CN104628834B - A kind of tuberculosis infection T cell immunodetection antigen and application thereof - Google Patents
A kind of tuberculosis infection T cell immunodetection antigen and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- 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
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- G01N33/5695—Mycobacteria
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
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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)
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Abstract
The invention provides a kind of antigen composition detected for tuberculosis infection cellular immunization, be made up of mycobacterium tuberculosis Rv3873 antigen and/or its derivative epitope peptide.In preferred embodiments, whole epitope peptides that antigen composition is comprised by mycobacterium tuberculosis Rv3873 antigen form.Present invention also offers the application of described antigen composition in the detection reagent of the Specific T cell immunity reaction caused for the preparation of vitro detection tuberculosis infection, comprise the lymphocyte of human or animal after the stimulation of described antigen composition, detect the cytokine of tuberculosis specific T-cells secretion.
Description
Technical field
The present invention relates to a kind of tuberculosis infection specific T cell immunodetection antigen and application thereof.Be exactly specifically a kind of antigen of mycobacterium tuberculosis and epitope peptide composition thereof, detect for tuberculosis infection cellular immunization, belong to diagnosis field.
Background technology
Tuberculosis is the chronic infectious disease of the infecting both domestic animals and human caused by m tuberculosis infection.According to the statistics of the World Health Organization, the current whole world has the population more than 1/3 to infect mycobacterium tuberculosis, has 2,000,000 people every year because of tuberculosis death.China is one of serious country of 22 TB endemic in the whole world, is also one of 27 popular countries seriously of multi-drug resistance tuberculosis in the whole world simultaneously.China's the 5th tuberculosis epidemiological random sampling survey report display, Chinese tuberculosis year neopathy number be about 1,300,000, account for 14.3% of global number of the infected, occupy global second; 15 years old and the phthisical morbidity of above crowd are 4,59/,100,000.Mycobacterium tuberculosis, except infection people, also infects more than 50 kind of Mammals and more than 20 kind of bird; Susceptibility because of animal species different and different with individuality, in domestic animal, ox is most Sensitivity animal.The popular development not only affecting aquaculture lungy, the more important thing is due to cross infection, and makes the health of the mankind be subject to serious threat.Therefore, tuberculosis has become a serious socioeconomic problem, brings serious harm to China citizen health, social stability and Economic development.
Early diagnosis lungy is most important to control lungy, and one of ultimate challenge of tuberculosis prevention and treatment is exactly lack early stage, quick, responsive and special diagnostic tool.The diagnostic method of currently used tuberculosis infection still also exists larger limitation.Classical tuberculin test (TST) is although reached a century for diagnosis of tuberculosis, and it is subject to the interference of BCG (Bacille Calmette-Guerin) vaccination and the infection of most of non-tuberculous mycobacteria, and specificity and susceptibility are all undesirable; The bacteriology gold standard cultivated as diagnosis is used for tuberculosis and also also exists that culture cycle is long, positive rate is lower and depend on the problems such as collection of specimens quality; And the method such as iconography, serology, because higher false negative or false positive, the reference value for diagnosis of tuberculosis is limited.Existing diagnosis reagent sensitivity, specific degree all have much room for improvement.
Antituberculotic protective immunity mechanism still imperfectly understands at present, and cellular immunization especially circulates and the T lymphocyte responses of infection site is considered to play an important role.Therefore the T cell of can excitating organism, especially CD4
+t cell immune response is the key that body success Antituberculous infects.By the T lymphocyte of pathogenic bacteria sensitization, be again subject to discharging IFN-γ after isoantigen stimulates, high-caliber IFN-γ reaction can point out the infection of this pathogenic bacteria.In recent years, along with the development of immunology and genomics, there is the external IFN-γ reaction assay method (IGRA) based on T cell, can be used for tuberculosis auxiliary diagnosis and tuberculosis infection examination.The commercialization IGRA reagent that to have had at present with tuberculosis specific antigens ESAT-6 and CFP-10 of M. tuberculosis genes group RD1 district coding be stimulator, as QuantiFERON-TBGoldtest and T-SPOT.TBtest, for detecting the T lymphocyte of tuberculosis specificity release IFN-γ in peripheral blood, present higher susceptibility and specificity.
Epitope is a part for polypeptide antigen, is antigenic key foundation; Antigen to be combined with corresponding lymphocyte surface receptors by epitope thus primed lymphocyte, cause immunne response, namely immunoreactive specificity is for epitope, instead of for whole antigen, in the process that epitope is replied at antigen induced immune, play decisive role.
