CN109406777A - Albumen Rv2824c for the infection of specific detection mycobacterium tuberculosis - Google Patents
Albumen Rv2824c for the infection of specific detection mycobacterium tuberculosis Download PDFInfo
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- CN109406777A CN109406777A CN201710710873.5A CN201710710873A CN109406777A CN 109406777 A CN109406777 A CN 109406777A CN 201710710873 A CN201710710873 A CN 201710710873A CN 109406777 A CN109406777 A CN 109406777A
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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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Abstract
The invention discloses the albumen Rv2824c infected for specific detection mycobacterium tuberculosis.The present invention provides application of the Rv2824c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv2824c is following albumen a) or b): a) protein of the composition of amino acid sequence shown in the sequence 1 in sequence table；B) by the amino acid residue sequence of the sequence 1 in sequence table by one or several amino acid residues substitution and/or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 1.The experiment proves that infection due to Mycobacterium tuberculosis recall rate can be improved more effectively.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to the albumen for the infection of specific detection mycobacterium tuberculosis
Rv2824c。
Background technique
Since multiple centuries, tuberculosis continues to cannot be neglected public health problem as one in the whole world.The whole world at present
The population of existing one third carries mycobacterium tuberculosis, only 1 year 2010 just newly-increased cases of tuberculosis 8,800,000, and dead 145
Ten thousand, average to have a people to die of pulmonary tuberculosis less than 22 seconds, tuberculosis is in first of Death of Infectious Diseases number.
Science proves to show easily propagated due to active tuberculosis, diagnosis to active tuberculosis and disappears as early as possible
Except the infection sources, controls TB endemic and have very great significance.However phthisical radical cure can't be accomplished completely at present,
Show that active tuberculosis patient after different short-term chemotherapies is cured, has the patient of 1%-9% that will recur according to the study, and
In some special populations, up to 20% (Hong Kong Chest Service/British Medical Research
Council.Am Rev Respir Dis, 1991,143:700-706), and drug-resistant tuberculosis is propagated further and recurs
The tubercular diagnosed not in time has very big relationship.So about tuberculosis cure patient active tuberculosis diagnosis for
The overall strategy of prevention and treatment tuberculosis has a very big significance.Activity judgement at present answers complex clinical, the performance of X line and sputum bacteria to determine,
And main foundation is sputum bacteria and x-ray.Sputum smear examination is simple and easy to do, and accuracy is higher, is the gold mark that active tuberculosis is made a definite diagnosis
Standard, but its recall rate is even lower than 10% in certain researchs.Diagnostic method based on nucleic acid amplification, such as real-time quantitative PCR and
The advantages of DNA chip is that sensitivity is costly and time consuming short, and problem is that specificity is relatively difficult to guarantee, and is easy to cause false positive results.
And the serodiagnosis based on antigen-antibody reaction has become important activity lung knot due to its simplicity, rapidity
Core clinic complementary diagnosis means, and it is able to satisfy the requirement for distinguishing tuberculosis rehabilitation crowd and active tuberculosis.Therefore, from
Long-range angle in general, should be dedicated to finding the preferable tuberculosis marker of sensibility and specificity.
Ideal Diagnosis of Tuberculosis marker should meet the following conditions: (1) sensibility is high；(2) specificity is high；(3) it is present in
In body fluid, especially blood, it is easy to detect.But the targeted antigen of Mycobacterium tuberculosis serodiagnosis is such as anti-at present
Original 5,38KD antigen, 30/31KD antigen and antigen 60 etc., have that positive rate is low, with intersecting for other mycobacteriums
The problems such as immunity, although there is certain valence in curative effect monitoring, prompt recurrence, judging prognosis and the generaI investigation of people at highest risk
Value, but still cannot be used for making a definite diagnosis for active tuberculosis at present.In order to realize the high sensitivity to active tuberculosis, Gao Te
Anisotropic diagnosis, urgent need search out more sensitive, more special active tuberculosis biomarker on a molecular scale.
Summary of the invention
It is an object of the present invention to provide the purposes of Rv2824c albumen.
The present invention provides application of the Rv2824c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；
The Rv2824c is following albumen a) or b):
A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；
B) by the amino acid residue sequence of the sequence 2 in sequence table by one or several amino acid residues substitution and/
Or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
Rv2824c is known albumen, and according to individual difference, which may have different sequences, they there may be
The difference of one or several amino acid, but be within, albumen used in one embodiment of the present of invention
Sequence be sequence 2.
