JP2672585B2 - MPB57 protein derived from BCG bacterium and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention relates to MPB5 derived from BCG bacteria produced by the recombinant DNA method.
7 protein (the 57th mycobacterial protein of BCG bacteria obtained by column operation is abbreviated as MPB57 protein), B
Culturing a transformant transformed with a plasmid containing a gene encoding a CG-derived MPB57 protein and a gene encoding a BCG-derived MPB57 protein to produce a target BCG-derived MPB57 protein. Manufacturing method.

<Prior Art> Even when a clinician is suspected to have tuberculosis based on chest photographs and medical conditions, it is often difficult to detect tubercle bacilli by microscopic examination from the sputum of a patient. In such a case, the bacteria are cultured to detect the bacteria, but this cultivation takes about 3 weeks. Also,
Due to the similarities between the symptoms of pulmonary tuberculosis and lung cancer, the number of cases in which lung cancer progresses and becomes too late is increasing while the lung cancer is misdiagnosed as pulmonary tuberculosis and an antituberculosis drug is administered on a trial basis. Therefore, the present inventors have focused on the protein MPB57 existing in BCG bacteria and Mycobacterium tuberculosis,
We started a study to obtain a large amount of 7 in a pure form by a genetic engineering method. This is because, if a diagnostic agent that makes full use of this antigen-antibody reaction (ELISA method) that utilizes MPB57 can be developed, lung cancer and tuberculosis can be distinguished extremely accurately and quickly.

However, in reality, the proteins secreted by Mycobacterium tuberculosis and BCG bacteria amount to about 300 species, and in order to extract MPB57 protein derived from Mycobacterium tuberculosis bacteria and BCG bacteria purely from these, electrophoresis and several kinds of complicated Column operation is required.

Therefore, it is extremely difficult to obtain MPB57 protein in a large amount and in a pure form in bacterial culture.

From this, by genetic engineering techniques, M. tuberculosis and
It is desired to provide a large amount of MPB57 protein secreted by BCG bacteria in pure form.

<Problems to be Solved by the Present Invention> An object of the present invention is to use a gene encoding a BCG-derived MPB57 protein and a BCG-derived MPB57 protein using a transformant transformed with a recombinant DNA containing the gene. And a MPB57 protein derived from BCG bacteria produced by the recombinant DNA method.

<Means for Solving the Problems> In order to solve the above-mentioned problems, the inventors of the present invention first performed MPB5
The N-terminal amino acid sequence of 7 protein was examined. As a result, 1984
It was identical to the 20 N-terminal amino acid sequences of the BCG-a protein derived from BCG bacteria reported by P. Mindon et al. In 1 year with one difference.

(INFECTION AND IMMUNITY 1984 Vol46 No.2519-525) However, it has not been reported thereafter that they succeeded in isolating the gene and expressing the gene. A chemically synthesized oligonucleotide probe corresponding to the N-terminal amino acid sequence of MPB57 determined there was prepared,
Based on this, a gene encoding the MPB57 protein was isolated from BCG bacteria secreting the MPB57 protein, and its nucleotide sequence was determined.
Next, by incorporating the gene into an appropriate expression vector, the MPB57 protein could be produced in large quantities in Escherichia coli, thus completing the present invention.

The present invention will be described below by dividing into (1) preparation of chromosomal DNA of BCG bacterium, (2) cloning of a gene encoding MPB57 protein, and (3) expression of a gene encoding MPB57.

(1) Chromosomal DNA Preparation BCG bacteria BCG bacteria chromosomal DNA, Suzuki et al's method (J. Bacteriol. 16
9 (2) 839 (1987)). Extracted DNA
Is purified by cesium chloride-ethidium bromide density gradient centrifugation.

(2) Cloning of gene encoding MPB57 protein Digestion products of the chromosomal DNA obtained in (1) above with various restriction enzymes were fractionated by agarose gel electrophoresis.
D by the method of Southern et al. [J. Mol. Biol. 98 , 503 (1975)].
Prepare a filter (nitrocellulose or nylon membrane) to which the NA fragment has been bound.

