KR20170011200A - Fusion protein of tuberculosis specific antigens, efficient method of producing thereof and uses thereof - Google Patents

Abstract

The present invention relates to a tuberculosis-specific antigen fusion protein composition, a method of efficiently producing the tuberculosis-specific antigen fusion protein, and a method for efficiently producing the tuberculosis-specific antigen fusion protein composition, The present invention relates to a diagnostic kit for a Mycobacterium tuberculosis that can effectively diagnose Mycobacterium tuberculosis and a method for detecting Mycobacterium tuberculosis using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a fusion protein of a tuberculosis-specific antigen, an efficient method for producing the fusion protein, and a use thereof.

A fusion protein of a tuberculosis specific antigen, an efficient production method thereof and a use thereof.

Tuberculosis is a disease caused by infection by Mycobacterium tuberculosis, which threatens humanity both in Korea and the world. In 2012, the World Health Organization has 8.6 million people worldwide, and tuberculosis Reported that 1.3 million people were killed. In Korea, the incidence, prevalence and mortality rate are the highest among OECD countries. According to the status of tuberculosis patient report by the CDC, 49,532 patients were reported in 2012, and more than 35,000 new patients are reported each year. Tuberculosis is becoming a serious refractory disease that threatens the public health. In the World Health Organization, failure to block tuberculosis is an inaccurate detection of tuberculosis infections, suggesting the need for improved tuberculosis detection through the development of simple and cost-effective new diagnostic methods. There are various tests to diagnose tuberculosis.

Mycobacterium tuberculosis is an absolute aerobic bacterium with a unique cell wall. The cell wall of Mycobacterium tuberculosis contains more than 60% of lipids containing lipoarabinomannan, mycolic acid, arabinogalactan, and the like. About 5% of Gram-positive bacteria and about 20% of Gram-negative bacteria contain lipids. Due to the nature of these cell walls, Gram staining, which is commonly used for the detection of other bacteria, is not well tolerated (graded as gram positive) and can be stained with dyes such as carbobupuccine or auramine. These dyes do not decolorize with acids and are called acid fast bacilli (AFB). Mycobacterium tuberculosis uses a characteristic of cell walls to perform Ziehl-Neelsen (ZN) staining and AFM smears such as Auramine-rhodamine fluorescence staining.

Mycobacterium tuberculosis has a slow growth rate. Escherichia coli has a break-up time of 15-20 minutes, whereas mycobacteria has a break-up time of 12-24 hours. Colonies of E. coli can be observed within one day, but colonization can be observed for days or weeks (~ 8 weeks) for M. tuberculosis. Solid medium and liquid medium are used for culture of Mycobacterium tuberculosis. Solid medium is egg-based Lowenstein-Jensen (LJ) medium, Ogawa medium (modified in LJ medium), and Middlebrook 7H10 / 11 based on agar. The liquid medium is Middlebrook 7H12 and Middlebrook 7H9 broth.

Because tuberculosis is a highly contagious disease, prompt treatment is required, so rapid and accurate diagnosis is needed. Accordingly, various methods have been performed for diagnosis of tuberculosis.

Microbiological diagnosis includes AFB smear, culture test, and TB test.

The use of Ziehl-Neelsen (ZN) staining and Auramine-rhodamine fluorescence staining method using the characteristics of specific cell wall of Mycobacterium tuberculosis, (Expert Rev. Anti Infect. Ther. 2009; 7: 1099-9). However, the sensitivity is not constant at 25% -80%, and it is difficult to differentiate between non-TB- 1108), the false negative rate is about 30%, and it is difficult to obtain sputum in young children.

Cultures are an important criterion for establishing tuberculosis as an absolute criterion and are required for drug susceptibility testing and are available in solid and liquid media. Solid medium culture takes 3-8 weeks, and some bacteria do not grow. Liquid culture is performed to shorten the time and increase the detection sensitivity compared to solid culture. Liquid culture method is able to detect Mycobacterium tuberculosis within 2-3 weeks and has a positive rate compared to solid culture. However, compared to solid culture, contamination rate It is difficult to distinguish it from non-tuberculous mycobacteria, and it has a disadvantage that it is expensive. It may also be cultured only in solid medium. Recently, automated liquid culture systems such as BACTEC MGIT960 (BD) and BacT / ALERT (bioMerieux) are available. Since MIGIT (Mycobacterial Growth Indicator Tubes), an automation system using a modified medium of Middlebrook 7H9, measures fluorescence emitted by oxygen consumed by oxygen in the liquid medium while oxygen is suppressed in the liquid culture medium, The bacteria detection time can be shortened. It is recommended that both solid and liquid media be used in combination to increase the time and sensitivity of culture assays.

The test for the amplification of mycobacterial nucleic acid is a molecular biology test that selectively amplifies and confirms the nucleic acid region specifically present in the Mycobacterium tuberculosis by PCR (PCR) on the mycobacterial nucleic acid (DNA). This method shows higher sensitivity and specificity than the smear test and can be obtained within 24 hours. It is also distinguishable from non - tuberculous antibiotics, and is also useful for detection of drug resistant gene and detection in extrapulmonary tuberculosis. However, it is costly compared with smear test, sensitivity and specificity of each product are various, and there is possibility of false positives in nucleic acid amplification process. Therefore, smear test and culture test can not be completely substituted and can be usefully supplemented. The amplification test of M. tuberculosis showed high sensitivity and specificity in smear - positive sputum but relatively low sensitivity and specificity in sputum - negative sputum.

If you have respiratory symptoms, first check your chest X-ray for tuberculosis. Chest radiography is useful for assessing the progression of disease in pulmonary tuberculosis. It is possible to observe the mass of granuloma in the lungs or the cavity. However, chest X - ray examination is difficult to differentiate from other pulmonary diseases, it can not be used in extra - pulmonary tuberculosis, it is difficult to detect in the early stage of pulmonary tuberculosis, and special equipment is needed. In order to distinguish between active tuberculosis and inactive tuberculosis, it is necessary to examine changes in chest x-ray photographs for 6 months. If you do not diagnose tuberculosis by suspicion of tuberculosis, you should perform a sputum test to confirm that tuberculosis is released.

Computed tomography (CT) is a diagnostic method of tuberculosis with higher accuracy than chest radiography. CT can distinguish active tuberculosis from inactive tuberculosis and can detect missed tuberculosis lesions on chest radiography. Chest computed tomography is helpful in diagnosing tuberculosis if it is difficult to accurately determine patients with active tuberculosis by chest radiography, negative in sputum smear, and difficult to distinguish between tuberculosis and other causative diseases. However, the cost of testing is considerably high and implies the risk of exposure to radiation. Even if there is evidence of active tuberculosis, efforts should be made to parallel the discharge of TB.

Tuberculin skin test (TST) and interferon gamma release assay (IGRA) have been used for tuberculosis infection. The tuberculin test, which has been used for a long time as an examination method for latent tuberculosis infection, is based on PPD (purified protein derivatives) prepared by concentrating mycobacterial proteins contained in the tubercle bacillus culture fluid, delayed 48-72 hours after intradermal injection type hypersensitivity). Since the positivity of the patient may be different according to the condition of the patient, the actual size is recorded without being simply displayed as positive or negative. In Korea, tuberculin test may show false positives in BCG vaccine and non-tuberculous mycobacterial infections. In addition, patients infected with the immune deficiency virus or those infected with tuberculosis may become negative after a long period of time. Tuberculin testing has the advantage of having a lot of accumulated clinical data and testing at low cost.

Interferon gamma assay is an examination method that examines tuberculosis infection by measuring interferon gamma secreted by stimulating T-lymphocytes sensitized to M. tuberculosis with TB-specific antigens such as ESAT-6 and CFP-10. Because BCG and most non-tuberculous antibiotics use antigens that are not present, they have higher specificity than tuberculin test, have no amplification effect on retesting, and have no side effects such as adverse reactions due to in vitro reactions. It is a useful method of screening people with latent tuberculosis in people with active TB. However, the cost of testing is high, and attention should be paid to the handling of specimens to maintain the viability of lymphocytes. As with TST, active tuberculosis and latent tuberculosis infection can not be distinguished.

There is a serological diagnostic method that uses an antigen-antibody reaction as a method of diagnosing active tuberculosis. In serological diagnostic methods, immunochromatography is simple and economical, easily detectable anywhere, and has the advantage of producing results within 10-15 minutes. About 90% of tuberculosis patients are known to produce antibodies against tuberculosis antigens. Antibodies reactive to various TB antigens are detected in the blood of patients with active tuberculosis who have tuberculosis. A number of antigens reacting in tuberculosis patients have been reported (Clin. Vaccine Immunol. 16: 260-276, Scand. J. Immunol. 66: 176-191). In countries with high rates of tuberculosis, commercial serologic methods have been used (Eur. Respir. J. 39: 502-505), but sensitivity is 25-75% due to the production of antibodies that react to different antigens, (Infect. Immun. 66: 3936-3940). Due to this low sensitivity problem, WHO has adopted a negative policy for the use of serological diagnosis (PLoS Med. 8: e1001062). However, the potential for improvement of sensitivity by the discovery of new antigens is suggested. Therefore, in order to increase the sensitivity of serologic diagnosis of tuberculosis, a combination of various antigens is searched for and there is a problem that the antigens with high significance are continuously discovered. In addition, non-responders should seek antigens that do not react in the control group.

Disclosure of the Invention The object of the present invention is to provide a Mycobacterium tuberculosis-specific antigen fusion protein composition using a tuberculosis-specific antigen combination having high sensitivity and specificity in order to solve the problems of the conventional tuberculosis diagnosis method.

