CN109406777B

CN109406777B - Protein Rv2824c for specific detection of mycobacterium tuberculosis infection

Info

Publication number: CN109406777B
Application number: CN201710710873.5A
Authority: CN
Inventors: 毕利军; 张先恩; 朱国峰; 陶生策; 邓教宇; 张泓泰; 侯剑; 王雅果
Original assignee: Tibikon Biotechnology Guangdong Co ltd; Institute of Biophysics of CAS
Current assignee: Tibikon Biotechnology Guangdong Co ltd; Institute of Biophysics of CAS
Priority date: 2017-08-18
Filing date: 2017-08-18
Publication date: 2022-02-18
Anticipated expiration: 2037-08-18
Also published as: CN109406777A

Abstract

The invention discloses a protein Rv2824c for specifically detecting mycobacterium tuberculosis infection. The invention provides an application of Rv2824c protein in preparation of a product for detecting or assisting in detecting mycobacterium tuberculosis; the Rv2824c is a protein of the following a) or b): a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; b) and (b) protein which is derived from the protein a) and has the same function with the protein shown in the sequence 1 by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence of the sequence 1 in the sequence table. Experiments prove that the detection rate of mycobacterium tuberculosis infection can be more effectively improved.

Description

Protein Rv2824c for specific detection of mycobacterium tuberculosis infection

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a protein Rv2824c for specifically detecting mycobacterium tuberculosis infection.

Background

Tuberculosis continues to be a non-negligible public health problem worldwide for many centuries. At present, one third of the population in the world carries mycobacterium tuberculosis, 880 ten thousands of new tuberculosis cases and 145 thousands of deaths are caused in one year 2010, one person can die of pulmonary tuberculosis in less than 22 seconds on average, and the tuberculosis is the first of the deaths caused by infectious diseases.

Scientific evidence shows that the diagnosis of active tuberculosis, the elimination of infectious sources as soon as possible and the control of tuberculosis epidemic are of great significance due to the extremely easy dissemination of active tuberculosis. However, the radical cure of tuberculosis cannot be completely achieved at present, and researches show that 1-9% of patients with active tuberculosis are cured by different short-range chemotherapies and the disease is recurrent, and in some special populations, the disease reaches 20% (Hong Kong Chest Service/British Medical Research council. am Rev Respir Dis,1991,143: 700-. Therefore, the diagnosis of active tuberculosis of tuberculosis-cured patients has great significance for the overall strategy of preventing and treating tuberculosis. At present, the judgment of activity should be made by combining clinical, X-ray manifestation and sputum bacteria determination, and the main basis is sputum bacteria and X-ray. The sputum smear examination is simple and easy to implement, has higher accuracy, is the gold standard for the confirmed diagnosis of active tuberculosis, but the detection rate of some studies is even lower than 10 percent. The diagnostic methods based on nucleic acid amplification, such as real-time quantitative PCR and DNA chips, have advantages of high sensitivity and short time consumption, and have problems in that specificity is difficult to guarantee and false positive results are easily caused. Serological diagnosis based on antigen-antibody reaction is increasingly becoming an important clinical auxiliary diagnosis means for active tuberculosis due to its simplicity and rapidity, and can meet the requirements of distinguishing tuberculosis rehabilitation people from active tuberculosis. Therefore, in the long term, the research is focused on finding tuberculosis markers with better sensitivity and specificity.

The ideal tuberculosis diagnosis marker should meet the following conditions: (1) the sensitivity is high; (2) the specificity is high; (3) is present in body fluids, particularly blood, and is easy to detect. However, the antigens such as antigen 5, 38KD antigen, 30/31KD antigen and antigen 60, etc. aimed by the existing serological diagnosis of the mycobacterium tuberculosis have the problems of low positive detection rate, cross immunity with other mycobacteria, etc., and although the antigens have certain values in the general investigation of curative effect monitoring, relapse prompting, prognosis judgment and high risk groups, the antigens can not be used for the accurate diagnosis of the active tuberculosis at present. In order to realize high sensitivity and high specificity diagnosis of active tuberculosis, more sensitive and specific active tuberculosis biomarkers are urgently needed to be found at a molecular level.

Disclosure of Invention

An object of the present invention is to provide the use of Rv2824c protein.

