CN106279382B

CN106279382B - Protein for specifically detecting mycobacterium tuberculosis infection

Info

Publication number: CN106279382B
Application number: CN201510293799.2A
Authority: CN
Inventors: 毕利军; 张先恩; 朱国峰; 邓教宇; 陶生策; 侯剑; 王雅果
Original assignee: GUANGDONG TIBIKANG BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Current assignee: Tibikon Biotechnology (Guangdong) Co.,Ltd.
Priority date: 2015-06-01
Filing date: 2015-06-01
Publication date: 2020-02-11
Anticipated expiration: 2035-06-01
Also published as: CN106279382A

Abstract

The invention relates to a protein for specifically detecting mycobacterium tuberculosis infection, wherein the protein source and mycobacterium tuberculosis have amino acid sequences shown in SEQ ID NO.2, can be used as a mycobacterium tuberculosis marker antigen for detecting tuberculosis, can be used for detecting specific T cell immunoreaction in vitro through the antigen, 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. Meanwhile, further animal experiments show that the protein antigen can induce immune response aiming at the mycobacterium tuberculosis and has the function of preparing corresponding vaccines.

Description

Protein for specifically detecting mycobacterium tuberculosis infection

Technical Field

The invention belongs to the technical field of medical products, and particularly relates to a protein for specifically detecting mycobacterium tuberculosis infection.

Background

The in vitro diagnostic industry in China is currently in a fast growing phase. However, the huge market demand is obviously different from the relatively backward independent research and development capability of the diagnostic reagent industry in China. How to develop independent innovation from the source and how to convert the scientific research achievements in the existing biological medicine field into products without being limited by individual overseas medicine huge on the supply of diagnostic reagent raw materials is a very much concerned hotspot in recent years in governments, scientific research institutions and medicine industries of China. And the market puts forward a rigid demand on a more sensitive and efficient diagnostic reagent nowadays, and the method can quickly and accurately diagnose tuberculosis patients and has important significance for medical workers and patients.

In recent years, data survey results show that 76.6% of patients with tuberculosis symptoms are subjected to relevant examination before tuberculosis, but only 35.8% of patients are diagnosed as tuberculosis patients, and the improvement of the patient discovery rate is still the key point of the current tuberculosis prevention and treatment work and is also difficult.

The in vitro diagnosis of pathogenic microorganism infection by using the cellular immune reaction of antigen-specific T cells is a new detection method developed in the present year. We isolated peripheral blood mononuclear cells from fresh whole blood and cultured in stimulation, and then tested the number of cells capable of secreting IFN-. gamma.using ELISPOT. The method is currently mainly applied to the diagnosis of mycobacterium tuberculosis infection. The current diagnosis of tuberculosis generally adopted in clinic mainly depends on clinical symptoms, influential diagnosis and etiological diagnosis, and is insensitive to the diagnosis of latent infection of mycobacterium tuberculosis. Meanwhile, in the tuberculosis screening process, the sensitivity and specificity for directly detecting pathogens or detecting mycobacterium tuberculosis antibodies are not ideal.

Disclosure of Invention

Aiming at the clinical practical working condition, protein fragments derived from mycobacterium tuberculosis are screened, a mycobacterium tuberculosis marker antigen for detecting tuberculosis is provided, and the antigen is used for detecting specific T cell immune response 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. Meanwhile, further animal experiments show that the protein antigen can induce immune response aiming at the mycobacterium tuberculosis and has the function of preparing corresponding vaccines.

Specifically, the invention firstly relates to a specific protein antigen Rv0472c of mycobacterium tuberculosis, the amino acid sequence of the antigen is shown as SEQ ID NO.2, and the amino acid sequence structure is as follows:

the nucleotide sequence of the coded protein antigen Rv0472c is shown in SEQ ID NO.1, and the DNA sequence structure is as follows:

the invention also relates to a preparation method of the protein antigen Rv0472c, which comprises the following steps,

the method comprises the following steps:

step (1) of

LR

Enzyme mix catalyzed Rv0472c entry vectors (freely available from PFGRC under the CraigVentor Institute, USA) and

pDEST ^TM17, generating an expression vector of Rv0472c by recombination;

transforming the expression vector of the Rv0472c in the step (1) into a target expression host to express a target Rv0472c protein;

and (3) breaking cells to collect protein and renaturating the target protein.

