CN104327160A - Mycobacterium tuberculosis specific CD8+T cell epitope peptide P15 and application thereof - Google Patents
Mycobacterium tuberculosis specific CD8+T cell epitope peptide P15 and application thereof Download PDFInfo
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Abstract
The invention discloses a mycobacterium tuberculosis specific CD8+T cell epitope peptide and an application thereof. The amino acid sequence of the epitope peptide is GTHPTTTYK, and the epitope peptide has high affinity with HLA-A*1101 molecules, and can be used for activating CD8+T cells and inducing the CD8+T cells to perform proliferation and secrete IFN-gamma. The determination of the epitope peptide disclosed by the invention can provide a theoretical basis and new solutions for the research and development of tuberculosis vaccines, diagnostic reagents and treatment medicaments, and has important significance in prevention and treatment of tuberculosis.
Description
Technical field
The invention belongs to immunological technique field, particularly mycobacterium tuberculosis specific C D8
+t cell epitope peptide and application thereof.
Background technology
Tuberculosis is that case fatality rate is only second to acquired immune deficiency syndrome (AIDS) by the transmissible disease of bringing out after m tuberculosis infection, global infection rate is the highest.Only 2012, the new tuberculosis patient of sending out in the whole world reached 8,600,000, dead 1,300,000.The new feature of tuberculosis epidemic situation is: resistant tuberculosis example occur and in the gesture spread; Tuberculosis and acquired immune deficiency syndrome (AIDS) coinfection situation grow in intensity.For this two classes patient, there is no effectively preventing measure at present.China is your pp of one of the heaviest country of tuberculosis burden m in the world, and research and development are applicable to the tuberculosis vaccine of Chinese population and medicine, and time and tide wait for no man.
Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is born of the same parents' endophytes, effectively controls to depend on cellular immunization, especially T cell immunity to it.In human body, T cell is mainly divided into CD4
+t (Th) cell and CD8
+the large subgroup of T (CTL) cell two, large quantity research proves, these two kinds of T cell subgroups all have vital role, wherein CD8 for control tubercle bacillus affection
+cTL removes the main force of tubercule bacillus especially.
The tubercule bacillus that T cell None-identified is complete and proteantigen, but identify the tuberculosis epitope peptide discharged after antigen presenting cell picked-up and processing.Epitope peptide is the core part causing immunne response in antigen, also referred to as antigenic determinant, it is combined with the HLA molecule on antigen presenting cell surface and forms peptide-HLA mixture and be delivered and express on antigen presenting cell surface, and then activating T cell, brings out t cell immune response.CD4
+the epitope peptide of T cell identification HLAII quasi-molecule submission, and CD8
+the epitope peptide of T cell identification HLAI quasi-molecule submission.T cell is to the identification of epitope peptide and activate request epitope peptide thereof and antigen presenting cell surface HLA molecules stable bond, and the avidity of peptide and HLA molecule is the key forming peptide-HLA mixture, therefore medicine and the vaccine of T cell immunity is brought out in preparation, first step is the peptide filtering out high-affinity, and then identifies and have immunocompetent epitope peptide.
Epitope peptide can be obtained by synthetic completely, and the R&D cycle is short; Owing to eliminating other objectionable constituent of pathogenic agent beyond epitope peptide, security is good; Multiple epitope peptide series connection can also be used, booster immunization effect and minimizing immunologic escape.Tubercule bacillus CTL epitope peptide known at present is mainly identified in American-European crowd, mostly is HLA-A*0201 restricted, and for the most serious Africa of TB endemic and South East Asia crowd, suitable epitope peptide can not use.In China, HLA-A*1101 is the HLA molecule that in crowd, gene frequency is the highest, and the epitope peptide vaccine that qualification HLA-A*1101 restricted CTL epitope is applicable to Chinese population for research and development has very important significance.
Summary of the invention
The object of the present invention is to provide mycobacterium tuberculosis specific C D8
+t cell epitope peptide.
Another object of the present invention is to provide this mycobacterium tuberculosis specific C D8
+t cell epitope peptide is preparing the application in tuberculosis vaccine, diagnostic reagent and medicine.
The technical solution used in the present invention is:
The invention provides with HLA-A*1101 molecule avidity high, can CD8 be activated
+t cell, induce its propagation and mycobacterium tuberculosis specific C D8 of secretion of gamma-IFN
+t cell epitope peptide, the aminoacid sequence of described epitope peptide is: GTHPTTTYK (SEQ ID NO.15) (that is: Gly-Thr-His-Pro-Thr-Thr-Thr-Tyr-Lys).
