CN106442983B - The application of antigen of mycobacterium tuberculosis albumen Rv3793 and its t cell epitope peptide - Google Patents
The application of antigen of mycobacterium tuberculosis albumen Rv3793 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 Rv3793 and its t cell epitope peptide in tuberculosis detection reagent, vaccine and drug is prepared, the epitope peptide is selected from P1, P10, P12 and P13, and amino acid sequence is respectively such as SEQ ID NO:Shown in 14.The present invention utilizes mycobacterium tuberculosis Rv3793 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 Rv3793 proteantigens and its epitope peptide can be widely used for the related fields such as auxiliary diagnosis lungy, epidemiological surveillance.Vaccinum Calmette-Guerini and antituberculotic prepared by Rv3793 proteantigens and its t cell epitope peptide 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 Rv3793 and its t cell epitope peptide in diagnosis reagent, vaccine and medicine preparation.
Background technology
Tuberculosis is the chronic infectious disease as 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 height not always
Under, according to WHO report, increase tuberculosis patient about 8,000,000 newly every year, there are about 200~3,000,000 people every year to die of tuberculosis.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 that 27, whole world resistant tuberculosis height bears one of country, 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
The hygienic issues important through becoming the whole world, need to cause the great attention of people.
The early diagnosis of tuberculosis and prophylactic treatment are most important to control lungy, and it is high that researcher should be dedicated to exploitation
Effect, sensitive, special Diagnosis of Tuberculosis detection method.Clinically tuberculosis symptoms show as cough, expectoration, high fever etc., easily with
Chronic bronchitis, bronchiectasis, pneumonia, flu etc. are obscured, and need by diagnosis sides such as iconography, bacteriology, immunologys
Method can be made a definite diagnosis.Clinically the most commonly used is bacteriological method Sputum smears microscopy and Sputum culturing, Sputum smears microscopy is in world wide
Most popular technology during tuberculosis checks due to this method simple equipments, is suitble to use in the area of economics of underdevelopment.But
High due to requiring the bacterial content in sample, this method sensitivity is not high, cause largely to apply cloudy patients be not found so as to
Turn sun, and apply cloudy patient there is infectiousness, can not be ignored, this method is without species specificity, poor sensitivity, by sputum sample and the state of an illness
Influence.Goldstandard of the Sputum culturing as diagnosis of tuberculosis, but it is long there are incubation time, have BACTEC now
The rapid culture systems such as MGIT960 systems can be separately cultured mycobacterium tuberculosis within 2 weeks, but due to the culture medium of its preparation,
Nourishing additive agent, miscellaneous bacteria inhibitor are expensive, can not be widely popularized in developing country, and fast culture and improvement L-J
Culture is significantly increased compared to pollution rate, and false positive results is caused to occur.The common detection method for Mass screening is skin
Tuberculin is tested, and uses tulase purified protein derivative (PPD), and due to containing in PPD, there are many mycobacterial species (to cause
Characteristic of disease mycobacteria, environment mycobacteria and BCG) common to antigen molecule, therefore PPD diagnosis of tuberculosis specificity compared with
It is poor, it is impossible to the effectively infection of differentiation mycobacterium tuberculosis and BCG vaccination, the imageological examination such as x-ray inspection of routine of tuberculosis,
CT examination, MRI inspections, ultrasonic examination etc. are expensive, and body is caused centainly to injure, and specificity is low, are not suitable for often
The inspection diagnosis of rule.
The detection method T-SPOT based on T cell is to be captured using IFN-γ specific antibody through tuberculosis antigen at present
The IFN-γ generated after the peripheral blood lymphocytes culture of stimulation, and showed in a manner that elisa develops the color, from
The quantity of spot determines the situation of cell secretion of cytokines, and cellular immune function is evaluated from individual cell level.Using T cell as
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 core Mycobacterium tuberculosis genes group RD1 areas coding are stimulant
Commercialization IGRA detection kits, such as QuantiFERON-TB Gold test and T-SPOT, present higher sensitive
Property and specificity.And Rv3793 (GI:15610929) it is arabinose transferase gene on H37Rv genomes, participates in tuberculosis
The synthesis of arabinose on Mycobacterial cell wall leads to mycobacterium tuberculosis to ethambutol drug resistance.Rv3793 full length genes
3285bp encodes 1094 amino acid, is possible tuberculosis virulence-associated protein, Rv3793 is in mycobacterium tuberculosis complex
In the presence of carrying out Epitope prediction analysis to it using bioinformatics software, it is found that there are more T cells for Rv3793 albumen
Epitope has potential diagnostic, and only exists in mycobacterium tuberculosis and a small number of environment mycobacterias, can effective district
Divide tuberculosis patient, Pulmonary Diseases patient and Healthy People.