The specific method of the step (1) is as follows:

a. cloning reaction system: 1.5. mu.L of the entry vector of Rv0472c, pDEST ^TM17 the carrier is 1 mu L, and the carrier is,

BP

II Enzyme mix 2.5. mu.L; reaction conditions are as follows: reacting at 25 ℃ overnight;

b. the transformation method comprises adding 100 μ L of Escherichia coli DH5 α competent (self-made), ice-cooling for 30min, heat-shocking at 42 deg.C for 90s, standing on ice for 10min, adding 200 μ L of LB culture medium for renaturation, spreading on LB solid culture medium containing ampicillin (100mg/L), and culturing at 37 deg.C for 20 h;

c. plasmid extraction: extracting plasmids by using an N96 high-purity plasmid miniextraction kit (DP114) to obtain an expression vector of Rv0472 c;

the specific method of the step (2) is as follows:

a. adding 1 μ L of the plasmid solution obtained in step (1) into 100 μ L of Escherichia coli Rosetta (DE3) for competence, ice-cooling for 30min, and heat-shocking at 42 deg.C for 90 s;

b. placing the system on ice for 10min, adding 200 μ L LB culture medium for renaturation, coating on LB solid culture medium containing ampicillin (100mg/L), culturing at 37 deg.C for 12 h;

c. induction of Rv0472c protein expression with 0.75mM IPTG;

the specific method of the step (3) is as follows:

a. and (3) adding a heavy suspension: 60mM tris pH 9.0, 0.15M EDTA resuspension of the bacteria, sonication of the bacteria at 4 ℃ using a sonicator;

b.4 ℃, centrifuging at 10000rpm for 15min, collecting the precipitate to obtain the solid form inclusion body, and the electrophoresis result of the target protein is shown in figure 2.

c. The inclusion bodies were solubilized with a solubilization solution (60mM Tris-HCl pH7.0, 10M urea, 15mM DTT, 1mM EDTA); first dialysis: dialyzing the protein by using a dialysis bag with the molecular weight cutoff of 3K overnight, wherein the components of dialysate are as follows: 20mM Tris-HCl pH 9.0, 1M Urea, 3mM L-Argine;

d. dialyzing for the second time, wherein the dialysate comprises the following components: 20mM Tris-HCl pH 9.0, 10mM NaCl, second dialysis: dialyzing the protein into dialysate by using a dialysis bag with the molecular weight cutoff of 3K overnight to obtain the protein which can be directly used for immunogen screening;

the second method comprises the following steps:

expressing a target fusion protein containing a polyhistidine tag and collecting expression host bacteria:

collecting target fusion protein containing a polyhistidine tag:

and (3) purifying the target fusion protein.

The specific method of the step (1) comprises the following steps:

1) coli BL21 strain was used to express 6 × His-nus.a-Rv0472c fusion protein;

2) LB plate streaking activation (adding corresponding antibiotics), standing overnight (about 12 h) at 37 ℃;

3) inoculating into 5ml liquid LB medium (adding corresponding antibiotic) at 37 deg.C and 200rpm, and culturing to OD greater than 0.6-0.8 (about 3 h);

4) inoculated in an amount of 1-3% into 300ml of LB liquid medium, cultured at 37 ℃ and 200rpm to OD 600. apprxeq.0.6-0.8 (about 3 h). Cooling to 16 ℃, and adding IPTG to a final concentration of 0.1 mM;

5) culturing at 16 deg.C and 200rpm for 16h, centrifuging at 4 deg.C and 4000rpm for 15min, and collecting thallus.