Based on the vitro detection technology of T cell secretion IFN-γ; main using intact antigen as corresponding stimulus; antigenic component composition comparatively complexity, sensitivity and specificity has much room for improvement; and corresponding reagent is due to the patent protection of offshore company; making China's cost when introducing related products application higher, being not suitable under present circumstances in China's widespread adoption.The present invention is under national transmissible disease key special subjects is subsidized, tubercle bacillus protein antigens and epi-position thereof are fully analyzed, verify and comparative studies basis on produce, have independent intellectual property right completely, the association areas such as auxiliary diagnosis lungy, epidemiological surveillance and infection examination can be widely used in based on detection reagent of the present invention.
Summary of the invention
The present invention is on the basis of existing T cell IFN-γ release test technology, apply the Rv3873 antigen coded by RD1 district gene Rv3873 and/or its epitope peptide comprised, form an epitope peptide combinations, and in conjunction with ELISA method, tuberculosis infection specific T-cells in tuberculosis case and healthy volunteer's body is detected, thus evaluate the sensitivity and specificity that this epitope peptide combinations detects for tuberculosis.
Rv3873 gene is guarded at Mycobacterium camber as the gene in mycobacterium core gene group, and its expression product has higher T cell immunogenicity; And Rv3873 antigen is present in pathogenic mycobacterium tuberculosis, and all lack this polypeptide antigen in bacille Calmette-Guerin vaccine (BCG) and most non-tuberculous mycobacteria.The present invention demonstrates Rv3873 antigen and also can be used for tuberculosis infection specific detection, and use corresponding epitope peptide to substitute complete polypeptide antigen as detection mark, provide the epitope peptide storehouse composition that a kind of antigen is derivative, and this antigen is derived epitope peptide storehouse be applied in the detection of tuberculosis case and healthy volunteer.
A first aspect of the present invention provides a kind of antigen composition detected for tuberculosis infection cellular immunization, is made up of mycobacterium tuberculosis Rv3873 antigen and/or its derivative epitope peptide.
Described antigen of mycobacterium tuberculosis Rv3873 aminoacid sequence is as shown in SEQIDNO:1.
The aminoacid sequence of the epitope peptide that described mycobacterium tuberculosis Rv3873 antigen derives is selected from one or more in SEQIDNO:2 ~ 59.
Epitope peptide in composition can be the aminoacid sequence of above-mentioned epitope peptide through replacing, disappearance or add 1 or several amino acid and there is derivative epitope peptide or its analogue of same antigen.
In a preferred embodiment, antigen composition is made up of mycobacterium tuberculosis Rv3873 antigen and whole epitope peptides of comprising thereof.
In another preferred embodiment, whole epitope peptides that antigen composition is comprised by mycobacterium tuberculosis Rv3873 antigen form.
In the most preferred embodiment, antigen composition by aminoacid sequence for the epitope peptide shown in SEQIDNO:2 ~ 59 forms.
Present invention also offers the DNA molecular of described antigen composition of encoding, and recombinant expression vector, expression cassette, transgenic cell line and the recombinant bacterium containing this DNA molecular.
A second aspect of the present invention provides the application of described antigen composition in the detection reagent of the Specific T cell immunity reaction caused for the preparation of vitro detection tuberculosis infection, it is characterized in that the lymphocyte of human or animal is after the stimulation of described antigen composition, detect the cytokine of tuberculosis specific T-cells secretion.
In certain embodiments, the cytokine of described tuberculosis specific T-cells secretion is selected from IFN-γ (IFN-γ), interleukin II (IL-2) and tumor necrosis factor alpha (TNF-α), preferred IFN-γ; The detection method of the cytokine of described tuberculosis specific T-cells secretion is selected from dyeing and T cell proliferation test in ELISpot test (ELISPOT), enzyme linked immunosorbent assay (ELISA), immunity-chromatography test, cytokine, preferred ELISA; Described lymphocyte derives from peripheral blood, cerebrospinal fluid, hydrothorax or ascites, preferred peripheral blood.
