CN106349350A - Protein capable of specifically detecting mycobacterium tuberculosis infection - Google Patents
Protein capable of specifically detecting mycobacterium tuberculosis infection Download PDFInfo
- Publication number
- CN106349350A CN106349350A CN201510420040.6A CN201510420040A CN106349350A CN 106349350 A CN106349350 A CN 106349350A CN 201510420040 A CN201510420040 A CN 201510420040A CN 106349350 A CN106349350 A CN 106349350A
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- Prior art keywords
- mycobacterium tuberculosis
- detection
- albumen
- product
- sequence
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Classifications
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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)
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Abstract
The invention discloses protein capable of specifically detecting mycobacterium tuberculosis infection, and provides application of Rv1438 protein to preparation of products for detecting or assisting in detecting mycobacterium tuberculosis. The experiment of the invention proves that mycobacterium tuberculosis-derived protein segments are screened, a mycobacterium tuberculosis marker antigen RV1438 for detecting tuberculosis is provided, and the antigen is used for in vitro detecting specific T cell immunoreaction to serve as a reference for diagnosing patients suffering from tuberculosis and can be used for diagnosing whether the patients are infected with the mycobacterium tuberculosis. The protein can be used for detecting mycobacterium tuberculosis infection, and the specificity reaches 70% at present.
Description
Technical field
The invention belongs to biomedicine field is and in particular to a kind of albumen of specific detection m tuberculosis infection.
Background technology
China's in-vitro diagnosis industry is now in rapid growth period.But the huge market demand and China's diagnostic reagent
The relatively backward independent research and development capacity of industry deposits obvious gap.How from source, to carry out autonomous innovation, how to examine
Without being bound by indivedual medical overseas giants in disconnected reagent raw material supply, how by the scientific research of existing biomedicine field
Achievements conversion is focus that product is that the Chinese government, scientific research institutions and pharmaceuticals industry are all paid special attention in recent years.And
On market today, rigid demand is proposed to sensitiveer, efficient diagnostic reagent, can fast and accurately make a definite diagnosis tuberculosis
Patient is significant for medical personnel and patient.
Data survey result shows in recent years, and the patient 76.6% having tuberculosis symptoms accepted related inspection before tuberculosis disease
Look into, but only 35.8% is diagnosed as lunger, improving Patient Detection rate as early as possible is still current tuberculosis prevention and treatment
The emphasis of work is also difficult point.
Cell immune response using antigenic specificity t cell carries out the in-vitro diagnosis of cause pathogeny imcrobe infection, is this year
A kind of new detection method growing up.Swash training by separating the peripheral blood lymphocytes in fresh whole blood and going forward side by side to assassinate
Support, then can secrete the quantity of the cell of ifn- γ using elispot detection.The method is mainly used in knot at present
The diagnosis of core mycobacterial infectionses.Clinical at present commonly used Diagnosis of Tuberculosis depends on clinical symptoms, impact is learned
Diagnosis and etiological diagnosis, the diagnosis to mycobacterium tuberculosis latent infection is insensitive.Meanwhile, in tuberculosis examination
During, direct detection pathogen or the detection sensitivity of mycobacterium tuberculosis antibody and specificity also undesirable.
T-spot.tb is a kind of simpler ELISpot (elispot) detection method.Elispot detects
Highly sensitive, before the cytokine diffusion dilution of cell secretion, it can capture secreted by cell peripheral immediately
Cytokine.This makes elispot detection more sensitive, exceedes traditional elisa experiment.T-spot.tb quilt
Design the effect t cell for detecting the stimulation activation of tuberculosis specific antigen.T-spot.tb counts what each activated
Tuberculosis specific response t cell.
Content of the invention
It is an object of the present invention to provide the purposes of rv1438 albumen.
The present invention provides application in preparation detection or auxiliary detection mycobacterium tuberculosis product for the rv1438 albumen；
Described rv1438 is following albumen a) or b):
A) protein of the aminoacid sequence composition shown in sequence 2 in sequence table；
B) by the amino acid residue sequence of the sequence 2 in sequence table through one or several amino acid residues replacement and/
Or disappearance and/or the protein derived from a) adding and having with albumen shown in sequence 2 identical function.