Another object of the present invention is to provide a diagnostic kit for effectively diagnosing Mycobacterium tuberculosis including the Mycobacterium tuberculosis-specific antigen fusion protein composition and a method for detecting Mycobacterium tuberculosis using the same.

The present invention provides a Mycobacterium tuberculosis-specific antigen fusion protein composition comprising Mycobacterium tuberculosis-specific antigen combination for serological diagnosis of tuberculosis.

In the Mycobacterium -specific antigen fusion protein composition of the present invention, the combination of CFP-10 and 38 kDa, ESAT-6 and 16 kDa, ESAT-6 and MTB48, MPT64 and CFP-21, Ag85B and Ag85A, CFP- ESAT-6, CFP-10 and HBHA and CFP-10, ESAT-6, 16 kDa and 38 kDa.

The present invention also relates to a method for producing a Mycobacterium tuberculosis-specific antigen-combining gene, Transforming the vector into which the gene is inserted into E. coli; And inducing IPTG of the transformed E. coli. The present invention also provides a method for producing a Mycobacterium tuberculosis-specific antigen fusion protein.

The present invention also provides a diagnostic kit for Mycobacterium tuberculosis comprising the Mycobacterium tuberculosis-specific antigen fusion protein composition.

The present invention also provides a method for detecting Mycobacterium tuberculosis, which comprises detecting an Mycobacterium in a sample through an antigen-antibody reaction using the Mycobacterium tuberculosis-specific antigen fusion protein composition.

A method for producing a Mycobacterium tuberculosis-specific antigen fusion protein composition and a Mycobacterium tuberculosis-specific antigen fusion protein including a Mycobacterium tuberculosis-specific antigen combination for serological diagnosis of tuberculosis according to the present invention, a tuberculosis diagnostic kit including the same, The sensitivity and specificity of the fusion protein using the fusion of the antigen is higher than that of the conventional method of diagnosing tuberculosis.

Figure 1 shows SDS-PAGE gel images of protein-expressing CFP-10 and ESAT-6 tuberculosis-specific antigens in Coomassie blue staining.
2 is a photograph showing the expression of the Mycobacterium tuberculosis-specific antigen fusion protein according to the vector through Western blot analysis.
FIG. 3 is a photograph showing Western blot analysis of the difference in the expression of the M. tuberculosis-specific antigen according to the restriction enzyme site.
FIG. 4 shows SDS-PAGE gel images of Coomassie blue stained proteins purified by affinity chromatography purification using a GST column.
FIG. 5 shows SDS-PAGE gel images of Coomassie blue stained proteins purified by affinity chromatography purification using GST or HIS columns.
Fig. 6 shows the results of experiments in which the expression of the fusion protein of Example 1 was confirmed by Western blotting.
Fig. 7 shows the results of experiments in which the expression of the fusion protein of Example 2 was confirmed by Western blotting.
Figure 8 shows the results of an experiment in which the expression of the fusion protein of Example 3 was confirmed by Western blotting.
Fig. 9 shows SDS-PAGE gel images of Coomassie blue staining of the fusion proteins of Example 4 and Example 5. Fig.
10 shows SDS-PAGE gel photographs of Coomassie blue staining of the expression of the fusion protein of Example 6. Fig.
Figure 11 shows SDS-PAGE gel photographs of Coomassie blue staining of the fusion protein of Example 8.
Fig. 12 shows the results of an experiment in which expression of the fusion protein of Example 8 was confirmed by Western blotting.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. In the following description of the present invention, a detailed description of related air technologies will be omitted if it is determined that the gist of the present invention may be obscured.

Hereinafter, the present invention will be described in detail.

The fusion protein composition of the Mycobacterium tuberculosis-specific antigen of the present invention is characterized by comprising a combination of Mycobacterium tuberculosis-specific antigens for serological diagnosis of tuberculosis.

In the present invention, the combination of Mycobacterium tuberculosis-specific antigens includes CFP-10 and 38 kDa, ESAT-6 and 16 kDa, ESAT-6 and MTB48, MPT64 and CFP-21, Ag85B and Ag85A, CFP-10 and ESAT-6, CFP- And a combination of CFP-10, ESAT-6, 16 kDa and 38 kDa.

According to a preferred embodiment, the Mycobacterium tuberculosis-specific antigen fusion protein may be characterized in that it is expressed from a gene sequence of one or more Mycobacteria-specific antigen combinations selected from the group consisting of SEQ ID NOS: 1 to 8.

According to a preferred embodiment, the M. tuberculosis-specific antigen fusion protein is at least one selected from the group consisting of SEQ ID NOS: 9 to 16.

More preferably, the nucleotide sequence of the combination of CFP-10 and the 38 kDa Mycobacterium tuberculosis-specific antigen can be represented by SEQ ID NO: 1, the fusion protein expressed from SEQ ID NO: 1 is SEQ ID NO: 9; The gene sequence of ESAT-6 and the 16 kDa Mycobacterium tuberculosis-specific antigen combination can be represented by SEQ ID NO: 2, the fusion protein expressed from SEQ ID NO: 2 is SEQ ID NO: 10, The nucleotide sequence of the combination of ESAT-6 and MTB48 of Mycobacterium tuberculosis-specific antigen can be represented by SEQ ID NO: 3, the fusion protein expressed from SEQ ID NO: 3 is represented by SEQ ID NO: 11; The gene sequence of the combination of M. tuberculosis-specific antigens of MPT64 and CFP-21 can be represented by SEQ ID NO: 4, the fusion protein expressed from SEQ ID NO: 4 is represented by SEQ ID NO: 12; The nucleotide sequence of the combination of Ag85B and Ag85A in the Mycobacterium tuberculosis-specific antigen can be represented by SEQ ID NO: 5, the fusion protein expressed from SEQ ID NO: 5 is represented by SEQ ID NO: 13; The gene sequence of the combination of Mycobacterium tuberculosis-specific antigen of CFP-10 and ESAT-6 can be represented by SEQ ID NO: 6, the fusion protein expressed from SEQ ID NO: 6 is SEQ ID NO: 14; The gene sequence of the combination of Mycobacterium tuberculosis specific antigen of CFP-10 and HBHA can be represented by SEQ ID NO: 7, the fusion protein expressed from SEQ ID NO: 7 is SEQ ID NO: 15; The nucleotide sequence of the combination of CFP-10, ESAT-6, 16 kDa and 38 kDa of Mycobacterium tuberculosis-specific antigen can be represented by SEQ ID NO: 8, and the fusion protein expressed from SEQ ID NO: 8 can be represented by SEQ ID NO:

The method for producing Mycobacterium tuberculosis specific antigen fusion protein of the present invention comprises the steps of: inserting a Mycobacterium tuberculosis specific antigen combination gene into a vector; Transforming the vector into which the gene is inserted into E. coli; And inducing IPTG of the transformed E. coli.

According to a preferred embodiment, the vector may be selected from the group consisting of a T7 promoter vector, a T5 promoter vector and a tac promoter vector.

More preferably, it may be selected from the group consisting of pET21a T7 promoter vector, pQE80L T5 promoter vector, and pGEX-4T tac promoter vector.

According to a preferred embodiment, the M. tuberculosis-specific antigen combination genes SEQ ID Nos. 1 to 5 may be inserted into a T7 promoter vector and expressed therein.

According to a preferred embodiment, the M. tuberculosis-specific antigen combination gene SEQ ID NO: 6 may be inserted into a T5 promoter vector and expressed.

In addition, the method for detecting Mycobacterium tuberculosis of the present invention is characterized by detecting mycobacteria in a sample through an antigen-antibody reaction using the Mycobacterium tuberculosis-specific antigen fusion protein composition.

According to a preferred embodiment, the antigen-antibody reaction may be induced by immunofluorescence staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, A Complement Fixation Assay (FACS), a Fluorescence-Activated Cell Sorter (FACS) and a protein chip assay.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to be illustrative of the present invention and the scope of the invention as defined by the appended claims. And it is clear that such modifications and variations are included in the scope of the appended claims.

< Experimental Example  1> Differences in Protein Expression of Tuberculous Antigen Gene by Expression Vector

In order to optimally express the tubercle antigen protein in E. coli , a vector with a promoter advantageous for expression and an appropriate restriction enzyme site should be selected. Therefore, in order to optimally express the TB antigen protein, a TB antigen gene is inserted into a vector having different promoters of pET21a (T7 promoter), pQE80L (T5 promoter) and pGEX-4T (tac promoter) The vector was transformed into E. coli and then expressed by IPTG. Then, the expressed protein was electrophoresed on 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and protein expression was confirmed by Coomassie blue dyeing and Western blotting.

< Experimental Example  1-1> pGEX -4T (tac promoter) Vector Expression of Tumor Antigen Gene

Mycobacterium tuberculosis ESAT-6-specific antigen gene In order to examine for the expression of the antigen according to the tuberculosis antigen gene type pGEX-4T (tac promoter) of the vector Bam The CFP-10 tuberculosis antigen gene was cloned into the HI / Xho I restriction site and expressed in the Bam of the pGEX-4T (tac promoter) vector HI / Eco RI restriction enzyme site. The experimental results are shown in Fig.

As shown in FIG. 1, the expression of ESAT-6 was comparatively well, but the expression was relatively weak as compared with CFP-10.