The present invention is established on the basis of reverse vaccinology, and it is possible immune to go out mycobacterium tuberculosis using computational screening
Then originality antigenic storehouse predicts tuberculosis antigen MHC-I class T cell tables using bioinformatics software TE predict and IEDB
Position, synthesizes these polypeptides by solid-state synthetic method, tuberculosis neoantigen is screened first with community immunity screening test, screens
Go out Immunodominant Antigenic and then its immunogenicity is verified by zoopery, last empirical tests obtain immune excellent
On the one hand gesture antigen can be used for Diagnosis of Tuberculosis, on the other hand available 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 Rv3793.
It is a further object of the present invention to provide antigen of mycobacterium tuberculosis albumen Rv3793T cell epitope peptides and its applications.
The present invention is based on following designs：Rv3793 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 process of cell wall, is possible tuberculosis virulence-associated protein.This
Invention encodes base using bioinformatics software TE predict and IEDB based on T cell IFN-γ release tech to Rv3793
Because upper T cell antigen epitope is predicted, epitope polypeptide is synthesized, then by T-SPOT methods to tuberculosis using solid-state synthetic method
People, lung other diseases patient, the T lymphocyte specific in healthy human body are detected, so as to evaluate the antigen for tying
The sensitivity and specificity of core detection, while the immunodominance on Rv2201 antigen proteins is selected by community immunity testing sieve
Epitope peptide, then by zoopery, determine the immunogenicity of immunodominant T cell epitope peptide.
In order to realize the object of the invention, the present invention provides antigen of mycobacterium tuberculosis albumen Rv3793 and is preparing tuberculosis detection
With the application in diagnostic reagent；Wherein, the amino acid sequence of the antigen protein Rv3793 such as SEQ ID NO:It or should shown in 5
Sequence is through replacing, lacking or adding one or several amino acids formed amino with identical immunogenicity and same antigen
Acid sequence.
The present invention also provides antigen of mycobacterium tuberculosis albumen Rv3793T cell epitope peptides, the epitope peptide be selected from P1,
P10, P12 and P13, amino acid sequence is respectively such as SEQ ID NO:Shown in 1-4.
The present invention also provides epitope peptide or its analogs as 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 contain the recombinant bacterium of DNA molecular and its recombination egg of expression and purification for encoding the epitope peptide
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 tuberculosis detection reagent, vaccine and drug is prepared.
The present invention also provides a kind of Diagnosis of Tuberculosis reagent, contain antigen of mycobacterium tuberculosis albumen in the diagnostic reagent
The weight that the DNA molecular or the recombinant bacterium by containing the DNA molecular of the Rv3793 or coding antigen protein Rv3793 generates
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 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 Rv3793 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 Rv3793 or compiles
The recombinant protein that the DNA molecular of the code antigen protein Rv3793 or the recombinant bacterium by containing the DNA molecular generate；With/
Or,
The epitope peptide, the DNA molecular of the coding epitope peptide and/or the recombinant protein.
The present invention also provides a kind of antituberculotic, active ingredient is included with antigen of mycobacterium tuberculosis albumen Rv3793
And/or the epitope peptide is as immunogene, is aided with adjuvant immunity experimental animal, the polyclonal antibody of preparation or with tuberculosis point
Branch bacteroides antigen albumen Rv3793 and/or the epitope peptide are aided with adjuvant immunity experimental animal, using hybridoma as immunogene
Technology or DNA recombinant techniques, the identification antigen of mycobacterium tuberculosis albumen Rv3793 and its t cell epitope peptide antigen of preparation
Humanized monoclonal antibodies.