The present invention is in conjunction with Bioinformatics Prediction method and uv induction peptide displacement experiment, utilize the antigen selection of mycobacterium tuberculosis self to go out to have the epitope peptide of anti-tubercular, detect the avidity between epitope peptide GTHPTTTYK (SEQ ID NO.15) and HLA-A*1101 molecule efficiently and accurately.This is the restricted mycobacterium tuberculosis epitope peptide of HLA-A*1101 that is new, that be not in the news, by tuberculosis patient CD8
+t cell extensively identifies.Because HLA-A*1101 genotype is the genotype of the most extensively carrying in Chinese, do not find the HLA-A*1101 restricted CTL epitope peptide that mycobacterium tuberculosis is special at present, the determination of epitope peptide of the present invention, research and development for tuberculosis vaccine, diagnostic reagent and medicine provide theoretical basis and new solution, are significant to control lungy.
Accompanying drawing explanation
Fig. 1 is candidate's epitope peptide and HLA-A*1101 molecule avidity schematic diagram.
Fig. 2 is that the epitope peptide of 22 high-affinities induces tubercular's T cell activation and the schematic diagram of secretion of gamma-IFN.
Fig. 3 is the schematic diagram that ELISPOT detects that P15 induces tubercular's T cell secretion of gamma-IFN.
Fig. 4 is the mass spectroscopy figure of P15.
Fig. 5 is that the lymphocyte proliferation assay of CFSE mark detects P15 induction tubercular CD8
+the result schematic diagram of T cell propagation.
Embodiment
The present invention is according to the primary structure of antigen, adopt Immunoinformatics means, the HLA-A*1101 restricted CTL epitope peptide of NetMHCcons Antigen Epitope Prediction software to antigen of mycobacterium tuberculosis albumen in CBS database is used to carry out forecast analysis, then the avidity adopting uv induction peptide displacement examination candidate peptide to be combined with HLA-A*1101 molecule and stability, screening obtains epitope peptide, identified by experiment in vitro, CTL epi-position CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS can induce CD8
+t cell produces strong cellullar immunologic response, secreting high levels IFN-γ, and induces CD8
+there is significantly propagation in T cell.
Content of the present invention is set forth further below in conjunction with specific embodiment.
Applying biological Informatics Method, prediction HLA-A*1101 restricted epitope peptide:
According to bibliographical information, choosing 94 has stronger immunogenic antigen of mycobacterium tuberculosis albumen (as shown in table 1), obtains the aminoacid sequence of each antigen protein from GeneBank.
Table 1 candidate antigen of mycobacterium tuberculosis albumen
| MTB antigen | |||
| Rv0079 | Rv1478 | Rv2030c | Rv3044 |
| Rv0288 | Rv1569 | Rv2031c | Rv3127 |
| Rv0315 | Rv1626 | Rv2032 | Rv3130c |
| Rv0350 | Rv1733c | Rv2034 | Rv3131 |
| Rv0440 | Rv1738 | Rv2108 | Rv3132c |
| Rv0467 | Rv1787 | Rv2220 | Rv3347c |
| Rv0475 | Rv1788 | Rv2244 | Rv3353c |
| Rv0496 | Rv1789 | Rv2324 | Rv3418c |
| Rv0577 | Rv1790 | Rv2380c | Rv3420c |
| Rv0685 | Rv1791 | Rv2389c | Rv3478 |
| Rv0733 | Rv1793 | Rv2450c | Rv3615c |
| Rv0754 | Rv1813c | Rv2468c | Rv3619c |
| Rv0824c | Rv1837c | Rv2608 | Rv3716c |
| Rv0831 | Rv1860 | Rv2620c | Rv3803c |
| Rv0867c | Rv1884c | Rv2623 | Rv3804c |
| Rv0932c | Rv1886c | Rv2626c | Rv3873 |
| Rv0934 | Rv1908c | Rv2627c | Rv3874 |
| Rv0978c | Rv1926c | Rv2628 | Rv3875 |
| Rv1009 | Rv1956 | Rv2629 | Rv3914 |
| Rv1130 | RV1966 | Rv2744c | |
| Rv1169c | Rv1980c | Rv2770c | |
| Rv1174c | Rv1996 | Rv2780 | |
| Rv1349 | Rv2005c | Rv2875 | |
| Rv1363c | Rv2006 | Rv2903c | |
| Rv1411 | Rv2029c | Rv3020c |
From HLA gene frequency database AFND (Allele Frequency Net Database, AFND) each genotypic frequency distribution information in upper acquisition Chinese population HLA-A site, wherein HLA-A*1101 gene frequency is about 28%, is the individual gene type that HLA-A site upper frequency is the highest.