< Experimental Example  1-2> Expression of fusion protein of Mycobacterium tuberculosis-specific antigen by vector

In order to investigate the expression of the fusion protein of the Mycobacterium tuberculosis-specific antigen according to the type of vector, the expression of pET21a (T7 promoter) vector Bam HI / Eco RI and Sac I / Hind III restriction site and removed the stop codon and the expression cloning of the ESAT-6 and CFP-10 to remove the stop codon, pQE80L (T5 promoter) of the vectors of the vector Bam CFP-10 with the stop codon removed and ESAT-6 without the stop codon removed were cloned into the HI / Eco RI and Sac I / Hind III restriction sites. Then, Rv2660c Mycobacterium tuberculosis-specific antigen was inserted and expressed in the front part of CFP-10 / ESAT-6 cloned in the pET21a vector, and protein expression was confirmed by Western blotting using anti-HIS antibody. The experimental results are shown in Fig.

As shown in FIG. 2, the expression of the CFP-10 / ESAT-6 fusion protein (FIG. 2 lines 3 and 4) cloned into the pET21a vector varied depending on the expression conditions and only a relatively small amount of protein was expressed. The cloned CFP-10 / ESAT-6 fusion protein (FIG. 2 line 2) expressed a relatively large amount regardless of expression conditions. In addition, the expression of Rv2660c / CFP-10 / ESAT-6 fusion protein (FIG. 2 line 5) expressed by inserting Rv2660c Mycobacterium tuberculosis specific antigen into the front part of CFP-10 / ESAT- / ESAT-6 &lt; / RTI &gt; fusion protein of the present invention.

< Experimental Example  1-3> Expression of Mycobacterium tuberculosis-specific antigens by restriction enzyme site

In order to examine the expression of Mycobacterium tuberculosis-specific antigens according to restriction enzyme sites, the MPT32 tuberculosis antigen gene was cloned into the Eco RI / Xho I restriction site and the Nhe I / Xho I restriction site of the pET21a vector to be expressed and cloned into the pQE80L vector And protein expression was confirmed by Western blot using anti-HIS antibody. The results of the experiment are shown in FIG.

3). Compared with the case of cloning into the Nhe I / Xho I restriction site (Fig. 3 line 3) as shown in Fig. 3, better expression was obtained when the Eco RI / Xho I restriction enzyme site was cloned (Fig. 3 line 2) . In addition, when cloned into the pQE80L vector, it was detected in the patient antibody, but it was confirmed that the HIS was cleaved by the protease between HIS and MPT32 and was not expressed as an anti-HIS antibody.

< Experimental Example  2> Differences in Protein Expression of Tuberculous Antigen Gene by Purification Method

In order to obtain a desired protein from Mycobacterium tuberculosis, various steps such as ion exchange chromatography, gel filtration, and affinity chromatography are required, resulting in high protein loss and high cost. For effective protein purification, protein purification was performed using affinity chromatography by attaching HIS-tag or GST-tag to the desired protein.

The expression of Rv3872, CFP-10 and ESAT-6 tuberculosis antigen genes was cloned into pGEX-4T vector and the ESAT-6 tuberculosis antigen gene was cloned into pET21a vector and expressed. The protein was purified by affinity chromatography using GST column or HIS column. The purified protein was electrophoresed on 12% SDS-PAGE gel and stained with Coomassie blue to confirm protein expression. The experimental results are shown in FIGS. 4 and 5. FIG.

< Example  1 to 8> Expression of a fusion protein of a Mycobacterium tuberculosis-specific antigen comprising a combination of Mycobacterium tuberculosis-specific antigens

Two or more tuberculosis-specific antigens with high sensitivity were used to express the fusion protein to increase the efficiency of antigen expression and effectively express antigens that were not expressed or expressed when expressed alone.

The fusion proteins were expressed by the combination of the Mycobacterium tuberculosis specific antigen shown in Table 1 below.

Mycobacterium tuberculosis-specific antigen combination Base sequence Amino acid sequence Example 1 CFP-10, 38 kDa SEQ ID NO: 1 SEQ ID NO: 9 Example 2 ESAT-6, 16 kDa SEQ ID NO: 2 SEQ ID NO: 10 Example 3 ESAT-6, MTB48 SEQ ID NO: 3 SEQ ID NO: 11 Example 4 MPT64, CFP21 SEQ ID NO: 4 SEQ ID NO: 12 Example 5 Ag85B, Ag85A SEQ ID NO: 5 SEQ ID NO: 13 Example 6 CFP-10, ESAT-6 SEQ ID NO: 6 SEQ ID NO: 14 Example 7 CFP-10, HBHA SEQ ID NO: 7 SEQ ID NO: 15 Example 8 CFP-10, ESAT-6, 16 kDa, 38 kDa SEQ ID NO: 8 SEQ ID NO: 16

< Example  1> CFP -10 and 38 kDa  Fusion protein of Mycobacterium tuberculosis-specific antigen

The Nde of the pET21a (T7 promoter) vector The pET21a-CFP-10 / 38kDa fusion protein was expressed by cloning the CFP-10 from which the stop codon was removed and the 38 kDa from which the stop codon was removed in the I / Xho I restriction enzyme site. The pET21a-CFP-10 / 38kDa fusion protein is CFP-10- Bam HI-38 kDa, and the CFP-10- Bam The gene sequence of HI -38 kDa is shown in SEQ ID NO: 1, and the amino acid sequence of the protein expressed from the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID NO:

The pET21a-CFP-10 / 38kDa fusion protein was confirmed by Western blotting using serum IgG of active tuberculosis patients P1, P17 and P37. The results of the experiment are shown in Fig.

As shown in Fig. 6, the same reaction was observed in the P1 patient sample as well as the pET21a-CFP-10 / 38kDa fusion protein of Example 1 as well as CFP-10 and 38kDa, and the P17 patient sample showed a very weak response to CFP-10 , 38 kDa and the pET21a-CFP-10/38 kDa fusion protein of Example 1 were good in reactivity. It was also found that the reactivity of the pET21a-CFP-10 / 38kDa fusion protein of Example 1 was better than that of CFP-10 and 38kDa in the P37 patient sample.

As a result of the above experiment, it was confirmed that the tuberculosis specific antigen fusion protein of Example 1 is more reactive than the specific antigen.

< Example  2> ESAT -6 and 16 kDa  Fusion protein of Mycobacterium tuberculosis-specific antigen

The Nde of the pET21a (T7 promoter) vector I / The pET21a-ESAT-6 / 16kDa fusion protein was expressed by cloning ESTA-6 with termination codon and initiation codon removed, and 16kDa with termination codon removed at the Xho I restriction site.

The pET21a-ESAT-6 / 16kDa fusion protein was identified as ESAT-6- Bam HI - 16 kDa, and the ESAT-6- Bam The gene sequence of HI-16 kDa is shown in SEQ ID NO: 2, and the amino acid sequence of the protein expressed from the nucleotide sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 10.

The reactivity of the pET21a-ESAT-6 / 16kDa fusion protein was confirmed by Western blotting using serum IgG of active tuberculosis patients P1, P17 and P99. The results of the experiment are shown in FIG.

As shown in FIG. 7, in the P1 patient sample, not only the ESAT-6 and 16 kDa but also the pET21a-ESAT-6/16 kDa fusion protein of Example 2 showed a reaction, whereas the P17 patient sample did not show any response in ESAT- In the P99 patient sample, the response was poor in ESAT-6, and the response was not observed at 16 kDa, but it was confirmed that the fusion protein of Example 2 was well reacted.

As a result of the above experiment, it was confirmed that the tuberculosis-specific antigen fusion protein of Example 2 had better reactivity than the case of each specific antigen.

< Example  3> ESAT -6 and MTB48  Fusion protein of Mycobacterium tuberculosis-specific antigen

The Nde of the pET21a (T7 promoter) vector The pET21a-ESAT-6 / MTB48 fusion protein was expressed by cloning ESAT-6 with the stop codon removed and MTB48 with the termination codon removed in the I / Xho I restriction site.

The pET21a-ESAT-6 / MTB48 fusion proteins ESAT-6 - Eco RI - gat - was fused in the form of a MTB48, the ESAT-6 - Eco RI - gat - gene sequence of MTB48 is showed in SEQ ID NO: 3, The amino acid sequence of the protein expressed from the nucleotide sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 11.

The reactivity of pET21a-ESAT-6 / MTB48 fusion protein was confirmed by Western blotting using serum IgG of active tuberculosis patients P1, P17 and P99. The results of the experiment are shown in FIG.

As shown in Fig. 8, in the P1 patient sample, not only the ESAT-6 and MTB48 but also the pET21a-ESAT-6 / MTB48 fusion protein of Example 3 showed the reaction, whereas the P17 patient sample did not show the ESTA- The reaction of MTB48 was insufficient, but the fusion protein of Example 3 was well reacted. In the P99 patient sample, ESAT-6 and MTB48 reacted weakly, but the fusion protein of Example 3 strongly reacted.

As a result of the above experiment, it was confirmed that the tuberculosis-specific antigen fusion protein of Example 3 had better reactivity than the case of each specific antigen.

< Example  4> MPT64 Wow CFP21  Fusion protein of Mycobacterium tuberculosis-specific antigen

The pET21a-MPT64 / CFP-21 fusion protein was expressed by cloning MPT64 and CFP-21 in which the N-terminal region was removed from the Eco RI / Xho restriction site of the pET21a (T7 promoter) vector and the initiation codon (ATG) .

The pET21a-MPT64 / CFP21 fusion protein MPT64 - Xho RI - were fused in the form of CFP21, the MPT64 - Xho RI - gene sequence of CFP21 is showed in SEQ ID NO: 4, the base of the SEQ ID NO: 4 The amino acid sequence of the protein expressed from the sequence is shown in SEQ ID NO: 12.

The pET21a-MPT64 / CFP21 fusion protein was electrophoresed on 12% SDS-PAGE gel and stained with Coomassie blue to confirm protein expression. The results of the experiment are shown in FIG.