The specific method of the step (2) is as follows:

1) and (3) resuspending the thallus: the collected cells were resuspended in about 40ml lysis buffer per 1L of medium (final concentration 0.5-1mM if PMSF, a protease inhibitor, is added)

2) And (3) crushing thalli: performing ultrasonic treatment for 3s at an interval of 9s and at a power of 38% for 30 min. (the suspension is transparent, and the total time of ultrasonic operation is adjusted as appropriate) for crushing for 1-2 times by using a high-pressure cell crusher, wherein the required time is about 10 min;

3) removing bacterial debris: centrifuging at 15000-18000rpm for 20 minutes at 4 ℃,

4) loading: the supernatant is incubated with Ni-NTA resin (Novagen Cat. NO 70691-5) as column material at 4 deg.C for about 1-1.5 h.

The specific method of the step (3) is as follows:

1) passing 10 times of lysine buffer through the gravity column, repeating for 2 times (adding liquid only after the liquid in the gravity column flows completely each time);

2) flowing 20mM imidazole lysine buffer about 10 column volumes through the gravity column, repeating 3 times (same as above);

3) collecting an elution and uniform mixing sample for detecting the impurity washing effect;

4) about 1-2 column volumes of Elution buffer were passed through the gravity column, repeated 3 times (as above), and tube collected. The eluate was examined with bradford reagent to determine whether the protein was completely eluted. If the protein is contained, continuing to elute until the protein is completely contained;

5) and (3) detection: SDS-PAGE detects whether the target protein exists;

6) dialyzing the eluate overnight at 4 deg.C with dialysis bag with molecular weight cutoff of 3K, and adding HRV3c protease restriction fusion protein to generate 6 × His-Nus.a fragment and target fragment Rv0472 c;

7) incubating the enzyme digestion product with Ni-NTA resin at 4 ℃ for 30min, collecting a penetrating sample to obtain a target fragment Rv0472c, and simultaneously combining 6 His-Nus.a, HRV3c and fusion protein which is not completely digested by enzyme with Ni-NTA resin so as to be eliminated;

8) the protein obtained above was purified by HiTrap Q HP (GE Cat. NO 17-1154-01) to obtain a high purity sample.

The invention also relates to the application of the Rv0472c protein antigen as an immunogen for detecting the mycobacterium tuberculosis, and the application comprises the following steps,

(1) the Rv0472c protein antigen is used for detecting mycobacterium tuberculosis alone;

or (2) the Rv0472c antigen is combined with the existing mycobacterium tuberculosis detection antigen for detecting mycobacterium tuberculosis.

The invention also relates to the application of the Rv0472c protein antigen in the preparation of a mycobacterium tuberculosis detection kit,

(1) preparing a detection kit by taking the Rv0472c protein antigen as a detection antigen;

or (2) the Rv0472c protein antigen and the existing mycobacterium tuberculosis detection antigen are cooperatively combined, so that the accuracy of the existing antigen immunodetection is improved.

The existing mycobacterium tuberculosis detection antigen is an ESAT-6 antigen, a CFP10 antigen, a PPD antigen, a BCG antigen, a LAM antigen and an ES-31 antigen which are derived from mycobacterium tuberculosis.

The invention also relates to a single antigenic type mycobacterium tuberculosis detection kit prepared from the Rv0472c protein antigen, which comprises,

(1) detecting an effective amount of Rv0472c protein antigen at a concentration (preferably at 75 ug/ml);

(2) necessary detection reagent and color reagent.

The invention also relates to a multi-antigen combined type mycobacterium tuberculosis detection kit containing the Rv0472c protein antigen, which comprises,

(1) detecting an effective amount of Rv0472c protein antigen at a concentration (preferably at 75ug/ml) and detecting an effective amount of other antigens;

(2) necessary detection reagent and color reagent.

The invention also relates to the application of the single type or combined type mycobacterium tuberculosis detection kit, which is characterized in that the detection method comprises the following steps,

(1) the detection sample is anticoagulated whole blood or PBMC cells prepared from a blood sample of a patient to be detected;

(2) rv0472c protein antigen as immunogen is incubated with anticoagulated whole blood or PBMC cells for 20-30 hours, preferably 20-24 hours

(3) Detecting the cytokine secreted by Rv0472c protein antigen, wherein the cytokine is IFN-gamma, IFN-alpha, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1 beta, IL-6, IL-8, MCP-1, IL-10 and IL-12, preferably IFN-gamma, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1 beta, further preferably IFN-gamma, TNF-alpha, IL-2, IL-13, most preferably IFN-gamma, and comparing with a positive reference to determine the detection result.

