JPH02169599A - Peptide including antigenic determinant region of mpb57 protein originating from bcg cell - Google Patents

Abstract

NEW MATERIAL:A peptide having the structure represented by the formula (n is 1 to 15), containing the antigen-decision zone of MPB57 protein originating from BCG. USE:A diagnostic for tuberculosis according to the antigen-antibody reaction (the ELISA method). PREPARATION:For example, the gene coding MPB57 protein is isolated from the chromosome DNA of BCG, cleaved with a restriction enzyme, bonded to an expression vector to prepare a recombinant DNA. The recombinant DNA is introduced into the host cells of, e.g., Escherichia coli, the transformed cells are cultured to form colonies and the colonies are screened to select clones. Subsequently, cloned DNA segment is prepared from the clone to take out the DNA containing the antigen-decision zone of MPB57 protein, which is bound to a vector. The vector is introduced into the host cells of Escherichia coli for transformation, the transformed cells are cultured to produce the peptide containing the antigen-decision zone of MPB57 protein.

Description

[Detailed description of the invention] Industrial application field> Mycobacterium? Bis BCG (Mycobae
t@riumbovia BCG (hereinafter referred to as BCG bacteria) contains the antigen-determining region of the white rice MPB 57 protein! zotide, a synthetic NA encoding the iptide,
The present invention relates to a recombinant DNA containing the synthetic NA, a transformant that has been transformed using the recombinant DNA, and a method for producing a peptide by culturing the transformant.

Prior Art> Even when a clinician suspects tuberculosis based on a chest photograph and stratification, it is often difficult to detect tuberculosis m by microscopic examination of the patient's sputum. In such cases, the cells are cultured to detect the bacteria, but this culture takes about three weeks. In addition, due to the similarity of symptoms between pulmonary tuberculosis and lung cancer, there are an increasing number of cases where lung cancer is misdiagnosed as pulmonary tuberculosis and anti-tuberculosis drugs are administered on a trial basis, but the cancer progresses until it is too late. Therefore, the present inventors.

MPB 57, a protein present in both BCG and Mycobacterium tuberculosis
Focusing on this, we began research to obtain a large amount of peptides containing the antigen-determining region of MPB 57 in pure form using synthetic methods or genetic engineering methods. Given the number of cases, the antigen-antibody reaction using the antigen-determining region of MPB 57 (
If a diagnostic agent that makes full use of the ELJSA method could be developed, it would be possible to diagnose tuberculosis extremely accurately and quickly.

However, in reality, about 300 proteins secreted by Mycobacterium tuberculosis and BCG are found in the bacteria.
Even the MPB 57 protein derived from G. bacterium requires electrophoresis and a number of complex column operations to extract it in its pure form. It is extremely difficult to obtain.

This means that MP can be produced by synthetic methods or genetic engineering methods.
B57! It is desired to obtain a large quantity of the 4-gutide containing the white antigen-determining region in pure form.

MIA problem to be solved by the present invention The problem of the present invention is to provide a peptide containing the antigen-determining region of the CG bacterium MPB 57 protein, which is effective in the diagnosis of tuberculosis.

Means to solve problems〉 The present inventors first attempted to solve the above problem.

The antigen-determining region of MPB 57 protein was determined.

In other words, big BI! MPB57i in the expression vector of J fungus
The white gene fragments are linked together and expressed as a fusion protein.
The protein was screened with an antibody, and the nucleotide sequence of the MPB 57 protein genetic pre-fragment of one reacting clone was determined, and based on this, the nucleotide sequence of the antigen-determining region was determined. and,
Gene t, which is a combination of the antigen-determining region gene and several fragments thereof, can be incorporated into an appropriate expression vector,
The protein can be produced in large quantities using Escherichia coli.

A peptide containing the antigen-determining region of MPB 57 could also be produced by a synthetic method.

The present invention will be described below (1) Determination of the antigen-determining region of the MPB 57 gene (
2) Synthesis of peptide containing antigen-determining region (3)
The expression of the resistance-determining region of the MPB 57 gene will be explained separately.

First, we extracted MPB 57 from the chromosomal DNA of BCG bacteria.
We have succeeded in isolating a gene encoding a protein (4! Face Show 63-205444).