< Example  5> Ag85B Wow Ag85A  Fusion protein of Mycobacterium tuberculosis-specific antigen

The pET21a-Ag85B / Ag85A fusion protein was expressed by cloning Ag85B and Ag85A in which the N-terminal region was removed from the Eco R / Not I restriction site of the pET21a (T7 promoter) vector and the initiation codon (ATG) was inserted.

The pET21a-Ag85B / Ag85A fusion protein Ag85B - were fused in the form of Ag85A, the Ag85B - - Sal I Sal I - gene sequence of Ag85A is showed in SEQ ID NO: 5, which is expressed from the nucleotide sequence of the SEQ ID NO: 5 The amino acid sequence of the protein is shown in SEQ ID NO: 13.

The cloned pET21a-Ag85B / Ag85A fusion protein was electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie blue to confirm protein expression. The results of the experiment are shown in FIG.

< Example  6> CFP -10 and ESAT -6 fusion protein of Mycobacterium tuberculosis-specific antigen

The pQE80L-CFP-10 / ESAT-6 fusion protein was expressed by cloning CFP-10 and ESAT-6 from which the stop codon was removed in the BamHI / HindIII restriction site of the pQE80L (T5 promoter) vector.

The pQE80L-CFP-10 / ESAT- 6 fusion protein CFP-10 - Eco RI - were fused in the form of the ESAT-6, the CFP-10 - - Sac I Eco RI - Sac I - gene sequence of ESAT-6 Is shown in SEQ ID NO: 6, and the amino acid sequence of the protein expressed from the nucleotide sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 14.

The pQE80L-CFP-10 / ESAT-6 fusion protein was electrophoresed on 12% SDS-PAGE gel and stained with Coomassie blue to confirm protein expression. The results of the experiment are shown in FIG.

< Example  7> CFP -10 and HBHA  Fusion protein of Mycobacterium tuberculosis-specific antigen

The Nde of the pET21a (T7 promoter) vector I / Sal I restriction enzyme sites were cloned into Mycobacterium tuberculosis specific antigen CFP-10 and HBHA to express pET21a-CFP-10 / HBHA fusion protein.

The pET21a-CFP-10 / HBHA fusion protein CFP-10 - was fused in the form of HBHA, the CFP-10 - - Eco RI Eco RI - gene sequence of the HBHA is showed in SEQ ID NO: 7, wherein SEQ ID NO: 7 The protein amino acid sequence expressed from the nucleotide sequence of SEQ ID NO: 15 is shown in SEQ ID NO:

< Example  8> CFP -10, ESAT -6, 16 kDa  And 38 kDa  Fusion protein of Mycobacterium tuberculosis-specific antigen

The Nde of the pET21a (T7 promoter) vector The pET21a-CFP-10 / ESAT-6 / 16kDa / 38kDa fusion protein was expressed by cloning the Mycobacterium tuberculosis specific antigen CFP-10, ESAT-6, 16kDa and 38kDa into the I / Xho I restriction enzyme site.

The pET21a-CFP-10 / ESAT- 6 / 16kDa / 38kDa fusion protein CFP-10 - Bam HI - ESAT -6 - Bam HI - 16kDa - Bam HI - Eco RI - were fused in the form of 38kDa, the CFP-10 - Bam HI - ESAT-6 - Bam HI - 16kDa - Bam HI - Eco RI - gene sequence of the 38kDa is showed in SEQ ID NO: 8, the amino acid sequence of the protein expressed from the nucleotide sequence of the SEQ ID NO: 8, SEQ ID NO: 16 Respectively.

The pET21a-CFP-10 / ESAT-6 / 16kDa / 38kDa fusion protein was electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie blue to confirm protein expression. Western blotting using serum IgG from patients with active tuberculosis .

The results of the experiment are shown in FIGS. 11 and 12. FIG.

< Experimental Example  3> Sensitivity test of fusion proteins to active TB patients

Among the patients with active tuberculosis, 63 patients (31.7% of patients with culture or smear positive patients) who had a high rate of negative culture or smear test, samples collected from cultured or smear positive patients were always subjected to bacterial smear test, culture test, Were used for the detection of Mycobacterium tuberculosis. The Mycobacteria-specific antigen protein and fusion protein used in the experiment are shown in Table 2 below, and the test results using the antigen proteins in Table 2 are shown in Table 3 below.

method of inspection Mycobacterium tuberculosis-specific antigen Example 2 Serological diagnostic method ESAT-6, 16 kDa fusion Example 3 ESAT-6, MTB48 fusion Example 4 MPT64, CFP21 fusion Example 5 Ag85B, Ag85A fusion Example 6 CFP-10, ESAT-6 fusion Example 7 CFP-10, HBHA fusion Example 8 CFP-10, ESAT-6, 16 kDa, 38 kDa fusion Comparative Example 1 Culture test - Comparative Example 2 Smear test - Comparative Example 3 Serological diagnostic method 16 kDa (Rv 2031c) Comparative Example 4 38 kDa (Rv 0934) Comparative Example 5 MTB81 (Rv 1837c) Comparative Example 6 MPT32 (Rv 1860) Comparative Example 7 Ag85B (Rv 1886c) Comparative Example 8 Rv1411

As shown in Table 3, it can be confirmed that Example 8 exhibits the highest sensitivity with a sensitivity of 61.9%.