Use the HLA-A*1101 restricted CTL epitope of bioinformatics prediction contained by antigen protein, the algorithm of employing is the NetMHCcons in CBS database.To predict the outcome according to IC50 value and sort, IC50 value is lower, illustrate epitope peptide and HLA molecule avidity higher.Thus, dope HLA-A*1101 restricted epitope peptide in 94 antigen proteins, and filter out 50 the highest epitope peptides of wherein avidity, alternatively, as shown in table 2.
The restricted candidate's epitope peptide of table 2 50 HLA-A*1101
| Candidate's epitope peptide is numbered | Aminoacid sequence | Antigen protein | IC50(nM) |
| p1 | MTNTLHSMLK(SEQ ID NO.1) | Rv3478 | 4.67 |
| p2 | KTVTFMPK(SEQ ID NO.2) | Rv2220 | 5.29 |
| p3 | TTLDAGFLK(SEQ ID NO.3) | Rv3130c | 5.9 |
| p4 | VVMTTTLSK(SEQ ID NO.4) | Rv1569 | 6.16 |
| p5 | ISSGVFLLK(SEQ ID NO.5) | Rv2029c | 6.4 |
| p6 | VSIPTLILFK(SEQ ID NO.6) | Rv3914 | 6.97 |
| p7 | SSMTRIAK(SEQ ID NO.7) | Rv1884c | 7.16 |
| p8 | MLFSMHGELYK(SEQ ID NO.8) | Rv1196c | 7.36 |
| p9 | TAMRVTTMK(SEQ ID NO.9) | Rv1009 | 7.69 |
| p10 | STIDEFAK(SEQ ID NO.10) | Rv0496 | 7.73 |
| p11 | TTSPIPLK(SEQ ID NO.11) | Rv3347c | 7.94 |
| p12 | AVMAGIVRAAK(SEQ ID NO.12) | Rv3130c | 8.07 |
| p13 | VSIARALLK(SEQ ID NO.13) | Rv1349 | 8.11 |
| p14 | AVAAPAFAEK(SEQ ID NO.14) | Rv1790 | 8.16 |
| p15 | GTHPTTTYK(SEQ ID NO.15) | Rv1980c | 8.25 |
| p16 | TTSNVSVAK(SEQ ID NO.16) | Rv0867c | 8.25 |
| p17 | AVNTLFEK(SEQ ID NO.17) | Rv3873 | 8.34 |
| p18 | KVNRFPDPK(SEQ ID NO.18) | Rv3131 | 8.34 |
| p19 | MTSGSSSGFK(SEQ ID NO.19) | Rv3347c | 8.34 |
| p20 | GTQAVVLK(SEQ ID NO.20) | Rv1980c | 8.52 |
| p21 | SVNNYQASK(SEQ ID NO.21) | Rv2006 | 8.61 |
| p22 | ATIAKFQK(SEQ ID NO.22) | Rv0467 | 8.8 |
| p23 | MTSLDFNK(SEQ ID NO.23) | Rv1363c | 8.8 |
| p24 | VVMPVLKK(SEQ ID NO.24) | Rv0824 | 8.94 |
| p25 | GTFKSVAVK(SEQ ID NO.25) | Rv0440 | 9.65 |
| p26 | AVARLVAISK(SEQ ID NO.26) | Rv3130c | 9.86 |
| p27 | TTLTAAITK(SEQ ID NO.27) | Rv0685 | 9.86 |
| p28 | RVLGANYK(SEQ ID NO.28) | Rv1908c | 10.07 |
| p29 | ATFAEIGHK(SEQ ID NO.29) | Rv2324 | 10.69 |
| p30 | KTFGFGFGR(SEQ ID NO.30) | Rv1908c | 11.11 |
| p31 | KTQGPGAWPK(SEQ ID NO.31) | Rv2389c | 11.6 |
| p32 | KTYCEELK(SEQ ID NO.32) | Rv1980c | 11.66 |
| p33 | SVQMTLSK(SEQ ID NO.33) | Rv1411 | 11.79 |
| p34 | TVFDYHNENAK(SEQ ID NO.34) | Rv2108 | 11.79 |
| p35 | VSNAVRHAK(SEQ ID NO.35) | Rv3132c | 12.24 |
| p36 | CVANMPASVPK(SEQ ID NO.36) | Rv2780 | 12.58 |
| p37 | RVQTTVLK(SEQ ID NO.37) | Rv2108 | 12.64 |
| p38 | VSNSLVAHMK(SEQ ID NO.38) | Rv2780 | 12.64 |
| p39 | AVAVVLHK(SEQ ID NO.39) | Rv2380c | 12.99 |
| p40 | HTVALFLDK(SEQ ID NO.40) | Rv2032 | 12.99 |
| p41 | AIAADPSFK(SEQ ID NO.41) | Rv1956 | 13.06 |
| p42 | MTQWLIEEK(SEQ ID NO.42) | Rv2030c | 13.13 |
| p43 | GTSDNFQK(SEQ ID NO.43) | Rv0932c | 13.2 |
| p44 | KAFAPAHAGK(SEQ ID NO.44) | Rv1130 | 13.71 |
| p45 | RLIPFAAPPK(SEQ ID NO.45) | Rv1787 | 13.71 |
| p46 | TVINASRFK(SEQ ID NO.46) | Rv2380c | 13.79 |
| p47 | VVNKIRGTFK(SEQ ID NO.47) | Rv0440 | 13.94 |
| p48 | ATTAFGAK(SEQ ID NO.48) | Rv1966 | 14.09 |
| p49 | QANAHGQK(SEQ ID NO.49) | Rv1037c | 326.55 |