Next, the 5 'phosphate group was added to this filter by ³² P
Hybridization with a synthetic oligonucleotide probe labeled with 〔[Method in Enzymology 6]
8 , 419 (1975)], it contains the MPB57 gene.
DNA fragments can be detected. This DNA fragment was fractionated by agarose gel electrophoresis and then collected with a recovery buffer (50%).
% Gliser running buffer)
Collect the desired DNA band.

Next, this DNA fragment was introduced into the plasmid pUC18 and introduced into a host cell (for example, E. coli JM109 strain) according to the method of Hanahan (J. Mol. Biol., 166 , 557 (1983)]. A DNA library is prepared by conversion and selection (in the case of E. coli JM109 strain, a strain which is ampicillin resistant and does not have β-galactosidase activity).

For this DNA library, colony hybridization test using the ³² P-labeled oligonucleotide probe described above (Method in Enzymology 68
379 (1979)) to screen the desired clone.

A cloned DNA fragment was prepared from the clone thus obtained, and the nucleotide sequence was analyzed by a method such as meshing [NAR, 9 , 309 (1981)] to determine the nucleotide sequence encoding the amino acid sequence of the amino terminal portion of MPB57 protein. ,Finally
A cloned DNA containing a nucleotide sequence corresponding to the gold translation region of MPB57 protein can be obtained.

(3) Expression of gene encoding MPB57 protein MPB using the gene encoding MPB57 protein obtained in (2) above
To produce the 57 protein, first, the substantial sequence of the cloned DNA was inserted into the expression vector to obtain MPB57.
Make recombinant DNA for protein production. Next, the recombinant DNA is introduced into a suitable host cell for transformation.

The target MPB57 protein can be obtained by culturing the transformant thus obtained in a medium.

In the present invention, when expressing the gene of the cloned MPB57 protein, a wide range of prokaryotes or,
Combinations of eukaryotic host cells and expression vectors can be employed.

When a prokaryote is used as a host cell, there is no particular limitation. For example, E. coli K12 strain (hereinafter, E. coli
E. coli X1776 strain and E. coli JM103 strain (NAR 9 , 309 (1981) E. coli JM109 strain, which are designated as i) are preferable. In addition to these, Bacillus subtilis can also be used. Although there is no particular limitation when using organisms, for example, Saccharomyces cerevisiae and the like are preferable.

In addition, as a vector for expression of a phenotype, a vector containing a replicon compatible with the above-mentioned host cell and a regulatory function and a promoter necessary for the host cell to express its own protein, or modified so as to contain it Those that have been processed are preferred.

Specific examples of these combinations are as follows.

-When the host cell is E. coli: pBR322 [Gene, 2 , 95 (1977)] as a plasmid and lactose (lae) promoter [Nat as a promoter]
ure, 281 , 544 (1979)], tryptophan (trp) promoter [NAR 8 , 4507 (1980)] and tac (tac).
Examples include those having a promoter [Pro.NAS, 78 , 21 (1983)] and the like.

As a vector for expression of a phenotype, a puc vector such as pUC18 containing the above promoter in pBR322 [Method in Enzymology 101 , 20 (1983)] or pKK233
-2 (Pharmacia, Sweden).

-When the host cell is Saccharomyces cerevisiae: YRp7 as a plasmid (Nature, 282 , 39 (1979))
But also as a promoter glyceraldehyde-3-
Phosphate dehydrogenase promoter [JB
C., 254 , 9839 (1979)] and α-factor promoter.

In the present invention, a prokaryotic cell or a eukaryotic cell may be used as a host cell, but it is more preferable to use a prokaryotic cell.

The thus obtained host cells (transformants) containing the recombinant DNA for MPB57 protein production are cultured in a liquid medium,
It may be done aerobically. As the medium, for example, a normal nutrient medium containing polypeptone, yeast extract, salt, glucose and the like, as well as an M9 minimal medium can be used. The cultivation is preferably performed at about 30 to 40 ° C.