The invention also relates to application of the Rv0472c antigen in preparation of a vaccine against mycobacterium tuberculosis.

Drawings

FIG. 1 shows the result of enzyme digestion verification of Pdest17-Rv0472c expression vector, and lane A1 is a band after enzyme digestion.

FIG. 2-SDS-PAGE of Rv0472c protein, Marker (unit Kd) on the left,

FIG. 3 is a photograph of lung of guinea pig with Mycobacterium tuberculosis challenge infection model

FIG. 4 photograph showing immunohistochemistry of each tissue section of Mycobacterium tuberculosis challenge infection model guinea pig

Detailed Description

Example 1 construction of expression vector Rv0472c-pDEST17 and expression and renaturation of target protein Rv0472c (method one)

To be provided with

LR

Enzyme mix catalysis Rv0472cAnd (c) an entry vector (freely available from PFGRC under Craig Ventorinstitute, USA) and

pDEST ^TM17 vector recombination to create an expression vector for Rv0472 c.

The specific method comprises the following steps:

BP

II Enzyme mix 2.5. mu.L; reaction conditions are as follows: the reaction was carried out at 25 ℃ overnight.

B. The transformation method comprises adding 100 μ L Escherichia coli DH5 α competent (self-made) into reaction system, ice-cooling for 30min, heat-shocking at 42 deg.C for 90s, standing on ice for 10min, adding 200 μ L LB culture medium for renaturation, spreading on LB solid culture medium containing ampicillin (100mg/L), and culturing at 37 deg.C for 20 h.

C. Plasmid extraction: extracting the plasmid by using an N96 high-purity plasmid miniprep kit (DP114) to obtain an expression vector of Rv0472 c.

D. Enzyme digestion verification: the restriction enzyme kit (R0575S NEB) with BsrG1 was used to confirm the completion of cloning, and the results are shown in FIG. 1

The result shows that the size of the enzyme digestion fragment is about 700bp, and the plasmid construction is successful.

The obtained expression vector of Rv0472c is introduced into a rosetta (DE3) expression vector, and the specific method comprises the following steps:

a. mu.L of plasmid solution was added to 100. mu.L of plasmid solution competent (in-house) by DE3, ice-cooled for 30min, heat-shocked for 90s at 42 ℃,

c. rv047c protein expression was induced with 0.75mM IPTG.

2. The method comprises the following steps of carrying out ultracentrifugation after ultrasonic disruption of bacteria, obtaining inclusion body solid precipitate, collecting protein and renaturing:

a. and (3) adding a heavy suspension: resuspending the bacteria in PBS, and ultrasonically crushing the bacteria by using an ultrasonic crusher at 4 ℃;

b.4 ℃, centrifuging at 10000rpm for 20min, collecting the precipitate to obtain the solid form inclusion body, and the electrophoresis result of the collected protein is shown in figure 2.

c. Dissolving the inclusion body by using a dissolving solution (PBS +10M urea); first dialysis: dialyzing the protein overnight with dialysis bag with cut-off molecular weight of 3.5K, wherein the dialysate comprises the following components: PBS, 1M Urea, 3mM L-Argine;

d. dialyzing for the second time, wherein the dialysate is PBS, and dialyzing for the second time: dialyzing the protein into dialysate by using a dialysis bag with the molecular weight cutoff of 3.5K overnight to obtain the protein which can be directly used for immunogen screening.