<110> ENGLISH NATIONAL TUBERCULOSIS ASSOCIATION <120> FUSION PROTEIN OF TUBERCULOSIS SPECIFIC ANTIGENS, EFFICIENT          METHOD OF PRODUCING THEREOF AND USES THEREOF <130> DPA-0653 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 1422 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > CFP-10/38 kDa <400> 1 atggcagaga tgaagaccga tgccgctacc ctcgcgcagg aggcaggtaa tttcgagcgg 60 atctccggcg acctgaaaac ccagatcgac caggtggagt cgacggcagg ttcgttgcag 120 ggccagtggc gcggcgcggc ggggacggcc gcccaggccg cggtggtgcg cttccaagaa 180 gcagccaata agcagaagca ggaactcgac gagatctcga cgaatattcg tcaggccggc 240 gtccaatact cgagggccga cgaggagcag cagcaggcgc tgtcctcgca aatgggcttc 300 ggatccatgc gtttgcatac gctgttggcc gtgttgaccg ctgcgccgct gctgctagca 360 gcggcgggct gtggctcgaa accaccgagc ggttcgcctg aaacgggcgc cggcgccggt 420 actgtcgcga ctacccccgc gtcgtcgccg gtgacgttgg cggagaccgg tagcacgctg 480 ctctacccgc tgttcaacct gtggggtccg gcctttcacg agaggtatcc gaacgtcacg 540 atcaccgctc agggcaccgg ttctggtgcc gggatcgcgc aggccgccgc cgggacggtc 600 aacattgggg cctccgacgc ctatctgtcg gaaggtgata tggccgcgca caaggggctg 660 atgaacatcg cgctagccat ctccgctcag caggtcaact acaacctgcc cggagtgagc 720 gagcacctca agctgaacgg aaaagtcctg gcggccatgt accagggcac catcaaaacc 780 tgggacgacc cgcagatcgc tgcgctcaac cccggcgtga acctgcccgg caccgcggta 840 gttccgctgc accgctccga cgggtccggt gacaccttct tgttcaccca gtacctgtcc 900 aagcaagatc ccgagggctg gggcaagtcg cccggcttcg gcaccaccgt cgacttcccg 960 gcggtgccgg gtgcgctggg tgagaacggc aacggcggca tggtgaccgg ttgcgccgag 1020 acaccgggct gcgtggccta tatcggcatc agcttcctcg accaggccag tcaacgggga 1080 ctcggcgagg cccaactagg caatagctct ggcaatttct tgttgcccga cgcgcaaagc 1140 attcaggccg cggcggctgg cttcgcatcg aaaaccccgg cgaaccaggc gatttcgatg 1200 atcgacgggc ccgccccgga cggctacccg atcatcaact acgagtacgc catcgtcaac 1260 aaccggcaaa aggacgccgc caccgcgcag accttgcagg catttctgca ctgggcgatc 1320 accgacggca acaaggcctc gttcctcgac caggttcatt tccagccgct gccgcccgcg 1380 gtggtgaagt tgtctgacgc gttgatcgcg acgatttcca gc 1422 <210> 2 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> ESAT-6/16 kDa <400> 2 atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60 aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120 gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180 acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240 caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcaggatc catggccacc 300 acccttcccg ttcagcgcca cccgcggtcc ctcttccccg agttttctga gctgttcgcg 360 gccttcccgt cattcgccgg actccggccc accttcgaca cccggttgat gcggctggaa 420 gacgagatga aagaggggcg ctacgaggta cgcgcggagc ttcccggggt cgaccccgac 480 aaggacgtcg acattatggt ccgcgatggt cagctgacca tcaaggccga gcgcaccgag 540 cagaaggact tcgacggtcg ctcggaattc gcgtacggtt ccttcgttcg cacggtgtcg 600 ctgccggtag gtgctgacga ggacgacatt aaggccacct acgacaaggg cattcttact 660 gtgtcggtgg cggtttcgga agggaagcca accgaaaagc acattcagat ccggtccacc 720 aac 723 <210> 3 <211> 1674 <212> DNA <213> Artificial Sequence <220> <223> ESAT-6 / MTB48 <400> 3 atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60 aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120 gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180 acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240 caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcagaatt cgatatgacg 300 cagtcgcaga ccgtgacggt ggatcagcaa gagattttga acagggccaa cgaggtggag 360 gccccgatgg cggacccacc gactgatgtc cccatcacac cgtgcgaact cacggcggct 420 aaaaacgccg cccaacagct ggtattgtcc gccgacaaca tgcgggaata cctggcggcc 480 ggtgccaaag agcggcagcg tctggcgacc tcgctgcgca acgcggccaa ggcgtatggc 540 gaggttgatg aggaggctgc gaccgcgctg gacaacgacg gcgaaggaac tgtgcaggca 600 gaatcggccg gggccgtcgg aggggacagt tcggccgaac taaccgatac gccgagggtg 660 gccacggccg gtgaacccaa cttcatggat ctcaaagaag cggcaaggaa gctcgaaacg 720 ggcgaccaag gcgcatcgct cgcgcacttt gcggatgggt ggaacacttt caacctgacg 780 ctgcaaggcg acgtcaagcg gttccggggg tttgacaact gggaaggcga tgcggctacc 840 gcttgcgagg cttcgctcga tcaacaacgg caatggatac tccacatggc caaattgagc 900 gctgcgatgg ccaagcaggc tcaatatgtc gcgcagctgc acgtgtgggc taggcgggaa 960 catccgactt atgaagacat agtcgggctc gaacggcttt acgcggaaaa cccttcggcc 1020 cgcgaccaaa ttctcccggt gtacgcggag tatcagcaga ggtcggagaa ggtgctgacc 1080 gaatacaaca acaaggcagc cctggaaccg gtaaacccgc cgaagcctcc ccccgccatc 1140 aagatcgacc cgcccccgcc tccgcaagag cagggattga tccctggctt cctgatgccg 1200 ccgtctgacg gctccggtgt gactcccggt accgggatgc cagccgcacc gatggttccg 1260 cctaccggat cgccgggtgg tggcctcccg gctgacacgg cggcgcagct gacgtcggct 1320 gggcgggaag ccgcagcgct gtcgggcgac gtggcggtca aagcggcatc gctcggtggc 1380 ggtggaggcg gcggggtgcc gtcggcgccg ttgggatccg cgatcggggg cgccgaatcg 1440 gtgcggcccg ctggcgctgg tgacattgcc ggcttaggcc agggaagggc cggcggcggc 1500 gccgcgctgg gcggcggtgg catgggaatg ccgatgggtg ccgcgcatca gggacaaggg 1560 ggcgccaagt ccaagggttc tcagcaggaa gacgaggcgc tctacaccga ggatcgggca 1620 tggaccgagg ccgtcattgg taaccgtcgg cgccaggaca gtaaggagtc gaag 1674 <210> 4 <211> 1275 <212> DNA <213> Artificial Sequence <220> <223> MPT64 / CFP21 <400> 4 atggcgccca agacctactg cgaggagttg aaaggcaccg ataccggcca ggcgtgccag 60 attcaaatgt ccgacccggc ctacaacatc aacatcagcc tgcccagtta ctaccccgac 120 cagaagtcgc tggaaaatta catcgcccag acgcgcgaca agttcctcag cgcggccaca 180 tcgtccactc cacgcgaagc cccctacgaa ttgaatatca cctcggccac ataccagtcc 240 gcgataccgc cgcgtggtac gcaggccgtg gtgctcaagg tctaccagaa cgccggcggc 300 acgcacccaa cgaccacgta caaggccttc gattgggacc aggcctatcg caagccaatc 360 acctatgaca cgctgtggca ggctgacacc gatccgctgc cagtcgtctt ccccattgtg 420 caaggtgaac tgagcaagca gaccggacaa caggtatcga tagcgccgaa tgccggcttg 480 gacccggtga attatcagaa cttcgcagtc acgaacgacg gggtgatttt cttcttcaac 540 ccgggggagt tgctgcccga agcagccggc ccaacccagg tattggtccc acgttccgcg 600 atcgactcga tgctggccct cgagatgact ccacgcagcc ttgttcgcat cgttggtgtc 660 gtggttgcga cgaccttggc gctggtgagc gcacccgccg gcggtcgtgc cgcgcatgcg 720 gatccgtgtt cggacatcgc ggtcgttttc gctcgcggca cgcatcaggc ttctggtctt 780 ggcgacgtcg gtgaggcgtt cgtcgactcg cttacctcgc aagttggcgg gcggtcgatt 840 ggggtctacg cggtgaacta cccagcaagc gacgactacc gcgcgagcgc gtcaaacggt 900 tccgatgatg cgagcgccca catccagcgc accgtcgcca gctgcccgaa caccaggatt 960 gtgcttggtg gctattcgca gggtgcgacg gtcatcgatt tgtccacctc ggcgatgccg 1020 cccgcggtgg cagatcatgt cgccgctgtc gcccttttcg gcgagccatc cagtggtttc 1080 tccagcatgt tgtggggcgg cgggtcgttg ccgacaatcg gtccgctgta tagctctaag 1140 accataaact tgtgtgctcc cgacgatcca atatgcaccg gaggcggcaa tattatggcg 1200 catgtttcgt atgttcagtc ggggatgaca agccaggcgg cgacattcgc ggcgaacagg 1260 ctcgatcacg ccgga 1275 <210> 5 <211> 1878 <212> DNA <213> Artificial Sequence <220> <223> Ag85b / Ag85a <400> 5 atgttctccc ggccggggct gccggtcgag tacctgcagg tgccgtcgcc gtcgatgggc 60 cgcgacatca aggttcagtt ccagagcggt gggaacaact cacctgcggt ttatctgctc 120 gacggcctgc gcgcccaaga cgactacaac ggctgggata tcaacacccc ggcgttcgag 180 tggtactacc agtcgggact gtcgatagtc atgccggtcg gcgggcagtc cagcttctac 240 agcgactggt acagcccggc ctgcggtaag gctggctgcc agacttacaa gtgggaaacc 300 ttcctgacca gcgagctgcc gcaatggttg tccgccaaca gggccgtgaa gcccaccggc 360 agcgctgcaa tcggcttgtc gatggccggc tcgtcggcaa tgatcttggc cgcctaccac 420 ccccagcagt tcatctacgc cggctcgctg tcggccctgc tggacccctc tcaggggatg 480 gggcctagcc tgatcggcct cgcgatgggt gacgccggcg gttacaaggc cgcagacatg 540 tggggtccct cgagtgaccc ggcatgggag cgcaacgacc ctacgcagca gatccccaag 600 ctggtcgcaa acaacacccg gctatgggtt tattgcggga acggcacccc gaacgagttg 660 ggcggtgcca acatacccgc cgagttcttg gagaacttcg ttcgtagcag caacctgaag 720 ttccaggatg cgtacaacgc cgcgggcggg cacaacgccg tgttcaactt cccgcccaac 780 ggcacgcaca gctgggagta ctggggcgct cagctcaacg ccatgaaggg tgacctgcag 840 agttcgttag gcgccggcgt cgacatgcag cttgttgaca gggttcgtgg cgccgtcacg 900 ggtatgtcgc gtcgactcgt ggtcggggcc gtcggcgcgg ccctagtgtc gggtctggtc 960 ggcgccgtcg gtggcacggc gaccgcgggg gcattttccc ggccgggctt gccggtggag 1020 tacctgcagg tgccgtcgcc gtcgatgggc cgtgacatca aggtccaatt ccaaagtggt 1080 ggtgccaact cgcccgccct gtacctgctc gacggcctgc gcgcgcagga cgacttcagc 1140 ggctgggaca tcaacacccc ggcgttcgag tggtacgacc agtcgggcct gtcggtggtc 1200 atgccggtgg gtggccagtc aagcttctac tccgactggt accagcccgc ctgcggcaag 1260 gccggttgcc agacttacaa gtgggagacc ttcctgacca gcgagctgcc ggggtggctg 1320 caggccaaca ggcacgtcaa gcccaccgga agcgccgtcg tcggtctttc gatggctgct 1380 tcttcggcgc tgacgctggc gatctatcac ccccagcagt tcgtctacgc gggagcgatg 1440 tcgggcctgt tggacccctc ccaggcgatg ggtcccaccc tgatcggcct ggcgatgggt 1500 gcgctggcg gctacaaggc ctccgacatg tggggcccga aggaggaccc ggcgtggcag 1560 cgcaacgacc cgctgttgaa cgtcgggaag ctgatcgcca acaacacccg cgtctgggtg 1620 tactgcggca acggcaagcc gtcggatctg ggtggcaaca acctgccggc caagttcctc 1680 gagggcttcg tgcggaccag caacatcaag ttccaagacg cctacaacgc cggtggcggc 1740 cacaacggcg tgttcgactt cccggacagc ggtacgcaca gctgggagta ctggggcgcg 1800 cgctcaacg ctatgaagcc cgacctgcaa cgggcactgg gtgccacgcc caacaccggg 1860 cccgcgcccc agggcgcc 1878 <210> 6 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> CFP-10 / ESAT-6 <400> 6 atggcagaga tgaagaccga tgccgctacc ctcgcgcagg aggcaggtaa tttcgagcgg 60 atctccggcg acctgaaaac ccagatcgac caggtggagt cgacggcagg ttcgttgcag 120 ggccagtggc gcggcgcggc ggggacggcc gcccaggccg cggtggtgcg cttccaagaa 180 gcagccaata agcagaagca ggaactcgac gagatctcga cgaatattcg tcaggccggc 240 gtccaatact cgagggccga cgaggagcag cagcaggcgc tgtcctcgca aatgggcttc 300 gaattcgagc tcatgacaga gcagcagtgg aatttcgcgg gtatcgaggc cgcggcaagc 360 gcaatccagg gaaatgtcac gtccattcat tccctccttg acgaggggaa gcagtccctg 420 accaagctcg cagcggcctg gggcggtagc ggttcggagg cgtaccaggg tgtccagcaa 480 aaatgggacg ccacggctac cgagctgaac aacgcgctgc agaacctggc gcggacgatc 540 agcgaagccg gtcaggcaat ggcttcgacc gaaggcaacg tcactgggat gttcgcatag 600                                                                          600 <210> 7 <211> 902 <212> DNA <213> Artificial Sequence <220> <223> CFP-10 / HBHA <400> 7 atgcagagat gaagaccgat gccgctaccc tcgcgcagga ggcaggtaat ttcgagcgga 60 tctccggcga cctgaaaacc cagatcgacc aggtggagtc gacggcaggt tcgttgcagg 120 gccagtggcg cggcgcggcg gggacggccg cccaggccgc ggtggtgcgc ttccaagaag 180 cagccaataa gcagaagcag gaactcgacg agatctcgac gaatattcgt caggccggcg 240 tccaatactc gagggccgac gaggagcagc agcaggcgct gtcctcgcaa atgggcttcg 300 aattcatggc tgaaaactcg aacattgatg acatcaaggc tccgttgctt gccgcgcttg 360 gagcggccga cctggccttg gccactgtca acgagttgat cacgaacctg cgtgagcgtg 420 cggaggagac tcgtacggac acccgcagcc gggtcgagga gagccgtgct cgcctgacca 480 agctgcagga agatctgccc gagcagctca ccgagctgcg tgagaagttc accgccgagg 540 agctgcgtaa ggccgccgag ggctacctcg aggccgcgac tagccggtac aacgagctgg 600 tcgagcgcgg tgaggccgct ctagagcggc tgcgcagcca gcagagcttc gaggaagtgt 660 cggcgcgcgc cgaaggctac gtggaccagg cggtggagtt gacccaggag gcgttgggta 720 cggtcgcatc gcagacccgc gcggtcggtg agcgtgccgc caagctggtc ggcatcgagc 780 tgcctaagaa ggctgctccg gccaagaagg ccgctccggc caagaaggcc gctccggcca 