CN106248936B - The application of antigen of mycobacterium tuberculosis albumen Rv2201 and its t cell epitope peptide - Google Patents
The application of antigen of mycobacterium tuberculosis albumen Rv2201 and its t cell epitope peptide Download PDFInfo
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Abstract
The present invention relates to the application of antigen of mycobacterium tuberculosis albumen Rv2201 and its t cell epitope peptide in preparing diagnosis reagent, vaccine and drug, the amino acid sequence of the antigen protein Rv2201 and its t cell epitope peptide is respectively such as SEQ ID NO:Shown in 13.The present invention utilizes mycobacterium tuberculosis Rv2201 proteantigens and its t cell epitope peptide as stimulant for specific T-cells and B cell immune response caused by mycobacterium tuberculosis infection, with in the past using comlete antigen compared with, can reduce due to antigen it is impure caused by false positive.The detection reagent prepared by the Rv2201 proteantigens and its epitope peptide can be widely used for related fields, the Vaccinum Calmette-Guerinis and antituberculotic prepared by Rv2201 proteantigens and its epitope peptide such as auxiliary diagnosis lungy, epidemiological surveillance and can be used for prevention and treatment lungy.
Description
Technical field
The present invention relates to molecular biology and field of immunology, specifically, being related to antigen of mycobacterium tuberculosis albumen
The application of Rv2201 and its t cell epitope peptide in diagnosis reagent, vaccine and medicine preparation.
Background technology
Tuberculosis is the chronic infectious disease caused by mycobacterium tuberculosis, investigation result show the whole world have three/
One population is in latent infection, and has 5%~10% will likely develop into active tuberculosis in following life.From
After the World Health Organization in 1993 announces that tuberculosis becomes global crisis, morbidity and mortality lungy occupy always height not
Under, according to WHO report, increase tuberculosis patient about 8,000,000 newly every year, there are about 200~3,000,000 people to die of tuberculosis every year.China is 22
The 2nd is occupied in a tuberculosis high burden country, national the 5th epidemiology sampling check result is shown:National 1,300,000 human hairs
Disease, accounts for the 14.3% of global incidence, and China is also one of 27 resistant tuberculosis high burden countries in the whole world, multi-drug resistance tuberculosis
Number of patients occupies the whole world first.The active tuberculosis patient of each untreated can infect 10~15 people.Tuberculosis is
Through as the important hygienic issues in the whole world, the great attention of people need to be caused.
The early diagnosis of tuberculosis and prophylactic treatment are most important to control lungy, and clinically the most commonly used is bacteriology sides
Method Sputum smears microscopy and Sputum culturing, Sputum smears microscopy be in world wide tuberculosis check in most popular technology, due to
This method simple equipments is suitble to the area in economics of underdevelopment to use.But it is high due to requiring the bacterial content in sample, it should
Method sensitivity is not high, causes the cloudy patient of largely painting not to be found to turn sun, and the cloudy patient of painting has infectiousness, does not allow to neglect
Depending on this method is influenced without species specificity, poor sensitivity by sputum sample and the state of an illness.Gold of the Sputum culturing as diagnosis of tuberculosis
Standard, long but there are incubation times, the rapid culture systems such as existing BACTEC MGIT960 systems can divide within 2 weeks now
From culture mycobacterium tuberculosis, but since the culture medium, nourishing additive agent, miscellaneous bacteria inhibitor of its preparation are expensive, Wu Fa
Developing country is widely popularized, and fast culture pollution rate compared with improvement L-J cultures significantly increases, and leads to false positive results
Occur.The common detection method for Mass screening is the experiment of cutaneous tuberculosis rhzomorph, and tulase pure protein is used to derive
Object (PPD), since mycobacterial species (pathogenic mycobacterium, environment mycobacteria and BCG) are common containing there are many in PPD
Antigen molecule, therefore PPD diagnosis of tuberculosis specificity it is poor, cannot effectively distinguish mycobacterium tuberculosis infection and BCG vaccine
Inoculation, imageological examination such as the x-ray inspection of routine, CT examination, MRI inspections, the ultrasonic examination of tuberculosis are expensive, and to body
Body causes centainly to injure, and specificity is low, is not suitable for conventional inspection diagnosis.
