CN104387450B

CN104387450B - Mycobacterium tuberculosis specific C D8+T cell epitope peptide P12 and its application

Info

Publication number: CN104387450B
Application number: CN201410598638.XA
Authority: CN
Inventors: 马骊; 刘苏东; 罗微
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2014-10-29
Filing date: 2014-10-29
Publication date: 2017-07-18
Anticipated expiration: 2034-10-29
Also published as: CN104387450A

Abstract

The invention discloses a kind of mycobacterium tuberculosis specific C D8⁺T cell epitope peptide and its application.The amino acid sequence of the epitope peptide is AVMAGIVRAAK, and the epitope peptide is high with HLA A*1101 molecules affinity, can activate CD8+T cells, induce it to breed and secretion IFN γ.The determination of epitope peptide of the present invention, theoretical foundation is provided and new solution for the research and development of tuberculosis vaccine, diagnostic reagent and medicine, and preventing and treating lungy is significant.

Description

Mycobacterium tuberculosis specific C D8+T cell epitope peptide P12 and its application

Technical field

The invention belongs to immunological technique field, more particularly to mycobacterium tuberculosis specific C D8⁺T cell epitope peptide and It is applied.

Background technology

Tuberculosis is induction after being infected by mycobacterium tuberculosis, global infection rate highest infectious disease, and case fatality rate is only second to AIDS.Only 2012, the new hair tuberculosis patient in the whole world was up to 8,600,000, and dead 1,300,000.The new feature of tuberculosis epidemic situation is：It is resistance to Medicine tuberculosis case occurs and is in the gesture that spreads；Tuberculosis grows in intensity with AIDS coinfection situation.For this two classes patient, at present Have no effectively preventing measure.China is that tuberculosis bears one of most heavy country in the world, and research and development are adapted to the knot of Chinese population Time and tide wait for no man for core disease vaccine and medicine.

Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is intracellular bacterial parasite, to its effective control Cellular immunity, especially T cell is depended on to be immunized.T cell is broadly divided into CD4 in human body⁺T (Th) cells and CD8⁺T (CTL) the big subgroup of cell two, numerous studies are it has been proved that both T cell subgroups all have weight for control tubercle bacillus affection Act on, wherein CD8⁺CTL is even more the main force for removing tulase.

The complete tulase of T cell None- identified and proteantigen, but identification is absorbed and added by antigen presenting cell The tuberculosis epitope peptide discharged after work.Epitope peptide is to cause the core of immune response in antigen partly, also referred to as antigenic determinant, It combines to form peptide-HLA compounds and is delivered and expresses in antigen presenting cell with the HLA molecules on antigen presenting cell surface Surface, and then activating T cell, induce t cell immune response.CD4⁺T cell recognizes the epitope peptide of HLA II quasi-molecule submissions, and CD8⁺T cell recognizes the epitope peptide of HLA I quasi-molecule submissions.Identification and its activation of the T cell to epitope peptide require epitope peptide with Antigen presenting cell surface HLA molecules stable bond, and the affinity of peptide and HLA molecules is to form the pass of peptide-HLA compounds Key, therefore induction T cell immune medicine and vaccine are prepared, first step is the peptide for filtering out high-affinity, Jin Erjian Make with immunocompetent epitope peptide.

Epitope peptide can be obtained by artificial synthesized completely, and the R＆D cycle is short；Due to eliminating pathogen beyond epitope peptide Other harmful components, security is good；Multiple epitope peptides can also be used in series, booster immunization effect and reduction are immunized and escaped Ease.The tulase CTL epitope peptides being currently known mainly identify that mostly HLA-A*0201 is restricted, and right in American-European crowd In the Africa of TB endemic most serious and Southeast Asia crowd, unsuitable epitope peptide can be used.In China, HLA-A* 1101 be gene frequency highest HLA molecules in crowd, during identification HLA-A*1101 restricted CTL epitopes are adapted to for research and development The epitope peptide vaccine of state crowd has very important significance.

The content of the invention

It is an object of the invention to provide mycobacterium tuberculosis specific C D8⁺T cell epitope peptide.

Another object of the present invention is to provide mycobacterium tuberculosis specific C D8⁺T cell epitope peptide is preparing tuberculosis Application in disease vaccine, diagnostic reagent and medicine.

The technical solution used in the present invention is：

The invention provides high with HLA-A*1101 molecules affinity, CD8 can be activated⁺T cell, induce its breed and secrete The mycobacterium tuberculosis specific C D8 of IFN-γ⁺T cell epitope peptide, the amino acid sequence of the epitope peptide is： AVMAGIVRAAK (SEQIDNO.12) is (i.e.：Ala-Val-Met-Ala-Gly-Ile-Val-Arg-Ala-Ala-Lys).