In addition, in the case of an appropriate inducer depending on the promoter used in the culture, such as the lae promoter, isopropyl-β-D-thiogalactoside can be added to the medium to more efficiently culture the transformants. It can be carried out.

 In the present invention, the following abbreviations are used.

A: adenine C: cytosine G: guanine T: thymine dCTP: deoxycytidine triphosphate EDTA: ethylenediaminetetraacetic acid kb: kilobase kbp: kilobase pair DEAE: diethylaminoethyl SDS: sodium dodecyl sulfate SSC: 0.15M sodium chloride 0.015M Sodium citrate (pH 7.0) buffer IPTG: isopropyl-β-D-thiogalactoside X-gal: 5-bromo-4-chloro-3-indolyl-β
-D-Galactopyranoside Hereinafter, the present invention will be described more specifically with reference to Examples.

<Example 1> (1) Preparation of chromosomal DNA of BCG bacterium Mycobacterium bovis BCG Tokyo strain was transformed into a soton medium (composition: asparagine 0.4%, citric acid 0.2%, sodium citrate 0.28%, potassium phosphate 0.05%, magnesium sulfate 0.05% , 0.005% ferrous ammonium citrate,
The cells were cultured at 37 ° C. in glycerin (6%) 1 and harvested by centrifugation when the logarithmic growth phase was reached. The cells were suspended in 5 ml of 10 mM Tris-base (pH 8.0) 1 mM EDTA buffer, 5 mg of lysozyme was added and incubated at 37 ° C for 15 minutes, and then 0.5 ml of 10% SDS aqueous solution was added. Phenol: chloroform: isoamyl alcohol = 25: 24: 1 mixture 6ml
After extracting three times with, the aqueous layer was taken, 10 ml of ethanol was added, and the precipitated DNA was spread on a glass rod. This DNA
It was dissolved in 6.6 ml of 10 mM Tris-hydrochloric acid (pH 8.0) 1 mM EDTA buffer, and dissolved by adding 7 g of cesium chloride and 0.7 ml of an ethidium bromide aqueous solution (5 mg / ml).

The solution was placed in a centrifuge tube (Quick Seal Tube manufactured by Beckman, USA) and centrifuged at 60,000 rpm for 6 hours.
The chromosomal DNA band was collected from above the centrifuge tube. The DNA solution was dialyzed against 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM EDTA buffer, and desalted to obtain purified BCG chromosomal DNA.

(2) Preparation of synthetic oligonucleotide probe 20 times from the determined N-terminal of MPB57 protein derived from BCG bacteria.
Amino acid sequence of ¹ Ala Lys Val Aan Ile Lys Pro Leu Glu Asp ¹⁰ Lys Iie
Three synthetic oligonucleotide probes for Leu Val Gln Ala Asn Glu Ala Glu ²⁰ were prepared. That is, the following probes I, II, and III.

Probe I (compatible with ¹⁴ Val- ²⁰ Glu): Probe II (compatible with ⁹ Giu- ¹⁷ Asn): Probe III (compatible with ² Lys- ¹⁹ Ala): The synthesis operation is specifically shown below. First, an automated DNA synthesizer (Applied Biosystems, USA, 380A
After synthesizing the target DNA probe using the above method, the protecting groups other than the dimethoxytrityl group are removed, and reversed-phase medium-pressure column chromatography (conditions: using a C18-silica gel column, 100 mM triethylamine acetate buffer (pH 7 .0), a gradient solution consisting of acetonitrile was used).

Then, the dimethoxytrityl group was removed using 80% acetic acid, followed by reverse phase HPLC (conditions: using a YMC PACK AM-314 ODS column, and using a concentration gradient solution consisting of acetonitrile in 100 mM triethylamine acetate buffer (pH 7.0) as a mobile phase). Used) and lyophilized.