In preparation detection or auxiliary, above-mentioned rv1438 albumen detects whether patient to be measured infects in mycobacterium tuberculosis product
Application be also the scope of protection of the invention.
It is a further object to provide whether a kind of detection or auxiliary detection sample to be tested infect tuberculosis branch bar
The product of bacterium.
The product that the present invention provides, including rv1438 albumen.
The said goods also include positive quality control and blank；
Described positive quality control is phytohaemagglutinin pha；
Described blank is nonantigenic cell culture fluid.
In the said goods, described product also includes the readable carrier being described below content:
If the speckle number in blank control wells and detection hole meets following standard: the speckle number of blank control wells is 0-5,
And speckle number >=5 of the speckle number-blank control wells in detection hole, then sample to be tested infection or candidate's infection tuberculosis branch bar
Bacterium；If the speckle number in blank control wells and detection hole does not meet above-mentioned standard, sample to be tested is uninfected by or candidate does not feel
Dye mycobacterium tuberculosis；
Described blank control wells are blank place hole,
Described detection hole is described rv1438 albumen place hole.
The said goods are test kit, and mentioned reagent box is specially t-spot test kit or elisa test kit.
Above-mentioned t-spot test kit also includes well plates in t-spot test kit, concentrates traget antibody, colour developing
Substrate solution.Mentioned reagent box is the product of oxford immunotec (Britain).
The 3rd purpose of the present invention be provide one kind be used for differentiating, diagnose, auxiliary diagnosis, examination and/or assist examination
The mark of mycobacterium tuberculosis.
The mark that the present invention provides, is rv1438 albumen.
The experiment proves that, the present invention has screened the protein fragments in mycobacterium tuberculosis source, there is provided Yi Zhongjian
Survey mycobacterium tuberculosis marker antigen rv1438 lungy, exempted from come vitro detection specific t cell by this antigen
Epidemic disease is reacted, can be as the reference of diagnosis of tuberculosis patient, for whether diagnosing patient by m tuberculosis infection.
This albumen can be used in infection due to Mycobacterium tuberculosis detection, and current specificity reaches 70%.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Portion of reagent is as follows:
Lysis buffer:20mm tris-hcl, 500mm sodium chloride, 10% glycerol, ph 8.0
Wash buffer:20mm tris-hcl, 20mm imidazoles, 500mm sodium chloride, 10% glycerol, ph 8.0
Elution buffer:20mm tris-hcl, 250mm imidazoles, 500mm sodium chloride, 10% glycerol, ph
8.0
Chromatography buffer: take potassium dihydrogen phosphate 13.609g (preparing 1000ml), plus 0.1mol/l sodium hydroxide solution,
Adjust ph to 7.5, obtain the pbs buffer for 7.5 for the ph value.
Solidification ni+ chromatographic resin novagen, ni-nta his, (cat.no 70691-5, Beijing is magnificent；2-8
Degree preserves)；Protein binding capacity is more than 5mg/ml resin chromatographic column (glass or polypropylene).Superdex 75:
(ge).
Following embodiments are oxford immunotec (Britain) using tuberculosis infection t cell detection kit
T-spot test kit, is 10 pairs of antigenic reagent box of esat-6 and cfp.
Embodiment 1, prokaryotic expression destination protein rv1438
The preparation of destination protein rv1438: by the recombinant vector pet28a-rv1438 of expression rv1438 protein coding gene
Import in escherichia coli bl21, obtain recombinant bacterium bl21/pet28a-rv1438；Iptg induction recombinant bacterium bl21/ again
Pet28a-rv1438, collects supernatant, as destination protein rv1438.
Specific as follows:
1st, the structure of express express target protein recombinant bacterium
The aminoacid sequence of rv1438 albumen is sequence 2, and the nucleotides sequence of its encoding gene is classified as sequence 1.
Rv1438 protein coding gene shown in sequence 1 is inserted ndei the and ecori site of pet28a carrier, obtain
To recombinant vector pet28a-rv1438；
Recombinant vector is imported in escherichia coli bl21, obtains recombinant bacterium bl21/pet28a-rv1438.