The present invention combines Bioinformatics Prediction method and the displacement experiment of uv induction peptide, utilizes mycobacterium tuberculosis itself Antigen selection go out the epitope peptide with anti-tubercular, efficiently and accurately detect epitope peptide AVMAGIVRAAK (SEQ ID NO.12) the affinity between HLA-A*1101 molecules.This is the restricted tuberculosis of HLA-A*1101 that is new, not being reported Mycobacterial epitope peptide, by tuberculosis patient CD8⁺T cell is recognized extensively.Due to HLA-A*1101 genotype be in Chinese it is most wide The genotype of general carrying, does not find the special HLA-A*1101 restricted CTL epitope peptides of mycobacterium tuberculosis, the present invention at present The determination of epitope peptide, theoretical foundation is provided and new solution party for the research and development of tuberculosis vaccine, diagnostic reagent and medicine Case, is significant to preventing and treating lungy.

Brief description of the drawings

Fig. 1 is candidate's epi-position peptide and HLA-A*1101 molecule affinity schematic diagrames.

Fig. 2 is the schematic diagram of the epitope inducing peptide tubercular T cell activation of 22 high-affinities and secretion of gamma-IFN.

Fig. 3 is that ELISPOT detects that P12 induces the schematic diagram of tubercular's T cell secretion of gamma-IFN.

Fig. 4 is P12 mass spectral analysis figure.

Fig. 5 is the lymphocyte proliferation assay detection P12 induction tuberculars CD8 that CFSE is marked⁺The result of T cell propagation Schematic diagram.

Embodiment

Primary structure of the invention according to antigen, using Immunoinformatics means, with CBS databases NetMHCcons Antigen Epitope Predictions software is carried out to the HLA-A*1101 restricted CTL epitopes peptide of antigen of mycobacterium tuberculosis albumen Forecast analysis, the affinity then combined using uv induction peptide displacement experimental check candidate peptide with HLA-A*1101 molecules and Stability, screening obtains epitope peptide, is identified by experiment in vitro, CTL epitope peptides P12 can induce CD8⁺T cell produces strong Cellullar immunologic response, secreting high levels IFN-γ, and induce CD8⁺Notable propagation occurs for T cell.

Present invention is expanded on further with reference to specific embodiment.

Using bioinformatics method, HLA-A*1101 restricted epitope peptides are predicted：

According to the literature, choosing 94 has more strongly immunogenic antigen of mycobacterium tuberculosis albumen (as shown in table 1), The amino acid sequence of each antigen protein is obtained from GeneBank.

The candidate's antigen of mycobacterium tuberculosis albumen of table 1

Chinese are obtained from HLA gene frequency databases AFND (AlleleFrequency NetDatabase, AFND) The frequency distribution information of each genotype in group HLA-A sites, wherein HLA-A*1101 gene frequencies are about 28%, are HLA-A sites Upper frequency highest individual gene type.

The HLA-A*1101 restricted CTL epitopes contained by antigen protein, the calculation of use are predicted using bioinformatics Method is the NetMHCcons in CBS databases.It will be predicted the outcome and be ranked up according to IC50 values, IC50 values are lower, illustrate epitope Peptide and HLA molecule affinity are higher.Thus, HLA-A*1101 restricted epitope peptides in 94 antigen proteins are predicted, and are sieved Wherein 50 epitope peptides of affinity highest are selected, as candidate, as shown in table 2.

The restricted candidate's epi-position peptides of 250 HLA-A*1101 of table

Experimental method, the affinity between detection candidate's epi-position peptide and HLA-A*1101 molecules are replaced using uv induction peptide And combination stability：

Experimentation is as follows：

Peptide-HLA the compounds of the epitope peptide containing photosensitivity are under ultraviolet irradiation, and photosensitivity epitope peptide is broken, Depart from from HLA molecules, at this moment toward candidate's epi-position peptide to be detected is added in reaction system, if candidate's epi-position peptide and HLA points Sub- affinity is high, then can form the epitope peptide-HLA compounds of new stabilization.Using anti-beta 2m antibody capture HLA molecules, Epitope peptide-HLA the complex concentrations that ELISA detections are newly formed, evaluate the affinity size between epitope peptide and HLA molecules.Altogether The epitope peptide (being more than 100%) 22 that there is high-affinity with HLA-A*1101 is selected, as shown in Fig. 1 and table 3.

Table 3 and HLA-A*1101 has 22 epitope peptides of high-affinity

ELISpot experiment 22 epitope peptide inducing T cell secretion of gamma-IFN of detection, the immune effect of preliminary identification epitope peptide Should：

Experimentation is as follows：

Using density gradient method separation HLA-A*1101 genotype tubercular's PMNCs (peripheral blood mononuclear,PBMC).The 20ml peripheral bloods of collection are diluted one times with PBS, separation of lymphocytes is slowly added to Above liquid, both volume ratios are 2:1,30min is then centrifuged with 800g/min rotating speeds, liquid is divided into three layers after centrifugation, wherein PBMC is between first layer and the second layer, into tunica albuginea shape, and PBMC is carefully drawn with dropper into new centrifuge tube, add 5 times with Upper volume PBS is washed, and 800g/min centrifugation 10min, repeated washing once, is frozen carefully with frozen stock solution (90%FBS+10%DMSO) Born of the same parents, liquid nitrogen storage.