Thus, the obtained three kinds of oligonucleotide probes were dissolved in 10 mM Tris-HCl (pH8.0) 1 mM EDTA buffer to a concentration of 0.002 OD / μ. 100 pmol of this oligonucleotide solution and a kinase reaction buffer [5
0 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 10 mM dithiothreitol, 0.66 μM ATP, 100 μ
A total amount of Ci [γ- ³² P] ATP (specific activity 3000 Ci / mmol), 15 units polynucleotide kinase] was 50 μm, and the mixture was reacted at 37 ° C. for 1 hour. The reaction solution was extracted with phenol, washed with chloroform, passed through a NEN SORE ^™ 20 DNA purification cartridge to remove unreacted ³² P, coexisting proteins and salts, and a labeled nucleotide probe was recovered as a 50% methanol solution. Then by measuring the count of ³² P After evaporation of the solvent, 10
Such that the ⁵ cpm / mu concentrations above 10mM Tris - HCl (p
H8.0) It was dissolved in 1 mM EDTA buffer.

(3) Southern hybridization To detect and fractionate a chromosomal DNA fragment carrying the MPB57 protein gene, Southern hybridization test was performed.
It was performed according to the method of hern et al. (J. Mol. Biol 98 503 (1975)). That is, about 3 μg of the chromosomal DNA obtained in (1) was completely digested with restriction enzymes BamHI, KpnI, and PstI, and fractionated by 0.8% agarose gel electrophoresis. Shake for 40 minutes in M sodium hydroxide aqueous solution.

Next, this gel was shaken for 1 hour in 3M sodium chloride, 0.5 Tris-HCl buffer (pH 7.0). Finally, shake in a 20-fold concentration SSC (3 M sodium chloride, 0.3 M sodium citrate buffer (pH 7.0) for 30 minutes. A nylon membrane filter (NEN, USA) dipped in a 20-fold concentration SSC solution is applied to the gel. Gene Screen Plus)
To adsorb the DNA. The filter was washed with double concentration SSC and dried at room temperature for 30 minutes and at 37 ° C. for 18 hours to prepare a DNA binding filter.

This filter was washed with a hybridization solution [5-fold concentration Denhardt (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 5-fold concentration SSC, 0.1S
DS solution] and allowed to stand at 65 ° C. for 6 hours. Next, 40 μ of the ³² P-labeled probe of the above (2) was added to 5 ml of the above hybridization solution, and the mixture was allowed to stand at 55 ° C. for 18 hours. The filters were then washed in 2x SSC 0.1% SDS at 55 ° C for 20 minutes.
This operation was repeated 3 times and then air-dried, and autoradiography was performed to detect a DNA fragment containing the gene of MPB57 protein (Fig. 1).

As can be seen from the results in FIG. 1, probes I, II and III
The DN that showed a band at about 9000 bp reacted with any of these.
Only the A fragment.

(4) Preparation of DNA library Preparation of Pat I DNA fragment Approximately 9000 bp restriction enzyme Pat I detected by (3)
20 μg BCG chromosomal DNA for cloning digested DNA fragments
Was completely digested with the restriction enzyme Pat I and then fractionated by 0.8% agarose gel electrophoresis. Make a groove downstream of the desired 9000 bp band and collect in recovery buffer (50% glycerose, 4%
0 mM Tris, 20 mM sodium acetate, 2 mM EDTA18 mM NaCl) was added, and the DNA was electrophoresed and collected. To this solution were added 2 volumes of ethanol and 0.05 volumes of 5M NaCl to precipitate DNA. After drying this precipitate under reduced pressure, 10 mM Tris-hydrochloric acid (pH 8.0) 1
It was dissolved in mM EDTA buffer to give a Pat I DNA fragment solution.

