CN106645733A - Protein for specific detection of mycobacterium tuberculosis infection - Google Patents
Protein for specific detection of mycobacterium tuberculosis infection Download PDFInfo
- Publication number
- CN106645733A CN106645733A CN201510420053.3A CN201510420053A CN106645733A CN 106645733 A CN106645733 A CN 106645733A CN 201510420053 A CN201510420053 A CN 201510420053A CN 106645733 A CN106645733 A CN 106645733A
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- CN
- China
- Prior art keywords
- detection
- sequence
- protein
- product
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- 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
-
- 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
- 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 invention discloses protein for specific detection of mycobacterium tuberculosis infection. The invention provides application of Rv0054 protein in preparation of products for detection or aided detection of mycobacterium tuberculosis. The Rv0054 is the protein as shown in the following a) or b): a) the protein composed of amino acid sequence and as shown in the sequence 1 in the sequence table; and b) the protein which is obtained after one or more amino acid residues substitution and/or deletion and/or addition of the amino acid residue sequence as shown in the sequence 1 in the sequence table and has the same functions as the protein as shown in the sequence 1 and is derived from a). It proves through experiments that the protein can better enhance detection rate of mycobacterium tuberculosis infection.
Description
Technical field
The invention belongs to biomedicine field, and in particular to for the 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 to carry out autonomous innovation from source, how to examine
Without being bound by overseas medical giant individually 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
Rigid demand is proposed to sensitiveer, efficient diagnostic reagent on market today, tuberculosis can be fast and accurately made a definite diagnosis
Patient is significant for medical personnel and patient.
In recent years data survey result shows that the patient 76.6% for having tuberculosis symptoms received related inspection before tuberculosis disease
Look into, but only 35.8% is diagnosed as lunger, and it is still current tuberculosis prevention and treatment work to improve Patient Detection rate
Emphasis be also difficult point.
The in-vitro diagnosis of cause pathogeny imcrobe infection are carried out using the cell immune response of T cells with antigenic specificity, is in recent years
A kind of new detection method for growing up.PMBC in by separating fresh whole blood is gone forward side by side, and assassination is sharp to train
Support, the quantity of the cell of secretion of gamma-IFN is then capable of using ELISPOT detections.The method is mainly used at present knot
The diagnosis of core mycobacterial infections.The Diagnosis of Tuberculosis that at present clinic is generally adopted depends on clinical symptoms, affects to learn
Diagnosis and etiological diagnosis, the diagnosis to Much's bacillus latent infection is insensitive.Meanwhile, in tuberculosis examination
During, the sensitivity of direct detection pathogen or detection mycobacterium tuberculosis antibody is also undesirable with specificity.
T-SPOT.TB is a kind of simpler ELISpot (ELISPOT) detection method.ELISPOT is detected
Highly sensitive, before the cell factor diffusion dilution of cell secretion, it can be captured immediately secreted by cell peripheral
Cell factor.This causes ELISPOT detections more sensitive, tests more than traditional ELISA.T-SPOT.TB quilts
Design for detecting that tuberculosis specific antigen stimulates the effector T cell of activation.T-SPOT.TB counts what each was activated
Tuberculosis specific response T cell.
The content of the invention
It is an object of the present invention to provide the purposes of Rv0054 albumen.
The present invention provides application of the Rv0054 albumen in preparing detection or aiding in detection Much's bacillus product；
The Rv0054 is following albumen a) or b)：
A) protein of the amino acid sequence composition shown in the 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 add and have with albumen shown in sequence 2 protein by derived from a) of identical function.
Above-mentioned Rv0054 albumen is preparing detection or is aiding in whether detection patient to be measured is infected in Much's bacillus 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 is provided, including Rv0054 albumen.
The said goods also include positive quality control and blank；
The positive quality control is PHA-P HA；
The blank is nonantigenic cell culture fluid.