The present invention further provides antigen of mycobacterium tuberculosis albumen Rv3793 and/or the epitope peptides and its derivative table
The application of position peptide or its analog in specific T-cells and B cell immune response caused by detection mycobacterium tuberculosis infection.
It is through the epitope peptide and its derivative epitope peptide or its analog antigen or their group by the lymphocyte of human or animal
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.
Include elisa experiment (T- for the detection method of the cell factor of tuberculosis specific T-cells secretion
SPOT), enzyme-linked immunosorbent assay (ELISA), immune colloid gold experiment, the interior dyeing of cell factor and T cell proliferation test etc..
The detection method of the cell factor of B cell secretion includes enzyme-linked immunosorbent assay (ELISA) etc..
Peripheral blood of the lymphocyte from human or animal, venous blood, cerebrospinal fluid, pleural effusion or hydrothorax etc..
The present invention has the following advantages：
(1) present invention utilizes mycobacterium tuberculosis Rv3793 proteantigens and its t cell epitope peptide to be used for as stimulant
Mycobacterium tuberculosis infect caused by specific T-cells and B cell immune response, 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, epitope provided by the invention can stimulate body to generate stronger T cell and be immunized instead
Should, therefore when above-mentioned epitope is used to carry out ex vivo T cell interferon release experiment as stimulant, sensitivity significantly carries
It is high.
(3) epitope polypeptide being synthesized using the method for synthesis in solid state, is conducive to quality control, and cost is relatively low, purity is high,
It is suitble to large-scale commercial production.
(4) antigen protein Rv3793 of the invention is tuberculosis virulence correlation factor, and the high intensity of t cell responses shows this
Antigen has stronger immunogenicity, and zoopery equally confirms that Rv3793 and its t cell epitope have good immunogene
Property, therefore can be used as novel Vaccinum Calmette-Guerini candidate antigens.
(5) detection reagent of the invention can be widely used for the related necks such as auxiliary diagnosis lungy, epidemiological surveillance
Domain.
Description of the drawings
Fig. 1 is the spot figure that albumen Rv3793 carries out T-SPOT experiments in the embodiment of the present invention 4.
Fig. 2 shows that the t cell epitope peptide P1 of antigen protein Rv3793 in the embodiment of the present invention 6 can the generation of effective stimulus body
IFN-γ (A), IL-2 (B), IL-4 (C) and IL-10 (D).
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 Rv3793 of embodiment and the expression and purification of albumen
Rv3793(GI:15610929) be Mycobacterium tuberculosis H37Rv genome encoding conservative memebrane protein, containing 1094
Amino acid, amino acid sequence such as SEQ ID NO:Shown in 5.According to its coding gene sequence, primer is designed, utilizes prokaryotic expression
System (such as Escherichia coli), which is expressed and purified, obtains antigen protein Rv3793.
The synthesis of 2 antigen of mycobacterium tuberculosis albumen Rv3793T cell epitope peptides of embodiment
Based on T cell IFN-γ release tech, Rv3793 is compiled using bioinformatics software TE predict and IEDB
T cell antigen epitope is predicted on code gene, and epitope polypeptide is synthesized, then by T-SPOT methods to tuberculosis using solid-state synthetic method
Patient, lung other diseases patient, the T lymphocyte specific in healthy human body are detected, and are used for so as to evaluate the antigen
The sensitivity and specificity of tuberculosis detection.
Antigen of mycobacterium tuberculosis albumen Rv3793T cell epitope peptides provided in this embodiment be selected from P1, P10, P12 and
P13, amino acid sequence is respectively such as SEQ ID NO:Shown in 1-4.
The preparation of 3 tuberculosis T-SPOT detection kits of embodiment
The kit forms as follows substantially：
1. the epitope peptide antigen that proteantigen Rv3793 and/or embodiment 2 prepared by embodiment 1 are synthesized：The epitope peptide
Selected from least one of P1, P10, P12 and P13.