Treatment of cloning vector pUC18 10 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothreitol, 4 μg vector pUC
A mixture of 18 (Takara Shuzo) and 20 units restriction enzyme Pat I at 37 ℃
After the reaction was carried out for 2 hours, the mixture was extracted once with phenol and once with chloroform and then precipitated with ethanol.
After the precipitate was dried under reduced pressure, it was dissolved in 194 µm of 50 mM Tris-HCl buffer (pH 8.4), 1.5 units of alkaline phosphatase (E. coli C75) was added, and the mixture was reacted at 65 ° C for 1 hour. Further, after adding 1.5 units of alkaline phosphatase and reacting at 65 ° C. for 1 hour, extraction was performed twice each with phenol and chloroform, and ethanol was added to the aqueous layer to precipitate DNA. This precipitate was dissolved in 20 mM 10 mM Tris-HCl buffer (pH 8.0) 1 mM EDTA to give about 2.7 kb.
A vector DNA solution of p was prepared.

Preparation of recombinant DNA and DNA library (4) -Pat I DNA fragment solution 4μ (0.1
μg), 1μ of vector DNA solution prepared in (4)-
(0.1 µg) and a mixture of 30 µl of A solution and 5 µm of B solution of DNA ligation kit (Takara Shuzo) were reacted at 16 ° C for 30 minutes, and 16 µl of the reaction mixture was added to 100 µl of E. coli JM109 competent cell (Takara Shuzo). And allowed to stand at 0 ° C. for 60 minutes. This is followed by a further 90 seconds at 42 ° C, then 5 ° C at 0 ° C.
Let stand for minutes.

This mixture is mixed with 1% bactotripton and 0.5% yeast extract.
%, Sodium chloride 0.5%, glycose 0.1%, 300 μL of L-broth medium was added, and the mixture was allowed to stand at 37 ° C. for 30 minutes. Next, centrifuge at 5000 rpm for 5 minutes, discard the supernatant by half, and resuspend it. Ampicillin 50 μg / ml, 0.1 mM IPTG, 0.004%
It was applied to an L-agar plate containing X-gal (1.3% agar added to L-broth) and cultured at 37 ° C for about 16 hours to obtain a clone of ampicillin-resistant, β-galactosidase activity-non-retaining bacterium. A DNA library consisting of the transformant was prepared.

(5) Selection of clone having MPB57 protein gene (colony hybridization test) MPB57 for BCG DNA library of (4)-
In order to select a transformant containing the gene, a colony hybridization test using the ³² P-labeled oligonucleotide probe prepared in (2) above was performed. That is, the DN prepared in the above (4)-was placed on a nitrocellulose membrane filter (HA filter manufactured by Millipore).
After culturing the transformant of the A library and treating the filter with an alkali according to a conventional method, neutralization with a 1 M Tris-HCl buffer (pH 7.5) is performed, and then 1 M Tris-HCl buffer (pH 7.5) with 1.5 M sodium chloride. Processed. Next, after soaking in SSC buffer at a concentration of 2 times, it was air dried at room temperature and then baked at 80 ° C. for 3 hours to prepare a DNA binding filter.

This filter was subjected to the same operation as in the above-mentioned Southern hybridization (3), and probe I, probe I
Recombinant D containing a base sequence that strongly binds to I and probe III
By selecting a transformant having an NA molecule, about 30
One colony was obtained from 0 colonies.

The plasmid DNA was extracted and purified from this transformant, and then separately digested with restriction enzymes Xho I and BamHI, respectively, and Southern hybridization was carried out by the same procedure as in (3). As a result, it was bound to both the 1.0 kb Xho I digested fragment and the 0.6 kb BamHI digested fragment. Therefore, both fragments were cloned.

That is, the plasmid DNA of the transformant obtained above was cleaved separately with the restriction enzymes Xho I and BamHI, and the restriction enzyme Sal I of the vector pUC18 (the same cleavage site as Xho I) and BamH I digestion product. Ligated with E. coli JM109 and transformed with host E. coli JM109.
A clone that binds to any of I was obtained.

(6) Determination of nucleotide sequence of cloned DNA A plasmid was prepared from the clone obtained in (5),
This DNA was prepared by the method of Meshing et al. (Gene, 33 103 (1985), P
ro.NAS, 74 , 5463 (1967)), the nucleotide sequences of the 1.0 kb Xho I digested fragment and the 0.6 kb BamHI digested fragment were determined.

