JP2624759B2 - MPB64 protein derived from BCG bacteria and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Applications> The present invention relates to BCG-derived MPB64 protein (the 64th mycobacterial protein of BCG is abbreviated as MPB64 protein), and BCG-derived MPB64 protein. A gene encoding
Culturing a transformant transformed with a plasmid containing a gene encoding the MPB64 protein from BCG,
The present invention relates to a method for producing a desired BCG-derived MPG64 protein.

<Prior Art> Even when a clinician is suspected of tuberculosis from a chest photograph and a medical condition, it is often difficult to find tuberculosis bacteria from a patient's sputum using a microscope. In such a case, the bacteria are cultured to detect the bacteria, but it takes about three weeks for the culture. In addition, due to the similarity between the symptoms of pulmonary tuberculosis and lung cancer (chest photographs), the number of cases in which lung cancer progresses and is too late during test administration of antituberculous drugs due to misdiagnosis of lung cancer as pulmonary tuberculosis is increasing. Furthermore, atypical mycobacteriosis (AM) showing symptoms very similar to tuberculosis recently
Patients are increasing. This AM disease is also at risk of being confused with lung cancer.

Currently, kits for the identification of acid-fast bacilli by biochemical techniques are commercially available, but the cultivation of the bacteria takes time. It is desired to develop a reliable identification method.

Under such circumstances, the present inventors have found that the α antigen derived from Mycobacterium tuberculosis is BCG bacteria or mycobacterium (Mycobacterium intracellulum).
are Mycobacterium kansasii), based on the finding that it is a cross-reacting material widely distributed (Tuberculosis, 61 , 663, (1986)). Research has begun to obtain a large amount of α antigen in a pure form by genetic engineering techniques. This is because the antigen-antibody reaction using this α antigen (ELISA
If a diagnostic agent that makes full use of the method can be developed, it is possible to distinguish lung cancer from tuberculosis and AM disease extremely accurately and quickly.

As a result of the intensive studies by the present inventors, it has become possible to produce an α antigen common to Mycobacterium tuberculosis, atypical mycobacteria and BCG bacteria by genetic engineering techniques (Japanese Patent Application No. Sho 62-3052).
No. 50). This allows accurate and quick lung cancer and tuberculosis and
It is now possible to identify AM syndrome.

However, this finding certainly made it possible to distinguish lung cancer from tuberculosis and AM disease,
No diagnostic agent has yet been developed to discriminate between tuberculosis and AM disease with similar symptoms. Therefore, there is an urgent need to develop a diagnostic agent for accurately and rapidly distinguishing between tuberculosis and AM disease.

On the other hand, M. Harboe et al. Isolated and purified the MPB64 protein produced by BCG bacteria, and the entire amino acid sequence has not been determined, but the amino acid sequence near its N-terminus has already been determined (INFECTION AND IMMUNITY). 1986 Vol 52 No.1 293-30
2). Unlike the α-antigen, the MPB64 protein does not produce atypical mycobacteria, but is a specific secreted protein produced by BCG bacteria and Mycobacterium tuberculosis.

Therefore, if this MPB64 protein could be obtained in a pure form, an antigen-antibody reaction using this MPB64 protein (ELISA
It is possible to accurately and quickly distinguish between tuberculosis and AM disease using the method.

However, in reality, about 300 kinds of proteins are secreted by M. tuberculosis and BCG, and in order to extract the MPB64 protein derived from M. tuberculosis and BCG purely, electrophoresis and several kinds of complicated Column operation is required.

Therefore, it is extremely difficult to obtain a large amount of pure MPB64 in pure form by culturing the bacteria.

From this, by genetic engineering techniques, M. tuberculosis and
It is desired to provide a large amount of the MPB64 protein secreted by BCG bacteria in a pure form.

<Problems to be solved by the present invention> The object of the present invention is to provide a gene encoding a BCG-derived MPB64 protein, and a BCG-derived MPB64 protein using a transformant transformed with a recombinant DNA containing the gene. And a target BCG-derived MPB64 protein.