Example 2 construction of expression vector containing polyhistidine tag Rv0472c and expression and renaturation of target protein Rv0472c (method two)

1. Expressing target fusion protein containing polyhistidine tag and collecting host thallus:

1) coli BL21 strain was used to express 6 × His-nus.a-Rv0472c fusion protein;

5) culturing at 16 deg.C and 200rpm for 16h, centrifuging at 4 deg.C and 4000rpm for 15min, and collecting thallus;

2. collecting target fusion protein containing polyhistidine tag:

4) loading: incubating the supernatant with Ni-NTA resin (Novagen Cat. NO 70691-5) at 4 deg.C for about 1-1.5 h;

3. purifying the target fusion protein:

3.1 washing protein:

3.2 elution of the protein of interest:

1) about 1-2 column volumes of Elution buffer were passed through the gravity column, repeated 3 times (as above), and tube collected. The eluate was examined with bradford reagent to determine whether the protein was completely eluted. If the protein is contained, continuing to elute until the protein is completely contained;

2) and (3) detection: SDS-PAGE detects whether the target protein exists;

3) dialyzing the eluate overnight at 4 deg.C with dialysis bag with molecular weight cutoff of 3K, and adding HRV3c protease restriction fusion protein to generate 6 × His-Nus.a fragment and target fragment Rv0472 c;

4) incubating the enzyme digestion product with Ni-NTA resin at 4 ℃ for 30min, collecting a penetrating sample to obtain a target fragment Rv0472c, and simultaneously combining 6 His-Nus.a, HRV3c and fusion protein which is not completely digested by enzyme with Ni-NTA resin so as to be eliminated;

5) the protein obtained above was purified by HiTrap Q HP (GE Cat. NO 17-1154-01) to obtain a high purity sample.

Example 3 immunogenicity testing of the protein of interest Rv0472c

In order to test the immunogenicity of Rv0472c and its enhanced effect on the existing antigen detection kit, further antigen detection screening was performed on multiple cases clinically confirmed from the Beijing thoracic hospital.

Testing blood samples: patients from Beijing thoracic hospital

Positive control reagents and consumables: T-SPOT kit (ESAT-6 and CFP10 dual antigen kit), Oxfordimemunotec (UK),

negative control: another randomly selected protein fragment Rv2327 of the Rv2327 protein antigen is derived from mycobacterium tuberculosis, and the nucleotide sequence and the amino acid sequence of the protein antigen are respectively shown as SEQ ID NO.3 and SEQ ID NO.4

SEQ ID No.4

SEQ ID No.3

The experimental method and the evaluation method are as follows:

1) heparin anticoagulation, namely uniformly mixing an anticoagulation sample with RPMI1640 according to the volume of 1: 1; carefully adding the blood sample to the upper layer of the separating liquid of the Ficoll lymphocyte according to the proportion of 2-3: 1;

2) centrifuging at 1000g for 22 min; the horizontal rotor slowly rises and slowly falls;

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

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

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

6) cells were counted and diluted, and 10. mu.l of cell suspension was added to 40. mu.l of 1% trypan blue to prepare 1: 5, counting living cells, calculating the volume required by cell dilution, adding corresponding AIM-V serum-free culture solution to prepare cell dilution solution, and detecting by using the kit, wherein the number of cells added into each hole (24-hole PVDF membrane plate) is 2.5 multiplied by 10 ⁵；

7) The plates were removed from the aluminum sealed bags and added in order:

50 μ l of AIM V to negative control wells; 50 μ l of antigen ESAT-6 to antigen A well; 50 μ l antigen CFP10 to antigen B well; 50 μ l positive control to positive control wells; 100. mu.l of the prepared cell dilution medium was added to each of the 4 wells. Mu.l of Sample protein (initial concentration of 60ug/ml) obtained in example 2 was added to the Sample well, in which the final concentration of Sample protein was 1/3 of the initial concentration, followed by addition of the medium and the cells.

8) The plates were incubated at 37 ℃ in a 5% CO2 incubator for 16-20 hrs.

9) Fresh enzyme-labeled antibody working solution was prepared with sterile PBS at a ratio of 1: 200. The plate was removed from the incubator and the well liquid was discarded. Wash 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. Unbound enzyme-labeled antibody was removed by washing 4 times with PBS.

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 plates were dried at 37 ℃ for 2-3hrs or overnight at room temperature.