840 agaaggcggc ggccaagaag gcgcccgcga agaaggcggc ggccaagaag gtcacccaga 900 ag 902 <210> 8 <211> 2157 <212> DNA <213> Artificial Sequence <220> <223> CFP-10 / ESAT-6/16 kDa / 38 kDa <400> 8 atggcagaga tgaagaccga tgccgctacc ctcgcgcagg aggcaggtaa tttcgagcgg 60 atctccggcg acctgaaaac ccagatcgac caggtggagt cgacggcagg ttcgttgcag 120 ggccagtggc gcggcgcggc ggggacggcc gcccaggccg cggtggtgcg cttccaagaa 180 gcagccaata agcagaagca ggaactcgac gagatctcga cgaatattcg tcaggccggc 240 gtccaatact cgagggccga cgaggagcag cagcaggcgc tgtcctcgca aatgggcttc 300 ggatctatga cagagcagca gtggaatttc gcgggtatcg aggccgcggc aagcgcaatc 360 cagggaaatg tcacgtccat tcattccctc cttgacgagg ggaagcagtc cctgaccaag 420 ctcgcagcgg cctggggcgg tagcggttcg gaggcgtacc agggtgtcca gcaaaaatgg 480 gacgccacgg ctaccgagct gaacaacgcg ctgcagaacc tggcgcggac gatcagcgaa 540 gccggtcagg caatggcttc gaccgaaggc aacgtcactg ggatgttcgc aggatccatg 600 gccaccaccc ttcccgttca gcgccacccg cggtccctct tccccgagtt ttctgagctg 660 ttcgcggcct tcccgtcatt cgccggactc cggcccacct tcgacacccg gttgatgcgg 720 ctggaagacg agatgaaaga ggggcgctac gaggtacgcg cggagcttcc cggggtcgac 780 cccgacaagg acgtcgacat tatggtccgc gatggtcagc tgaccatcaa ggccgagcgc 840 accgagcaga aggacttcga cggtcgctcg gaattcgcgt acggttcctt cgttcgcacg 900 gtgtcgctgc cggtaggtgc tgacgaggac gacattaagg ccacctacga caagggcatt 960 cttactgtgt cggtggcggt ttcggaaggg aagccaaccg aaaagcacat tcagatccgg 1020 tccaccaaca gatccgaatt catgcgtttg catacgctgt tggccgtgtt gaccgctgcg 1080 ccgctgctgc tagcagcggc gggctgtggc tcgaaaccac cgagcggttc gcctgaaacg 1140 ggcgccggcg ccggtactgt cgcgactacc cccgcgtcgt cgccggtgac gttggcggag 1200 accggtagca cgctgctcta cccgctgttc aacctgtggg gtccggcctt tcacgagagg 1260 tatccgaacg tcacgatcac cgctcagggc accggttctg gtgccgggat cgcgcaggcc 1320 gccgccggga cggtcaacat tggggcctcc gacgcctatc tgtcggaagg tgatatggcc 1380 gcgcacaagg ggctgatgaa catcgcgcta gccatctccg ctcagcaggt caactacaac 1440 ctgcccggag tgagcgagca cctcaagctg aacggaaaag tcctggcggc catgtaccag 1500 ggcaccatca aaacctggga cgacccgcag atcgctgcgc tcaaccccgg cgtgaacctg 1560 cccggcaccg cggtagttcc gctgcaccgc tccgacgggt ccggtgacac cttcttgttc 1620 acccagtacc tgtccaagca agatcccgag ggctggggca agtcgcccgg cttcggcacc 1680 accgtcgact tcccggcggt gccgggtgcg ctgggtgaga acggcaacgg cggcatggtg 1740 accggttgcg ccgagacacc gggctgcgtg gcctatatcg gcatcagctt cctcgaccag 1800 gccagtcaac ggggactcgg cgaggcccaa ctaggcaata gctctggcaa tttcttgttg 1860 cccgacgcgc aaagcattca ggccgcggcg gctggcttcg catcgaaaac cccggcgaac 1920 caggcgattt cgatgatcga cgggcccgcc ccggacggct acccgatcat caactacgag 1980 tacgccatcg tcaacaaccg gcaaaaggac gccgccaccg cgcagacctt gcaggcattt 2040 ctgcactggg cgatcaccga cggcaacaag gcctcgttcc tcgaccaggt tcatttccag 2100 ccgctgccgc ccgcggtggt gaagttgtct gacgcgttga tcgcgacgat ttccagc 2157 <210> 9 <211> 474 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > CFP-10/38 kDa <400> 9 Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly   1 5 10 15 Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val              20 25 30 Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly          35 40 45 Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys      50 55 60 Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly  65 70 75 80 Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser                  85 90 95 Gln Met Gly Phe Gly Ser Met Arg Leu His Thr Leu Leu Ala Val Leu             100 105 110 Thr Ala Pro Leu Leu Leu Ala Ala Aly Gly Cys Gly Ser Lys Pro         115 120 125 Pro Ser Gly Ser Pro Glu Thr Gly Ala Gly Ala Gly Thr Val Ala Thr     130 135 140 Thr Pro Ala Ser Ser Val Thr Leu Ala Glu Thr Gly Ser Thr Leu 145 150 155 160 Leu Tyr Pro Leu Phe Asn Leu Trp Gly Pro Ala Phe His Glu Arg Tyr                 165 170 175 Pro Asn Val Thr Ile Thr Ala Gln Gly Thr Gly Ser Gly Ala Gly Ile             180 185 190 Ala Gln Ala Ala Aly Gly Thr Val Asn Ile Gly Ala Ser Asp Ala Tyr         195 200 205 Leu Ser Glu Gly Asp Met Ala Ala His Lys Gly Leu Met Asn Ile Ala     210 215 220 Leu Ala Ile Ser Ala Gln Gln Val Asn Tyr Asn Leu Pro Gly Val Ser 225 230 235 240 Glu His Leu Lys Leu Asn Gly Lys Val Leu Ala Ala Met Tyr Gln Gly                 245 250 255 Thr Ile Lys Thr Trp Asp Asp Pro Gln Ile Ala Ala Leu Asn Pro Gly             260 265 270 Val Asn Leu Pro Gly Thr Ala Val Val Pro Leu His Arg Ser Asp Gly         275 280 285 Ser Gly Asp Thr Phe Leu Phe Thr Gln Tyr Leu Ser Lys Gln Asp Pro     290 295 300 Glu Gly Trp Gly Lys Ser Pro Gly Phe Gly Thr Thr Val Asp Phe Pro 305 310 315 320 Ala Val Pro Gly Ala Leu Gly Glu Asn Gly Asn Gly Gly Met Val Thr                 325 330 335 Gly Cys Ala Glu Thr Pro Gly Cys Val Ala Tyr Ile Gly Ile Ser Phe             340 345 350 Leu Asp Gln Ala Ser Gln Arg Gly Leu Gly Glu Ala Gln Leu Gly Asn         355 360 365 Ser Ser Gly Asn Phe Leu Leu Pro Asp Ala Gln Ser Ile Gln Ala Ala     370 375 380 Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn Gln Ala Ile Ser Met 385 390 395 400 Ile Asp Gly Pro Ala Pro Asp Gly Tyr Pro Ile Ile Asn Tyr Glu Tyr                 405 410 415 Ala Ile Val Asn Asn Arg Gln Lys Asp Ala Ala Thr Ala Gln Thr Leu             420 425 430 Gln Ala Phe Leu His Trp Ala Ile Thr Asp Gly Asn Lys Ala Ser Phe         435 440 445 Leu Asp Gln Val His Phe Gln Pro Leu Pro Pro Ala Val Val Lys Leu     450 455 460 Ser Asp Ala Leu Ile Ala Thr Ile Ser Ser 465 470 <210> 10 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> ESAT-6/16 kDa <400> 10 Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser   1 5 10 15 Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly              20 25 30 Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser          35 40 45 Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu      50 55 60 Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly  65 70 75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Gly                  85 90 95 Ser Met Ala Thr Thr Leu Pro Val Gln Arg His His Arg Ser Leu Phe             100 105 110 Pro Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu         115 120 125 Arg Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys     130 135 140 Glu Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp 145 150 155 160 Lys Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala                 165 170 175 Glu Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr             180 185 190 Gly Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp         195 200 205 Asp Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Ala     210 215 220 Val Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr 225 230 235 240 Asn     <210> 11 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> ESAT-6 / MTB48 <400> 11 Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser   1 5 10 15 Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly              20 25 30 Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser          35 40 45 Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu      50 55 60 Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly  65 70 75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Glu                  85 90 95 Phe Asp Met Thr Gln Ser Gln Thr Val Thr Val Asp Gln Gln Glu Ile             100 105 110 Leu Asn Arg Ala Asn Glu Val Glu Ala Pro Met Ala Asp Pro Pro Thr         115 120 125 Asp Val Pro Ile Thr Pro Cys Glu Leu Thr Ala Ala Lys Asn Ala Ala     130 135 140 Gln Gln Leu Val Leu Ser Ala Asp Asn Met Arg Glu Tyr Leu Ala Ala 145 150 155 160 Gly Ala Lys Glu Arg Gln Arg Leu Ala Thr Ser Leu Arg Asn Ala Ala                 165 170 175 Lys Ala Tyr Gly Glu Val Asp Glu Glu Ala Ala Thr Ala Leu Asp Asn             180 185 190 Asp Gly Glu Gly Thr Val Gln Ala Glu Ser Ala Gly Ala Val Gly Gly         195 200 205 Asp Ser Ser Ala Glu Leu Thr Asp Thr Pro Arg Val Ala Thr Ala Gly     210 215 220 Glu Pro Asn Phe Met Asp Leu Lys Glu Ala Ala Arg Lys Leu Glu Thr 225 230 235 240 Gly Asp Gln Gly Ala Ser Leu Ala His Phe Ala Asp Gly Trp Asn Thr                 245 250 255 Phe Asn Leu Thr Leu Gln Gly Asp Val Lys Arg Phe Arg Gly Phe Asp             260 265 270 Asn Trp Glu Gly Asp Ala Ala Thr Ala Cys Glu Ala Ser Leu Asp Gln         275 280 285 Gln Arg Gln Trp Ile Leu His Met Ala Lys Leu Ser Ala Ala Met Ala     290 295 300 Lys Gln Ala Gln Tyr Val Ala Gln Leu His Val Trp Ala Arg Arg Glu 305 310 315 320 His Pro Thr Tyr Glu Asp Ile Val Gly Leu Glu Arg Leu Tyr Ala Glu                 325 330 335 Asn Pro Ser Ala Arg Asp Gln Ile Leu Pro Val Tyr Ala Glu Tyr Gln             340 345 350 Gln Arg Ser Glu Lys Val Leu Thr Glu Tyr Asn Asn Lys Ala Ala Leu         355 360 365 Glu Pro Val Asn Pro Pro Lys Pro Pro Pro Ala Ile Lys Ile Asp Pro     370 375 380 Pro Pro Pro Pro Gln Glu Gln Gly Leu Ile Pro Gly Phe Leu Met Pro 385 390 395 400 Pro Ser Asp Gly Ser Gly Val Thr Pro Gly Thr Gly Met Pro Ala Ala                 405 410 415 Pro Met Val Pro Pro Thr Gly Ser Pro Gly Gly Gly Leu Pro Ala Asp             420 425 430 Thr Ala Gla Leu Thr Ser Gla Aly Gla Ala Ala Ala Leu Ser         435 440 445 Gly Asp Val Ala Val Lys Ala Ala Ser Leu Gly Gly Gly Gly Gly Gly     450 455 460 Gly Val Pro Ser Ala Pro Leu Gly Ser Ala Ile Gly Gly Ala Glu Ser 465 470 475 480 Val Arg Pro Ala Gly Ala Gly Asp Ile Ala Gly Leu Gly Gln Gly Arg                 485 490 495 Ala Gly Gly Gly Ala Ala Leu Gly Gly Gly Gly             500 505 510 Gly Ala Ala His Gln Gly Gln Gly Gly Ala Lys Ser Lys Gly Ser Gln         515 520 525 Gln Glu Asp Glu Ala Leu Tyr Thr Glu Asp Arg Ala Trp Thr Glu Ala     530 535 540 Val Ile Gly Asn Arg Arg Arg Gln Asp Ser Lys Glu Ser Lys 545 550 555 <210> 12 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> MPT64 / CFP21 <400> 12 Met Ala Pro Lys Thr Tyr Cys Glu Glu Leu Lys Gly Thr Asp Thr Gly   1 5 10 15 Gln Ala Cys Gln Ile Gln Met Ser Asp Pro Ala Tyr Asn Ile Asn Ile              20 25 30 Ser Leu Pro Ser Tyr Tyr Pro Asp Gln Lys Ser Leu Glu Asn Tyr Ile          35 40 45 Ala Gln Thr Arg Asp Lys Phe Leu Ser Ala Ala Thr Ser Ser Thr Pro      50 55 60 Arg Glu Ala Pro Tyr Glu Leu Asn Ile Thr Ser Ala Thr Tyr Gln Ser  65 70 75 80 Ala Ile Pro Pro Arg Gly Thr Gln Ala Val Val Leu Lys Val Tyr Gln                  85 90 95 Asn Ala Gly Gly Thr His Pro Thr Thr Thr Tyr Lys Ala Phe Asp Trp             100 105 110 Asp Gln Ala Tyr Arg Lys Pro Ile Thr Tyr Asp Thr Leu Trp Gln Ala         115 120 125 Asp Thr Asp Pro Leu Pro Val Val Phe Pro Ile Val Gln Gly Glu Leu     130 135 140 Ser Lys Gln Thr Gly Gln Gln Val Ser Ile Ala Pro Asn Ala Gly Leu 145 150 155 160 Asp Pro Val Asn Tyr Gln Asn Phe Ala Val Thr Asn Asp Gly Val Ile                 165 170 175 Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro Glu Ala Ala Gly Pro Thr             180 185 190 Gln Val Leu Val Pro Arg Ser Ala Ile Asp Ser Met Leu Ala Leu Glu         195 200 205 Met Thr Pro Arg Ser Leu Val Arg Ile Val Gly Val Val Ala Thr     210 