Present invention also offers the application of described antigen composition in preparation tuberculosis detection reagent, and provide a kind of tuberculosis detection kit, its essentially consist is as follows:
Antigen composition of the present invention;
The mouse IgG monoclonal antibody (primary antibodie) of anti-human or animal IFN-γ;
Enzyme marking reagent: the different epi-position of anti-human or animal IFN-γ of horseradish peroxidase-labeled another kind of mouse IgG monoclonal antibody (two resist);
Calibration object: containing the positive raw material of IFN-γ;
Culture tube: the antigen composition of test cultures Guan Zhonghan synthetic, containing tuberculosis nonspecific stimulation antigen in positive control culture tube, containing matrix liquid in Background control culture tube;
Other ELISA detect required reagent and consumptive material.
Preferably, primary antibodie is fixed on Sptting plate.
What the present invention adopted is the ELISA detection method of double-antibody sandwich principle detectable antigens.Primary process is: the primary antibodie of Sptting plate wrapping quilt catches the IFN-γ in sample, and same IFN-γ molecule and then anti-ly to be caught by enzyme target two, develop the color.Although primary antibodie fixing on Sptting plate and enzyme target two resist be IFN-γ monoclonal antibody, these two kinds of antibody are the monoclonal antibody identifying epitopes different on IFN-γ, therefore can not influence each other.
A third aspect of the present invention provides a kind of Vaccinum Calmette-Guerini candidate material, it is characterized in that containing described antigen and epitope polypeptide composition, DNA molecular, recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium.
The present invention has following advantage relative to prior art: the present invention is combined with can by the epitope peptide antigen of the mycobacterium tuberculosis specific antigen Rv3873 of T cell identification, compared with adopting the detection means of intact antigen in the past, the interference that the non-epi-position identified region due to redundancy causes specific T-cells can be reduced, thus improve detection sensitivity; On the other hand owing to being combined with the epitope sequence of multiple sensitivity, the response intensity of single detect aperture obviously increases, and the judgement of detected result is more prone to compared with single intact antigen; 3rd, the whole derivative all studied proof of epitope peptide adopted in the present invention can stimulate body to produce stronger t cell immune response, therefore, when using above-mentioned epitope peptide to carry out ex vivo T cell IFN-γ release test as stimulator, detected result is true and reliable; 4th, the amino acid epitope of synthetic, as detection reagent, can adjust the melting concn of each epi-position artificially and then strengthen the intensity of detection reaction; 5th, adopt the method synthetic antigen epitope peptide of solid phase synthesis, be conducive to quality control, and cost is lower, be applicable to large-scale commercial and produce.
Embodiment
Further illustrate the present invention below by embodiment, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
The formation of embodiment 1 epitope peptide combinations
The epitope peptide derived by Rv3873 antigen for the epitope peptide combinations of tuberculosis detection is formed.The aminoacid sequence of described antigen Rv3873 is as shown in SEQIDNO:1; The aminoacid sequence of the epitope peptide that described Rv3873 antigen derives is as shown in SEQIDNO:2 ~ 59.
Embodiment 2 epitope peptide combinations is applied to the exploitation of detection kit
Epitope peptide combinations of the present invention can be used for the detection of tuberculosis infection, forms clinical or laboratory diagnosis test kit.
Detection kit comprises:
1) the epitope peptide combinations described in embodiment 1;
2) the mouse IgG monoclonal antibody (primary antibodie) of anti-human or animal IFN-γ, described primary antibodie is fixed on Sptting plate;
3) enzyme marking reagent: the another kind of mouse IgG monoclonal antibody (two resist) of the different epi-position of anti-human or animal IFN-γ of horseradish peroxidase-labeled;
4) calibration object: containing the positive raw material of IFN-γ;
5) culture tube: the epitope polypeptide that the plurality of antigens of test cultures Guan Zhonghan synthetic is derivative, containing tuberculosis nonspecific stimulation antigen in positive control culture tube, containing matrix liquid in Background control culture tube
6) other ELISA detect required reagent and consumptive material.
Embodiment 3 antigen epitope polypeptide is applied to tuberculosis infection clinical detection
A. the separation of peripheral blood lymphocyte:
The screening criteria of volunteer's case involved in the present invention is:
According to " diagnosis of pulmonary tuberculosis standard " national standard (WS288-2008) that the Ministry of Health issues, clinical manifestation symptom, sign and imaging examination of chest are diagnosed as phthisical patient, wherein bacteriology positive or negative tuberculosis patient refers to that clinical diagnosis is in patient lungy, through the tuberculosis patient of bacteriology checking (Sputum smears acid-fast stain microscopy and/or Roche solid medium bacterial solids are cultivated) positive or negative; And outer tuberculosis (as bone tuberculosis, renal tuberculosis, tuberculosis of intestine, the lymphoid tuberculosis etc.) patient of lung.