It is resided in macrophage after mycobacterium tuberculosis intrusion human body, human body is main to mycobacterium tuberculosis immune anti-
Should be cellullar immunologic response, the cell being primarily involved in is CD4+ and CD8+T cells.CD8+T cells can pass through perforin, particle
The macrophage infected by mycobacterium tuberculosis or Dendritic Cells are killed to reach by the approach such as enzyme removes tuberculosis branch bar
The purpose of bacterium.When the people of infection mycobacterium tuberculosis is contacted again Specific Antigen of Mycobacterium Tuberculosis, the production of T lymphopoiesis
Raw IFN-γ, detects that IFN-γ can determine once or infecting mycobacterium tuberculosis by monoclonal antibody.
The detection method T-SPOT based on T cell is captured through tuberculosis antigen using IFN-γ specific antibody at present
The IFN-γ generated after the peripheral blood lymphocytes culture of stimulation, and showed in such a way that elisa develops the color, from
The quantity of spot evaluates cellular immune function come the case where determining cell secretion of cytokines, from individual cell level.It is with T cell
The detection of the external interferon on basis is used for auxiliary diagnosis lungy, which can not only filter out activity knot
Core patient, while can also detect incubation period patient, so as to preferably prevent and control incubation period tuberculosis, have at present with knot
The holoprotein or polypeptide of tuberculosis the specific antigen ESAT-6 and CFP-10 of the areas core Mycobacterium tuberculosis genes group RD1 coding are stimulant
The IGRA detection kits of commercialization presented higher sensitive such as QuantiFERON-TB Gold test and T-SPOT
Property and specificity.
Rv2201(GI:15609338) it is asparagine synthetase gene on H37Rv genomes, full length gene
1959bp encodes the albumen containing 652 amino acid, is primarily involved in the building-up process of asparagine, Rv2201 is a kind of secretion
Albumen can detected in cell conditioned medium, and Epitope prediction analysis is carried out to it using bioinformatics software, find
There are more t cell epitopes for Rv2201 albumen, have potential diagnostic.
The present invention establishes on the basis of reverse vaccinology, and it is possible immune to go out mycobacterium tuberculosis using computational screening
Then originality antigenic storehouse utilizes bioinformatics software TE predict and IEDB to predict tuberculosis antigen MHC-I class T cell tables
Position, synthesizes these polypeptides by solid-state synthetic method, is screened to tuberculosis neoantigen first with community immunity screening test, screens
Go out Immunodominant Antigenic and then its immunogenicity verified by zoopery, it is last it is verified obtain it is immune excellent
On the one hand gesture antigen can be used for Diagnosis of Tuberculosis, on the other hand can be used for the transformation of BCG vaccine.
Invention content
The object of the present invention is to provide the applications of antigen of mycobacterium tuberculosis albumen Rv2201.
It is a further object of the present invention to provide antigen of mycobacterium tuberculosis albumen Rv2201T cell epitope peptides and its applications.
The present invention is based on following designs:Rv2201 is the conservative memebrane protein on mycobacterium tuberculosis, is that can be cultivated in H37Rv
The secretory protein that detected in supernatant is primarily involved in the synthesis of asparagine, and Rv2201 is only in mycobacterium tuberculosis complex
Middle presence.The present invention is based on T cell IFN-γ release tech, utilize bioinformatics software TE predict and IEDB pairs
T cell antigen epitope is predicted on Rv2201 encoding genes, synthesizes epitope polypeptide using solid-state synthetic method, then pass through T-SPOT
Method (Enzyme linked immunospot) to the T lymphocyte specific in tuberculosis patient, lung other diseases patient, healthy human body into
Row detection to evaluate sensitivity and specificity of the antigen for tuberculosis detection, while passing through community immunity experiment screening
Go out the immunodominant epitope peptide on Rv2201 antigen proteins, then by zoopery, determines exempting from for immunodominant T cell epitope peptide
Epidemic focus.
In order to realize that the object of the invention, the present invention provide antigen of mycobacterium tuberculosis albumen Rv2201 and preparing tuberculosis detection
Application in reagent, vaccine and drug;Wherein, the amino acid sequence of the antigen protein Rv2201 such as SEQ ID NO:Shown in 3,
Or the sequence is one or several amino acids formed with identical immunogenicity and same antigen through replacement, missing or addition
Amino acid sequence.
