CN106248934B - Antigen of mycobacterium tuberculosis albumen Rv0446c and its t cell epitope peptide application - Google Patents
Antigen of mycobacterium tuberculosis albumen Rv0446c and its t cell epitope peptide application Download PDFInfo
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- CN106248934B CN106248934B CN201610729305.5A CN201610729305A CN106248934B CN 106248934 B CN106248934 B CN 106248934B CN 201610729305 A CN201610729305 A CN 201610729305A CN 106248934 B CN106248934 B CN 106248934B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Abstract
The present invention relates to the application of antigen of mycobacterium tuberculosis albumen Rv0446c and its t cell epitope peptide in diagnosis reagent, vaccine and medicine is prepared, the amino acid sequence of the antigen protein Rv0446c and its t cell epitope peptide are respectively such as SEQ ID NO:Shown in 15.The present invention utilizes mycobacterium tuberculosis Rv0446c proteantigens and its t cell epitope peptide to be used for specific T-cells and B cell immune response caused by mycobacterium tuberculosis infection as stimulant, 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 Rv0446c proteantigens and its epitope peptide can be widely used for association area, the Vaccinum Calmette-Guerinis and antituberculotic prepared by Rv0446c 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, is related to antigen of mycobacterium tuberculosis albumen
The application of Rv0446c 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 the life in future.From
After the World Health Organization in 1993 announces that tuberculosis turns into global crisis, morbidity and mortality lungy occupy height not always
Under, according to WHO report, newly-increased tuberculosis patient about 8,000,000, there are about 200~3,000,000 people and dies from tuberculosis every year every year.China is 22
The 2nd is occupied in the high burden country of individual tuberculosis, national the 5th epidemiology sampling check result is shown:National 1,300,000 human hairs
Disease, the 14.3% of global incidence is accounted for, China, which is also that 27, whole world resistant tuberculosis is high, 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
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 it is high that researcher should be directed 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, it is necessary to by diagnosis sides such as iconography, bacteriology, immunologys
Method can be made a definite diagnosis.Clinically conventional is bacteriological method Sputum smears microscopy and Sputum culturing, and Sputum smears microscopy is in world wide
Most popular technology during tuberculosis checks, due to this method simple equipments, it is adapted to use in the area of economics of underdevelopment.But
Due to requiring high to the bacterial content in sample, therefore this method sensitivity is not high, cause largely to apply cloudy patient 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 incubation time to be present, now existing BACTEC
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, causes false positive results to occur.The conventional detection method for Mass screening is skin
Tuberculin is tested, and uses tulase purified protein derivative (PPD), (is caused due to containing many mycobacterial species in PPD
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 necessarily to injure, and specificity is low, are not suitable for often
The inspection diagnosis of rule.
Resided in after mycobacterium tuberculosis intrusion human body in macrophage, immune anti-main to mycobacterium tuberculosis of human body
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 BMDC are killed and remove tuberculosis branch bar so as to reach by the approach such as enzyme
The purpose of bacterium.Wherein MHC-I quasi-molecules are present in the surface of all karyocytes, and MHC- class Ⅱmolecules exist only in full-time resist
Former presenting cell such as macrophage, BMDC etc., therefore CD8+T cell epitopes have in the diagnosis of tuberculosis and vaccine construction
There is wider purposes.
The detection method T-SPOT based on T cell is to be captured using IFN-γ specific antibody through tuberculosis antigen at present
Caused IFN-γ after the PBLC culture of stimulation, and showed in a manner of ELISpot 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, and the detection method can not only filter out activity knot
Core patient, while incubation period patient can be also detected, it is existing with knot at present so as to preferably prevent and control incubation period tuberculosis
Tuberculosis the specific antigen ESAT-6 and CFP-10 of core Mycobacterium tuberculosis genes group RD1 areas coding holoprotein or polypeptide are stimulant
Commercialization IGRA detection kits, such as QuantiFERON-TB Gold test and T-SPOT, present higher sensitive
Property and specificity.And Rv0446c (GI:15607587) be Mycobacterium tuberculosis H37Rv genome encoding conservative memebrane protein, contain
256 amino acid, the secretory protein that can be detected in H37Rv culture supernatant, are primarily involved in the process of cell membrane, are
Possible tuberculosis virulence-associated protein.Rv0446c is that mycobacterium tuberculosis guards memebrane protein, in mycobacterium tuberculosis culture supernatant
The middle secretory protein that can be detected, is primarily involved in the process of cell membrane and cell, and it is entered using bioinformatics software
Row Epitope prediction is analyzed, it is found that Rv0446c albumen has more t cell epitope, have potential diagnostic.
