CN103172703A - Tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL (Cytotoxic T Lymphocyte) epitope peptide and application thereof - Google Patents
Tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL (Cytotoxic T Lymphocyte) epitope peptide and application thereof Download PDFInfo
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- CN103172703A CN103172703A CN2013101014825A CN201310101482A CN103172703A CN 103172703 A CN103172703 A CN 103172703A CN 2013101014825 A CN2013101014825 A CN 2013101014825A CN 201310101482 A CN201310101482 A CN 201310101482A CN 103172703 A CN103172703 A CN 103172703A
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Abstract
The invention discloses a tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL (Cytotoxic T Lymphocyte) epitope peptide which is a nonapeptide, wherein the amino acid sequence of the nonapeptide is P10: MLIAGLPCL. According to the tuberculosis-resisting-drug-related efflux-protein-sourced tuberculosis resisting CTL epitope peptide, immunological informatics means are adopted according to a primary structure of an antigen, an HLA-A*0201-restrictive CTL epitope of a tuberculosis-resisting-drug-related protein antigen is subjected to predictive analysis by using databases, namely SYFPEITHI, BIMAS and NetCTL1.2, the epitope peptide is obtained through screening, and assayed nonapeptides are not reported in documents. The epitope peptide is assayed through in-vitro ELISPOT experiments, and the results provide a theoretical basis for the development of tuberculosis vaccines based on resisting-drug-associated protein antigens and provide more information for designing tuberculosis multi-epitope peptide vaccines based on mixed T cell epitopes.
Description
Patent application of the present invention is number of patent application: 201110138646.2, and the applying date: on 05 26th, 2011, denomination of invention was divided an application for " the relevant efflux protein source anti-tuberculosis CTL epitope peptide of Drug Resistance for Tuberculosis and application thereof ".
Technical field
The present invention relates to the tuberculotherapy polypeptide vaccine, especially relate to the anti-tuberculosis CTL epitope peptide with therapeutic activity that utilizes tubercule bacillus self resistance related antigen to filter out, the invention still further relates to the application of this peptide in preparation tuberculotherapy polypeptide vaccine.
Background technology
Day by day increasing of Drug-Resistant Mycobacterium tuberculosis quantity is the major reason that causes in recent years that tuberculosis revives, stages a comeback.The main minute natural drug resistance of drug resistance of Mycobacterium tuberculosis mechanism is machine-processed and acquired resistance is machine-processed.Acquired resistance is mainly the point mutation due to drug resistance related gene, and natural drug resistance mechanism is mainly due to the permeability obstacle of cell walls with enliven extra-pumping system.Therefore, the drug efflux system can become important resistance tuberculosis treatment target spot.
Find in recent years CD8 in animal model and clinical study
+CTL plays a part important and unique in the resisting tuberculosis infection protective response, CD8
+CTL brings into play lethal effect to target cell, must be able to identify the surperficial specific antigens epi-position of being combined with corresponding MHC-I quasi-molecule of target cell, i.e. cytotoxic T lymphocyte (Cytotoxic T lymphocyte, CTL) epi-position.The CD8 that studies at present
+The CTL Protein Epitopes mainly concentrates on the special secretory protein of tubercule bacillus, as ESAT-6, CFP-10, CFP-21, MPT64, Ag85 mixture etc., and for membranin especially drug efflux PROTEIN C D8+CTL epi-position report seldom.
Summary of the invention
The object of the present invention is to provide the anti-tuberculosis CTL epitope peptide in the relevant efflux protein of paratuberculosis resistance source, the present invention also provides the application of such peptide in preparation tuberculotherapy polypeptide vaccine.
For achieving the above object, the present invention can take following technical proposals:
The relevant efflux protein of Drug Resistance for Tuberculosis of the present invention source anti-tuberculosis CTL epitope peptide is nonapeptide, and the aminoacid sequence of described nonapeptide is:
P10：Met-Leu-Ile-Ala-Gly-Leu-Pro-Cys-Leu。
The application of the relevant efflux protein of Drug Resistance for Tuberculosis of the present invention source anti-tuberculosis CTL epitope peptide in preparation tuberculotherapy polypeptide vaccine.
The present invention is the primary structure according to antigen, adopt the Immunoinformatics means, use SYFPEITHI, BIMAS and NetCTL 1.2 databases to carry out forecast analysis to the HLA-A*0201 restricted CTL epitope of Drug Resistance for Tuberculosis associated protein antigen, screening obtains epitope peptide.Then by external ELISPOT experiment, the prediction epitope peptide is identified.The present invention adopt method preliminary evaluation theoretical and that experiment combines go out can external evoked CTL epitope peptide, the nonapeptide of identifying has no bibliographical information, for the Vaccinum Calmette-Guerini of developing based on drug resistance-associated proteins antigen provides theoretical basis, and provide more information for designing based on the tuberculosis multi-epitope peptide vaccine that mixes t cell epitope.
Description of drawings
Fig. 1-8th, the mass spectroscopy collection of illustrative plates of epitope peptide of the present invention.