The present invention also provides antigen of mycobacterium tuberculosis albumen Rv2201T cell epitope peptides, the epitope peptide is selected from P136
And P137, amino acid sequence is respectively such as SEQ ID NO:Shown in 1-2.
The present invention also provides epitope peptide or its analogs derived from the t cell epitope peptide.
The present invention also provides the DNA moleculars for encoding the epitope peptide.
The present invention also provides expression cassettes and expression vector containing the DNA molecular for encoding the epitope peptide.
The present invention also provides the transgenic cell lines containing the DNA molecular for encoding the epitope peptide.
The present invention also provides the recombinant bacterium containing the DNA molecular for encoding the epitope peptide and its recombination eggs of expression and purification
In vain.
The present invention also provides the epitope peptide, the DNA molecular of the coding epitope peptide, the transgenic cell lines or described
Application of the recombinant protein of recombinant bacterium and its expression and purification in preparing tuberculosis detection reagent, vaccine and drug.
The present invention also provides a kind of Diagnosis of Tuberculosis reagent, contain antigen of mycobacterium tuberculosis albumen in the diagnostic reagent
Rv2201, or the DNA molecular of the antigen protein Rv2201 is encoded, or by the weight of the recombinant bacterium generation containing the DNA molecular
Histone;And/or
The epitope peptide, the DNA molecular of the coding epitope peptide and/or the recombinant protein.
The present invention also provides the tuberculosis T-SPOT detection kits containing above-mentioned diagnostic reagent.The kit further include with
Lower material or reagent:
1. primary antibody:The mouse IgG monoclonal antibody of anti-human or animal's IFN-γ.
2. enzyme marking reagent:Another mouse of anti-human or animal's IFN-γ different epitopes of horseradish peroxidase-labeled
IgG monoclonal antibody.
3. standard items:
Culture plate:The 96 hole micro reaction plates containing pvdf membrane or nitrocellulose filter contain tuberculosis in Positive control wells
Nonspecific stimulation antigen (such as PHA), negative control hole contain PBS or substrate liquid.
4. reagent and consumptive material needed for other T-SPOT detections.
Preferably, primary antibody is fixed on above-mentioned micro reaction plate.
The present invention is to be based on double-antibody sandwich principle, detects antigen using T-SPOT methods, experimentation is:It is wrapped on pvdf membrane
The primary antibody of quilt can be in combination cell supernatant as capture antibody IFN-γ, and IFN-γ can be captured by ELIAS secondary antibody, be shown
Color.Two kinds of antibody are the monoclonal antibody for identifying IFN-γ difference epitope.
The present invention also provides antigen of mycobacterium tuberculosis albumen Rv2201 and/or the epitope peptide, the coding epitope peptides
DNA molecular, the transgenic cell line or the recombinant protein of the recombinant bacterium and its expression and purification prepare Vaccinum Calmette-Guerini and
Application in antituberculotic.
The present invention also provides a kind of Vaccinum Calmette-Guerini, active ingredient is antigen of mycobacterium tuberculosis albumen Rv2201, or is compiled
The DNA molecular of the code antigen protein Rv2201, or the recombinant protein by the recombinant bacterium generation containing the DNA molecular;With/
Or,
The epitope peptide, the DNA molecular of the coding epitope peptide and/or the recombinant protein.
Invention also provides a kind of antituberculotic, and active ingredient includes with antigen of mycobacterium tuberculosis albumen Rv2201
And/or the epitope peptide is aided with adjuvant immunity experimental animal, the polyclonal antibody of preparation, or divide with tuberculosis as immunogene
Branch bacteroides antigen albumen Rv2201 and/or the epitope peptide are aided with adjuvant immunity experimental animal, using hybridoma as immunogene
Technology and DNA recombinant techniques, the identification antigen of mycobacterium tuberculosis albumen Rv2201 and its t cell epitope peptide antigen of preparation
Humanized monoclonal antibodies.
The present invention further provides antigen of mycobacterium tuberculosis albumen Rv2201 and/or the epitope peptides and its derivative table
Application in position peptide or its analog specific T-cells and B cell immune response caused by detection mycobacterium tuberculosis infection.