The screening criteria of healthy volunteer is:
Without tuberculosis clinical symptom, without other diseases or infection.
Selected tuberculosis patient is Zi random selecting in the continuous time samples of going to a doctor to tuberculosis hospital; Volunteer is healthy students; Tuberculosis patient and volunteer's age are between 18 ~ 60 years old.
The present invention's T lymphocyte used is from the peripheric venous blood of individuality.Before on-test, inform the object of volunteer/tuberculosis patient detailed programs, meaning, experiment process and required collect specimen and quantity thereof by trier, through agreeing to and signing Informed Consent Form, by Clinical Laboratory teacher, it is taken a blood sample.This project screening final 138 routine tuberculosis volunteers and 32 routine healthy volunteers, use during blood sampling and gather fresh whole blood without endotoxic anticoagulant heparin vacuum test tube, and every volunteer takes a blood sample about 20ml.Afterwards:
1) be dispensed into (blank pipe, sample culture tube, positive control pipe) in different culture tubes after whole blood sample gentle inversion being mixed 3 ~ 5 times within 2 hours, 1ml/ manages.
2) 37 DEG C of incubators are put into rapidly in the mixing of culture tube gentle inversion to cultivate 22 ± 2 hours, in culturing process, keep cultivation shop upright.
3) whole blood after cultivating is centrifugal 10 minutes with 3000 ~ 5000 revs/min, gets blood plasma and carries out ELISA detection.Attention can not be drawn onto cellular layer.
B. the preparation of epitope peptide
With the method chemosynthesis epitope peptide of solid phase synthesis.Each synthetic peptide is dissolved with DMSO.Amount to mole mixing such as 58 epitope peptides by SEQIDNO:2 ~ 59 in embodiment 1 afterwards, form epitope peptide storehouse (Rv3873); The RPIM1640 substratum of epitope peptide storehouse containing 10% foetal calf serum is diluted to final concentration.
C.ELISA detectable antigens epitope peptide Al Kut specific T cell
Elisa plate shifts to an earlier date the mouse IgG monoclonal antibody bag quilt of the anti-human IFN-γ that more than 12 hours dilute with phosphate buffered saline buffer (pH7.4), 4 DEG C keep flat, before adding detection sample, close 1 hour under containing the RPIM1640 substratum room temperature condition of 10% foetal calf serum.
In well, add diluent 20 μ l (except blank control wells) respectively, and then add testing sample 50 μ l (except blank control wells), each epitope peptide storehouse stimulates establishes diplopore, separately establishes Positive control wells, Background control wells; Respectively add diplopore after calibration object gradient dilution to detect.
After shrouding film shrouding, be placed in 37 DEG C of incubator incubations 60 minutes (retain liquid in micropore after incubation, do not wash).
D.ELISA washes plate and result obtains:
1) every hole adds enzyme marking reagent 50 μ l, except blank well, shakes mixing gently.
2) with after shrouding film shrouding, 37 DEG C of incubator incubations are placed in 60 minutes.
3) carefully taking shrouding film off, washing 5 times with washing trigger, last button is as far as possible dry.
4) develop the color: shake mixing gently after every hole adds nitrite ion, 37 DEG C of lucifuges develop the color 15 minutes.
5) every hole adds stop buffer 50 μ l, shakes mixing gently, is 450nm/600-650nm detection with microplate reader wavelength in 10 minutes.
Power matched curve and typical curve is done with the absorbancy average of the antigenic content of calibration object and correspondence thereof, obtain equation of linear regression, after blank pipe (N), sample culture tube (T), positive control pipe (P) three kinds of culture tubes being cultivated, the absorbance of plasma specimen substitutes into regression equation respectively, tries to achieve plasma specimen corresponding IFN-γ content in three kinds of pipes.
E. interpretation of result:
Relate to altogether in this research through amounting to 138 examples, healthy volunteer 32 example in strict accordance with the tuberculosis patient of standard screening.
The existing major antigen carrying out m tuberculosis infection detection based on IGRA principle is ESAT-6 and the CFP-10 antigen coded by mycobacterium tuberculosis RD1 regional gene.The present invention uses epitope peptide storehouse (Rv3873) to detect 138 routine tuberculosis patients, wherein 91 examples are positive, detection sensitivity is 65.94%: detect 32 routine healthy volunteers, and wherein 27 examples are negative reaction, and specificity is 84.38% (as shown in table 1).