215 220 Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Gly Arg Ala Ala His Ala 225 230 235 240 Asp Pro Cys Ser Asp Ile Ala Val Val Phe Ala Arg Gly Thr His Gln                 245 250 255 Ala Ser Gly Leu Gly Asp Val Gly Glu Ala Phe Val Asp Ser Leu Thr             260 265 270 Ser Gln Val Gly Gly Arg Ser Ile Gly Val Tyr Ala Val Asn Tyr Pro         275 280 285 Ala Ser Asp Asp Tyr Arg Ala Ser Ala Ser Asn Gly Ser Asp Asp Ala     290 295 300 Ser Ala His Ile Gln Arg Thr Val Ala Ser Cys Pro Asn Thr Arg Ile 305 310 315 320 Val Leu Gly Gly Tyr Ser Gln Gly Ala Thr Val Ile Asp Leu Ser Thr                 325 330 335 Ser Ala Met Pro Ala Val Ala Asp His Ala Val Ala Leu             340 345 350 Phe Gly Glu Pro Ser Ser Gly Phe Ser Ser Met Leu Trp Gly Gly Gly         355 360 365 Ser Leu Pro Thr Ile Gly Pro Leu Tyr Ser Ser Lys Thr Ile Asn Leu     370 375 380 Cys Ala Pro Asp Asp Pro Ile Cys Thr Gly Gly Gly Asn Ile Met Ala 385 390 395 400 His Val Ser Tyr Val Gln Ser Gly Met Thr Ser Gln Ala Ala Thr Phe                 405 410 415 Ala Ala Asn Arg Ala Asp His Ala Gly             420 425 <210> 13 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> Ag85b / Ag85a <400> 13 Met Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Ser Ser   1 5 10 15 Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn              20 25 30 Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp          35 40 45 Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln      50 55 60 Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr  65 70 75 80 Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr                  85 90 95 Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala             100 105 110 Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met         115 120 125 Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe     130 135 140 Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Ser Ser Gln Gly Met 145 150 155 160 Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys                 165 170 175 Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn             180 185 190 Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu         195 200 205 Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn     210 215 220 Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys 225 230 235 240 Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn                 245 250 255 Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu             260 265 270 Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly Val Asp         275 280 285 Met Gln Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg     290 295 300 Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val 305 310 315 320 Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly                 325 330 335 Leu Pro Val Glu Tyr Leu Gln Val Ser Ser Ser Ser Gly Arg Asp             340 345 350 Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr         355 360 365 Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile     370 375 380 Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser Val Val 385 390 395 400 Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro                 405 410 415 Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu             420 425 430 Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val Lys Pro         435 440 445 Thr Gly Ser Ala Val Ser Gia Le Ser Ser Ala Leu     450 455 460 Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr Ala Gly Ala Met 465 470 475 480 Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly                 485 490 495 Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly             500 505 510 Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu Asn Val         515 520 525 Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn     530 535 540 Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe Leu 545 550 555 560 Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn                 565 570 575 Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr             580 585 590 His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp         595 600 605 Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln     610 615 620 Gly Ala 625 <210> 14 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> CFP-10 / ESAT-6 <400> 14 Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly   1 5 10 15 Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val              20 25 30 Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly          35 40 45 Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys      50 55 60 Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly  65 70 75 80 Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser                  85 90 95 Gln Met Gly Phe Glu Phe Glu Leu Met Thr Glu Gln Gln Trp Asn Phe             100 105 110 Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser         115 120 125 Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr Lys Leu Ala     130 135 140 Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val Gln Gln 145 150 155 160 Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu                 165 170 175 Ala Arg Thr Ile Ser Glu Ala Gly             180 185 190 Asn Val Thr Gly Met Phe Ala         195 <210> 15 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> CFP-10 / HBHA <400> 15 Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly   1 5 10 15 Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val              20 25 30 Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly          35 40 45 Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys      50 55 60 Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly  65 70 75 80 Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser                  85 90 95 Gln Met Gly Phe Glu Phe Met Ala Glu Asn Ser Asn Ile Asp Asp Ile             100 105 110 Lys Ala Pro Leu Leu Ala Ala Leu Gla Ala Ala Asp Leu Ala Leu Ala         115 120 125 Thr Val Asn Glu Leu Ile Thr Asn Leu Arg Glu Arg Ala Glu Glu Thr     130 135 140 Arg Thr Asp Thr Arg Ser Arg Val Glu Glu Ser Arg Ala Arg Leu Thr 145 150 155 160 Lys Leu Gln Glu Asp Leu Pro Glu Gln Leu Thr Glu Leu Arg Glu Lys                 165 170 175 Phe Thr Ala Glu Glu Leu Arg Lys Ala Ala Glu Gly Tyr Leu Glu Ala             180 185 190 Ala Thr Ser Arg Tyr Asn Glu Leu Val Glu Arg Gly Glu Ala Ala Leu         195 200 205 Glu Arg Leu Arg Ser Glu Gln Ser Phe Glu Glu Val Ser Ala Arg Ala     210 215 220 Glu Gly Tyr Val Asp Glu Ala Val Glu Leu Thr Gln Glu Ala Leu Gly 225 230 235 240 Thr Val Ala Ser Gln Thr Arg Ala Val Gly Glu Arg Ala Ala Lys Leu                 245 250 255 Val Gly Ile Glu Leu Pro Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala             260 265 270 Pro Ala Lys Lys Ala Ala Pro Ala Lys Ala Lys Ala Ala Ala Lys Lys Ala         275 280 285 Pro Ala Lys Lys Ala Ala Ala Lys Lys Val Thr Gln Lys     290 295 300 <210> 16 <211> 719 <212> PRT <213> Artificial Sequence <220> <223> CFP-10 / ESAT-6/16 kDa / 38 kDa <400> 16 Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly   1 5 10 15 Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val              20 25 30 Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly          35 40 45 Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys      50 55 60 Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly  65 70 75 80 Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser                  85 90 95 Gln Met Gly Phe Gly Ser Met Thr Glu Gln Gln Trp Asn Phe Ala Gly             100 105 110 Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser Ile His         115 120 125 Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala     130 135 140 Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp 145 150 155 160 Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg                 165 170 175 Thr Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val             180 185 190 Thr Gly Met Phe Ala Gly Ser Met Ala Thr Thr Leu Pro Val Gln Arg         195 200 205 His Pro Arg Ser Leu Phe Pro Glu Phe Ser Glu Leu Phe Ala Ala Phe     210 215 220 Pro Ser Phe Ala Gly Leu Arg Pro Thr Phe Asp Thr Arg Leu Met Arg 225 230 235 240 Leu Glu Asp Glu Met Lys Glu Gly Arg Tyr Glu Val Arg Ala Glu Leu                 245 250 255 Pro Gly Val Asp Pro Asp Lys Asp Val Asp Ile Met Val Arg Asp Gly             260 265 270 Gln Leu Thr Ile Lys Ala Glu Arg Thr Glu Gln Lys Asp Phe Asp Gly         275 280 285 Arg Ser Glu Phe Ala Tyr Gly Ser Phe Val Arg Thr Val Ser Leu Pro     290 295 300 Val Gly Ala Asp Glu Asp Asp Ile Lys Ala Thr Tyr Asp Lys Gly Ile 305 310 315 320 Leu Thr Val Ser Val Ala Val Ser Glu Gly Lys Pro Thr Glu Lys His                 325 330 335 Ile Gln Ile Arg Ser Thr Asn Arg Ser Glu Phe Met Arg Leu His Thr             340 345 350 Leu Leu Ala Val Leu Thr Ala Ala Pro Leu Leu Leu Ala Ala Ala Gly         355 360 365 Cys Gly Ser Lys Pro Pro Ser Gly Ser Pro Glu Thr Gly Ala Gly Ala     370 375 380 Gly Thr Val Ala Thr Thr Pro Ala Ser Ser Val Thr Leu Ala Glu 385 390 395 400 Thr Gly Ser Thr Leu Leu Tyr Pro Leu Phe Asn Leu Trp Gly Pro Ala                 405 410 415 Phe His Glu Arg Tyr Pro Asn Val Thr Ile Thr Ala Gln Gly Thr Gly             420 425 430 Ser Gly Ala Gly Ale Ala Ala Gly         435 440 445 Ala Ser Asp Ala Tyr Leu Ser Glu Asp Met Ala Ala His Lys Gly     450 455 460 Leu Met Asn Ile Ala Leu Ala Ile Ser Ala Gln Gln Val Asn Tyr Asn 465 470 475 480 Leu Pro Gly Val Ser Glu His Leu Lys Leu Asn Gly Lys Val Leu Ala                 485 490 495 Ala Met Tyr Gln Gly Thr Ile Lys Thr Trp Asp Asp Pro Gln Ile Ala             500 505 510 Ala Leu Asn Pro Gly Val Asn Leu Pro Gly Thr Ala Val Val Pro Leu         515 520 525 His Arg Ser Asp Gly Ser Gly Asp Thr Phe Leu Phe Thr Gln Tyr Leu     530 535 540 Ser Lys Gln Asp Pro Glu Gly Trp Gly Lys Ser Pro Gly Phe Gly Thr 545 550 555 560 Thr Val Asp Phe Pro Ala Val Pro Gly Ala Leu Gly Glu Asn Gly Asn                 565 570 575 Gly Gly Met Val Thr Gly Cys Ala Glu Thr Pro Gly Cys Val Ala Tyr             580 585 590 Ile Gly Ile Ser Phe Leu Asp Gln Ala Ser Gln Arg Gly Leu Gly Glu         595 600 605 Ala Gln Leu Gly Asn Ser Ser Gly Asn Phe Leu Leu Pro Asp Ala Gln     610 615 620 Ser Ile Gln Ala Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn 625 630 635 640 Gln Ala Ile Ser Met Ile Asp Gly Pro Ala Pro Asp Gly Tyr Pro Ile                 645 650 655 Ile Asn Tyr Glu Tyr Ala Ile Val Asn Asn Arg Gln Lys Asp Ala Ala             660 665 670 Thr Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala Ile Thr Asp Gly         675 680 685 Asn Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro Leu Pro Pro     690 695 700 Ala Val Val Lys Leu Ser Asp Ala Leu Ile Ala Thr Ile Ser Ser 705 710 715