Above-mentioned Rv2824c albumen detects whether patient to be measured infects in mycobacterium tuberculosis product in preparation detection or auxiliary
Application be also the scope of protection of the invention.
It is a further object to provide a kind of detections or auxiliary detection sample to be tested whether to infect tuberculosis branch bar
The product of bacterium.
Product provided by the invention, including Rv2824c albumen.
The said goods further include positive quality control and negative control hole；
The positive quality control is phytohemagglutinin HA；
The negative control hole is nonantigenic cell culture fluid.
In the said goods, the product further includes the readable carrier (can be specification) for being described below content:
If testing result meets following condition: negative control hole spot number is 0-5, and Rv2824c detection hole spot number-
Negative control hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2824c detection hole spot number-yin
Property control wells spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, for the positive
Sample；If testing result meets condition as described below, sample to be tested candidate is uninfected by mycobacterium tuberculosis；
The negative control hole is hole where negative control,
The Rv2824c detection hole is hole where the Rv2824c albumen.
The said goods are kit, and mentioned reagent box is specially T-SPOT kit, or ELISA kit.
Above-mentioned T-SPOT kit further includes well plates in T-SPOT kit, concentration labelled antibody, colour developing bottom
Object solution.Mentioned reagent box is the product of Oxford immunotec (Britain).
Third purpose of the present invention be to provide it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or auxiliary screening knot
The marker of core mycobacteria.
Marker provided by the invention is Rv2824c albumen.
The experiment proves that the present invention has screened the protein fragments in mycobacterium tuberculosis source, a kind of inspection is provided
Mycobacterium tuberculosis marker antigen RV2824c lungy is surveyed, by the antigen come vitro detection Specific T cell immunity
Reaction, can be used as the reference of diagnosis of tuberculosis patient, for diagnosing whether patient is infected by mycobacterium tuberculosis.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
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: potassium dihydrogen phosphate 13.609g (preparing 1000ml) is taken, adds 0.1mol/l sodium hydroxide solution, adjusts
PH to 7.5 is saved, the PBS buffer solution that pH value is 7.5 is obtained.
Solidify Ni+ chromatographic resin Novagen, Ni-NTA His, (Cat.NO 70691-5, Beijing are magnificent；2-8 degree is protected
It deposits)；Protein binding capacity is greater than 5mg/ml resin chromatographic column (glass or polypropylene).Superdex 75:(GE).
Inclusion body buffer: 50mM tris, 0.05M EDTA；
Inclusion body cleaning solution: 50mM tris, 0.05M EDTA, 2%DOC, 1M urea；
Solubilization of inclusion bodies liquid: 50mM Tris-HCl, 8M urea；
It is the T- of Oxford immunotec (Britain) that following embodiments, which use tuberculosis infection T cell detection kit,
SPOT kit is 10 pairs of antigenic reagent box of ESAT-6 and CFP.
Embodiment 1, prokaryotic expression destination protein Rv2824c
The preparation of destination protein Rv2824c: the recombinant vector pET28a- of Rv2824c protein coding gene will be expressed
Rv2824c is imported in e. coli bl21, obtains recombinant bacterium BL21/pET28a-Rv2824c；IPTG induces recombinant bacterium again
BL21/pET28a-Rv2824c collects supernatant, as destination protein Rv2824c.
It is specific as follows:
1, the building of express express target protein recombinant bacterium
The amino acid sequence of Rv2824c albumen is sequence 2, and the nucleotides sequence of encoding gene is classified as sequence 1.
By the site NdeI and EcoRI of the insertion pET28a carrier of Rv2824c protein coding gene shown in sequence 1, obtain
Recombinant vector pET28a-Rv2824c；
Recombinant vector is imported in e. coli bl21, recombinant bacterium BL21/pET28a-Rv2824c is obtained.
2, protein expression
1) recombinant bacterium BL21/pET28a-Rv2824c stands overnight (16h or so) at 37 DEG C.
2) it is inoculated in 5ml LB liquid medium (5ul kan is added), 200rpm, 37 DEG C are incubated overnight, 12h or so.
3) bacterium solution being incubated overnight is inoculated in the 200ml LB liquid that corresponding resistant has been added by inoculum concentration 150-200ul
In body culture medium, being cultivated at 37 DEG C, under the conditions of 200rpm to bacterium solution OD600 is 0.6-0.8；
4) final concentration 0.4mM IPTG is added and starts inducing expression, in 200rpm, 37 DEG C of conditions are down toward OD600 on 1.5 left sides
The right side, 4000rpm centrifugation 10min start to receive bacterium.