The invention provides an application of Rv2824c protein in preparation of a product for detecting or assisting in detecting mycobacterium tuberculosis;

the Rv2824c is a protein of the following a) or b):

a) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

b) and (b) protein which is derived from the protein a) and has the same function with the protein shown in the sequence 2 by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence of the sequence 2 in the sequence table.

Rv2824c is a known protein, which may have different sequences depending on individual differences, which may differ by one or several amino acids, but is within the scope of the present invention, and the sequence of the protein used in one embodiment of the present invention is sequence 2.

The application of the Rv2824c protein in preparing a product for detecting or assisting in detecting whether a patient to be detected is infected with mycobacterium tuberculosis is also within the protection scope of the invention.

The invention also aims to provide a product for detecting or assisting in detecting whether a sample to be detected is infected with mycobacterium tuberculosis.

The product provided by the invention comprises Rv2824c protein.

The product also comprises a positive quality control hole and a negative control hole;

the positive quality control is Phytohemagglutinin (PHA);

the negative control wells were cell culture without antigen.

In the above product, the product further comprises a readable carrier (which may be a specification) describing:

if the detection result meets the following conditions: the number of the negative control hole speckles is 0-5, and the number of the negative control hole speckles is more than or equal to 6 when the number of the negative control hole speckles is detected by the Rv2824 c; or if the number of the negative control hole spots is more than or equal to 6, and (the number of the Rv2824c detection hole spots-the number of the negative control hole spots) is more than or equal to 2 x (the number of the negative control hole spots); the candidate infected mycobacterium tuberculosis of the sample to be detected is a positive sample; if the detection result meets the following conditions, the sample to be detected is candidate to be not infected with mycobacterium tuberculosis;

the negative control hole is the hole where the negative control is positioned,

the Rv2824c detection hole is a hole in which the Rv2824c protein is located.

The product is a kit, and the kit is specifically a T-SPOT kit and can also be an ELISA kit.

The T-SPOT kit also comprises a micropore culture plate, a concentrated labeled antibody and a chromogenic substrate solution in the T-SPOT kit. The kit is a product of Oxford immunotec (UK).

The third object of the present invention is to provide a marker for identification, diagnosis, auxiliary diagnosis, screening and/or auxiliary screening of Mycobacterium tuberculosis.

The marker provided by the invention is Rv2824c protein.

Experiments prove that the protein fragment derived from mycobacterium tuberculosis is screened, the mycobacterium tuberculosis marker antigen RV2824c for detecting tuberculosis is provided, and the antigen is used for detecting specific T cell immune reaction in vitro, can be used as a reference for diagnosing patients with tuberculosis, and is used for diagnosing whether the patients are infected by the mycobacterium tuberculosis.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Some reagents were as follows:

lysine buffer: 20mM Tris-HCl, 500mM sodium chloride, 10% glycerol, pH8.0

Wash buffer: 20mM Tris-HCl, 20mM imidazole, 500mM sodium chloride, 10% glycerol, pH8.0

Elution buffer: 20mM Tris-HCl, 250mM imidazole, 500mM sodium chloride, 10% glycerol, pH8.0

Chromatography buffer: 13.609g of potassium dihydrogen phosphate (prepared 1000ml) was added to 0.1mol/l sodium hydroxide solution to adjust the pH to 7.5, thereby obtaining a PBS buffer solution with a pH of 7.5.

Solidifying Ni + chromatographic resin Novagen, Ni-NTA His, (Cat. NO 70691-5, Beijing Huamei; preservation at 2-8 deg); the protein binding capacity is more than 5mg/ml resin chromatographic column (glass or polypropylene). superdex 75: (GE).

Inclusion body buffer: 50mM tris, 0.05M EDTA;

inclusion body wash: 50mM tris, 0.05M EDTA, 2% DOC, 1M urea;

inclusion body dissolution solution: 50mM Tris-HCl, 8M urea;

the following examples employ a T cell detection kit for tuberculosis infection which is the T-SPOT kit for Oxford immunotec (UK) as the ESAT-6 and CFP 10 dual antigen kit.

Example 1 prokaryotic expression of the protein of interest Rv2824c

Preparation of target protein Rv2824 c: introducing a recombinant vector pET28a-Rv2824c for expressing an Rv28 2824c protein coding gene into escherichia coli BL21 to obtain a recombinant bacterium BL21/pET28a-Rv2824 c; and then IPTG induces the recombinant bacteria BL21/pET28a-Rv2824c, and the supernatant is collected, namely the target protein Rv2824 c.