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：
The lung's other diseases of Sputum culturing and Sputum smears for 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 between 15-80 Sui, from the continuous time gone to a doctor to tuberculosis ward
It is randomly selected in sample.50 tuberculosis volunteers, 49 Pulmonary Disease patients and 55 healthy volunteer's blood are acquired altogether
Sample acquires peripheric venous blood during 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, adds in the separation of certain volume in centrifuge tube
Liquid, by the blood sample tiling after dilution to separating liquid ullage, two liquid level interfaces of holding are clear, and separating liquid, anti-freezing are not diluted
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, and plasma layer is with detaching
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, adds in 1640 culture mediums of RPMI to 10ml, at room temperature 10 points of 800g centrifugations
Clock.
4) it discards supernatant, 1640 culture mediums of 7ml RPMI is added in after resuspension, 700g is centrifuged 10 minutes.
5) it discards supernatant plus precipitation is resuspended in 0.5ml AIM-V culture mediums.
6) using automated cell calculating instrument to cell count, with AIM-V culture mediums prepare 500 μ L cell concentrations for 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 in into the microwell plate of pre-coated primary antibody, each patient sets respectively
6 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), 4 detection holes (are separately added into a concentration of 20 μ g/ml's of 100 μ L in 4 holes
One in 4 epitope peptides P1, P10, P12, P13), the good PBMC of the 100 above-mentioned dilutions of μ L is added in each hole, is made in every hole
The quantity of PBMC is placed in 37 DEG C up to 250,000, by antigen and PBMC cells, 5%CO2Incubator in cultivate 20 hours.
4. board-washing and result judgement
PBMC cells and antigenic stimulus object are washed away, 100 μ L primary antibodies is added to be incubated at room temperature 1 hour, is washed 5 times with PBS, adds secondary antibody
Incubation at room temperature 1 hour, then washed 5 times with PBS, after substrate is added 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
Wherein four epitope peptides detect the result of 50 tuberculosis patients, 49 lung other diseases patients and 55 Healthy Peoples
Statistics is shown in Table 2.
2 Rv3793 proteantigens of table, four epitope peptide detection sensitivities and specificity statistics
| Epitope peptide | Sensitivity (%) | Specificity (%) |
| P1 | 12 | 99.04 |
| P10 | 6 | 100 |
| P12 | 10 | 100 |
| P13 | 4 | 100 |
| 4 polypeptide joints | 24 | 99.04 |
Detection sensitivity=(tuberculosis patient detection 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 obtaining Rv3793 proteantigens P1, P10,
The sensitivity that tetra- epitope peptide joint-detections of P12, P13 go out tuberculosis patient is 24%, specificity 99.04%.
The spot figure that T-SPOT experiments are carried out using albumen Rv3793 is shown in Fig. 1.
The immunogenicity detection of 5 Rv3793T cell epitope peptides of embodiment
1st, in order to verify the immunogenicity of Rv3793T cell epitope peptides, it is contemplated that naked peptide is degradable and is not easy to be passed by antigen
In the characteristic of cell recognition, naked peptide and the hemocyanin (KLH) of synthesis are coupled, the t cell epitope peptide that coupling is obtained
Carry out immune BALB/c mouse.
2nd, mouse is immunized
Female BAl BIc/c mouse 36 of 6 week old are screened, are divided into 6 groups, PBS negative control groups (PBS), vehicle control group blood
Azurin (KLH) control group, 20 μ g low dose groups (Ag85b-L) of antigen of mycobacterium tuberculosis albumin A g85B, 50 μ g of Ag85B
High dose group (Ag85B-H), 50 μ g low dose groups (P1-L) of Rv3793P1 polypeptides, 100 μ g high dose groups of Rv3793P1 polypeptides
(P1-H).By every group of antigen and corresponding adjuvant -- poly poly (I:C) 50 μ L and double octadecyldimethyl brominations
It is immune primary every 3 weeks using mouse is immunized by the way of inoculating after 100 μ L of ammonium (DDA) are fully emulsified, it is common to be immunized three times,
Final immunization is detected after 4 weeks.
3rd, the separation of mice serum
Eyeball of mouse 500 μ L of blood sampling, blood is placed in 37 DEG C of incubators 2 hours, is then transferred to 4 DEG C of refrigerator overnights.It is secondary
Day, supernatant is drawn into blood 3000rpm centrifugations after five minutes.