In the said goods, the product also includes being described below the readable carrier of content：
If the spot number in blank control wells and detection hole meets following standard：The spot number of blank control wells is 0-5,
And spot number >=5 of the spot number-blank control wells in detection hole, then sample to be tested infection or candidate infect tuberculosis branch bar
Bacterium；If the spot 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 Much's bacillus；
The blank control wells are blank place hole,
The detection hole is the Rv0054 albumen place hole.
The said goods are kit, and mentioned reagent box is specially T-SPOT kits, or ELISA kit.
Above-mentioned T-SPOT kits also include well plates, concentration labelled antibody, colour developing in T-SPOT kits
Substrate solution.Mentioned reagent box is the product of Oxford immunotec (Britain).
The 3rd purpose of the present invention be to provide it is a kind of for differentiating, diagnosing, auxiliary diagnosis, examination and/or auxiliary examination
The mark of Much's bacillus.
The mark that the present invention is provided, is Rv0054 albumen.
The experiment proves that, the present invention has screened the protein fragments in Much's bacillus source, there is provided Yi Zhongjian
Much's bacillus marker antigen RV0054 lungy is surveyed, is exempted from come vitro detection specific T-cells by the antigen
Epidemic disease is reacted, can be as the reference of diagnosis of tuberculosis patient, for whether diagnosing patient by m tuberculosis infection.
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, commercially obtain.
Portion of reagent is as follows：
lysis buffer：20mM Tris-HCl, 500mM sodium chloride, 10% glycerine, pH 8.0
Wash buffer：20mM Tris-HCl, 20mM imidazoles, 500mM sodium chloride, 10% glycerine, pH 8.0
Elution buffer：20mM Tris-HCl, 250mM imidazoles, 500mM sodium chloride, 10% glycerine, pH
8.0
Chromatography buffer：Potassium dihydrogen phosphate 13.609g (preparing 1000ml), plus 0.1mol/l sodium hydroxide solutions are taken, pH is adjusted to 7.5,
Obtain the PBS that pH value is 7.5.
Solidification Ni+ chromatographic resin Novagen, Ni-NTA His, (Cat.NO 70691-5, Beijing is magnificent；2-8 degree
Preserve)；Protein binding capacity is more than 5mg/ml resin chromatographic columns (glass or polypropylene).superdex 75：
(GE)。
Following embodiments are Oxford immunotec (Britain) using tuberculosis infection T cell detection kit
T-SPOT kits are ESAT-6 and 10 pairs of antigenic reagent box of CFP.
Embodiment 1, prokaryotic expression destination protein Rv0054
The preparation of destination protein Rv0054：By the recombinant vector pET28a-Rv0054 of expression Rv0054 protein coding genes
In importing e. coli bl21, recombinant bacterium BL21/pET28a-Rv0054 is obtained；Again IPTG induces recombinant bacterium BL21/
PET28a-Rv0054, collects supernatant, as destination protein Rv0054.
It is specific as follows：
1st, the structure of express express target protein recombinant bacterium
The amino acid sequence of Rv0054 albumen is sequence 2, and the nucleotides sequence of its encoding gene is classified as sequence 1.
Rv0054 protein coding genes shown in sequence 1 are inserted into NdeI the and EcoRI sites of pET28a carriers, is obtained
To recombinant vector pET28a-Rv0054；
Recombinant vector is imported in e. coli bl21, recombinant bacterium BL21/pET28a-Rv0054 is obtained.
2nd, protein expression
1) recombinant bacterium BL21/pET28a-Rv0054 stands overnight (16h or so) at 37 DEG C.
2) 37 DEG C of 200rpm of 5ml LB liquid mediums (adding 5ul kan) are inoculated in, are cultivated to OD and is more than
0.6-0.8 (about 3h).
3) it is inoculated in 300ml LB fluid nutrient mediums by inoculum concentration 5ml, 37 DEG C, 200rpm is cultivated to OD600
≈ 0.6-0.8 (about 2h).16 DEG C are cooled to, final concentration 0.4mM IPTG are added；
4) 16 DEG C, 200rpm culture culture 16h, 4 DEG C, 4000rpm centrifugation 10min collects thallines.