The method comprises the following specific steps:

1. construction of recombinant bacteria for expressing target protein

The amino acid sequence of the Rv2824c protein is sequence 2, and the nucleotide sequence of the coding gene is sequence 1.

Inserting the Rv2824c protein coding gene shown in the sequence 1 into NdeI and EcoRI sites of a pET28a vector to obtain a recombinant vector pET28a-Rv2824 c;

the recombinant vector is introduced into Escherichia coli BL21 to obtain recombinant strain BL21/pET28a-Rv2824 c.

2. Protein expression

1) The recombinant strain BL21/pET28a-Rv2824c is kept at 37 ℃ overnight (about 16 h).

2) Inoculated into 5ml of liquid LB medium (5 ul kan added), cultured at 200rpm and 37 ℃ overnight for about 12 h.

3) Inoculating the overnight cultured bacterial liquid into 200ml LB liquid culture medium added with corresponding resistance according to the inoculum size of 150-;

4) the induction of expression was started by adding IPTG to a final concentration of 0.4mM, and the cells were harvested by centrifugation at 200rpm and 37 ℃ until the OD600 was around 1.5 and 4000rpm for 10 min.

3. Protein purification

1) And (3) resuspending the thallus: every 300ml of culture medium collected thalli is resuspended by 20ml of inclusion body buffer solution;

2) and (3) crushing thalli: performing ultrasound for 15min at the interval of 6S and the power of 38 percent for 3S;

3) collecting inclusion bodies: centrifuging at 4 deg.C 10000g for 20 min;

4) washing the inclusion bodies: resuspending the precipitate with 15ml of inclusion body buffer solution, and centrifuging at 4 ℃ and 10000g for 10min at high speed; washing for 3 times;

5) dissolving the inclusion body: resuspending the inclusion body with 15ml inclusion body solution, blowing, uniformly mixing and vibrating on a vortex instrument for 3-5 min;

6) 5ml of the supernatant was placed in a dialysis bag and sealed, and placed in a container containing 10 times the volume of the supernatant in a peripheral dialysis solution (200mM Tris-HCl, 8M urea) (i.e., 100: 1) the mixture was left at 4 ℃ and fully equilibrated. Then gradually and slowly dripping clear water into the peripheral dialysate by using a peristaltic pump until the peripheral dialysate is diluted by 10 times;

7) putting into secondary dialysate, and dialyzing until the concentration of urea in protein solution is lower than 0.04M. If no obvious precipitation is generated, the renaturation is successful.

The product after renaturation is detected by electrophoresis, and is the target protein Rv2824c (the size is 34.4 KDa).

The concentration of the renatured product is 0.2mg/ml, wherein the solvent consists of 20mM Tris-HCl, pH8.0 and 10% glycerol, and the solute is the target protein Rv2824 c.

Example 2 application of Mycobacterium tuberculosis marker antigen Rv2824c in detection of Mycobacterium tuberculosis

1. T-SPOT kit detection

The specific procedure was as follows, some of the solutions therein were obtained from the T-SPOT kit from Oxford immunotec (UK):

1) directly collecting peripheral venous blood from different patients to be detected (45 shown in table 1 are tuberculosis patients and 13 clinical non-tuberculosis patients shown in table 2, and informed) by using a heparin vacuum blood collection tube to obtain an anticoagulation sample;

2) uniformly mixing the anticoagulation sample with RPMI1640 culture solution according to the volume of 1: 1; the blood samples were carefully applied to the top of the Ficoll lymphocyte isolate (GE Healthcare 17-1440-02) (using Ficoll-Paque (TM)) at a 3:1 ratio^plusIsolation of PBMCs according to the protocol); centrifuging at room temperature of 25 ℃ for 22 minutes at 1000 g; the horizontal rotor slowly rises and slowly falls;

3) transferring a mononuclear cell layer (which is located in the middle layer of a centrifuge tube and is in a thin white film shape) from a Ficoll separating tube to a sterile 15ml centrifuge tube added with 10ml AIM-V culture solution (Invitrogen 12055091), gently mixing, and centrifuging at the room temperature of 600g for 7 min;

4) carefully removing the supernatant, adding 1ml of AIM-V culture solution, gently suspending the cells, adding AIM-V culture solution to 10ml, and centrifuging for 7min at 350 g;