3, protein purification
1) thallus is resuspended: thallus collected by every 300ml culture medium is resuspended with the inclusion body buffer of 20ml；
2) bacterial cell disruption: ultrasonic 3S is spaced 6S, power 38%, ultrasonic 15min；
3) inclusion body: 4 DEG C of 10000g high speed centrifugation 20min is collected；
4) wash inclusion body: precipitating is resuspended with 15ml inclusion body buffer, 4 DEG C of 10000g high speed centrifugation 10min；3 are washed altogether
It is secondary；
5) solubilization of inclusion bodies: inclusion body is resuspended with 15ml solubilization of inclusion bodies liquid, and piping and druming mixes and vibrates 3- on mediation instrument
5min；
6) 5ml supernatant is placed in bag filter and is sealed, be put into the peripheral dialyzate equipped with 10 times of amount supernatant volumes
(200mM Tris-HCl, 8M urea) (being 100:1 with liquid volume ratio in bag filter), is placed in 4 DEG C, sufficiently balances.Then
Clear water is gradually slowly added dropwise in peristaltic pump to the periphery dialyzate, until peripheral dialyzate is diluted 10 times；
7) it is put into dialysis urea concentration into protein liquid in secondary dialyzate and is lower than 0.04M.If no obvious sediment generates,
Then renaturation success.
Product after electrophoresis detection renaturation is purpose albumen Rv2824c (size 34.4KDa).
The concentration of product is 0.2mg/ml after renaturation, and wherein solvent is by 20mM Tris-HCl, pH8.0,10%
Glycerol composition, solute are purpose albumen Rv2824c.
The application of embodiment 2, mycobacterium tuberculosis marker antigen Rv2824c in detection mycobacterium tuberculosis
1, T-SPOT kit detects
The specific method is as follows, and some solution of the inside are all from the T-SPOT reagent of Oxford immunotec (Britain)
Box:
1) using heparin vacuum blood collection tube, to different patients to be measured, (45 be tuberculosis patient example clinical detection knot shown in table 1
13 clinical detection non-tuberculosis people shown in the patient and Biao 2 of core disease, and know) peripheric venous blood is directly adopted, it obtains anticoagulant
Blood sample；
2) anticoagulation sample is mixed by volume 1:1 and RPMI1640 culture solution；Carefully blood sample is added in 3:1 ratio
Ficoll lymphocyte separation medium upper layer (GE Healthcare 17-1440-02) (utilizes Ficoll-PaqueTMplusAccording to
Operating instruction separates PBMCs)；25 DEG C of room temperature, 1000g is centrifuged 22 minutes；Horizontal rotor delays and rises slow drop；
3) by mononuclear cell layer (being located at centrifuge tube middle layer, be the tunica albuginea shape of layer) from Ficoll separating pipe
It is transferred to the sterile 15ml centrifuge tube for having added 10ml AIM-V culture solution (Invitrogen 12055091), is mixed gently, room
Warm 600g is centrifuged 7min；
4) supernatant is carefully removed, 1ml AIM-V culture solution is added, AIM-V culture solution is added extremely in light and slow resuspension cell
10ml, 350g are centrifuged 7min；
5) supernatant is carefully abandoned, 1ml AIM-V culture solution is added, cell is resuspended, obtain cell suspension (PBMCs)；
6) it takes 10 μ l cell suspensions that the 1%trypan blue of 40 μ l is added, is diluted with AIM-V culture solution, obtain cell
Dilute working solution；
7) take out 24 hole pvdf membrane plates from aluminium envelope, be sequentially added into: 50 μ l phytohemagglutinin HA (sigma) are extremely
The Rv2824c protein solution that Positive control wells, 50 μ l are prepared (initial concentration 60ug/ml, solvent are AIM V culture solution)
It is added to detection hole, 50 μ l AIM V culture solutions to negative control hole；It is separately added into above-mentioned 3 hole and has prepared cell dilution
Working solution 100 μ l (every hole cell quantity 2.5 × 105), and in sample well Rv2824c protein solution final concentration of 20ug/
ml；
8) culture plate is put into 37 DEG C, 5%CO2Incubator is incubated for 16 hours；
9) fresh enzyme labelled antibody working solution is prepared by 1:200 with sterile PBS；Culture plate is taken out from incubator, is discarded in hole
Liquid.It is washed 4 times with the sterile PBS of 200 μ l/well；
10) 50 μ l/well enzyme labelled antibody working solutions are added, culture plate is incubated for 1 hour at 2-8 DEG C.It is washed 4 times with PBS
Remove unbonded enzyme labelled antibody；
11) the 50 μ l of substrate working solution that equilibrium at room temperature is crossed is added in every hole, reacts at room temperature 7 minutes.It is terminated with distilled water flushing
Reaction, in 37 DEG C of 2-3hrs or ambient temperature overnight desiccation culture plate, obtains the culture plate containing reaction product.