It is by the lymphocyte of human or animal through the epitope peptide and its derivative epitope peptide or its analog antigen or their group
After closing object stimulation, T cell or the cell factor of B cell secretion are detected.
Wherein, the cell factor of tuberculosis specific T-cells secretion includes:Interferon (IFN-γ), interleukin 2
(IL-2), interleukin-4 (IL-4), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α) etc..B cell is secreted
Cell factor include antibody.
Detection method for the cell factor of tuberculosis specific T-cells secretion includes elisa experiment
(ELISPOT), enzyme-linked immunosorbent assay (ELISA), immune colloid gold experiment, the interior dyeing of cell factor and T cell proliferation examination
It tests.The detection method of the cell factor of B cell secretion includes enzyme-linked immunosorbent assay (ELISA) etc..
Peripheral blood, venous blood, cerebrospinal fluid, pleural effusion or hydrothorax etc. of the lymphocyte from human or animal.
The present invention has the following advantages:
(1) present invention utilizes mycobacterium tuberculosis Rv2201 proteantigens and its t cell epitope peptide to be used for as stimulant
Specific T-cells and B cell immune response caused by mycobacterium tuberculosis infection, in the past using comlete antigen compared with, can
Reduce due to antigen it is impure caused by false positive.
(2) research has shown that, it is immune anti-that epitope provided by the invention can stimulate body to generate stronger T cell
It answers, therefore when using above-mentioned epitope to carry out ex vivo T cell interferon release experiment as stimulant, sensitivity significantly carries
It is high.
(3) method for using synthesis in solid state synthesizes epitope polypeptide, is conducive to quality control, and cost is relatively low, and purity is high,
It is suitble to large-scale commercial production.
Embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The clone of 1 antigen of mycobacterium tuberculosis gene Rv2201 of embodiment and the expression and purification of albumen
Rv2201(GI:15609338) be Mycobacterium tuberculosis H37Rv genome encoding conservative memebrane protein, contain 652
Amino acid, amino acid sequence such as SEQ ID NO:Shown in 3.According to its coding gene sequence, design primer utilizes prokaryotic expression
System (such as Escherichia coli), which is expressed and purified, obtains antigen protein Rv2201.
The synthesis of 2 antigen of mycobacterium tuberculosis albumen Rv2201T cell epitope peptides of embodiment
Based on T cell IFN-γ release tech, Rv2201 is compiled using bioinformatics software TE predict and IEDB
T cell antigen epitope is predicted on code gene, synthesizes epitope polypeptide using solid-state synthetic method, then by T-SPOT methods to tuberculosis
Patient, lung other diseases patient, the T lymphocyte specific in healthy human body are detected, and are used for evaluate the antigen
The sensitivity and specificity of tuberculosis detection.
Antigen of mycobacterium tuberculosis albumen Rv2201T cell epitope peptides provided in this embodiment are selected from P136 and P137,
Amino acid sequence is respectively such as SEQ ID NO:Shown in 1-2.
The preparation of 3 tuberculosis T-SPOT detection kits of embodiment
The kit forms as follows substantially:
1. the epitope peptide antigen that proteantigen Rv2201 and/or embodiment 2 prepared by embodiment 1 are synthesized:The epitope peptide
Selected from least one of P136 and P137.
2. primary antibody:The mouse IgG monoclonal antibody of anti-human or animal's IFN-γ.
Enzyme marking reagent:Another mouse IgG of anti-human or animal's IFN-γ different epitopes of horseradish peroxidase-labeled
Monoclonal antibody.
3. standard items:
Culture plate:The 96 hole micro reaction plates containing pvdf membrane or nitrocellulose filter contain tuberculosis in Positive control wells
Nonspecific stimulation antigen (such as PHA), negative control hole contain PBS or substrate liquid.
4. reagent and consumptive material needed for other T-SPOT detections.
Primary antibody is fixed on above-mentioned micro reaction plate.
The kit is designed based on double-antibody sandwich principle, detects antigen using T-SPOT methods, experimentation is:
The IFN-γ that coated primary antibody can be in combination cell supernatant as capture antibody on pvdf membrane, and IFN-γ can be by enzyme mark two
Anti- capture, colour developing.Two kinds of antibody are the monoclonal antibody for identifying IFN-γ difference epitope.