Immunogen test results and analysis:

immunogenicity testing is carried out in molecular biology laboratories of thoracic hospitals, a certain number of tuberculosis patients which are clinically confirmed are selected for testing, blood samples of the tuberculosis patients are detected by using various protein antigens, and the immunogenicity and application direction of the Rv0472c protein antigen are analyzed according to the results, specific test result statistics are shown in the following table 1,

TABLE 1 Mycobacterium tuberculosis detection results of Rv0472c antigen for hospitalized patients with confirmed tuberculosis

Injecting: non-responsive patients are different from T-spot kits for detecting non-responsive subjects.

In 12 cases, the T-spot kit has no detection result, but Rv0472c can be screened out. The result of Rv0472c is obviously better than the detection result of T-spot.

Compared with the detection result of a commercially available T-spot kit, the high response rate and the low no-response rate of the Rv0472c protein antigen are higher, so that the Rv0472c immunogen can more effectively detect the mycobacterium tuberculosis compared with the commercially available double-antigen detection kit, and the detection rate can be more effectively improved if the immunogen is combined with other positive antigens.

Example 4 immunogenicity testing of the protein of interest Rv0472c

1. Establishment of guinea pig infection model

Injecting Mycobacterium tuberculosis infection source into guinea pig by subcutaneous injection in groin with injection dose of 0.2ml and toxin-counteracting dose of 4 × 10 ⁶～4x10 ⁷CFU。

After the completion of the challenge, the guinea pigs were sacrificed, lung organ infection of the guinea pigs injected with the infection source was detected, and relevant tissue sections were taken for immunohistochemical analysis.

A photograph of the lungs of a guinea pig infected with the model is shown in FIG. 3, and it can be seen that the lungs of the challenged guinea pig showed a marked inflammatory response.

The photograph of immunohistochemical analysis of each tissue section of the infection model guinea pig is shown in FIG. 4, and the results of the organ section are shown,

interstitial pneumonia (30-40%) and interstitial congestion (40-50%) appear in the lung of the guinea pig after virus attack;

epithelial cell nodules can be seen in the partial red marrow of the spleen, a small amount of lymphocytes at the periphery can infiltrate and moisten, and a cheese-like necrotic focus can be seen in the center;

liver congestion (20-30%) occurs in liver, and small granulation (1-5%) is observed locally.

The results show that the guinea pig after the mycobacterium tuberculosis challenge has obvious infection symptoms, and the model is successfully constructed

2. Immunogenicity testing

Two guinea pigs after successful challenge modeling are taken, different concentrations of the antigen protein prepared in example 2 are injected subcutaneously, and the sizes of immunoreaction inflammatory plaques caused by the antigen are observed after 48 and 72 hours, and the results are shown in the following table, and the results show that, similar to positive control, the Rv0472c antigen can also cause immunoreaction of the guinea pig infected with mycobacterium tuberculosis, which indicates that the antigen has the capacity of being used as a mycobacterium tuberculosis vaccine.

Sample (I)	48h transverse diameter X longitudinal diameter (mm)	72h transverse diameter X longitudinal diameter (mm)
			PBS (negative control)	-	-
BCG-PPD 20ug/ml (Positive control)	14x16	17x15
			Rv1098c 25ug/ml	10x11	10x11

Finally, it should be noted that the above examples are only for those skilled in the art to understand the essence of the present invention, and should not be used as a limitation to the scope of the present invention.

Claims

1. The application of the specific protein antigen of the mycobacterium tuberculosis in preparing the kit for detecting the mycobacterium tuberculosis is that the specific protein antigen of the mycobacterium tuberculosis is independently used for detecting the mycobacterium tuberculosis, and the specific protein antigen sequence of the mycobacterium tuberculosis is shown as SEQ ID NO. 2.

2. A combined type Mycobacterium tuberculosis detection kit comprising a specific protein antigen of Mycobacterium tuberculosis, the kit comprising,

(1) a specific protein antigen of said mycobacterium tuberculosis at a concentration of 75 μ g/ml;

(2) necessary detection reagents and color reagents;

the specific protein antigen sequence of the mycobacterium tuberculosis is shown as SEQ ID NO. 2.