The nucleotide sequence of the gene encoding the MPB57 protein derived from BCG bacteria thus determined is shown in FIG.

The 1.0 kb Xho I cleavage fragment is 246 shown in FIG.
The fragment containing a base of about 1.0 kb downstream from the Xho I site from the 2nd to 252nd, and a 0.6 kb BamH I cleavage fragment is a fragment of about 0.6 kb upstream from the 395th to 400th BamH I site shown in FIG. It is a fragment having a base.

The nucleotide sequences shown in FIG. 2 were determined in consideration of the nucleotide sequences of both fragments.

By comparing the determined nucleotide sequence with the amino acid sequence near the N-terminal of the already known MPB57 protein, the 226th to 228th GCGs in FIG.
Corresponding to a, it was determined that the 523rd to 525th TAGs correspond to the stop codon.

Therefore, the gene encoding the MPB57 protein derived from BCG bacteria,
That is, it was found that the structural gene of MPB57 protein corresponds to Nos. 226 to 522 in Fig. 2 (underlined part in the figure).

The number of amino acid residues translated from the nucleotide sequence encoding the MPB57 protein was 99, and its calculated molecular weight was 10.818, and its amino acid sequence is shown in FIG.

<Example 2> Using a trait expression vector pKK233-2 (Pharmacia, Sweden) and a chemically synthesized oligonucleotide,
The MPB57 protein gene was ligated to produce MPB57 protein.

 The details of the production of this MPB57 protein are shown below.

(1) Preparation of a fragment containing the gene of MPB57 protein About this, 1.0 kb Xh obtained in Example 1- (5)
o I fragment was used.

(2) Preparation of chemically synthesized oligonucleotide Were synthesized in the same manner as in <Example 1>-(2), respectively.
Purified.

This linker (top), taken by 100pmol each (lower), the presence ATP, T ₄ polynucleotide kinase (E. coli
A19) 2 units of (manufactured by Takara Shuzo) were added and reacted at 37 ° C. for 30 minutes.

After completion of the reaction, the mixture was treated with phenol and chloroform, and then heated at 65 ° C. for 5 minutes.

After heating, the mixture was gradually cooled to obtain the desired linker as shown below.

The thus-prepared linker is the lacking part of the gene of the MPB57 protein of the Xho I fragment of (1) of <Example 2>, that is, the 226th to 247th nucleotide sequences (second
In addition to supplementing (see the figure), it also had a structure linked to the Nco I site of pKK233-2.

(3) Treatment of trait expression vector pKK233-2 Vector pKK233-22 μg was added to 10 mM Tris-HCl pH8.5,7 mM
Restriction enzyme Nco I (12unita) in MgCl ₂ , 80 mM NaCl
At 37 ° C for 2 hours.

Next, 3 units of alkaline phosphatase (E. coli A19) (manufactured by Takara Shuzo) were added, and the mixture was reacted at 65 ° C for 1 hour, and further added with 3 units of alkaline phosphatase, and reacted at 65 ° C for 1 hour.

Then, phenol treatment, chloroform treatment, ethanol precipitation, washing with 80% ethanol water, and drying were performed to prepare the desired Nco I-cleaved pKK233-2.

(4) Construction of expression vector pKKM57 Vector pKK233-2 (Nc obtained in (3) of <Example 2>
o I cleavage) 0.1 μg, 0.12 μg of Xho I fragment obtained in (1) of <Example 2>, 3.3 pmol of linker obtained in (2) of <Example 2> and DNA ligation kit (Takara Shuzo)
A mixture of 24 .mu.A solution and 4 .mu.B solution was reacted at 16.degree. C. for 30 minutes. That is, an expression vector pKKM57 containing the structural gene of MPB57 protein was constructed by this operation (Fig. 4).

Now, by adding 16 μ of this reaction mixture to 100 μ of E. coli JM109 strain competent cell (manufactured by Takara Shuzo), the mixture was allowed to stand for 60 minutes, then at 42 ° C. for 90 seconds and then at 0 ° C. for 5 minutes. Transformed.