The present invention is established on the basis of reverse vaccinology, and it is possible immune to go out mycobacterium tuberculosis using computational screening
Originality antigenic storehouse, then predict 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 then, then its immunogenicity is verified by zoopery, last empirical tests obtain immune excellent
On the one hand gesture antigen and its epitope can be used for Diagnosis of Tuberculosis, on the other hand the transformation available for BCG vaccine.
The content of the invention
It is an object of the invention to provide antigen of mycobacterium tuberculosis albumen Rv0446c application.
It is a further object of the present invention to provide antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitopes peptide and its answer
With.
The present invention is based on following design:Rv0446c is the conservative memebrane protein on mycobacterium tuberculosis, based on T cell IFN-
γ release tech, using bioinformatics software TE predict and IEDB to T cell antigen epitope on Rv0446c encoding genes
Be predicted, using solid-state synthetic method synthesize t cell epitope peptide, then by T-SPOT (T cell spot test) to tuberculosis patient,
T lymphocyte specific in lung other diseases patient, healthy human body is detected, and is examined so as to evaluate the antigen for tuberculosis
The sensitivity and specificity of survey, while select by community immunity testing sieve the immunodominance table on Rv0446c antigen proteins
Position 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 Rv0446c and is preparing tuberculosis inspection
Application in test agent, vaccine and medicine;Wherein, the amino acid sequence of the antigen protein Rv0446c such as SEQ ID NO:5 institutes
Show, or the sequence one or several amino acids formed has identical immunogenicity and same antigen through replacing, lacking or add
The amino acid sequence of property.
The present invention also provides antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitope peptides, and the epitope peptide is selected from
P120, P121, P122 and P123, its amino acid sequence is respectively such as SEQ ID NO:Shown in 1-4.
The present invention also provides epitope peptide or its analog as derived from the t cell epitope peptide.
The present invention also provides the DNA molecular for encoding the epitope peptide.
The present invention also provides expression cassette and expression vector containing the DNA molecular for encoding the epitope peptide.
The present invention also provides the transgenic cell line containing the DNA molecular for encoding the epitope peptide.
The present invention is also provided containing the recombinant bacterium of DNA molecular and its restructuring egg of expression and purification for encoding the epitope peptide
In vain.
The present invention also provides the epitope peptide, DNA molecular, the transgenic cell line or described of the coding epitope peptide
Application of the recombinant protein of recombinant bacterium and its expression and purification in tuberculosis detection reagent, vaccine and medicine is prepared.
The present invention also provides a kind of tuberculosis detection reagent, contains antigen of mycobacterium tuberculosis albumen in the detection reagent
Rv0446c, or coding antigen protein Rv0446c DNA molecular, or as caused by the recombinant bacterium containing the DNA molecular
Recombinant protein;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 detection reagent.The kit also 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), Background control 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:Wrapped on pvdf membrane
The primary antibody of quilt can combine the IFN-γ in cell conditioned medium as capture antibody, and IFN-γ and can is captured by ELIAS secondary antibody, shown
Color.Two kinds of antibody are the monoclonal antibody of identification IFN-γ difference epitope.
The present invention also provides antigen of mycobacterium tuberculosis albumen Rv0446c and/or the epitope peptide, the coding epitope peptide
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, and its active ingredient is antigen of mycobacterium tuberculosis albumen Rv0446c, or compiles
Code antigen protein Rv0446c DNA molecular, or the recombinant protein as caused by the recombinant bacterium containing the DNA molecular;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, and its active ingredient is included with antigen of mycobacterium tuberculosis albumen
Rv0446c and/or the epitope peptide are aided with adjuvant immunity experimental animal as immunogene, the polyclonal antibody of preparation, or with
Antigen of mycobacterium tuberculosis albumen Rv0446c and/or the epitope peptide are aided with adjuvant immunity experimental animal, adopted as immunogene
With hybridoma technology or DNA recombinant techniques, the identification antigen of mycobacterium tuberculosis albumen Rv0446c and its T cell of preparation
The Humanized monoclonal antibodies of epitope peptide antigen.