This gene was cut with a restriction enzyme (for example, Siu 3AI), and the expression vector pUR278, 288, 289
(Ruth*r, O, and Muller-Hllll
, B,, EMBOJ, 2 +1791~1794
(1983)) and Hanahan's method (J, Mo1
．． BloI, 166, 557 (1983)].
strain) for transformation and colony formation by ampicillin selection.

Colony immunoassay (M
l l l*r, D, M, , 5toakdal
E., F., E., and Karn, J.

Proc, Natl, Acad, Set, US
(1985)) to screen for colonies that react with the anti-MPB 57 polyclonal antibody. A cloned DNA fragment vI4 was prepared from the clone obtained in this way, and the method of Metzing (N, A, R, , 9, 30
9 (1981)), and finally the nucleotide sequence corresponding to the antigen-determining region of MPB 57 protein can be determined.

(2) Chemical synthesis of peptide of antigen-determining region The peptide is chemically synthesized by solid phase method.

(3) Expression of the antigen-determining region of the MPB 57 protein gene Using the gene encoding the antigen-determining region of the MPB 57 protein obtained in t1) above, in order to produce the peptide, Considering stability, MPB5
The gene encoding the peptide was inserted n times (n = 1 to 15) into the BamHI site, which is a gene in the 7 genes.
The repeated genes are linked to tabulate recombinant DNA for producing the peptide of the antigen-determining region of MPB 57 protein. This recombinant DNA 1 is then introduced into appropriate host cells for transformation.

By culturing the thus obtained transformed strain in a medium, a fusion peptide containing the target antigen-determining region of MPB 57 protein can be obtained. Even if the peptide is produced in the form of a fusion peptide, in the present invention,
When expressing the cloned peptide gene containing the antigen-determining region of MPB57 protein, a wide range of prokaryotes or organisms can be used.

Combinations of eukaryotic host cells and expression vectors can be employed.

When using prokaryotes as host cells, there are no particular restrictions, but for example, 12 strains in the large intestine (hereinafter).

E, co, which belongs to the Escherichia coli (expressed as E, colt)
ilX1776 strain, B, colt JM103 strain (N
,A,R,9.

309 (1981) E, coll JM109 strain is preferred. In addition to this, a hayride can also be used.

There are also particular restrictions when using eukaryotes.

For example, Sazucharomyces cerevisiae and the like are preferred.

In addition, vectors for expression include vectors that contain reglicons and regulatory functions that are compatible with the above-mentioned host cells, and promoters necessary for the host cells to express their own proteins. Improved versions are preferred.

Specific examples of each of these combinations are shown below.

・If the host cell is E, call, pBR322 (Gon level, 2.95 (
1977)], lactose (lae
) promoter (Nature, 281.544(1
979) 1 trigthophan (trp) promoter (
N.A.R.

s, 4507 (1980)) and tack (tILc
) Promoter (Pro, N, A, S,, 78.21
(1983) E and the like.

As vectors for expression, pUC Baekme [ such as pUC18 containing the above-mentioned promoter in pBR322] can be used.
Method in endemology 101゜20 (19
83)) or PKK233-2 (manufactured by Pharmacia, Sweden).

・If the m-king cell is Sakuryoku Romaisenu cerevisiae, YRp 7 as nigerasmid (Nature, 282
．． 39 (1979)), but also glyceraldehyde-3-phosphate dehydrognase glomotor (J, B, C, 254, 9839 (1979)) as a promoter.
(1979)) and α-factor promoter.

In the present invention, prokaryotic cells are used as host cells? Although eukaryotes may be used, it is more preferable to use prokaryotes.

The host cell (transformant) containing the thus obtained recombinant DNA for producing 1e7°tide containing the antigen-determining region of MPB 57 protein may be cultured aerobically in a liquid medium. As a medium, for example, polyheptone,
In addition to the usual nutrient medium containing 9 mother extract, 1 common salt, gluco-7, etc., M9 minimal medium etc. can be used.

Cultivation is preferably carried out at about 30-40°C.

In addition, depending on the promoter used during culture, an appropriate forging agent (for example, in the case of the laa promoter, inglovir-β-D-thiogalactoside may be added to the medium) may be used to increase the efficiency of culturing the transformants. It can be done in a specific manner.