<Means for Solving the Problems> The present inventors have conducted intensive studies to solve the above problems, and as a result, have determined that MPB64 light white N
A chemically synthesized oligonucleotide probe corresponding to the amino acid sequence near the terminal was prepared, and based on this, a gene encoding the MPB64 protein was isolated from BCG bacteria secreting the MPB64 protein, the nucleotide sequence was determined, and then the nucleotide sequence was determined. By incorporating the gene into an appropriate expression vector, it was possible to produce and provide the MPB64 protein in a large amount in Escherichia coli, thereby completing the present invention.

 Hereinafter, the present invention will be described with respect to (1) preparation of chromosomal DNA of BCG bacteria, (2) cloning of a gene encoding MPB64 protein, and (3) expression of a gene encoding MPB64 protein.

(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 MPB64 protein Digests 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 (1979)] to include the MPB64 gene
DNA fragments can be detected. This DNA fragment was fractionated by agarose gel electrophoresis and then collected with a recovery buffer (50%).
% Glycerol running buffer) to collect the target DNA band.

Next, this DNA fragment was introduced into plasmid pUC18, and introduced into host cells (for example, E. coli JM109 strain) according to the method of Hanahan (J. Mol. Biol., 166 , 557 (1983)). A DNA library is prepared by inverting and selecting (in the case of E. coli JM109 strain, selecting a strain having no β-galactosidase activity with ampicillin resistance).

For this DNA library, a colony hybridization test using the aforementioned ³² P-labeled oligonucleotide probe (Mezzot In Enzymology 68)
379 (1979)) and screen the desired clone.

A cloned DNA fragment was prepared from the clone thus obtained, and its nucleotide sequence was analyzed by a method such as meshing [NAR, 9 , 309 (1981)] to determine a nucleotide sequence encoding the amino acid sequence of the amino terminal portion of MPB64 protein. ,Finally
A cloned DNA containing a nucleotide sequence corresponding to the entire translation region of the MPB64 protein can be obtained.

(3) Expression of Gene Encoding MPB64 Protein To produce MPB64 protein using the gene encoding MPB64 protein obtained in (2) above, first, a substantial sequence of the cloned DNA is converted into an expression vector. By incorporating
Produce recombinant DNA for MPB64 protein production. Next, the recombinant DNA is introduced into a suitable host cell for transformation.

The desired MPB64 protein can be obtained by culturing the thus obtained transformant in a medium.

In the present invention, when expressing the gene of the cloned MPB64 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
i represent) belonging (E.ColiX1776 strain, E. coli JM103 strain (NAR 9, 309 (1981) ) E.coli JM109 strain is preferred.
In addition, Bacillus subtilis can also be used. There are no particular restrictions on the use of eukaryotes, but for example, Saccharomyces cerevisiae is preferred.

The expression vector may be a vector containing a replicon and a regulatory function compatible with the host cell and a promoter necessary for the host cell to express its own protein, or may contain the promoter. Improved ones 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, lactose (lac) promoter [Nat
ure, 281 , 544 (1979)], tryptophan (trp) promoter [NAR 8 , 4507 (1980)] and tac (tac).
Promoter [Pro.NAS, 78 21 (1983)] can be mentioned those having like.

Examples of the expression vector include 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.

Culture of the host cells (transformants) containing the recombinant DNA for MPB64 protein production thus obtained is performed 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 a suitable inducing agent depending on the promoter used in the culture, for example, in the case of a lac promoter, the culture of the transformant can be more efficiently performed by adding isoprol-β-D-thiogalactoside to the medium. 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-HCl (pH 8.0), 1 mM EDTA buffer, 5 mg of lysozyme was added, the mixture was incubated at 37 ° C. for 15 minutes, and then 0.5 ml of a 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 aqueous solution of ethidium bromide (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.
A 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 BCG-derived M already determined by M. Harboe et al.
30 amino acid sequences from the N-terminus of PB64 protein, Three types of synthetic oligonucleotide probes were prepared.

 That is, the following probes I, II and III.

Probe I (corresponding to ⁷ Glu to Gly ¹⁵ ): Probe II (for ¹³ Asp to ²² Met): Probe III (for ²⁴ Asp to ³⁰ Asn): 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, after removing the dimethoxytrityl group using 80% acetic acid, a concentration gradient solution consisting of acetonitrile in 100 mM triethylamine acetate buffer (pH 7.0) as a mobile phase using a reverse phase PHPLC (condition: YMC PACK AM-314 ODS column). And lyophilized.