| p50 | NNMAQTDSAV(SEQ ID NO.50) | Rv1037c | 29745 |
Application uv induction peptide displacement experimental technique, detects the avidity between candidate's epitope peptide and HLA-A*1101 molecule and combination stability:
Experimentation is as follows:
Containing the peptide-HLA mixture of sensitivity of light epitope peptide under uviolizing, sensitivity of light epitope peptide ruptures, depart from from HLA molecule, at this moment in reaction system, candidate's epitope peptide to be detected is added, if candidate's epitope peptide and HLA molecule avidity high, then can form new stable epitope peptide-HLA mixture.Use anti-beta 2m antibody capture HLA molecule, ELISA detects the new epitope peptide-HLA complex concentration formed, and evaluates the avidity size between epitope peptide and HLA molecule.Select the epitope peptide (being greater than 100%) 22 with HLA-A*1101 with high-affinity altogether, see shown in Fig. 1 and table 3.
Table 3 and HLA-A*1101 have 22 epitope peptides of high-affinity
ELISpot tests detection 22 epitope peptide inducing T cell secretion of gamma-IFN, the immunological effect of preliminary identification epitope peptide:
Experimentation is as follows:
Density gradient method is adopted to be separated HLA-A*1101 genotype tubercular's peripheral blood mononuclear cell (peripheral blood mononuclear, PBMC).The 20ml peripheral blood PBS of collection is diluted one times, slowly add above lymphocyte separation medium, both volume ratios are 2:1, then with the centrifugal 30min of 800g/min rotating speed, centrifugal rear liquid is divided into three layers, wherein PBMC is between the first layer and the second layer, become tunica albuginea shape, PBMC is carefully drawn in new centrifuge tube with dropper, add 5 times with upper volume PBS washing, the centrifugal 10min of 800g/min, repeated washing once, with frozen storing liquid (90%FBS+10%DMSO) freeze-stored cell, liquid nitrogen storage.
ELISpot tests: recovery HLA-A*1101 genotype patient PBMC, at 37 DEG C, and 5%CO
2hold over night in incubator.Spend the dead cell in dead cell magnetic bead removal PBMC.Cell counting, perfect medium re-suspended cell to 1.25 × 10
6200 μ L/ porocyte suspensions are spread into ELISpot plate (namely 2.5 × 10 by individual/mL
5individual/hole), totally 48 holes.Add in 44 holes by the candidate peptide of 22 shown in table 3 respectively by 10 μ g/mL concentration, every hole adds a kind of peptide, and often kind of peptide spreads 2 holes, and 2 Positive control wells add 4 μ g/mLPHA respectively, and 2 negative control holes do not add any stimulation, fill full training.After each hole has added sample respectively, culture plate is placed in cell culture incubator, 37 DEG C, 5%CO
2cultivate 24h.Take out ELISpot plate, PBS washs 5 times, and add enzyme mark anti-IFN-gamma antibodies 100ul after patting dry, hatch 2h, PBS washs 5 times, adds developer 100ul after patting dry, and occurs, after obvious spot, using ultrapure water termination reaction until Positive control wells; Dry, read plate, counting, correct.Peptide-specific T-cell frequency is with SFC/10
6represent.