2, spot counts
1) culture plate containing reaction product is taken out from incubator discard cell culture fluid；
2) 200ul PBS buffer solution (pH 7.5) is added in each reacting hole；
3) PBS buffer solution is discarded；It is washed repeatedly at least 3 times with fresh PBS buffer solution；
4) with 200 times of PBS buffer solution dilution kit in concentration labelled antibody reagents (alkali phosphatase enzyme mark it is small
The anti-human gamma interferon monoclonal antibody of mouse)；
5) 50ul labelled antibody working solution is added in each reacting hole, and 2-8 DEG C is incubated for 1 hour；
6) labelled antibody working solution is discarded, is washed by above-mentioned step 2 and 3；
7) each reacting hole is added 50ul substrate chromophoric solution and is incubated at room temperature 7 minutes；
8) culture plate is thoroughly washed with distilled water or deionized water terminate reaction；
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 negative control hole spot number is 0-5, and Rv2824c detection hole spot number-negative control hole spot number >=6；
If or negative control hole spot number is more than or equal to 6, and (Rv2824c detection hole spot number-negative control hole spot
Number) >=2 × (negative control hole spot number)；
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and Rv2824c do not generate immune response, and sample to be tested candidate is uninfected by knot
Core mycobacteria.
45 tuberculosis patient samples to be tested in table 1 are detected in aforementioned manners, and the results are shown in Table 1.
The sample to be tested of 13 Healthy Peoples in table 2 is detected in aforementioned manners, and the results are shown in Table 2.
Comparative example:
45 tuberculosis patients shown in T-SPOT kit detection table 1 using Oxford immunotec (Britain) wait for
The sample to be tested of 13 Healthy Peoples in test sample sheet and table 2, method is same as Example 1, unique the difference is that two detections of setting
Hole, and A detection hole is antigen A hole, addition is antigen ESAT-6, and B detection hole is the hole antigen B, and addition is antigens c FP 10.
According to the reaction and judgement result of antigen A and the hole antigen B:
If negative control hole spot number is 0-5, and (antigen A spot number)-(negative control hole spot number) >=6 or (antigen
B spot number)-(negative control hole spot number) >=6；
Or work as negative control hole spot number >=6, and (antigen A spot number) >=2 × (negative control hole spot number) or (anti-
Former B spot number) >=2 × (negative control hole spot number).
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and antigen A or antigen B do not generate immune response, and sample to be tested candidate does not feel
Contaminate mycobacterium tuberculosis.
Table 1 is sample and its testing result
In table ,-it is negative control ,+it is Positive control wells (culture medium for having added PHA), X is Rv2824c Protein Detection
Hole, A are antigen A detection hole, and B is antigen B detection hole.
Table 2 is Healthy People sample and its testing result
| Sample number | - | A | B | + | Rv2824c |
| 1 | 0 | 0 | 1 | 405 | 4 |
| 2 | 0 | 0 | 0 | 672 | 1 |
| 3 | 0 | 0 | 0 | 1092 | 3 |
| 4 | 0 | 0 | 0 | 772 | 3 |
| 5 | 0 | 1 | 0 | 32 | 2 |
| 6 | 0 | 0 | 0 | 515 | 0 |
| 7 | 0 | 45 | 20 | 252 | 3 |
| 8 | 0 | 3 | 58 | 334 | 0 |
| 9 | 0 | 29 | 12 | 736 | 1 |
| 10 | 1 | 0 | 0 | 952 | 3 |
| 11 | 0 | 25 | 4 | 1238 | 11 |
| 12 | 0 | 2 | 0 | 958 | 2 |
| 13 | 0 | 2 | 2 | 937 | 2 |
In table ,-it is negative control (culture medium for being added without any antigen) ,+it is that Positive control wells (have added the culture of PHA
Base), X is Rv2824c Protein Detection hole.
