expression values of 10 marker mRNAs as the activation score of the TCR, and screening the first three TCRs with the highest scores as TCRs corresponding to tumor specific T cells according to the TCR activation score;
the 10 markers are respectively: IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF 1;
the method for calculating the activation score of each TILs in the step (3) comprises the following steps: inputting the normalized expression values of 10 marker mRNAs of each TILs into Seurat AddModule software, calculating the activation score of each TILs according to default parameters of the software, wherein the activation score is the average expression value of the normalized expression values of the 10T cell markers subtracted by a background value, each TILs can calculate an activation score, and then averaging the activation scores of all TILs with the same TCR to obtain the value of the TCR activation score.
2. Use of a TCR selected for use according to claim 1 in the preparation of a TCR-T cell.
3. A marker composition for screening tumor specific TCRs, consisting of the following 10 genes: IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF 1.
4. Use of the marker composition of claim 3 for screening tumor-specific TCRs in vitro and/or for increasing the efficiency and accuracy of tumor-specific TCR screening.
5. Use of a marker composition according to claim 3 in the preparation of TCR-T cells.
6. Use of a reagent composition for detecting the expression level of each marker in the marker composition of claim 3 for screening tumor-specific TCRs.
7. Use of a reagent composition for detecting the expression level of each marker in a marker composition according to claim 3 in the preparation of TCR-T cells.
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