2nd, protein expression
1) recombinant bacterium bl21/pet28a-rv1438 stands overnight (16h about) at 37 DEG C.
2) it is inoculated in 37 DEG C of 200rpm of 5ml liquid lb culture medium (adding 5ul kan), cultivate and be more than to od
0.6-0.8 (about 3h).
3) it is inoculated in 300ml lb fluid medium by inoculum concentration 5ml, 37 DEG C, 200rpm cultivates to od600
≈ 0.6-0.8 (about 2h).Cool to 16 DEG C, add final concentration 0.4mm iptg；
4) 16 DEG C, 200rpm culture culture 16h, 4 DEG C, 4000rpm is centrifuged 10min collects thalline.
3rd, protein purification
1) thalline is resuspended: every thalline collected by 1l culture medium with 40ml about lysis buffer is resuspended (adds
1% protease inhibitor pmsf, then final concentration of 0.5-1mm)
2) bacterial cell disruption: ultrasonic 6s/ time, power be 38%, ultrasonic 20min.(suspension bright for standard, drink
Sentiment whole ultrasound works total time).
3) bacterial debris are removed: 4 DEG C, 12000g is centrifuged 30 minutes.
4) ni post is processed: 3mlni post first uses water process 8cv (8 times of column volumes), then with 8cv lysis buffer conduct
Balance liquid balances, and flow velocity is 30cm/h.
5) loading: broken bacterium centrifugation supernatant carries out loading with the flow velocity of 30cm/ml, collects and flows through peak.
6) balance: after sample loading, balance 5cv with balance liquid；
7) wash: wash 5cv with wash buffer；
8) eluting: with elution buffer eluting, be in charge of collection.Detect eluent with bradford reagent, understand egg
White whether completely eluting.As contained albumen, then continue eluting, until completely, obtaining wash-out protein.
9) dialyse: in the bag filter of wash-out protein loading 7kd, put into the ion exchange equipped with 100 times of wash-out protein volumes
Carry out dialysed overnight in the beaker of buffer a (formula 10mm pb, 45mm nacl, ph 8.0).Centrifuging and taking supernatant.
10) sieve chromatography
A) chromatographic column: superdex 75 (amount according to sample to determine the volume of chromatography media)
B) flow velocity；30cm/h
C) chromatography process: first use water process 5cv, then process half an hour with 0.6m naoh aqueous solution, more slow with chromatography
Rush liquid (pbs, ph 7.5) balance chromatography, until chromatographic column conductance is consistent with chromatography buffer with ph effluent.Will
The supernatant loading that above-mentioned 9) obtain, with chromatography buffer eluting (flow velocity 30cm/h), collects corresponding eluting peak, is
Destination protein rv1438 (size is 19.516kda).
Embodiment 2, the mycobacterium tuberculosis marker antigen rv1438 application in detection mycobacterium tuberculosis
1st, t-spot test kit detection
Specific as follows:
Specific as follows:
1) heparin vacuum test tube is used directly to adopt periphery to different patients' (shown in table 1, and patient knows the inside story) to be measured quiet
Arteries and veins blood, obtains anticoagulant blood sample；
2) anticoagulant blood sample is pressed volume 1:1 to mix with rpmi1640 culture fluid；Carefully blood sample is added in 3:1 ratio
Ficoll lymphocyte separation medium upper strata (utilizes ficoll-paquetmplusSeparate pbmcs according to operating instruction)；Room
25 DEG C of temperature, 1000g is centrifuged 22 minutes；Horizontal rotor, delays and rises slow fall；
3) by mononuclear cell layer (positioned at centrifuge tube intermediate layer, be the tunica albuginea shape of layer) from ficoll separating pipe
In transfer to plus 10ml aim-v culture fluid aseptic 15ml centrifuge tube, gently mix, room temperature 600g be centrifuged 7min；
4) carefully remove supernatant, add 1ml aim-v culture fluid, light and slow re-suspended cell, add aim-v culture fluid extremely
10ml, 350g are centrifuged 7min；
5) carefully abandon supernatant, add 1ml aim-v culture fluid re-suspended cell, obtain cell suspension (pbmcs)；