3rd, protein purification
1) thalline is resuspended：(added with 40ml or so lysis buffer are resuspended per the thalline collected by 1L culture mediums
1% protease inhibitors PMSF, then final concentration of 0.5-1mM)
2) bacterial cell disruption：Ultrasonic 6s/ time, power is 38%, ultrasonic 20min.(suspension is bright for standard, meal with wine
Sentiment whole ultrasound works total time).
3) bacterial debris are removed：4 DEG C, 12000g is centrifuged 30 minutes.
4) Ni posts are processed：3mlNi posts first use water process 8cv (8 times of column volumes), then with 8cv lysis buffer conducts
Equilibrium liquid is balanced, and flow velocity is 30cm/h.
5) loading：Broken bacterium centrifugation supernatant carries out loading with the flow velocity of 30cm/ml, and collection flows through peak.
6) balance：5cv is balanced after sample loading with equilibrium liquid；
7) wash：5cv is washed with wash buffer；
8) elute：Eluted with elution buffer, be in charge of collection.Eluent is detected with bradford reagents, egg is understood
Whether elute completely in vain.Such as contain albumen, then continue to elute, until completely, obtain wash-out protein.
9) dialyse：Wash-out protein is fitted in the bag filter of 7KD and is put into the ion equipped with 100 times of wash-out protein volumes and hands over
Dialysed overnight is carried out in the beaker for changing buffer A (formula 10mM PB, 50mM NaCl, pH 7.5).Centrifugation
Take supernatant.
10) sieve chromatography
A) chromatographic column：Superdex 75 (determines the volume of chromatography media according to the amount of sample)
B) flow velocity；30cm/h
C) chromatography process：Water process 5cv is first used, then half an hour is processed with the 0.6M NaOH aqueous solution, then it is slow with chromatography
Liquid (PBS, pH 7.5) balance chromatography is rushed, until chromatographic column conductance and pH effluxes are consistent with chromatography buffer.Will
The above-mentioned supernatant loading for 9) obtaining, with chromatography buffer (flow velocity 30cm/h) is eluted, and collects corresponding eluting peak, is
Destination protein Rv0054 (size is 19.516KDa).
The application of embodiment 2, Much's bacillus marker antigen Rv0054 in detection Much's bacillus
1st, T-SPOT kits detection
It is specific as follows：
1) using heparin vacuum test tube to different patients to be measured (shown in table 1, and patient knows the inside story, clinical definite)
Peripheric venous blood is directly adopted, anti-freezing blood sample is obtained；
2) anti-freezing blood sample is pressed into volume 1:1 mixes with RPMI1640 nutrient solutions；By 3:1 ratio is carefully added in blood sample
Ficoll lymphocyte separation mediums upper strata (utilizes Ficoll-PaqueTMplusPBMCs is separated according to operating instruction)；Room
25 DEG C of temperature, 1000g is centrifuged 22 minutes；Horizontal rotor, delays and rises slow drop；
3) by mononuclear cell layer (being located at centrifuge tube intermediate layer, be the tunica albuginea shape of layer) from Ficoll separating pipes
In be transferred to plus 10ml AIM-V nutrient solutions aseptic 15ml centrifuge tubes, gently mix, room temperature 600g centrifugation 7min；
4) supernatant is carefully removed, adds 1ml AIM-V nutrient solutions, light and slow re-suspended cell to add AIM-V nutrient solutions extremely
10ml, 350g are centrifuged 7min；
5) supernatant is carefully abandoned, 1ml AIM-V nutrient solution re-suspended cells are added, cell suspension (PBMCs) is obtained；
6) the 1%trypan blue that 10 μ l cell suspensions add 40 μ l are taken, is diluted with AIM-V nutrient solutions, obtained
Cell dilutes working solution；
7) 24 hole pvdf membrane plates are taken out from aluminium envelope, is sequentially added into：50 μ l PHA-P HA Zhiyang
Property control wells, the 50 μ l Rv0054 protein solutions that prepare (initial concentration is 60ug/ml, and solvent is AIM V