The ability of the specific CTL secretion of gamma-IFN that induce Fig. 9-16th, epitope peptide of the present invention.
Embodiment
The relevant efflux protein of Drug Resistance for Tuberculosis of the present invention source anti-tuberculosis CTL epitope peptide is nonapeptide, and the aminoacid sequence of described nonapeptide is:
P5:Tyr-Leu-Gly-Gly-Thr-Thr-Gly-Pro-Val (YLGGTTGPV) molecular weight: 864.6(theoretical value: 863.44)
Or P6:Tyr-Ile-Val-Gly-Phe-Cys-Leu-Leu-Val (YIVGFCLLV) molecular weight: 1026.7(theoretical value: 1025.86)
Or P7:Thr-Leu-Thr-Trp-Leu-Phe-Ala-Phe-Val (TLTWLFAFV) molecular weight: 1097.7(theoretical value: 1097.32)
Or P8:Gly-Leu-Val-Ala-Gly-Leu-Ser-Ala-Val (GLVAGLSAV) molecular weight: 786.7(theoretical value: 786.7)
Or P9:Ala-Leu-Gly-Met-Leu-Ile-Ala-Gly-Leu (ALGMLIAGL) molecular weight: 858.7(theoretical value: 857.81)
Or P10:Met-Leu-Ile-Ala-Gly-Leu-Pro-Cys-Leu (MLIAGLPCL) molecular weight: 930.6(theoretical value: 930.24)
Or P11:Leu-Leu-Cys-Ala-Ile-Phe-Ala-Glu-Val (LLCAIFAEV) molecular weight: 978.6(theoretical value: 977.52)
Or P12:Arg-Leu-Trp-Pro-Thr-Val-Gly-Cys-Leu (RLWPTVGCL) molecular weight: 1044.6(theoretical value: 1043.56).
The present invention mainly adopts the method for integrating theory with practice, primary structure according to antigen, adopt the Immunoinformatics means, use SYFPEITHI, BIMAS with NetCTL 1.2 databases, the HLA-A*0201 restricted CTL epitope of the relevant efflux protein of resistance to be carried out forecast analysis, screening obtains epitope peptide and adopts the Fmoc scheme of standard to synthesize, after the HPLC purifying, its purity is greater than 90%, and mass spectroscopy also confirms that its molecular weight meets theoretical value.Each peptide Mass Spectrometric Identification figure sees Fig. 1-8.
Synthetic and the preparation of epitope peptide of the present invention: adopt the synthetic CTL epitope peptide of solid-phase synthesis.Basic procedure is as follows: at first an amino is connected on insoluble solid phase carrier Wang resin by the amino acid of Fmoc radical protection, and the protecting group of desamidizate then, first amino acid namely is connected on solid phase carrier; Secondly amino is activated with condensing agent by second amino acid whose carboxyl of Fmoc radical protection; amino acid after activation forms peptide bond with first the amino acid whose amino reaction that is connected on solid phase carrier again, has just generated a dipeptides with protecting group this moment on solid phase carrier.Repeat above-mentioned peptide bond and form reaction, make peptide chain from the C end to the growth of N end, until reach needed peptide chain length, cutting at last obtains the purpose peptide.After the HPLC purifying, its purity is greater than 90%, and mass spectroscopy also confirms that its molecular weight meets theoretical value.(referring to: 1. yellow only moral, Chen Changqing show, and polypeptide is synthetic, Science Press, 1985.2. .N. Xiu Ede, H.D. Jia Kubuke are outstanding, and Liu Keliang etc. translate, peptide: chemistry and biology, Science Press, 2005.）
The separation of human peripheral blood single nucleus cell (PBMCs) and ELISPOT experiment detect:
The peripheral blood that extracts health donors separates through density gradient centrifugation, obtains PBMCs, adds interleukin II (IL-2, Peprotech company) and people β
2Microglobulin is induced differentiation CTL cell, further verifies at external ELISPOT.Method is as follows:
The separation of PBMCs with induce: (1) is with 40ml PBS(PH 7.2) peripheral blood 40ml after the dilution anti-freezing is processed; (2) add 4ml Lymphoprep parting liquid (Axis-Shield company) in centrifuge tube; (3) add in 8ml step (1) peripheral blood after dilution on 4ml Lymphoprep parting liquid liquid level; (4) 20 ℃ of centrifugal (2000rmp * 20min); (5) after centrifugal, be divided into four layers, discard the superiors, drawing the second layer with glass pipette is tunica albuginea layer (being rich in PBMCs); (6) the tunica albuginea layer of sucking-off PBS(pH 7.2) centrifuge washing is twice; (7) with 24 orifice plate bed boards, the concentration of cell is 1 * 10
6/ ml, every hole 1ml; (8) the every hole of second day adds 3 μ g people β
2Microglobulin and 10 μ g epitope peptides set up PBS group (substituting epitope peptide with PBS) as negative control group simultaneously; Every hole added 50u IL-2 in (9) the 3rd days; Changed liquid in every 2～3 days, carrying out the second wheel load peptide after Yu Qitian (is that 50u IL-2,10 μ g people β are added in every hole
2Microglobulin and 10 μ g epitope peptides/PBS), carry out third round lotus peptide after fortnight.After third round lotus peptide 3 days, obtain effector cell CTL, then carry out the ELISPOT experiment and detect IFN-γ and discharge.