6) take the 1%trypan blue that 10 μ l cell suspension add 40 μ l, with the dilution of aim-v culture fluid, obtain
Cell dilutes working solution；
7) 24 hole pvdf lamina membranaceas are taken out from aluminum envelope, be sequentially added into: 50 μ l phytohaemagglutinin pha Zhiyang
Property the rv1438 protein solution for preparing of control wells, 50 μ l (initial concentration is 60ug/ml, and solvent is aim v
Culture fluid) it is added to detection hole, 50 μ l aim v culture fluid to blank control wells；Above-mentioned 3 holes are separately added into
Prepare cell dilution working solution 100 μ l (every hole cell quantity 2.5 × 105), and rv1438 protein solution in sample well
Final concentration of 20ug/ml；
8) culture plate is put into 37 DEG C, 5%co2Incubator is incubated 16 hours；
9) press 1:200 with aseptic pbs and prepare fresh enzyme labelled antibody working solution.Take out culture plate from incubator, discard
In the hole liquid.Washed 4 times with the aseptic pbs of 200 μ l/well；
10) add 50 μ l/well enzyme labelled antibody working solutions, culture plate is incubated 1 hour at 2-8 DEG C.Washed with pbs
Wash 4 times and remove uncombined enzyme labelled antibody；
11) every hole adds the substrate working solution 50 μ l that equilibrium at room temperature is crossed, room temperature reaction 7 minutes.With distilled water flushing eventually
Only react, in 37 DEG C of 2-3hrs or ambient temperature overnight desiccation culture plate, obtain the culture plate containing product.
2nd, speckle numeration
1) take out the culture plate containing product from incubator and discard cell culture fluid；
2) each reacting hole adds 200ul pbs buffer (ph 7.5)；
3) discard pbs buffer；With fresh pbs buffer repeated washing at least 3 times；
4) use concentration traget antibody reagent in 200 times of pbs buffer dilution test kits (alkali phosphatase enzyme mark little
The anti-human gamma interferon monoclonal antibody of Mus)；
5) each reacting hole adds 50ul traget antibody working solution, and 2-8 DEG C is incubated 1 hour；
6) discard traget antibody working solution, by above-mentioned step 2 and 3 washings；
7) each reacting hole adds 50ul substrate chromophoric solution incubated at room 7 minutes；
8) thoroughly wash culture plate terminating reaction with distilled water or deionized water；
9) in ventilation or 37 DEG C of incubator desiccation culture plates；
10) counting, each reacts in the hole navy blue clearly speckle.
Result judges:
If blank control wells speckle number is 0-5, and (the speckle number-blank control wells speckle number in detection hole) >=5,
Then there is reaction, that is, sample to be tested and rv1438 produce immunoreation, and sample to be tested candidate infects mycobacterium tuberculosis.
If being unsatisfactory for above-mentioned, sample to be tested and rv1438 do not produce immunoreation, and sample to be tested candidate is uninfected by tuberculosis
Mycobacteria.
(the clinical identification specifically disease and all infect tuberculosis branch bar of 30 samples to be tested in detection table 1 in aforementioned manners
Bacterium), result is as shown in table 1.
Also 30 samples of table 1 are detected using the antigen esat-6 carrying in test kit and antigen cfp 10,
Criterion is according in kit specification: if blank control wells speckle number is 0-5, and (the speckle number in detection hole-
Blank control wells speckle number) >=6, then there is reaction, that is, sample to be tested and antigen produce immunoreation, and sample to be tested infects
Or candidate's infection mycobacterium tuberculosis.If being unsatisfactory for above-mentioned, sample to be tested and antigen do not produce immunoreation, to be measured
Sample is uninfected by or candidate is uninfected by mycobacterium tuberculosis.
Table 1 is sample and its testing result
In table ,-it is blank ,+it is positive quality control.