The present invention further provides antigen of mycobacterium tuberculosis albumen Rv0446c and/or the t cell epitope peptide and its spread out
Raw epitope peptide or its analog are in specific T-cells and B cell immune response caused by detection mycobacterium tuberculosis infection
Application.It is through the mycobacterium tuberculosis Rv0446c proteantigens, its t cell epitope by the lymphocyte of human or animal
After peptide and its derivative epitope peptide or its analog antigen or their compositions stimulate, the thin of T cell or B cell secretion is detected
Intracellular cytokine.
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.
Tested for the detection method of the cell factor of tuberculosis specific T-cells secretion including ELISpot
(ELISPOT), EUSA (ELISA), immune colloid gold experiment, the interior dyeing of cell factor and T cell propagation examination
Test.The detection method of the cell factor of B cell secretion includes EUSA (ELISA) etc..
Peripheral blood of the lymphocyte from human or animal, venous blood, cerebrospinal fluid, pleural effusion or hydrothorax etc..
The present invention has advantages below:
(1) present invention is used by the use of mycobacterium tuberculosis Rv0446c proteantigens and its t cell epitope peptide as stimulant
In mycobacterium tuberculosis infection caused by specific T-cells and B cell immune response, with the past using comlete antigen compared with, energy
It is enough 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 produce stronger T cell
Should, therefore when using above-mentioned epitope peptide to carry out ex vivo T cell interferon release experiment as stimulant, sensitivity significantly carries
It is high.
(3) using the method synthesis t cell epitope peptide of synthesis in solid state, quality control is advantageous to, and cost is relatively low, purity
Height, it is adapted to large-scale commercial production.
(4) antigen protein Rv0446c of the invention is tuberculosis virulence correlation factor, cell immune response in zoopery
Show that the antigen has stronger immunogenicity with humoral immune reaction, can be used as new 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.
Brief description of the drawings
Fig. 1 shows that two t cell epitope the peptides P120 and P123 of antigen protein Rv0446c in the embodiment of the present invention 6 can have
Effect stimulates body to produce IFN-γ (A), IL-4 (B) and IL-10 (C).
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The antigen of mycobacterium tuberculosis gene Rv0446c of embodiment 1 clone and the expression and purification of albumen
Rv0446c(GI:15607587) be Mycobacterium tuberculosis H37Rv genome encoding conservative memebrane protein, containing 256
Amino acid, its 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 Rv0446c.
The synthesis of the antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitope peptides of embodiment 2
Based on T cell IFN-γ release tech, Rv0446c is compiled using bioinformatics software TE predict and IEDB
T cell antigen epitope is predicted on code gene, synthesizes t cell epitope peptide using solid-state synthetic method, then pass through T-SPOT methods pair
Tuberculosis patient, lung other diseases patient, the T lymphocyte specific in healthy human body are detected, so as to evaluate the antigen
Sensitivity and specificity for tuberculosis detection.
The present embodiment provide antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitopes peptide be selected from P120, P121,
P122 and P123, its amino acid sequence is respectively such as SEQ ID NO:Shown in 1-4.
The preparation of the tuberculosis T-SPOT detection kits of embodiment 3
The kit forms as follows substantially:
1. embodiment 1 prepare proteantigen Rv0446c and/or embodiment it is 2-in-1 into epitope peptide antigen:The epitope peptide
Selected from least one of P120, P121, P122 and P123.
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), Background control 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:
Coated primary antibody can combine the IFN-γ in cell conditioned medium as capture antibody on pvdf membrane, and IFN-γ and can is by enzyme mark two
Anti- capture, colour developing.Two kinds of antibody are the monoclonal antibody of identification IFN-γ difference epitope.