4 epitope peptide of embodiment is used for the clinical detection of tuberculosis infection
1. the separation of peripheral blood lymphocytes
1.1 study subject
1. the screening criteria of volunteer's case:
Clinical manifestation symptom, sign and imaging examination of chest are diagnosed as phthisical, and Sputum culturing is positive lung knot
Core patient.
2. the screening criteria of Pulmonary Disease patients:
Sputum culturing and lung's other diseases that Sputum smears are feminine gender, such as pneumoconiosis, chronic obstructive pulmonary disease Pulmonary Disease patients.
3. the screening criteria of healthy volunteer:
Without tuberculosis clinical symptoms, without tuberculosis patient close contact history, without other diseases or infection.
Selected tuberculosis patient and volunteer's age is between 15-80 Sui, from the continuous time gone to a doctor to tuberculosis ward
It is randomly selected in sample.50 tuberculosis volunteers, 40 Pulmonary Disease patients and 55 healthy volunteer's blood are acquired altogether
Sample acquires peripheric venous blood when blood sampling using the anticoagulant heparin vacuum blood collection tube of endotoxin-free, and every volunteer takes a blood sample about 5ml
~10ml.
1) sample in 4 hours use Ficoll-Hypaque separating liquids detach PBMCs.
2) first by whole blood RPMI-1640 culture mediums 1:1 dilution mixing, is added the separation of certain volume in centrifuge tube
Liquid keeps two liquid level interfaces clear, separating liquid, anti-freezing are not diluted by the blood sample tiling after dilution to separating liquid ullage
Whole blood, 1640 culture volumes of RPMI are 1:1:1, room temperature (18~26 DEG C), 800g is centrifuged 20 minutes.
3) after centrifuging, tube bottom is red blood cell, and middle layer is separating liquid, and top layer is plasma layer, plasma layer with detach
It is nebulous mononuclearcell (including the lymphocyte and monocyte) layer of white between liquid layer.White cloud and mist is drawn with suction pipe
Shape cellular layer is simultaneously transferred in 15ml sterile centrifugation tubes, and 1640 culture mediums of RPMI are added to 10ml, at room temperature 10 points of 800g centrifugations
Clock.
4) it discards supernatant, 1640 culture mediums of 7ml RPMI is added after resuspension, 700g is centrifuged 10 minutes.
5) it discards supernatant plus precipitation is resuspended in 0.5ml AIM-V culture mediums.
6) utilize automated cell calculating instrument to cell count, with AIM-V culture mediums prepare 500 μ L cell concentrations be 2.5 ×
106The cell suspension of/ml.
2. the preparation of epitope peptide
The epitope peptide that solid-phase synthesis in embodiment 2 synthesizes is dissolved with DMSO, each epitope peptide is respectively with containing
The RPIM1640 culture mediums of 10% fetal calf serum use after being diluted to a certain concentration.
3.T-SPOT detects epitope peptide-specific T-cell
Using the kit of embodiment 3, following reagent is added into the microwell plate of pre-coated primary antibody, each patient sets respectively
4 detection holes:Positive control wells (adding the phytohemagglutinin HA of a concentration of 15 μ g/ml of 100 μ L as positive stimulus object), feminine gender
Control wells (adding 100 μ L PBS as negative control), 2 detection holes (are separately added into a concentration of 20 μ g/ml of 100 μ L in two holes
Polypeptide P136, P137), the good PBMC of the 100 above-mentioned dilutions of μ L is added in each hole, makes the quantity of PBMC in every hole up to 250,000
It is a, antigen and PBMC cells are placed in 37 DEG C, 5%CO2Incubator in cultivate 20 hours.
4. board-washing and result judgement
PBMC cells and antigenic stimulus object are washed away, adds 100 μ L primary antibodies to be incubated at room temperature 1 hour, is washed 5 times with PBS, add secondary antibody
Incubation at room temperature 1 hour, then washed 5 times with PBS, after adding substrate to be protected from light colour developing 7 minutes, with purified water color development stopping, culture plate is put
The spot number on access panel is dried at ventilation opening.