Note that the following abbreviations are used in the present invention.

dcTP: deoxycytidine trisunate EDTA: ethylenediaminetetraacetic acid kb: kilobase kbp: kilobase pair DIAE Nidiethylaminoethyl SDS Sodium nidodecyl sulfate SSC: 0.15M sodium chloride 0.015M sodium citrate (d47.0) buffer IPTG: Isozorobyl-β-D-thiogalactoside

<Example 1> (i) Creation of DNA library ■ PKKM5, in which MPB 57 genes were cloned
Add 10 μg of 7f lasmid to 20 units of restriction enzyme NaoI and 1
0 mM) Lis-HCl buffer (pi(7,5>
, 7mM magnesium chloride, and 80mM sodium chloride were reacted at 37°C for 2 hours, and then fractionated by 0.8% agarose gel electrophoresis. Create a groove downstream of the band of interest (~1000 bp) in recovery buffer (50% glycerol, 40 mM).

20mM sodium acetate, 2mM EDTA,
Add 18 mM Na(j) and collect the DNA by electrophoresis. This solution VC 2 times the amount of ethanol, 0.05 times the amount of 5
Add MNaCt.

DNA 1 was precipitated. After drying this precipitate under reduced pressure, 10 m
M Tris-salt IR(p)I 8. O) 1rytM
It was dissolved in EDTA buffer 7- to obtain an MPB 57 gene fragment. This fragment (approximately 2 pi) and restriction enzyme X Sau
10 units of 3AI, 10mM Tris salt bucket (pH 7,5), 7mM magnesium chloride, 100
After reacting a mixture of mM sodium chloride at 37°C for 2 hours, phenol 1. After each sample was extracted once with chloroform, it was precipitated with ethanol. After drying this precipitated IN under reduced pressure, 10 mM) lith-hydrochloric acid (Pk'18.0
) in 1 mM EDTA buffer.

MPB 57 Sau 3 people! It was made into a fragment solution.

■ Treatment of expression vector pUR27g, 288.289 10 mM) Rinu salt @ (pH 8,0),
7 mM Macnesia A, 100 mM sodium chloride, 2 mM 2-mercaptoethanol, 2μ9 vector pUR278.

A mixture of 10 units of restriction enzyme BamH1 was incubated at 30°C for 2 hours, and then incubated with phenol and chloroform for 1 hour each.
After each extraction, ethanol precipitation was performed.

After drying this precipitate fM under reduced pressure, 194μIVc of 50mM) Lys-Hanasaka buffer (PI(8,4) was dissolved and 1
．． 5 units alkaline phosphatase (E, collc7
5) was added and reacted at 65°C for 1 hour. moreover.

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 extract the DNA.
was precipitated. Transfer this precipitate to 1° Tris-HCl buffer 7
- (pHs, o) 1 It was dissolved in 20 μ7 of 1 mM EDTA to prepare a vector DNA fy solution of about 5.2 kb. A vector DNA solution t-- was prepared for pUR288 and pUR289 using exactly the same procedure.

■ Creation of recombinant DNA and DNA library (1) - Dissolution of the Sau 3AI DNA fragment prepared in ■
(0,1 μl, +1) - ■ 14&! vector DN
After reacting a mixture of A I'M (0.1μI) and 30μJ of A solution (manufactured by Zenshuzo Co., Ltd.) and 5μJ of B solution at 16°C for 30 minutes, 1 Reaction mixture 1
6μl of E, coil EQ192 strain competent cells 2
00 μl and allowed to stand at 0° C. for 60 minutes. After this, another 90 seconds at 42℃ and then 2 minutes at 0℃! ! 1 remained silent.

This mixture contains 1% bactodryptone, yeast extract go,
s%, sodium chloride 0.5%, glucose 0, 1%
Add 300 μl of L-gross medium consisting of
Let stand for 0 minutes. Next, centrifuge at 5000 rpm for 5 minutes, discard half of the supernatant, resuspend, and add 50 μl of ampicillin/
1141 on an L-agar medium (1.3% agar is added to L-Pros) and cultured at 37°C for about 16 hours to obtain ampicillin-resistant clones and create a DNA library consisting of transformed strains. Created.

(2) MPB 57! Selection of clones expressing the -e notation of the white antigen-determining region (colony immunoassay) Regarding the BCG DNA library in (1)-① above.