The epitope peptide of embodiment 4 is used for the clinical detection of tuberculosis infection
1. the separation of PBLC
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 between 15-80 year, to tuberculosis ward go to a doctor continuous time
Randomly selected in sample.50 tuberculosis volunteers, 39 Pulmonary Disease patients and 55 healthy volunteer's blood are acquired altogether
Sample, peripheric venous blood is gathered using the anticoagulant heparin vacuum blood collection tube of endotoxin-free during blood sampling, every volunteer takes a blood sample about 5ml
~10ml.
1) sample in 4 hours use Ficoll-Hypaque separating liquids separation PBMCs.
2) first by whole blood RPMI-1640 culture mediums 1:1 dilution is mixed, and the separation of certain volume is added in centrifuge tube
Liquid, separating liquid ullage is arrived into the blood sample tiling after dilution, two liquid level interfaces of holding are clear, and separating liquid, anti-freezing are not diluted
Whole blood, the culture volumes of RPMI 1640 are 1:1:1, room temperature (18~26 DEG C), 800g is centrifuged 20 minutes.
3) after centrifugation terminates, ttom of pipe is red blood cell, and intermediate layer is separating liquid, and the superiors are plasma layers, and plasma layer is with separating
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, the addition culture mediums of RPMI 1640 to 10ml, and 800g is centrifuged under room temperature condition
10 minutes.
4) supernatant discarding, adds the culture mediums of 7ml RPMI 1640 after resuspension, 700g is centrifuged 10 minutes.
5) supernatant discarding adds 0.5ml AIM-V culture mediums that precipitation is resuspended.
6) using automated cell calculating instrument to cell count, with AIM-V culture mediums prepare 500 μ L cell concentrations be 2.5 ×
106/ ml cell suspension.
2. the preparation of epitope peptide
Epitope peptide prepared by solid-phase synthesis in embodiment 2 is dissolved with DMSO, each bar epitope peptide is respectively with containing
10% hyclone RPIM1640 culture mediums use after being diluted to finite 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
6 detection holes:Positive control wells (adding the phytohemagglutinin HA that 100 μ L concentration are 15 μ g/ml as positive stimulus thing), feminine gender
Control wells (adding 100 μ L PBS as negative control), (it is 20 μ g/ml's that 100 μ L concentration are separately added into 4 holes to 4 detection holes
One in 4 epitope peptides P120, P121, P122, P123), the good PBMC of the 100 above-mentioned dilutions of μ L is added in each hole, is made every
Antigen and PBMC cells are placed in 37 DEG C, 5%CO by PBMC quantity up to 250,000 in hole2Incubator in cultivate 20 hours.
4. T-SPOT board-washings and result judgement
PBMC cells and antigenic stimulus thing 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 lucifuge to develop the color 7 minutes, with purified water color development stopping, culture plate is put
The spot number on access panel is dried at ventilating opening.
Result judgement (table 1):Blank control wells spot number=N, detection hole spot number=T, positive quality control hole spot number=
P。
The T-SPOT result criterions of table 1
Wherein four t cell epitope peptides detect 50 tuberculosis patients, 39 lung other diseases patients and 55 Healthy Peoples
Result statistics be shown in Table 2.
2 Rv0446c proteantigens of table, four t cell epitope peptide detection sensitivities and specificity statistics
Epitope peptide | Sensitivity (%) | Specificity (%) |
P120 | 30 | 96.81 |
P121 | 18 | 98.94 |
P122 | 6 | 100 |
P123 | 22 | 96.81 |
4 polypeptide joints | 50 | 93.62 |
Detection sensitivity=(tuberculosis patient detection number positive/tuberculosis patient sum) × 100%
Detect specificity=1- (consumptive detects number positive+healthy volunteer and detects number positive)/(PUD D
Patient populations+healthy volunteer's sum)
Detect that positive is positive principle according to wall scroll polypeptide, be computed drawing Rv0446c proteantigens P120,
The sensitivity that tetra- epitope peptide joint-detections of P121, P122, P123 go out tuberculosis patient is 50%, specificity 93.62%.Wall scroll
T cell epitope peptide detection sensitivity is up to 30%.