As can be seen that
Rv2824c is detected as antigen, 37 in the identified sample to be tested for infecting mycobacterium tuberculosis of 45 clinics
The sample of mycobacterium tuberculosis is infected for Rv2824c Protein Detection, this is consistent with clinical identification result.But remaining 8 sample
It is not detected and, therefore, the present invention uses Rv2824c to detect whether that the recall rate of infection mycobacterium tuberculosis reaches as antigen
82%.
12 are not detected mycobacterium tuberculosis in 13 Healthy People samples, this is consistent with clinical identification result, and there are also 1
A detection mycobacterium tuberculosis.
Comparative example detects the identified sample to be tested for infecting mycobacterium tuberculosis of 45 clinics, wherein 35 are comparison
The sample of example detection infection mycobacterium tuberculosis, this is consistent with clinical identification result；But remaining 10 sample is not detected
Come.Therefore, comparative example detects whether that the recall rate of infection mycobacterium tuberculosis is only 77%, well below Rv2824c as anti-
Original detection.
In comparative example in 13 Healthy People samples 9 be not detected mycobacterium tuberculosis, 4 detection mycobacterium tuberculosis, this
It is different from clinical identification result.
Can be seen that Rv2824c albumen from above-mentioned experiment can detecte or assist whether detection patient to be measured infects tuberculosis
Mycobacteria, it is specific as follows: using Rv2824c albumen as antigen, to be detected by tuberculosis infection T cell detection kit, with thin
Born of the same parents' culture medium is as negative control hole, if negative control hole spot number is 0-5, and Rv2824c detection hole spot number-feminine gender is right
According to hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2824c detection hole spot number-negative control
Hole spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and Rv2824c do not generate immune response, and sample to be tested candidate is uninfected by tuberculosis branch bar
Bacterium.
Sequence table
<110>Institute of Biophysics, Academia Sinica, Guangdong Ti Bikang Biotechnology Co., Ltd
<120>the albumen Rv2824c for the infection of specific detection mycobacterium tuberculosis
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 945
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
atggctgctc gccgaggcgg catcagaagg acggatctgc tacgtcggag tggccagccc 60
cgaggccgac atcgcgcgag tgcggcggaa tcaggcttga cctggatttc accaacgctt 120
attctggtgg gcttttctca ccgaggagat cgccggatga cggaacactt gtcgcgattg 180
acgctgactc tagaggttga tgccccgctc gaacgcgcga gggtggcgac cctggggccg 240
catcttcatg gcgtcctcat ggagtcgatc ccggccgatt atgtgcagac cctccacacc 300
gtgccggtga acccgtacag tcagtacgcg ctggcccgct cgaccacatc tttggagtgg 360
aagatcagca cgctgacgaa cgaggcgcgg cagcagatcg tcggacctat caacgacgcg 420
gcgtttgcgg gttttcggct ccgtgcgagc gggatagcga cacaggtcac gtcgcgatcg 480
ctggagcaga acccgctaag tcaattcgcg cgcattttct acgcgcggcc cgagacgcgc 540
aagttccggg tcgagttcct gacgcctacc gcattcaagc aatccggcga gtacgtgttc 600
tggccggatc cgcggctcgt gtttcagagt ctcgcgcaga agtacggtgc aattgtcgac 660
ggcgaagagc ccgatcctgg cctcatcgcc gaattcggtc agtcggttcg cctctccgcg 720
ttccgggtgg cgtcggcccc gttcgcggtg ggcgcggcgc gtgttcccgg cttcaccggc 780
tcggccacgt tcaccgtccg cggtgtggat acttttgcga gctatatcgc ggcgctgttg 840
tggttcgggg agttctcggg atgcggaata aaggcatcca tggggatggg cgcgatccgg 900
gtccagccac tggcaccgag ggaaaaatgc gtaccgaagc catga 945
<210> 2
<211> 314
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 2
Met Ala Ala Arg Arg Gly Gly Ile Arg Arg Thr Asp Leu Leu Arg Arg
1 5 10 15
Ser Gly Gln Pro Arg Gly Arg His Arg Ala Ser Ala Ala Glu Ser Gly
20 25 30
Leu Thr Trp Ile Ser Pro Thr Leu Ile Leu Val Gly Phe Ser His Arg
35 40 45
Gly Asp Arg Arg Met Thr Glu His Leu Ser Arg Leu Thr Leu Thr Leu
50 55 60
Glu Val Asp Ala Pro Leu Glu Arg Ala Arg Val Ala Thr Leu Gly Pro
65 70 75 80
His Leu His Gly Val Leu Met Glu Ser Ile Pro Ala Asp Tyr Val Gln
85 90 95
Thr Leu His Thr Val Pro Val Asn Pro Tyr Ser Gln Tyr Ala Leu Ala
100 105 110
Arg Ser Thr Thr Ser Leu Glu Trp Lys Ile Ser Thr Leu Thr Asn Glu
115 120 125