ELISpot is tested：Recovery HLA-A*1101 genotype patient PBMC, in 37 DEG C, 5%CO₂Stood overnight in incubator. Spend the dead cell in dead cell magnetic bead removal PBMC.Cell count, complete medium re-suspended cell to 1.25 × 10⁶Individual/mL, 200 μ L/ holes cell suspensions are spread into ELISpot plates (i.e. 2.5 × 10⁵Individual/hole), totally 48 hole.By 22 candidate peptides shown in table 3 It is separately added into by 10 μ g/mL concentration in 44 holes, a kind of peptide is added per hole, every kind of peptide spreads 2 holes, and 2 Positive control wells are separately added into 4 μ G/mLPHA, 2 negative control holes are not added with any stimulation, fill full training.Each hole has added after sample respectively, and culture plate is placed in into cell In incubator, 37 DEG C, 5%CO₂Cultivate 24h.ELISpot plates are taken out, PBS is washed 5 times, the anti-IFN-γ of enzyme mark is added after patting dry and is resisted Body 100ul, is incubated 2h, and PBS is washed 5 times, and developer 100ul is added after patting dry, after obvious spot occur in Positive control wells, is used Ultra-pure water terminating reaction；Dry, read plate, count, correction.Peptide-specific T-cell frequency is with SFC/10⁶Represent.

Positive reaction criterion is：①SFC/10⁶>50,2. peptide stimulation hole spot number is more than or equal to negative control hole Twice.Meet 1. simultaneously 2., reaction and judgement is the positive.

Count identifications of 23 HLA-A*1101 type tubercular PBMC to epitope peptide to react, i.e. patient P BMC is to epitope peptide Identification frequency.The detection Ren Shuo ╳ 100% of peptide discrimination=positive reaction number/total.Filtering out at least can be by 20% patient The epitope peptide of identification, as a result as shown in Figure 2.Fig. 3 is ELISPOT detection epitope peptide P12 induction tuberculosis patient T cell secretions IFN- γ result schematic diagram.

The lymphocyte proliferation assay detection epitope peptide P12 inductions CD8 of CFSE marks⁺T cell is bred：

Experimentation is as follows：

Recovery HLA-A*1101 type tubercular PBMC, stand overnight.Dead cell, meter are removed using dead cell magnetic bead is removed Number, with PBS re-suspended cells to 1 ╳ 10 after centrifugation⁷Individual/mL, adds 5 μM of CFSE marks, lucifuge is incubated 10min after mixing.Add 5 The complete medium (10%FBS+RPMI-1640) of times volume precooling terminates dye marker.300g centrifuges 10min, with complete training Base washing is supported, is repeated twice.Count again, with complete medium re-suspended cell to 1 ╳ 10⁶Individual/mL.Cell suspension is spread into 96 In the U floor cells culture plates of hole, 200 μ L/ holes, totally 8 hole, each hole of experimental group is separately added into 10 μ g/mL epitope peptides P12, and (Fig. 4 is P12 Mass spectral analysis figure), Positive control wells add 4 μ g/mLPHA, and negative control hole is not added with any stimulant, supplies full training.Glimmering Viewed under light microscopy, determines that cell is marked (green fluorescence) by CFSE.37 DEG C, 5%CO₂48h is cultivated in incubator.Each Kong Zhongfen 100IUIL-2 is not added, continues to cultivate to 7d, takes out cell, and anti-CD8 antibody, flow cytometry inspection are marked after washing Survey.

According to the characteristics of CFSE dye markers, dyestuff is equably assigned to daughter cell with cell propagation from parent cell In, fluorescence intensity halves.So, the cell of weak CFSE fluorescence, the cell after as breeding.Epitope inducing peptide CD8⁺T cell increases Grow CD8 of the ability by weak CFSE fluorescence⁺T cell/CD8⁺T cell is represented.Testing result is as shown in Figure 5.

Epitope peptide P12 by above scheme Screening and Identification, specifically can be recognized by HLA-A*1101 types tubercular, And induce CD8⁺T cell is bred and secretion of gamma-IFN, and reason is provided for the research and development of tuberculosis vaccine, diagnostic reagent and medicine By basic and new solution, preventing and treating lungy is significant.

<110>Nanfang Medical Univ

<120>Mycobacterium tuberculosis specific C D8+T cell epitope peptides P12 and its application

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Claims

1. mycobacterium tuberculosis specific C D8⁺T cell epitope peptide P12 is preparing diagnostic reagent of tuberculosis and tuberculotherapy medicine In application, the amino acid sequence of the epitope peptide is： AVMAGIVRAAK（SEQ ID NO.12）.

2. mycobacterium tuberculosis specific C D8⁺Applications of the t cell epitope peptide P12 in tuberculosis prophylaxis vaccine is prepared, it is described The amino acid sequence of epitope peptide is： AVMAGIVRAAK（SEQ ID NO.12）.