5) carefully abandoning the supernatant, adding 1ml of AIM-V culture solution to resuspend the cells to obtain Cell Suspension (PBMCs);

6) adding 10 μ l of cell suspension into 40 μ l of 1% trypan blue, and diluting with AIM-V culture solution to obtain cell dilution working solution;

7) the 24-well PVDF membrane plate was removed from the aluminum envelope and added in the order: 50 μ l of phytohemagglutinin PHA (sigma) to the positive control well, 50 μ l of the prepared Rv2824c protein solution (initial concentration 60ug/ml, solvent AIM V culture solution) to the detection well, 50 μ l of AIM V culture solution to the negative control well; adding 100 μ l of prepared cell dilution solution (1: (A))Cell number per well 2.5X 10⁵) And the final concentration of the Rv2824c protein solution in the sample hole is 20 ug/ml;

8) the plates were placed at 37 ℃ in 5% CO₂Incubating in an incubator for 16 hours;

9) preparing fresh enzyme-labeled antibody working solution by sterile PBS according to the ratio of 1: 200; the plate was removed from the incubator and the well liquid was discarded. Washed 4 times with 200. mu.l/well sterile PBS;

10) add 50. mu.l/well enzyme-labeled antibody working solution and incubate the plate at 2-8 ℃ for 1 hour. Washing with PBS for 4 times to remove unbound enzyme-labeled antibody;

11) 50. mu.l of the substrate working solution equilibrated at room temperature was added to each well, and reacted at room temperature for 7 minutes. The reaction was stopped by rinsing with distilled water, and the plate was dried at 37 ℃ for 2-3hrs or overnight at room temperature to obtain a plate containing the reaction product.

2. Blob counting

1) Taking out the culture plate containing the reaction product from the incubator and discarding the cell culture solution;

2) 200ul PBS buffer (pH 7.5) was added to each reaction well;

3) discarding the PBS buffer solution; washing with fresh PBS buffer was repeated at least 3 times;

4) diluting the concentrated labeled antibody reagent (anti-human gamma-interferon monoclonal antibody of alkaline phosphatase labeled mouse) in the kit with PBS buffer solution 200 times;

5) adding 50ul of labeled antibody working solution into each reaction hole, and incubating for 1 hour at the temperature of 2-8 ℃;

6) discarding the labeled antibody working solution, and washing according to the steps 2 and 3;

7) 50ul of substrate chromogenic solution is added into each reaction hole and incubated for 7 minutes at room temperature;

8) thoroughly washing the culture plate with distilled water or deionized water to terminate the reaction;

9) drying the culture plate in a ventilated place or an incubator at 37 ℃;

10) the dark blue clear spots in each reaction well were counted.

And (5) judging a result:

if the number of the negative control hole spots is 0-5, and the number of the Rv2824c detection hole spots is not less than 6;

or if the number of the negative control hole spots is more than or equal to 6, and (the number of the Rv2824c detection hole spots-the number of the negative control hole spots) is more than or equal to 2 x (the number of the negative control hole spots);

the candidate to-be-detected sample is infected with the mycobacterium tuberculosis and is a positive sample;

if the result does not meet the requirement, the sample to be detected does not have an immune reaction with Rv2824c, and the sample to be detected is candidate to be infected with the mycobacterium tuberculosis.

The test samples of 45 tuberculosis patients in Table 1 were tested by the above method, and the results are shown in Table 1.

The test samples of 13 healthy persons in Table 2 were tested by the above method, and the results are shown in Table 2.

Comparative example:

the samples to be tested for 45 tuberculosis patients shown in Table 1 and the samples to be tested for 13 healthy persons in Table 2 were tested using T-SPOT kit of Oxford immunotec (UK) in the same manner as in example 1, except that two test wells were provided, and the test well A was the antigen A well to which the antigen ESAT-6 was added, and the test well B was the antigen B well to which the antigen CFP 10 was added.

Judging the result according to the reaction of the antigen A and the antigen B:

if the number of the negative control hole spots is 0-5, and the number of (antigen A spots) - (negative control hole spots) is more than or equal to 6 or the number of (antigen B spots) - (negative control hole spots) is more than or equal to 6;

or when the number of the negative control hole spots is more than or equal to 6, and the number of the antigen A spots is more than or equal to 2 x (the number of the negative control hole spots) or the number of the antigen B spots is more than or equal to 2 x (the number of the negative control hole spots).

if the antigen A and the antigen B do not meet the requirement, the sample to be detected does not generate immune reaction with the antigen A or the antigen B, and the sample to be detected is candidate without being infected with the mycobacterium tuberculosis.