Result judgement (table 1):Blank control wells spot number=N, detection hole spot number=T, positive quality control hole spot number=
P。
1 T-SPOT result criterions of table
Two of which epitope peptide detect 50 tuberculosis patients, 40 lungs other diseases patients and 55 Healthy Peoples result
Statistics is shown in Table 2.
2 Rv2201 proteantigens of table, two epitope peptide detection sensitivities and specificity statistics
Epitope peptide | Sensitivity (%) | Specificity (%) |
P136 | 28 | 100 |
P137 | 6 | 100 |
Two polypeptide joints | 32 | 100 |
Detection sensitivity=(tuberculosis patient detects number positive/tuberculosis patient sum) × 100%
Detect specificity=1- (consumptive detects number positive+healthy volunteer and detects number positive)/(pulmonary disease
Patient populations+healthy volunteer's sum)
Detect that positive is positive principle according to single polypeptide, be computed obtain Rv2201 proteantigens P136,
The sensitivity that two epitope peptide joint-detections of P137 go out tuberculosis patient is 32%, specificity 100%.
Preferably, tuberculosis can be improved using ESAT6 and CFP10 joint antigens in the reagent box for tuberculate diagnosis of the present invention
The diagnosis efficiency of disease, tuberculosis patient PBMC has a cellullar immunologic response to Rv2201 and its epitope polypeptide in the present embodiment, and lung
Portion other illness patient and the PBMC of Healthy People are to Rv2201 and its epitope polypeptide non-responsiveness, it was demonstrated that Rv2201 protein epitopes
Polypeptide can be by tuberculosis patient specific recognition, and diagnosis efficiency of the Combining diagnosis efficiency of four polypeptides relative to single polypeptide
It improves, therefore Rv2201 and its epitope peptide have potential diagnosis performance, it may be considered that as joint antigen, for lungy
In detection reagent.In addition, crowd's screening test also demonstrates the IFN-γ that Rv2201 epitope polypeptides can stimulate body to generate, IFN-
γ can with activating macrophage to promote removing of the macrophage to mycobacterium tuberculosis, it can be considered to by Rv2201 and
Structure and preparation of its epitope polypeptide for the vaccine of tuberculosis.Although above having used general explanation and specific embodiment party
The present invention is described in detail for case, but on the basis of the present invention, can be made some modifications or improvements to it, this is to this field
It is obvious for technical staff.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention,
Belong to the scope of protection of present invention.
Claims (7)
1. applications of the antigen of mycobacterium tuberculosis albumen Rv2201 in preparing tuberculosis detection reagent and vaccine;Wherein, described anti-
The amino acid sequence of former albumen Rv2201 such as SEQ ID NO:Shown in 3.
2. antigen of mycobacterium tuberculosis albumen Rv2201T cell epitope peptides, which is characterized in that the epitope peptide is selected from such as SEQ ID
NO:Amino acid sequence shown in 1 or 2.
3. application of the epitope peptide described in claim 2 in preparing tuberculosis detection reagent.
4. a kind of Diagnosis of Tuberculosis reagent, which is characterized in that containing epitope peptide described in claim 2 in the diagnostic reagent, or compile
The DNA molecular of the code epitope peptide, or the recombinant protein by the recombinant bacterium generation containing the DNA molecular for encoding the epitope peptide.
5. the tuberculosis T-SPOT detection kits containing diagnostic reagent described in claim 4.
6. kit according to claim 5, which is characterized in that contain in the kit:
1. primary antibody:The mouse IgG monoclonal antibody of anti-human or animal's IFN-γ;
2. enzyme marking reagent:Another mouse IgG list of anti-human or animal's IFN-γ different epitopes of horseradish peroxidase-labeled
Clonal antibody;
3. standard items:
Culture plate:The 96 hole micro reaction plates containing pvdf membrane or nitrocellulose filter contain the non-spy of tuberculosis in Positive control wells
Anisotropic stimulator antigen, negative control hole contain PBS;
4. reagent and consumptive material needed for other T-SPOT detections;
Wherein, primary antibody is fixed on above-mentioned micro reaction plate.
7. applications of the antigen of mycobacterium tuberculosis albumen Rv2201 in preparing Vaccinum Calmette-Guerini.
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