4th, serum antibody titer detects
1) antigen coat liquid is prepared, the epitope polypeptide P1 and Ag85B of antigen protein Rv3793 are delayed respectively with carbonate
After fliud flushing (pH=9.6) dilution, the Rv3793 epitope polypeptide P1 coating buffers of 5 μ g/ml and the Ag85B antigen coats of 2 μ g/ml are obtained
Liquid, ELISA Plate are coated with overnight with two kinds of coating buffers in 4 DEG C respectively, and next day is washed 5 times with PBST.
2) it is closed 2 hours with 37 DEG C of the PBS liquid containing 2%BSA, is washed 5 times with PBST.
3) with PBS by each group serum from 1:10 proceed by doubling dilution, and the serum after 100 μ L dilutions is added in every hole,
After 37 DEG C are incubated 1 hour, washed 5 times with PBST.
4) IgG, IgG1, IgG2a antibody of 5000 times of diluted HRP labels are separately added into, per 100 μ L of hole, 37 DEG C are incubated 1
After hour, washed 5 times with PBST.
5) 50 μ L 2M sulfuric acid are added in after adding in 37 DEG C of TMB colour developings 15 minutes, in every hole and terminate color development stopping.
6) the suction pipe shading value of microplate reader Detection wavelength 450nm.
7) criterion：OD≥2.1×OD(negative control)Judgement for the positive.
8) interpretation of result：The Specific antibody titre of each group serum is shown in Table 3.
Specific antibody titre in 3 mice serum of table
| Group/antibody | IgG | IgG1 | IgG2a |
| PBS groups | 1 | 1 | 1 |
| KLH groups | 1 | 1 | 1 |
| Ag85B-L | 8063489 | 142543.8 | 452548 |
| Ag85B-H | 1015936 | 285087.6 | 640000 |
| P1-L | 226.3 | 56.6 | 25.1 |
| P1-H | 254 | 14.1 | 22.4 |
The results show that the specificity of epitope polypeptide P1 is not detected in blank control group PBS groups and negative control group KLH groups
Antibody.IgG, IgG1 and IgG2a antibody can be detected in P1 polypeptide high and low dose immune groups.Distinguished using the above method
Immunogenicity detection is carried out to Rv3793T cell epitope peptides P10, P12 and P13, in P10, P12 and P13 immune group mice serum
In be able to detect that P10, P12 and P13 specific antibody.
The cell immunogenicity detection of 6 Rv3793T cell epitope peptides of embodiment
1st, it after the mouse being immunized in embodiment 5 is put to death, impregnates 5 minutes, mouse is fixed on ultra-clean in 75% alcohol
On cystosepiment in platform, peritonaeum is cut off, isolates spleen, is placed it in equipped in 1640 plate.
2nd, on the nylon wire of 200 mesh, mouse spleen is lightly ground with plunger, the cell of grinding is passed through into strainer
Filtering.1000r/min, which is centrifuged, abandons supernatant for 5 minutes, collects cell precipitation.
3rd, gently vibrating cell precipitation makes its loosening, only adds in erythrocyte cracked liquid according to 2mL/, after mixing, is placed in 37
It is incubated in DEG C incubator and after ten minutes, adds in 1640 culture mediums for being equivalent to 2 times of volumes of erythrocyte cracked liquid and terminate reaction, 1000r/
Min, which is centrifuged, abandons supernatant for 5 minutes, collects splenocyte precipitation.
4th, splenocyte is only resuspended with 1640 culture medium 2ml/, 1000r/min, which is centrifuged, abandons supernatant for 5 minutes, collects splenocyte and sinks
It forms sediment.Cell concentration is measured with cell counter.