Nutrient solution) detection hole, 50 μ l AIM V nutrient solutions are added to blank control wells；It is separately added in above-mentioned 3 hole
The cell dilution μ l of working solution 100 are prepared (per hole cell quantity 2.5 × 105), and Rv0054 protein solutions in sample well
Final concentration of 20ug/ml；
8) culture plate is put into into 37 DEG C, 5%CO2Incubator is incubated 16 hours；
9) 1 is pressed with aseptic PBS:200 prepare fresh enzyme labelled antibody working solution.Culture plate is taken out from incubator, is discarded
Liquid in hole.Washed 4 times with the aseptic PBS of 200 μ l/well；
10) 50 μ l/well enzyme labelled antibody working solutions are added, culture plate is incubated 1 hour at 2-8 DEG C.Washed with PBS
Wash 4 times and remove uncombined enzyme labelled antibody；
11) the μ l of substrate working solution 50 for adding equilibrium at room temperature to cross per hole, room temperature reaction 7 minutes.With distilled water flushing end
Only react, in 37 DEG C of 2-3hrs or ambient temperature overnight desiccation culture plate, obtain the culture plate containing product.
2nd, spot numeration
1) culture plate containing product is taken out from incubator and discards cell culture fluid；
2) each reacting hole adds 200ul PBSs (pH 7.5)；
3) PBS is discarded；With fresh PBS repeated washing at least 3 times；
4) with 200 times of PBS dilution kits concentration labelled antibody reagent (alkali phosphatase enzyme mark it is little
The anti-human gamma interferon monoclonal antibody of mouse)；
5) each reacting hole adds 50ul labelled antibody working solutions, and 2-8 DEG C is incubated 1 hour；
6) labelled antibody working solution is discarded, by above-mentioned step 2 and 3 washings；
7) each reacting hole adds 50ul substrates chromophoric solution to be incubated at room temperature 7 minutes；
8) culture plate terminating reaction is thoroughly washed with distilled water or deionized water；
9) in ventilation or 37 DEG C of incubator desiccation culture plates；
10) navy blue that counts in each reacting hole clearly spot.
As a result judge：
If blank control wells spot number is 0-5, and (the spot number-blank control wells spot number in detection hole) >=5,
Then there are reaction, i.e. sample to be tested to produce immune response, sample to be tested candidate infection Much's bacillus with Rv0054.
If being unsatisfactory for above-mentioned, sample to be tested and Rv0054 do not produce immune response, and sample to be tested candidate is uninfected by tuberculosis
Mycobacterium.
18 samples to be tested (the concrete diseases of clinical identification and infection tuberculosis branch bar in table 1 are detected in aforementioned manners
Bacterium), as a result as shown in table 1.
Also 18 samples of table 1 are detected using the antigen ESAT-6 and antigens c FP 10 carried in kit,
Criterion is according in kit specification：If blank control wells spot number is 0-5, and (the spot number in detection hole-
Blank control wells spot number) >=6, then there are reaction, i.e. sample to be tested to produce immune response, sample to be tested infection with antigen
Or candidate's infection Much's bacillus.If being unsatisfactory for above-mentioned, sample to be tested and antigen do not produce immune response, to be measured
Sample is uninfected by or candidate is uninfected by Much's bacillus.
Table 1 is sample and its testing result
In table ,-it is blank ,+it is positive quality control.
As can be seen that
Rv0054 as antigen detect, in 11 samples to be tested 8 for infection Much's bacillus sample, this with face
Bed qualification result is consistent；But remaining 3 sample is not detected (with existing ESAT-6 albumen and CFP-10
Albumen and clinical detection, it can be determined that this 5 samples are also m tuberculosis infection), therefore, the present invention is used
Rv0054 detects whether the recall rate for infecting Much's bacillus up to 73% as antigen.