The immunogenicity detection of the Rv0446c t cell epitope peptides of embodiment 5
1st, in order to verify the immunogenicity of Rv0446c t cell epitope peptides, it is contemplated that naked peptide is degradable and is not easy by antigen
The characteristic of presenting cell identification, naked peptide and the hemocyanin (KLH) of synthesis are coupled, the t cell epitope that coupling is obtained
Peptide carries out immune BALB/c mouse.
2nd, mouse is immunized
Female BAl BIc/c mouse 48 of 6 week old are screened, are divided into 8 groups, PBS negative control groups (PBS), vehicle control group blood
Azurin (KLH) control group, the μ g low dose groups (Ag85b-L) of antigen of mycobacterium tuberculosis albumin A g85B 20, the μ g of Ag85B 50
High dose group (Ag85B-H), the μ g low dose groups (P120-L) of Rv0446c P120 polypeptides 50, the μ g of Rv0446c P120 polypeptides 100
High dose group (P120-H), the μ g low dose groups (P123-L) of Rv0446c P123 polypeptides 50, the μ g of Rv0446c P123 polypeptides 100 are high
Dosage group (P123-H).By every group of antigen and corresponding adjuvant -- poly poly (I:C) 50 μ L and double octadecyls two
Mouse is immunized after the μ L of methyl bromide ammonium (DDA) 100 are fully emulsified by the way of subcutaneous vaccination, was immunized once every 3 weeks, exempts from altogether
Three times, final immunization is detected epidemic disease 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, draw supernatant after blood 3000rpm is centrifuged 5 minutes.
4th, serum antibody titer detects
1) antigen coat liquid is prepared, by antigen protein Rv0446c two epitope polypeptide P120 and P123 and Ag85B points
Not Yong carbonate buffer solution (pH=9.6) dilution after, obtain 5 μ g/ml Rv0446c epitope peptide P120 and P123 coating buffers and
2 μ g/ml Ag85B antigen coat liquid, ELISA Plate are coated with overnight with two kinds of coating buffers in 4 DEG C respectively, next day, 5 are washed with PBST
Time.
2) closed 2 hours with 37 DEG C of the PBS liquid containing 2%BSA, washed 5 times with PBST.
3) with PBS by each group serum from 1:10 proceed by doubling dilution, the serum added in every hole after 100 μ L dilutions,
After 37 DEG C are incubated 1 hour, washed 5 times with PBST.
4) IgG, IgG1, IgG2a antibody of the HRP marks of 5000 times of dilutions are separately added into, per the μ L of hole 100,37 DEG C are incubated 1
After hour, washed 5 times with PBST.
5) 50 μ L 2M sulfuric acid are added after adding 37 DEG C of TMB colour developings 15 minutes, in every hole and terminate color development stopping.
6) ELIASA Detection wavelength 450nm suction pipe shading value.
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 the mice serum of table 3
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 |
P120-L | 126.99 | 80 | 22.45 |
P120-H | 226.27 | 113.14 | 31.75 |
P123-L | 253.98 | 142.54 | 31.75 |
P123-H | 403.17 | 142.57 | 40 |
As a result show, P120 and P123 specificity is not detected by blank control group PBS groups and negative control group KLH groups
Antibody.IgG, IgG1 and IgG2a antibody can be detected in P120 and P123 polypeptide height dose immunization groups, wherein P120 is more
Peptide IgG antibody high dose group titre is higher than low dose group, and the high low dose group antibody titer indifference of P123 polypeptides.Using above-mentioned
Method carries out immunogenicity detection to Rv0446c t cell epitope peptides P121 and P122 respectively, in P120 and P123 immune groups
It is able to detect that P120 and P123 specific antibodies.
The cell immunogenicity detection of the Rv0446c t cell epitope peptides of embodiment 6
1st, after the mouse being immunized in embodiment 5 is put to death, soak 5 minutes, mouse is fixed on ultra-clean in 75% alcohol
On cystosepiment in platform, peritonaeum is cut off, isolates spleen, is placed on equipped with 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 filter screen
Filtering.1000r/min, which is centrifuged, abandons supernatant for 5 minutes, collects cell precipitation.