Ala Arg Gln Gln Ile Val Gly Pro Ile Asn Asp Ala Ala Phe Ala Gly
130 135 140
Phe Arg Leu Arg Ala Ser Gly Ile Ala Thr Gln Val Thr Ser Arg Ser
145 150 155 160
Leu Glu Gln Asn Pro Leu Ser Gln Phe Ala Arg Ile Phe Tyr Ala Arg
165 170 175
Pro Glu Thr Arg Lys Phe Arg Val Glu Phe Leu Thr Pro Thr Ala Phe
180 185 190
Lys Gln Ser Gly Glu Tyr Val Phe Trp Pro Asp Pro Arg Leu Val Phe
195 200 205
Gln Ser Leu Ala Gln Lys Tyr Gly Ala Ile Val Asp Gly Glu Glu Pro
210 215 220
Asp Pro Gly Leu Ile Ala Glu Phe Gly Gln Ser Val Arg Leu Ser Ala
225 230 235 240
Phe Arg Val Ala Ser Ala Pro Phe Ala Val Gly Ala Ala Arg Val Pro
245 250 255
Gly Phe Thr Gly Ser Ala Thr Phe Thr Val Arg Gly Val Asp Thr Phe
260 265 270
Ala Ser Tyr Ile Ala Ala Leu Leu Trp Phe Gly Glu Phe Ser Gly Cys
275 280 285
Gly Ile Lys Ala Ser Met Gly Met Gly Ala Ile Arg Val Gln Pro Leu
290 295 300
Ala Pro Arg Glu Lys Cys Val Pro Lys Pro
305 310
Claims (7)
- Application of the 1.Rv2824c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv2824c is following albumen a) or b):A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；B) amino acid residue sequence of the sequence 2 in sequence table by the substitution of one or several amino acid residues and/or is lacked Lose and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
- 2.Rv2824c albumen detects whether patient to be measured infects answering in mycobacterium tuberculosis product in preparation detection or auxiliary With.
- 3. whether a kind of detection or auxiliary detection sample to be tested infect the product of mycobacterium tuberculosis, including Rv2824c albumen.
- 4. product according to claim 3, it is characterised in that: the product further includes positive quality control and negative control hole；The positive quality control is phytohemagglutinin HA；The negative control hole is nonantigenic cell culture fluid.
- 5. product according to claim 3 or 4, it is characterised in that: the product further includes be described below content readable Carrier:If testing result meets following condition: negative control hole spot number is 0-5, and Rv2824c detection hole spot number-feminine gender is right According to hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2824c detection hole spot number-negative control hole Spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；If inspection It surveys result and meets condition as described below, then sample to be tested candidate is uninfected by mycobacterium tuberculosis；The negative control hole is hole where negative control,The Rv2824c detection hole is hole where the Rv2824c albumen.
- 6. according to the product any in claim 3-5, it is characterised in that: the product is kit.
- 7. it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or assist screening mycobacterium tuberculosis marker, be Rv2824c albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710710873.5A CN109406777B (en) | 2017-08-18 | 2017-08-18 | Protein Rv2824c for specific detection of mycobacterium tuberculosis infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710710873.5A CN109406777B (en) | 2017-08-18 | 2017-08-18 | Protein Rv2824c for specific detection of mycobacterium tuberculosis infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109406777A true CN109406777A (en) | 2019-03-01 |
| CN109406777B CN109406777B (en) | 2022-02-18 |
Family
ID=65455247
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710710873.5A Active CN109406777B (en) | 2017-08-18 | 2017-08-18 | Protein Rv2824c for specific detection of mycobacterium tuberculosis infection |
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| EP1350839A1 (en) * | 2002-04-05 | 2003-10-08 | Institut Pasteur | Identification of virulence associated regions RD1 and RD5 enabling the development of improved vaccines of M. bovis BCG and M. microti |
| CN102004155A (en) * | 2010-02-12 | 2011-04-06 | 复旦大学附属华山医院 | Kit and method for detecting mycobacterium tuberculosis infection and application |
| CN103698511A (en) * | 2013-11-25 | 2014-04-02 | 广东体必康生物科技有限公司 | Application of mycobacterium tuberculosis protein in preparation of products used for diagnosis of latent tuberculosis infection |
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