Positive reaction judging criterion is: 1. SFC/10
6>50,2. peptide stimulates hole spot number to be more than or equal to the twice of negative control hole.Meet 1. 2., reaction and judgement is positive simultaneously.
Add up 23 HLA-A*1101 type tubercular PBMC to the recognition reaction of epitope peptide, namely patient P BMC is to the identification frequency of epitope peptide.The detection Ren Shuo ╳ 100% of peptide recognition rate=positive reaction number/total.Filtering out at least can by the epitope peptide of 20% patient identification, and result as shown in Figure 2.Fig. 3 is the result schematic diagram that ELISPOT detects epi-position CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS induction tuberculosis patient T cell secretion of gamma-IFN.
The lymphocyte proliferation assay of CFSE mark detects epi-position CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS induction CD8
+t cell is bred:
Experimentation is as follows:
Recovery HLA-A*1101 type tubercular PBMC, hold over night.Make to spend dead cell magnetic bead and remove dead cell, counting, centrifugal rear PBS re-suspended cell to 1 ╳ 10
7individual/mL, add 5 μMs of CFSE marks, after mixing, lucifuge hatches 10min.The perfect medium (10%FBS+RPMI-1640) adding 5 times of volume precoolings stops dye marker.The centrifugal 10min of 300g, with perfect medium washing, repeats twice.Again count, with perfect medium re-suspended cell to 1 ╳ 10
6individual/mL.Cell suspension is spread in 96 hole U floor cells culture plates, 200 μ L/ hole, totally 8 holes, the each hole of experimental group adds 10 μ g/mL epi-position CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYSs (Fig. 4 is the mass spectroscopy figure of P15) respectively, Positive control wells adds 4 μ g/mLPHA, and negative control hole does not add any stimulator, supplies full training.At fluorescence microscopy Microscopic observation, determine that cell is by CFSE mark (green fluorescence).37 DEG C, 5%CO
248h is cultivated in incubator.Add 100IUIL-2 respectively in each hole, continue to be cultured to 7d, take out cell, mark anti-CD8 antibody after washing, Flow cytometry.
According to the feature of CFSE dye marker, dyestuff is assigned to daughter cell from parent cell with cell proliferation equably, and fluorescence intensity reduces by half.So the cell of weak CFSE fluorescence, is the cell after propagation.Epitope peptide induction CD8
+t cell multiplication capacity is by the CD8 of weak CFSE fluorescence
+t cell/CD8
+t cell represents.Detected result as shown in Figure 5.
Through the epi-position CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS that above option screening is identified, can be identified by HLA-A*1101 type tubercular specifically, and induce CD8
+t cell propagation and secretion of gamma-IFN, the research and development for tuberculosis vaccine, diagnostic reagent and medicine provide theoretical basis and new solution, are significant to control lungy.
<110> Nanfang Medical Univ
<120> mycobacterium tuberculosis specific C D8+T cell epitope CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS and application thereof
<130>
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Claims (3)
1. mycobacterium tuberculosis specific C D8+T cell epitope peptide, the aminoacid sequence of described epitope peptide is: GTHPTTTYK(SEQ ID NO.15).
2. mycobacterium tuberculosis specific C D8+T cell epitope CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS according to claim 1 is preparing the application in diagnostic reagent of tuberculosis and treatment tubercular drugs.
3. mycobacterium tuberculosis specific C D8+T cell epitope CYS-TRP-MET-SER-PRO-ARG-HIS-LEU-GLY-THR-CYS according to claim 1 is preparing the application in tuberculosis prophylaxis vaccine.
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| CN117304284A (en) * | 2023-08-07 | 2023-12-29 | 梅州市人民医院(梅州市医学科学院) | Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009039854A2 (en) * | 2007-09-27 | 2009-04-02 | Dako Denmark A/S | Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009039854A2 (en) * | 2007-09-27 | 2009-04-02 | Dako Denmark A/S | Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117304284A (en) * | 2023-08-07 | 2023-12-29 | 梅州市人民医院(梅州市医学科学院) | Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide and application thereof |
| CN117304284B (en) * | 2023-08-07 | 2024-03-19 | 梅州市人民医院(梅州市医学科学院) | Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide and application thereof |
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