3rd, gently vibrating cell precipitation makes its loosening, only adds erythrocyte cracked liquid according to 2mL/, after mixing, is placed in 37
After being incubated 10 minutes in DEG C incubator, the 1640 culture medium terminating reactions equivalent to 2 times of volumes of erythrocyte cracked liquid, 1000r/ are added
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
Form sediment.Cell concentration is determined with cell counter.
5th, splenocyte is diluted with 1640 culture mediums containing 10%FBS, it is 2 × 10 to make its concentration6/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), Rv0446c
T cell epitope peptide P120 (5 μ g/mL are not coupled the naked peptide of KLH albumen), P123 (5 μ g/mL are not coupled the naked peptide of KLH albumen)
It is incubated jointly with splenocyte, Ag85B-L and Ag85B-H groups are common with splenocyte with 500 μ L Ag85B albumen (5 μ g/mL) respectively
It is incubated, P120-L and P120-H groups are total to 500 μ L P120 polypeptides (5 μ g/mL are not coupled the naked peptide of KLH albumen) with splenocyte
With being incubated, P123-L and P123-H groups are with 500 μ L P123 polypeptides (5 μ g/mL are not coupled the naked peptide of KLH albumen) and splenocyte
Common to be incubated, every group of μ L of addition 500 sterile PBS and 500 μ L phytohemagglutin phytolectins (PHA) (5 μ g/mL) are used as negative control and sun
Property control, each detection hole does 2 repetitions.By splenocyte and corresponding stimulant at 37 DEG C, 5%CO2It is incubated in incubator
After 72 hours, cell conditioned medium is collected, using the IFN-γ in ELISA method detection supernatant, IL-2, IL-4, IL-10.
6th, IFN-γ monoclonal antibody, IL-2 monoclonal antibodies, IL-4 monoclonal antibodies etc. are used with carbonate buffer solution (pH=9.6), L-10 monoclonal antibodies
Carbonate buffer solution (pH=6.5) is according to 1:After 500 times of dilutions, it is added to by 100 μ L/ holes in 96 microwell plates, 4 DEG C were coated with
Night.
7th, after washing 3 times with PBTS, pat dry, add the PBS liquid room 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 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-γs, IL-2, IL-4, IL-10+
The secondary antibody of HRP marks, is incubated at room temperature 1 hour.
10th, after washing 7 times with PBST, the μ L of TMB nitrite ions 100 are added, after being incubated at room temperature 30 minutes, add the μ L of terminate liquid 50
Terminating reaction.
11st, ELIASA measure wavelength 450nm light absorption value, while Detection wavelength 570nm light absorption value 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, calculate the final concentration of every group of various cell factors of mouse.As a result it is as shown in Figure 1.
As a result show, compared with PBS groups and KLH groups, Rv0446c t cell epitope peptide P120 can stimulate mouse produce compared with
IFN-γ (P < 0.001), IL-4 (P < 0.001), the IL-10 (P < 0.001) of high concentration, and P120 high doses group produces
The amount of IFN-γ (P < 0.001) is higher than low dose group, and Rv0446c t cell epitope peptide P123 can stimulate mouse to produce IFN-γ
(P < 0.001), and the amount of P123 high doses group generation IFN-γ (P < 0.05) is higher than low dose group.IFN-γ is that Th1 types are thin
Intracellular cytokine, removing of the macrophage to mycobacterium tuberculosis can be promoted by activating macrophage.IL-4 and IL-10 are
Th2 cytokines, the antibody during tuberculosis infection is promoted to produce.Immunoregulation effect is played in tuberculosis infection.Using
The above method carries out cell immunogenicity detection to Rv0446c t cell epitope peptides P121 and P122 respectively, the results showed that, P120
Mouse can be also stimulated to produce corresponding cell factor with P123.