Add 300μ of L-broth culture to this reaction solution, and
For 30 minutes.

Next, the mixture was centrifuged at 5000 rpm for 5 minutes, half of the supernatant was discarded and then resuspended, and L-agar medium containing 50 μg / ml of ampicillin (L
-Broth to which 1.3% agar was added) and the mixture was cultured at 37 ° C for about 16 hours to obtain ampicillin-resistant clones.

That is, a clone containing the expression vector pKKM57 was thus obtained.

(5) Production of MPB57 protein by Escherichia coli E. coli JM109 strain (E.coli A containing the expression vector pKKM57
J12407, FERM P-10151) and pKK233 as control
-2 containing E. coli JM109 strain was added to double concentration of YT medium (tryptone 16 g /, extract yeast extract 10 g /, NaC
After culturing in 10 ml of 15 g /) (containing 50 μg / ml of ampicillin) at 37 ° C. for 5 hours, IPTG was added to 200 μg / ml and further culturing for 15 hours. Centrifuge this culture (12,000
After 10 minutes of rpm) to collect the cells, the cells were suspended in 1 ml of sterilized water and sonicated. Then, the cells were collected again, and an equal volume of a 2 × sample buffer was added to the supernatant.
The mixture was boiled for a minute to obtain a cell extract. After performing SDS-polyacrylamide electrophoresis on the two kinds of cell extract and standard MPB57 protein, transfer the protein to a nitrocellulose filter,
The reaction with the anti-MPB57 protein polyclonal antibody was examined (Fig. 5).

As shown in FIG. 5, the band generated at exactly the same position as the standard sample was E. coli containing the pKKM57 vector (FERM P
-10151), the gene of MPB57 protein was expressed in E. coli and
It was confirmed that 57 protein was produced.

<Effect> The MPB57 protein derived from BCG bacteria provided by the present invention can be used as a diagnostic agent for tuberculosis by an antigen-antibody reaction (ELISA method).

Further, the method for producing the MPB57 protein derived from BCG bacteria by the gene encoding the BCG bacteria MPB57 protein and the genetic engineering method using the same provided by the present invention is a large amount and pure MPB57 protein useful as a diagnostic agent for tuberculosis. Important to serve.

Further, D encoding the MPB57 protein provided by the present invention
If a preferred sequence is selected from the NA sequences and a DNA probe labeled with piotin or a radioisotope is prepared, the diagnosis of tuberculosis can be performed by this.

[Brief description of the drawings]

Fig. 1 shows the restriction enzymes BamHI, KpnI of BCG chromosomal DNA and
It is a figure which shows the Southern hybridization of the probe I, II, and III with respect to the digested material by Pat I, It showed by the arrow.
The 9 kb Pat I fragment was cloned. FIG. 2 shows the nucleotide sequence of DNA encoding MPB57 protein. FIG. 3 shows the amino acid sequence of MPB57 protein. FIG. 4 is a diagram showing the construction of the expression vector pKKM57. FIG. 5 is a diagram in which the cell extract of Escherichia coli JM109 containing pKK233-2 and E. coli JM109 containing pKKM57 was analyzed by Western blotting.

 ─────────────────────────────────────────────────── ─── Continuation of front page Examiner Kazuo Hirata (56) References Infect. Immun. 46 [2] (1984) P. 519-525

Claims (3)

(57) [Claims] 1. A gene encoding an MPB57 protein derived from Mycobacterium bovis BCG (hereinafter referred to as BCG bacterium) having the following amino acid sequence (I). Amino acid sequence (I): 2. The gene according to claim 1, wherein the gene is represented by the following nucleotide sequence (a). Nucleotide sequence (a): 3. A BCG bacterium-derived strain, which comprises culturing a transformant transformed with the plasmid into which the gene according to claim 1 has been incorporated in a medium and collecting the produced MPB57 protein. MPB57 protein manufacturing method.