Thus, the obtained three kinds of oligonucleotide probes were dissolved in 10 mM Tris-HCl (pH 8.0) and 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
The reaction was carried out at 37 ° C. for 1 hour with a total amount of 50 μm of Ci [γ- ³² P] ATP (specific activity 3000 Ci / mmol), 15 units, polynucleotide kinase]. The reaction solution was extracted with phenol, washed with chloroform, and passed through a cartridge for NEN SORB ^™ 20 DNA purification 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 In order to detect and fractionate chromosomal DNA fragments containing the gene for the MPB64 protein, a Southern hybridization test was performed using Southern hybridization.
This was performed according to the method of rn et al. (J. Mol. Biol 98 503 (1975)). That is, about 3 μg of the chromosomal DNA obtained in (1) was completely digested with the restriction enzyme KpnI, fractionated by 0.8% agarose gel electrophoresis, and then the agarose gel was dissolved in 1.5 M sodium chloride 0.5 M sodium hydroxide aqueous solution. , Shaken for 40 minutes.

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 μm of the ³² P-labeled probe of the above (2) was added to 5 ml of the 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.
After repeating this operation three times, the resultant was air-dried and subjected to autoradiography to detect a DNA fragment containing the gene of MPB64 protein (FIG. 1).

As can be seen from the results in FIG. 1, probes I, II and III
Only the DNA fragment which showed a band at about 4400 bp reacted with any of the above.

(4) Preparation of DNA Library Preparation of KpnI DNA Fragment To clone a DNA fragment digested with KpnI of about 4400 bp, which was detected by (3), 20 μg of BCG chromosomal DNA was completely digested with KpnI and then 0.8% agarose. Fractionation was performed by gel electrophoresis. Create a groove downstream of the target 4400 bp band and use a recovery buffer (50% glycerol, 40m
M Tris, 20 mM sodium acetate, 2 mM EDTA and 18 mM NaCl) were 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 obtain a KpnI 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 (manufactured by Takara Shuzo) and 20 units of KpnI restriction enzyme was reacted at 37 ° C. for 2 hours, and then extracted once with phenol and chloroform, respectively, 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)
Was prepared.

Preparation of recombinant DNA and DNA library KpnI DNA fragment solution prepared in (3)-
μg), 1 μm of the vector DNA solution prepared in (3)-
(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%, and glucose 0.1%, 300 μL of an 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 spread on an L-agar medium containing X-gal (1.3% agar was added to L-broth) and cultured at 37 ° C. for about 16 hours to obtain a clone of ampicillin-resistant, β-galactosidase non-retaining bacterium. A DNA library consisting of the transformed strain was prepared.

(5) Selection of clone having MPB64 protein gene (colony hybridization test) The 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, the sample was immersed in a double concentration SSC buffer, air-dried at room temperature, and 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 10
One colony was obtained from 0 colonies.

The plasmid DNA was extracted and purified from the transformant, digested with the restriction enzymes KpnI and PstI, and subjected to Southern hybridization in the same manner as in (3).

This resulted in a 4.4 kb Kpn I cleavage fragment and a 1.5 k
Bound to the Pst I cleavage fragment. MPB6 for both fragments
Although it is thought to contain four protein genes, we decided to clone a 1.5 kb PatI-cleaved fragment for ease of nucleotide sequence determination.

That is, the plasmid DNA of the transformant obtained above was cut with the restriction enzyme PstI, ligated with the restriction enzyme PstI cut product of the vector pUC18, and the host E. coli JM109 was transformed. Clones that bind to all of the probes I, II and III obtained were obtained.

(6) Determination of base sequence of cloned DNA A plasmid was isolated and purified from the clone of the above (5),
Those cut with the restriction enzyme Pst I were fractionated by agarose gel electrophoresis, and then Pst I 1.5 kb
The fragment was separated and purified.