It can be seen that antigen of mycobacterium tuberculosis albumen Rv0446c four t cell epitope peptides, can stimulate tuberculosis patient to produce
Raw IFN-γ, it can effectively distinguish tuberculosis patient and lung other illness patient and healthy population, and four polypeptide Combining diagnosis effects
Rate improves.Rv0446c and its t cell epitope peptide can be used for diagnosis lungy as a kind of detection reagent.In zoopery
In, Rv0446c two t cell epitope peptides P120 and P123 can effective stimulus body generation IFN-γ (P < 0.001), IL-4
(P < 0.001), IL-10 (P < 0.001), and mouse can be stimulated to produce specific IgG antibody, IgG1 and IgG2a, thus demonstrate,prove
Bright Rv0446c and its t cell epitope peptide has good immunogenicity, and body can be stimulated to produce cellullar immunologic response and body fluid
Immune response, it is a kind of Immunodominant Antigenic, 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 (11)
1. antigen of mycobacterium tuberculosis albumen Rv0446c is in tuberculosis detection reagent, Vaccinum Calmette-Guerini or antituberculotic is prepared
Using;Wherein, the amino acid sequence of the antigen protein Rv0446c such as SEQ ID NO:Shown in 5.
2. antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitope peptides, it is characterised 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 is prepared.
4. a kind of tuberculosis detection reagent, it is characterised in that contain antigen of mycobacterium tuberculosis albumen in the detection reagent
Rv0446c, or coding antigen protein Rv0446c DNA molecular, or as caused by the recombinant bacterium containing the DNA molecular
Recombinant protein;And/or
Epitope peptide described in claim 2, or the DNA molecular of the coding epitope peptide, or by containing the DNA for encoding the epitope peptide
Recombinant protein caused by the recombinant bacterium of molecule.
5. the tuberculosis T-SPOT detection kits containing detection reagent described in claim 4.
6. kit according to claim 5, it is characterised 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, Background control 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. antigen of mycobacterium tuberculosis albumen Rv0446c and/or its application of t cell epitope peptide in Vaccinum Calmette-Guerini is prepared;Its
In, the amino acid sequence such as SEQ ID NO of the antigen protein Rv0446c:Shown in 5;The amino acid sequence of its t cell epitope peptide
Such as SEQ ID NO:Shown in 1 or 4.
8. a kind of Vaccinum Calmette-Guerini, it is characterised in that its active ingredient is antigen of mycobacterium tuberculosis albumen Rv0446c, or coding
The DNA molecular of the antigen protein Rv0446c, or the recombinant protein as caused by the recombinant bacterium containing the DNA molecular;And/or
Antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitope peptides, or the DNA molecular of the coding epitope peptide, or by containing
There is recombinant protein caused by the recombinant bacterium for the DNA molecular for encoding the epitope peptide;
Wherein, the amino acid sequence such as SEQ ID NO of the antigen of mycobacterium tuberculosis albumen Rv0446c t cell epitope peptides:1
Or shown in 4.
9. antigen of mycobacterium tuberculosis albumen Rv0446c and/or its application of t cell epitope peptide in antituberculotic is prepared;
Wherein, the amino acid sequence of the antigen protein Rv0446c such as SEQ ID NO:Shown in 5;The amino acid sequence of its t cell epitope peptide
Row such as SEQ ID NO:Shown in 1 or 4.
10. a kind of antituberculotic, it is characterised in that its active ingredient is included with antigen of mycobacterium tuberculosis albumen Rv0446c
And/or its t cell epitope peptide is as immunogene, it is aided with adjuvant immunity experimental animal, the polyclonal antibody of preparation, or with tuberculosis
Antigen of mycobacterium albumen Rv0446c and/or its t cell epitope peptide are aided with adjuvant immunity experimental animal, used as immunogene
Hybridoma technology or DNA recombinant techniques, the identification antigen of mycobacterium tuberculosis albumen Rv0446c and its t cell epitope peptide of preparation
The Humanized monoclonal antibodies of antigen;Wherein, the amino acid sequence of the antigen protein Rv0446c such as SEQ ID NO:Shown in 5;
The amino acid sequence of its t cell epitope peptide such as SEQ ID NO:Shown in 1 or 4.
11. application of the epitope peptide described in claim 2 in Vaccinum Calmette-Guerini or antituberculotic is prepared, wherein the epitope peptide
Amino acid sequence such as SEQ ID NO:Shown in 1 or 4.
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