5th, with containing 10%FBS 1640 culture mediums dilute splenocyte, make its a concentration of 2 × 106/mL.Every group takes 500 μ L
Splenocyte liquid is placed in 24 well culture plates, and PBS groups and DP adjuvant groups splenocyte are respectively with Ag85B albumen (5 μ g/mL), Rv3793
T cell epitope peptide P1 (5 μ g/mL are not coupled the naked peptide of KLH albumen) be incubated jointly with splenocyte, Ag85B-L and Ag85B-H groups
It is incubated jointly with splenocyte with the Ag85B albumen of 500 μ L (5 μ g/mL) respectively, P1-L and P1-H groups are with 500 μ L P1 polypeptides (5
μ g/mL are not coupled the naked peptide of KLH albumen) it is incubated jointly with splenocyte, P1-L and P1-H groups are with P1 polypeptides (the 5 μ g/ of 500 μ L
ML is not coupled the naked peptide of KLH albumen) it is incubated jointly with splenocyte, the sterile PBS and 500 μ L plant blood clottings of every group of 500 μ L of addition
Plain (PHA) (5 μ g/mL) as negative control and positive control, each detection hole does 2 repetitions.By splenocyte with it is corresponding
Stimulant is at 37 DEG C, 5%CO2After being incubated 72 hours in incubator, cell conditioned medium is collected, using in ELISA method detection supernatant
IFN-γ、IL-2、IL-4、IL-10。
6th, by carbonate buffer solution (pH=9.6), the L-10 monoclonal antibodies use such as IFN-γ monoclonal antibody, IL-2 monoclonal antibodies, IL-4 monoclonal antibodies
Carbonate buffer solution (pH=6.5) is according to 1:After 500 times of dilutions, it is added in 96 microwell plates by 100 μ L/ holes, 4 DEG C were coated with
Night.
7th, it after washing 3 times with PBTS, pats dry, adds in the PBS liquid chambers temperature containing 10%FBS and close 1 hour.
8th, after washing 5 times with PBST, by IFN-γ, IL-2, IL-4, IL-10 standard items according to corresponding dilution proportion
Into respective concentration, it is added separately on each elisa plate, as standard curve.Remaining adds in 100 μ L cell conditioned mediums to be detected,
Incubation at room temperature 2 hours.
9th, after being diluted 5 times with PBST, be separately added into the respective detection antibody of 100 μ L IFN-γ, IL-2, IL-4, IL-10+
The secondary antibody of HRP labels, is incubated at room temperature 1 hour.
10th, after washing 7 times with PBST, 100 μ L of TMB developing solutions are added in, after being incubated at room temperature 30 minutes, add in 50 μ L of terminate liquid
Terminate reaction.
11st, microplate reader measures the light absorption value of wavelength 450nm, while the light absorption value of Detection wavelength 570nm is as control.
12nd, interpretation of result：Standard curve is made according to cytokine standards product, is then substituted into the OD values of detection hole public
Formula calculates the final concentration of every group of various cell factors of mouse.The results are shown in Figure 2.
The results show that compared with blank control PBS groups and negative control KLH groups, the t cell epitope peptide P1 of Rv3793 can be pierced
The IFN-γ (P ＜ 0.001) of sharp mouse generation higher concentration, IL-2 (P ＜ 0.05), IL-4 (P ＜ 0.001), IL-10 (P ＜
0.001), and P1 high dose groups generate IFN-γ (P ＜ 0.001), the amount of IL-10 (P ＜ 0.001) is higher than low dose group.IFN-
γ is Th1 cytokines, can promote removing of the macrophage to mycobacterium tuberculosis by activating macrophage.
IL-4 and IL-10 is Th2 cytokines, and the antibody during tuberculosis infection is promoted to generate.It is played in tuberculosis infection immune
Adjustment effect.Cell immunogenicity detection is carried out to Rv3793T cell epitope peptides P10, P12 and P13 using the above method respectively,
The result shows that P10, P12 and P13 can also stimulate mouse to generate corresponding cell factor.As it can be seen that antigen of mycobacterium tuberculosis egg
Four t cell epitope peptides of white Rv3793, can stimulate tuberculosis patient to generate IFN-γ, can effectively distinguish tuberculosis patient and lung
Other illness patient and healthy population, and four polypeptide Combining diagnosis efficiency improve.Rv3793 and its t cell epitope peptide can be made
Diagnosis lungy is used for for a kind of detection reagent.In zoopery, the t cell epitope peptide P1 of Rv3793 can effective stimulus machine
Body generates IFN-γ (P ＜ 0.001), IL-2 (P ＜ 0.05), IL-4 (P ＜ 0.001), IL-10 (P ＜ 0.001), and can stimulate
Mouse generates IgG antibody, IgG1 and the IgG2a of specificity, thus proves that Rv3793 and its t cell epitope peptide have good exempt from
Epidemic focus can stimulate body to generate cellullar immunologic response and body is stimulated to generate humoral immune response, be a kind of immune excellent
Gesture antigen, it may be considered that structure and preparation as mtb vaccine.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. applications of the antigen of mycobacterium tuberculosis albumen Rv3793 in tuberculosis detection reagent, vaccine and drug is prepared；Wherein, institute
State the amino acid sequence such as SEQ ID NO of antigen protein Rv3793:Shown in 5.