Claims (12)

A fusion protein composition of Mycobacterium tuberculosis specific antigen comprising a combination of Mycobacterium tuberculosis specific antigen for serological diagnosis of tuberculosis. The method according to claim 1,
The combinations of the Mycobacterium tuberculosis-specific antigens include CFP-10 and 38 kDa, ESAT-6 and 16 kDa, ESAT-6 and MTB48, MPT64 and CFP-21, Ag85B and Ag85A, CFP-10 and ESAT-6, CFP-10 and HBHA and CFP -10, ESAT-6, a combination of 16 kDa and 38 kDa, and a fusion protein composition of Mycobacterium tuberculosis-specific antigen. The method according to claim 1,
Wherein the fusion protein of the Mycobacterium tuberculosis-specific antigen is expressed from the nucleotide sequence of at least one Mycobacterium-specific antigen combination selected from the group consisting of SEQ ID NOS: 1-8. The method according to claim 1,
Wherein the fusion protein of the Mycobacterium tuberculosis specific antigen is at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 9 to 16. Inserting a combination gene of Mycobacterium tuberculosis specific antigen into a vector;
Transforming the vector into which the gene is inserted into E. coli;
And inducing IPTG of the transformed E. coli. 6. The method of claim 5,
Wherein the Mycobacterium tuberculosis-specific antigen combination gene is at least one selected from the group consisting of SEQ ID NOS: 1 to 8. 6. The method of claim 5,
Wherein said vector is selected from the group consisting of T7 promoter vector, T5 promoter vector, and tac promoter vector. 6. The method of claim 5,
Wherein the Mycobacterium tuberculosis-specific antigen combination genes SEQ ID NOS: 1 to 5 are inserted into a T7 promoter vector. 6. The method of claim 5,
Wherein the Mycobacterium tuberculosis-specific antigen combination gene SEQ. ID. NO. 6 is inserted into a T5 promoter vector. A diagnostic kit for mycobacterium tuberculosis comprising the Mycobacterium tuberculosis specific antigen fusion protein according to any one of claims 1 to 4. A method for detecting Mycobacterium tuberculosis in a sample through an antigen-antibody reaction using the Mycobacterium tuberculosis specific antigen fusion protein according to any one of claims 1 to 4. 12. The method of claim 11,
The antigen-antibody reaction may be assayed by immunofluorescence assay using tissue immuno staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, Complement Fixation Assay (FACS), Fluorescence-Activated Cell Sorter (FACS) and protein chip analysis.

Images