Can be seen that Rv0054 albumen and can detect or aid in detection patient to be measured whether to infect tuberculosis from above-mentioned experiment and divide
Branch bacillus, it is specific as follows：Using Rv0054 albumen as antigen, detected by tuberculosis infection T cell detection kit,
Using cell culture medium as blank, if blank control wells spot number is 0-5, and (the spot number in detection hole-
Blank control wells spot number) >=5, then there are reaction, i.e. sample to be tested to produce with Rv0054 or candidate produce immune response,
Sample to be tested infects or candidate's infection Much's bacillus.If being unsatisfactory for above-mentioned, sample to be tested is not produced with Rv0054
Immune response, sample to be tested is uninfected by or candidate is uninfected by Much's bacillus.
Claims (7)
- Application of the 1.Rv0054 albumen in preparing detection or aiding in detection Much's bacillus product；The Rv0054 is following albumen a) or b)：A) protein of the amino acid sequence composition shown in the 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 add and have with albumen shown in sequence 2 protein by derived from a) of identical function.
- 2.Rv0054 albumen is preparing detection or is aiding in whether detection patient to be measured is infected in Much's bacillus product Using.
- 3. whether a kind of detection or auxiliary detection sample to be tested infect the product of Much's bacillus, including Rv0054 eggs In vain.
- 4. product according to claim 3, it is characterised in that：The product also includes positive quality control and blank Control；The positive quality control is PHA-P HA；The blank is nonantigenic cell culture fluid.
- 5. the product according to claim 3 or 4, it is characterised in that：The product is also including in being described below The readable carrier for holding：If the spot number in blank control wells and detection hole meets following standard：The spot number of blank control wells is 0-5, And spot number >=5 of the spot number-blank control wells in detection hole, then sample to be tested infection or candidate infect tuberculosis branch bar Bacterium；If the spot 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 Much's bacillus；The blank control wells are blank place hole,The detection hole is the Rv0054 albumen place hole.
- 6. according to arbitrary described product in claim 3-5, it is characterised in that：The product is kit.
- 7. it is a kind of for differentiating, diagnosing, auxiliary diagnosis, examination and/or auxiliary examination Much's bacillus mark, For Rv0054 albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510420053.3A CN106645733A (en) | 2015-07-16 | 2015-07-16 | Protein for specific detection of mycobacterium tuberculosis infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510420053.3A CN106645733A (en) | 2015-07-16 | 2015-07-16 | Protein for specific detection of mycobacterium tuberculosis infection |
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| Publication Number | Publication Date |
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| CN106645733A true CN106645733A (en) | 2017-05-10 |
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ID=58815206
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510420053.3A Pending CN106645733A (en) | 2015-07-16 | 2015-07-16 | Protein for specific detection of mycobacterium tuberculosis infection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109030826A (en) * | 2017-06-12 | 2018-12-18 | 广东体必康生物科技有限公司 | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101687027A (en) * | 2007-04-04 | 2010-03-31 | 传染性疾病研究院 | immunogenic compositions comprising mycobacterium tuberculosis polypeptides and fusions thereof |
| CN103063837A (en) * | 2011-10-18 | 2013-04-24 | 复旦大学附属华山医院 | Reagent, method and kit for detecting mycobacterial infection |
-
2015
- 2015-07-16 CN CN201510420053.3A patent/CN106645733A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101687027A (en) * | 2007-04-04 | 2010-03-31 | 传染性疾病研究院 | immunogenic compositions comprising mycobacterium tuberculosis polypeptides and fusions thereof |
| CN103063837A (en) * | 2011-10-18 | 2013-04-24 | 复旦大学附属华山医院 | Reagent, method and kit for detecting mycobacterial infection |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109030826A (en) * | 2017-06-12 | 2018-12-18 | 广东体必康生物科技有限公司 | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis |
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