Table 1. each antigen epitope polypeptide storehouse detection specificity and sensitivity statistics
| Epitope peptide storehouse | Tuberculosis case | Sensitivity | Healthy volunteer | Specificity |
| Rv3873 | 91/138 | 65.94％ | 27/32 | 84.38％ |
Note: detection sensitivity is expressed as: (tuberculosis patient detects number positive/tuberculosis patient sum) × 100%; Detection specificity is expressed as: (1-healthy volunteer detects number positive/healthy volunteer's sum) × 100%.
Therefore the antigen epitope polypeptide storehouse (Rv3873) of applying in the present invention presents higher sensitivity and specificity in the detection of tuberculosis case.
Positive reaction judging criterion: test cultures pipe (T) content value=T, Background control culture tube (N) content value=N, positive control culture tube (P) content value=P.(as shown in table 2)
Table 2. positive reaction judging criterion
Claims (18)
1. the antigen composition detected for tuberculosis infection cellular immunization, wherein, whole epitope peptides that described composition is comprised by mycobacterium tuberculosis Rv3873 antigen form, and the aminoacid sequence of the epitope peptide that wherein said mycobacterium tuberculosis Rv3873 antigen comprises is as shown in SEQIDNO:2 ~ 59.
2. the DNA molecular of coding antigen composition according to claim 1.
3. the recombinant expression vector containing DNA molecular according to claim 2.
4. the transgenic cell line containing DNA molecular according to claim 2.
5. the recombinant bacterium containing DNA molecular according to claim 2.
6. the application of antigen composition according to claim 1 in the detection reagent of the Specific T cell immunity reaction caused for the preparation of vitro detection tuberculosis infection.
7. application according to claim 6, is characterized in that the lymphocyte of human or animal is after described antigen composition stimulates, and detects the cytokine of tuberculosis specific T-cells secretion.
8. application according to claim 7, wherein said animal is selected from ox, sheep or pig.
9. application according to claim 7, the cytokine of wherein said tuberculosis specific T-cells secretion is selected from IFN-γ (IFN-γ), interleukin II (IL-2) and tumor necrosis factor alpha (TNF-α).
10. application according to claim 9, the cytokine of wherein said tuberculosis specific T-cells secretion is IFN-γ (IFN-γ).
11. application according to claim 7, the detection method of the cytokine of wherein said tuberculosis specific T-cells secretion is selected from dyeing and T cell proliferation test in ELISpot test (ELISPOT), enzyme linked immunosorbent assay (ELISA), immune colloid gold test, cytokine.
12. application according to claim 11, the detection method of the cytokine of wherein said tuberculosis specific T-cells secretion is enzyme linked immunosorbent assay (ELISA).
13. application according to claim 7, wherein said lymphocyte derives from peripheral blood, cerebrospinal fluid, hydrothorax or ascites.
14. application according to claim 13, wherein said lymphocyte derives from peripheral blood.
15. 1 kinds of Diagnosis of Tuberculosis test kits, it is composed as follows:
(1) antigen composition according to claim 1;
(2) the mouse IgG monoclonal antibody of anti-human or animal IFN-γ;
(3) enzyme marking reagent: the different epi-position of anti-human or animal IFN-γ of horseradish peroxidase-labeled another kind of mouse IgG monoclonal antibody;
(4) calibration object: containing the positive raw material of IFN-γ;
(5) epitope peptide of the external synthesis of culture tube: test cultures Guan Zhonghan, containing tuberculosis nonspecific stimulation antigen in positive control culture tube, containing matrix liquid in Background control culture tube
(6) other ELISA detect required reagent and consumptive material.
16. Diagnosis of Tuberculosis test kits according to claim 15, wherein, the mouse IgG monoclonal antibody of the anti-human or animal IFN-γ described in (2) is fixed on Sptting plate.
17. 1 kinds of Vaccinum Calmette-Guerinis, containing antigen composition according to claim 1, DNA molecular according to claim 2, recombinant expression vector according to claim 3, transgenic cell line according to claim 4 or recombinant bacterium according to claim 5.
The application of 18. antigen compositions according to claim 1 in preparation tuberculosis Detection and diagnosis reagent.
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