This DNA was prepared by the method of Meshing et al. (Gene, 33 103 (198
5)), the nucleotide sequence of the 1.5 kb fragment was determined by Pro. NAS, 74 , 5463 (1977)).

FIG. 2 shows the nucleotide sequence of the gene encoding the MPB64 protein derived from BCG bacteria determined in this manner.

By comparing the determined nucleotide sequence with the amino acid sequence near the N-terminus of the already known MPB64 protein,
GCG at positions 212 to 214 in the figure is the N-terminal amino acid Al of MPB64 protein.
Corresponding to a, it was determined that TAGs 827-829 corresponded to the stop codon.

Therefore, a gene encoding MPB64 protein derived from BCG bacteria,
That is, it was found that the structural gene of the MPB64 protein corresponds to 212 to 826 in FIG. 2 (underlined part in the figure).

In addition, the nucleotide sequence at positions 143 to 212 corresponds to a signal sequence required for secretion of MPB64 protein.

The number of amino acid residues translated from the nucleotide sequence encoding MPB64 protein is 205, and the calculated molecular weight is 22,400.
The amino acid sequence is shown in FIG.

From the determination of the entire amino acid sequence, the present inventors have noticed that the amino acid sequence near the N-terminus of the MPB64 protein previously reported by M. Harboe et al. Is weakly incorrect.

That is, they found that the sixth and twelfth amino acids from the N-terminal were erroneously reported.

<Example 2> An MP was prepared using the expression vector pKK233-2 (Pharmacia, Sweden) and a chemically synthesized oligonucleotide.
The gene of B64 protein was ligated to produce MPB64 protein.

 Details of the production of this MPB64 protein are shown below.

(1) Cleavage of the PatI 1.5 kb fragment containing the MPB64 protein gene by restriction enzyme BanαI GGCAC from the 242nd position in the nucleotide sequence shown in FIG.
C is the recognition site for restriction enzyme Ban I.

Therefore, a fragment containing most of the MPB64 gene can be obtained by cutting the Pst I 1.5 kb fragment with Ban I.

That is, the plasmid isolated from the clone prepared in (5) of <Example 1> is cleaved with the restriction enzyme PstI, and the target PstI 1.5 kb is obtained by an operation such as agarose electrophoresis.
Fragments were prepared.

10 μg of this Pst I 1.5 kb fragment was added to 10 mM Tris-HC
l (pH 8.0), 7 mM MgCl ₂ , restriction enzyme Ban I in the presence of 7 mM DTT
The reaction was carried out at 50 ° C. for 3 hours at 12 units (manufactured by Toyobo).

Thereafter, the target Ban I-Pst I fragment was recovered by separation and purification by agarose electrophoresis.

The Ban I-Pst I fragment was dissolved (0.12 μg / μ) in 20 μ of TE buffer.

Preparation of chemically synthesized oligonucleotides 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 described below.

The linker thus prepared is described above in Example 2
> (1) not only complements the deficient part of the MPB64 protein gene of the Ban I-Pst I fragment, that is, the base sequence at positions 212 to 241 (see FIG. 2).
It also had a structure linked to the Nco I site.

(3) Treatment of expression vector pKK233-2 2 μg of the vector pKK233-2 was added to 20 mM Tris-HCl pH7.5,10
Restriction enzyme Pst I (16 units) in mM MgCl ₂ and 100 mM NaCl
s), treated with Nco I (12 units) 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.

Thereafter, the mixture is subjected to phenol treatment, chloroform treatment, ethanol precipitation, and further washed with 80% ethanol water and then dried to cut the target PstI-NcoI, pKK233-.
2 was prepared.

(4) Construction of Expression Vector pKKM64 The vector pKK233-2 (Ps) obtained in (3) of <Example 2>
(tI-NcoI fragment) 0.1 μg, B obtained in (1) of <Example 2>
A mixture of 0.12 μg of the an I-Pst I fragment, 3,3 pmol of the linker obtained in (2) of <Example 2> and 24 μm of A solution of DNA ligation kit (manufactured by Takara Shuzo) and 4 μm of B solution were reacted at 16 ° C. for 30 minutes. I let it. In other words, MP
An expression vector pKKM64 containing the structural gene of B64 protein was constructed.