Table 1 shows the samples and the results of the measurements

In the table, the-is a negative control, the + is a positive control well (medium with PHA added), the X is an Rv2824c protein detection well, the A is an antigen A detection well, and the B is an antigen B detection well.

Table 2 shows the results of the examination of healthy human specimens

Sample numbering	-	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 the table, the-is negative control (medium without any antigen added), + is positive control well (medium with PHA added), and X is Rv2824c protein detection well.

As can be seen,

the Rv2824c is used as an antigen for detection, 37 samples of 45 clinically identified samples to be detected infected with the mycobacterium tuberculosis are samples of Rv2824c protein for detecting the mycobacterium tuberculosis, and the result is consistent with the clinical identification result. However, the other 8 samples are not detected, so that the detection rate of whether the mycobacterium tuberculosis is infected or not by using the Rv2824c as the antigen can reach 82%.

No M.tuberculosis was detected in 12 of the 13 healthy human specimens, which is consistent with the clinical identification results, and 1 of the 13 healthy human specimens was detected.

45 samples to be detected which are clinically identified to be infected with the mycobacterium tuberculosis are detected in the comparative example, wherein 35 samples are detected to be infected with the mycobacterium tuberculosis in the comparative example, which is consistent with the clinical identification result; but the remaining 10 samples were not detected. Therefore, the detection rate of detecting whether the mycobacterium tuberculosis is infected by the comparative example is only 77 percent and is far lower than that of Rv2824c as an antigen for detection.

In the comparative example, 9 of the 13 healthy human specimens were not detected for M.tuberculosis, and 4 were detected for M.tuberculosis, which is different from the clinical identification results.

From the above experiments, it can be seen that the Rv2824c protein can detect or assist in detecting whether the patient to be detected is infected with mycobacterium tuberculosis, which is as follows: detecting by using an Rv2824c protein as an antigen through a tuberculosis infection T cell detection kit, and using a cell culture medium as a negative control hole, wherein if the number of spots of the negative control hole is 0-5, and the number of spots of the Rv28 2824c detection hole-the number of spots of the negative control hole is more than or equal to 6; or if the number of the negative control hole spots is more than or equal to 6, and (the number of the detection hole spots-the number of the negative control hole spots) of the Rv2824c is more than or equal to 2 x (the number of the negative control hole spots); the candidate infected mycobacterium tuberculosis of the sample to be detected is a positive sample; if the result does not meet the requirement, the sample to be detected does not generate immune reaction with Rv2824c, and the sample to be detected is candidate to be not infected with the mycobacterium tuberculosis.

Sequence listing

<110> institute of biophysical research of Chinese academy of sciences, Guangdong Shuikang Biotech Co., Ltd

<120> protein Rv2824c for specific detection of Mycobacterium tuberculosis infection

<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

The application of the Rv2824c protein, positive quality control, negative control and readable carriers recorded in the following contents in preparing products for detecting or assisting in detecting whether a sample to be detected is infected with mycobacterium tuberculosis;

the Rv2824c is a protein shown as a sequence 2 in a sequence table;

the positive quality control is Phytohemagglutinin (PHA);

the negative control hole is the hole where the negative control is positioned,

the negative control is a cell culture solution without antigen;

the Rv2824c detection hole is a hole in which the Rv2824c protein is located;

the content is as follows:

if the detection result meets the following conditions: the number of the negative control hole speckles is 0-5, and the number of the negative control hole speckles is more than or equal to 6 when the number of the negative control hole speckles is detected by the Rv2824 c; or if the number of the negative control hole spots is more than or equal to 6, and (the number of the Rv2824c detection hole spots-the number of the negative control hole spots) is more than or equal to 2 x (the number of the negative control hole spots); the candidate to-be-detected sample is infected with the mycobacterium tuberculosis and is a positive sample; if the detection result does not meet the conditions, the sample to be detected is candidate to be not infected with the mycobacterium tuberculosis.
2. Use according to claim 1, characterized in that: the product is a kit.