2. antigen of mycobacterium tuberculosis albumen Rv3793T cell epitope peptides, which is characterized in that the epitope peptide is selected from such as SEQ ID
NO:One kind in amino acid sequence shown in 1-4.
3. application of the epitope peptide described in claim 2 in tuberculosis detection reagent, Vaccinum Calmette-Guerini or antituberculotic is prepared.
4. a kind of Diagnosis of Tuberculosis reagent, which is characterized in that contain antigen of mycobacterium tuberculosis albumen in the diagnostic reagent
The weight that the DNA molecular or the recombinant bacterium by containing the DNA molecular of the Rv3793 or coding antigen protein Rv3793 generates
Histone；And/or
Epitope peptide described in claim 2 encodes the DNA molecular of the epitope peptide or the DNA of the epitope peptide is encoded by containing
The recombinant protein that the recombinant bacterium of molecule generates.
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
Different in nature 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. the answering in Vaccinum Calmette-Guerini is prepared of epitope peptide described in antigen of mycobacterium tuberculosis albumen Rv3793 and/or claim 2
With.
8. a kind of Vaccinum Calmette-Guerini, which is characterized in that its active ingredient is antigen of mycobacterium tuberculosis albumen Rv3793 or coding institute
State the recombinant protein that the DNA molecular of antigen protein Rv3793 or the recombinant bacterium by containing the DNA molecular generate；And/or
Epitope peptide described in claim 2 encodes the DNA molecular of the epitope peptide or the DNA of the epitope peptide is encoded by containing
The recombinant protein that the recombinant bacterium of molecule generates.
9. epitope peptide is in antituberculotic is prepared described in antigen of mycobacterium tuberculosis albumen Rv3793 and/or claim 2
Using.
10. a kind of antituberculotic, which is characterized in that its active ingredient is included with antigen of mycobacterium tuberculosis albumen Rv3793
And/or epitope peptide described in claim 2 is as immunogene, is aided with adjuvant immunity experimental animal, the polyclonal antibody of preparation or
Using epitope peptide described in antigen of mycobacterium tuberculosis albumen Rv3793 and/or claim 2 as immunogene, it is aided with adjuvant immunity reality
Animal is tested, using hybridoma technology or DNA recombinant techniques, the identification antigen of mycobacterium tuberculosis albumen Rv3793 and right of preparation
It is required that the Humanized monoclonal antibodies of the 2 epitope peptide antigens.
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| CN102608333B (en) * | 2012-03-30 | 2013-06-05 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
| CN104628834A (en) * | 2015-01-23 | 2015-05-20 | 中国疾病预防控制中心传染病预防控制所 | Antigen used for immunodetection of tuberculosis infected T cell and application thereof |
| CN105294844A (en) * | 2015-09-28 | 2016-02-03 | 上海交通大学 | Mycobacterium tuberculosis Rv3457c recombinant protein as well as preparation method and application thereof |
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| CN102947465A (en) * | 2009-12-21 | 2013-02-27 | 贝克顿·迪金森公司 | Methods for identifying drug resistant mycobacterium |
| CN102608333B (en) * | 2012-03-30 | 2013-06-05 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
| CN104628834A (en) * | 2015-01-23 | 2015-05-20 | 中国疾病预防控制中心传染病预防控制所 | Antigen used for immunodetection of tuberculosis infected T cell and application thereof |
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