Next, 76 µ of the reaction mixture was added to 100 µ of E. coli JM109 strain Compilant Cell (manufactured by Takara Shuzo), and allowed to stand for 60 minutes, further left at 42 ° C for 90 seconds, and then left at 0 ° C for 5 minutes. It was transformed.

This reaction solution was added to 300 μL of L-broth medium,
For 30 minutes.

Next, the suspension was centrifuged at 5,000 rpm for 5 minutes, the supernatant was discarded in half, and then resuspended.
The resultant was applied to an L-agar medium (1.3% agar was added to L-broth) and cultured at 37 ° C. for about 16 hours to obtain a clone of an ampicillin-resistant, β-galactosidase non-retaining bacterium.

That is, a clone containing the expression vector pKKM64 was thus obtained.

 This process is shown in FIG.

(5) Production of MPB64 protein by Escherichia coli E. coli JM109 strain containing the expression vector pKKM64 (E. coli A
J12369, FERM P-9846) and pKK233 as control
E. coli JM109 strain containing -2 was added to a double-concentration YT medium (tryptone 16 g /, extract yeast extract 10 g /, NaC
l 5 g /) 37 ml in 10 ml (containing 50 μg / ml ampicillin)
After culturing for 5 hours, IPTG was added to a concentration of 200 μg / ml, and the cells were further cultured for 15 hours. Centrifuge this culture (12,0
(00 rpm for 10 minutes), and after collecting the cells, the cells were suspended in 1 ml of sterilized water and subjected to ultrasonic treatment. 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 SDS-polyacrylamide electrophoresis of the two bacterial cell extracts and the standard MPB64 protein, the proteins were transferred to a nitrocellulose filter,
The reaction with the anti-MPB64 protein polyclonal antibody was examined (FIG. 5).

As shown in FIG. 5, the band generated at the same position as that of the standard sample was the E. coli containing the pKKM64 vector (FERM P.
-9846), the MPB64 protein gene was expressed in Escherichia coli,
It was confirmed that protein was produced.

<Effect> The BCG-derived MPB64 protein provided by the present invention can be used as a diagnostic agent for distinguishing tuberculosis from AM disease by an antigen-antibody reaction (ELISA).

Further, the gene encoding the MPB64 protein of BCG bacteria provided by the present invention and the method for producing MPB64 protein derived from BCG bacteria by genetic engineering techniques using the same provide a large amount of pure MPB64 protein useful as a diagnostic agent for tuberculosis. It is important to provide

Further, D encoding the MPB64 protein provided by the present invention
If a preferable sequence is selected from the NA sequence and a DNA probe labeled with biotin or radioisotope is prepared, tuberculosis can be diagnosed by this.

[Brief description of the drawings]

FIG. 1 shows the restriction enzymes BamHI, KpnI and Ps of chromosomal DNA of BCG bacteria.
It is a figure showing Southern hybridization of probes I, II, III to digests by tI, 4.4k indicated by the arrow.
b The KpnI fragment was cloned. FIG. 2 shows the determined 919 bp DNA base sequence. FIG. 3 shows the amino acid sequence of the MPB64 protein. FIG. 4 is a diagram showing the construction of a mature MPB64 expression vector, pKKM64. FIG. 5 is a diagram showing the results of Western blot analysis of cell extracts of Escherichia coli JM109 containing pKK233-2 and Escherichia coli JM109 containing pKKM64.

 ──────────────────────────────────────────────────続 き Continued from the front page (72) Inventor Nagai Sada 4-5-54 Ueno Nishi, Toyonaka-shi, Osaka (72) Inventor Takeshi Yamada 1-140 Hagiwaradaito, Kawanishi-shi, Hyogo Examiner Eiji Takahori (56) Reference Literature Infect, Immun, 52 [1] (1986) p. 293-302

Claims (3)

(57) [Claims] 1. A Mycobacterium bovis BCG having the following amino acid sequence (I):
(Hereinafter referred to as BCG bacterium). 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-derived BCG bacterium, which comprises culturing a transformant transformed with the plasmid into which the gene according to (1) has been incorporated in a medium, and collecting the produced MPB64 protein; Method for producing MPB64 protein.