2. ELISPOT experiment: concrete experimental procedure is as follows: (1) sealing: take out required lath, with the substratum sealing that contains 5% FCS RPMI 1640 substratum, (FCS can seal the FCS acceptor of institute's coated antibody to reduce nonspecific reaction) after (25 ℃) standing 5 ~ 10 min deducted it in 200 μ L/holes under room temperature; (2) cell upper plate: the CTL action effect cell (1 * 10 of inducing
5/ hole), the T2A2 cell of lotus peptide is as irritation cell (1 * 10
5/ hole) bed board.100 μ L/ holes.The distribution of cell in the hole will try one's best evenly (after adding cell, do not shake again or bounce the ELISPOT plate); ● positive control: cell concn is 1 * 10
5/ hole, add 10 μ L PHA, the secretion that this concentration can effective stimulus IFN-γ; ● the negative contrast of background: the RPMI that contains 5%FCS 1640 substratum that add 100 μ L; (3) add all samples after, cover plate lid, put into CO
2Incubator is cultivated 18h for 37 ℃; (4) lysing cell: topple over cell and substratum in the hole, 200 μ L/ holes add ice-cold deionized water, 4 ℃ of ice baths reaction 10 min (Low Osmotic Method lysing cell); (5) washing: topple over the liquid the hole in, 1 * Washing Buffer, wash 5 ~ 7 times in 200 μ L/ holes, stops 30 ~ 60 s at every turn, for the last time, buckles dried on thieving paper; (6) add the detection antibody incubation: every hole adds 100 μ L to dilute good biotin labeling detection antibody, hatches 1h for 37 ℃; (7) washing: topple over the liquid the hole in, 1 * Washing Buffer, wash 5 times in 200 μ L/ holes, each residence time is 30 ~ 60 s, for the last time, buckles dried on thieving paper; (8) enzyme connection avidin is hatched: will dilute good enzyme and join the avidin working fluid and join experimental port, 1h is hatched for 37 ℃ in 100 μ L/ holes; (9) washing: topple over the liquid the hole in, 1 * Washing Buffer, wash 5 times in the 200uL/ hole, each residence time is 30 ~ 60 s, for the last time, buckles dried on thieving paper; (10) colour developing: the AEC nitrite ion that thaws and prepared.Every hole adds the nitrite ion of 100 μ L, and the standing 15-45min of room temperature (at 20-25 ° of C, 25min is more suitable in colour developing) notes lucifuge; (11) color development stopping: topple over liquid in the hole, open the plate base, with deionized water wash 3 ~ 5 times, the color development stopping process.Plate is tipped upside down on thieving paper, pat dry the tiny globule, take off afterwards protective layer, be placed on the place of ventilation, the standing 10-30min of room temperature allows film naturally dry; Spot number with every hole formation in ELISPOT Quantimet counting 96 orifice plates.
External ELISPOT experimental result shows: candidate's peptide all can be induced in the peripheral blood of health donors and be obtained CTLs, and the CTLs that obtains compares with positive peptide after stimulating the IFN-of higher amount γ secretion (as shown in Fig. 9-16) is all arranged.
The present invention utilizes the relevant efflux protein antigen selection of the resistance of tubercule bacillus self to go out to have antiphthisic therapeutic bioactive peptide, for the Vaccinum Calmette-Guerini of developing based on epitope provides theoretical basis, and provide more information for designing based on the tuberculosis multi-epitope peptide vaccine that mixes t cell epitope.
Claims (2)
1. the relevant efflux protein of Drug Resistance for Tuberculosis source anti-tuberculosis CTL epitope peptide, it is characterized in that: described epitope peptide is nonapeptide, and the aminoacid sequence of described nonapeptide is:
P10：Met-Leu-Ile-Ala-Gly-Leu-Pro-Cys-Leu。
2. the application of the relevant efflux protein of Drug Resistance for Tuberculosis according to claim 1 source anti-tuberculosis CTL epitope peptide in preparation tuberculotherapy polypeptide vaccine.
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| CN114349829A (en) * | 2022-01-14 | 2022-04-15 | 华南农业大学 | Identification of ALV-J MHC-B2 restricted epitope peptide and application thereof |
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| CN1858059A (en) * | 2006-06-06 | 2006-11-08 | 中国人民解放军第二军医大学 | Antigen epitope for exciting human anti-tubercle bacillus protective immunoreaction and its use |
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| CN1858059A (en) * | 2006-06-06 | 2006-11-08 | 中国人民解放军第二军医大学 | Antigen epitope for exciting human anti-tubercle bacillus protective immunoreaction and its use |
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