As can be seen that
Rv1438 as Detection of antigen, in 26 samples to be tested 19 be infection mycobacterium tuberculosis sample, this with
Clinical identification result is consistent；But remaining 7 sample does not detect (with existing esat-6 albumen and cfp-10
Albumen and Clinical detection are it can be determined that this 7 samples are also m tuberculosis infection), therefore, the present invention uses
The recall rate whether rv1438 infects mycobacterium tuberculosis as Detection of antigen reaches 70%.And with market on test kit
From the point of view of testing result contrast, it is capable of detecting when the result that other immunogens cannot detect, improve recall rate (as sample
400).
Can be seen that rv1438 albumen and can detect or assist detection patient to be measured whether to infect tuberculosis from above-mentioned experiment and divide
Branch bacillus, specific as follows: using rv1438 albumen as antigen, detected by tuberculosis infection t cell detection kit,
Using cell culture medium as blank, if blank control wells speckle number is 0-5, and (the speckle number in detection hole-
Blank control wells speckle number) >=5, then there is reaction, that is, sample to be tested and rv1438 produce immunoreation, sample to be tested
Candidate infects mycobacterium tuberculosis.If being unsatisfactory for above-mentioned, sample to be tested and rv1438 do not produce immunoreation, to be measured
Sample candidate is uninfected by mycobacterium tuberculosis.
Claims (7)
- Application in preparation detection or auxiliary detection mycobacterium tuberculosis product for the 1.rv1438 albumen；Described rv1438 is following albumen a) or b):A) protein of the aminoacid sequence composition shown in sequence 2 in sequence table；B) by the amino acid residue sequence of the sequence 2 in sequence table through one or several amino acid residues replacement and/ Or disappearance and/or the protein derived from a) adding and having with albumen shown in sequence 2 identical function.
- In preparation detection or auxiliary, 2.rv1438 albumen detects whether patient to be measured infects in mycobacterium tuberculosis product Application.
- 3. a kind of detection or auxiliary detect whether sample to be tested infects the product of mycobacterium tuberculosis, including rv1438 egg In vain.
- 4. product according to claim 3 it is characterised in that: described product also includes positive quality control and blank Comparison；Described positive quality control is phytohaemagglutinin pha；Described blank is nonantigenic cell culture fluid.
- 5. the product according to claim 3 or 4 it is characterised in that: in described product also includes being described below The readable carrier holding:If the speckle number in blank control wells and detection hole meets following standard: the speckle number of blank control wells is 0-5, And speckle number >=5 of the speckle number-blank control wells in detection hole, then sample to be tested infection or candidate's infection tuberculosis branch bar Bacterium；If the speckle number in blank control wells and detection hole does not meet above-mentioned standard, sample to be tested is uninfected by or candidate does not feel Dye mycobacterium tuberculosis；Described blank control wells are blank place hole,Described detection hole is described rv1438 albumen place hole.
- 6. the product according to claim 3 or 4 it is characterised in that: described product be test kit.
- 7. a kind of for differentiating, diagnosing, auxiliary diagnosis, examination and/or auxiliary examination mycobacterium tuberculosis mark, For rv1438 albumen.
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| CN201510420040.6A CN106349350A (en) | 2015-07-16 | 2015-07-16 | Protein capable of specifically detecting mycobacterium tuberculosis infection |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510420040.6A CN106349350A (en) | 2015-07-16 | 2015-07-16 | Protein capable of specifically detecting mycobacterium tuberculosis infection |
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| CN107422130A (en) * | 2017-07-20 | 2017-12-01 | 南通表源生物技术有限公司 | The detection kit and its detection method of tuberculosis infection T cell |
| CN109030825A (en) * | 2017-06-12 | 2018-12-18 | 广东体必康生物科技有限公司 | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis |
| CN113373162A (en) * | 2020-03-09 | 2021-09-10 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv novel gene Rv0229A and encoding protein and application thereof |
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Cited By (4)
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| CN107422130B (en) * | 2017-07-20 | 2019-03-19 | 南通表源生物技术有限公司 | The detection kit and its detection method of tuberculosis infection T cell |
| CN113373162A (en) * | 2020-03-09 | 2021-09-10 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv novel gene Rv0229A and encoding protein and application thereof |
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