Colony immunoassay was performed to select transformants containing the antigen-determining region of MPB 57 protein.

Sokuchi, nitrocellulose membrane filter (5ch
laicher & 5chuel1 ao, 45μm
BA) K.

A colony of DNA type 2li1 indicated by fE+1)-○ was transferred, and the filter was placed on an L-agar medium with the colony facing upward, and cultured at 37° C. for 5 hours.

Next, the filter was treated with alkaline, neutralized with 1M Tris-HCl buffer (voice 7.5), immersed in SSC buffer with 2 times the lid strength, and air-dried at room temperature. After air drying, proyfinf solution Q (50mM Tris-#i acid buffer (pH 7,5), 150
A paper soaked with 5 mM sodium chloride, 5 mM magnesium chloride) was placed on top of the filter, and the protein was bound to the filter by turning the paper from above. ? ) degree of dryness, blocking solution (1% bovine serum albumin, 30 mM Tris-HCl buffer (P) 47.4), 75 mM
Soak in sodium chloride).

The filter was shaken for 300 minutes to block the part of the filter where protein was not adsorbed.

Add anti-MPB57 protein polyclonal antibody to the plotted filter and react at room temperature for 4 hours, then add washing buffer (25mM), Lys-HCl buffer (
After washing with pH 7.4), 150-sodium chloride) and postmortem, the secondary antibody (-2 oxidase-conjugated anti-rabbit IgG
antibody) for 4 hours at room temperature.

After the reaction, it was washed with washing buffer.

After washing, base lJM solution (0.96 m9/ml 4-
1-naphthol, 0.02% hydrogen peroxide, 100mM sodium chloride, 50mM Trinu hydrochloride buffer (P)17. '5)) was added and reacted to obtain a positive clone.

The nucleotide sequence of the foreign gene of the clone was determined, and the antigen-determining region that reacts with Tayoshi anti-MPB 57 *white polyclonal antibody was determined.

The antigen-determining region of MPB57 protein is as follows.

IIs -Lye-Tyr-Asn-Gly-Glu-
Glu-Tyr-L4u Therefore, a peptide containing the above antigen-determining region by chemical synthesis or genetic engineering techniques, in particular,
lu-Glu-Tyr-L@u) (n=1-15)
The antigen-determining region or its polymer represented by or @
(11*-Lys-Tyr-Amn-Gly-Glu
-Glu-Tyr-L@u)n (n=1 to +5) can be prepared for early diagnosis of tuberculosis.

In the above description, n''=1 to 15 is shown, but n is not necessarily limited to 1 to 15. Therefore, n) of 16 or more is also acceptable.However.

n) If it is 16 or more, 1) Separation n production is time-consuming; 2)
) A first problem arises in which the expression level definitely decreases. This is it υ
It is preferable that n=1 to 15.

To reiterate, in the present invention, the antigen-determining region, ie, Ile-Lye-Tyr-Amn-Gly-Glu-
Any peptide having at least Glu-Tyr-Lau can achieve the object of the present invention.

<Example 2> 1) Chemical synthesis of peptide containing antigen-determining region of MPB 57 protein Zle-Lys-Tyr-Aan-Gly-Glu-G
Synthesis of the peptide having the lu-Tyr-Lau-11s tD structure was performed as follows.

Fmoc-IIs 4111 fat (Fmoe-II・
is o, 54 mmol/y (p-alkoxybenzyl alcohol resin introduced at the proportion of fat) 0.3251
719t - Merrif1 equipped for shaking
The mixture was placed in a @ld solid-phase method reactor, suspended in dimethylformamide (20m), shaken for 300 minutes, and Fry+p
q-fle fallen fat was swollen.

This was subjected to the following p'mOe group removal cycle.

a) After shaking for 1 minute in 20 nLl of DMF, it was filtered (twice).

b) Fried for 3 minutes in 20% piperidine-DMF #r solution and filtered.

c) 20% piperidine:y-DMF solution IO was shaken for 0 min in 20 IXl and filtered to remove the Fm 6 c group.

d) Wash twice with 0MF20d.

・)+-Prono? Wash twice with Nord 20M.

Here, it was confirmed that the Fmoc group was completely removed by the Kalaer method, and if it was incomplete, the above removal cycle was repeated. If it was completely removed, it was subjected to the synthesis cycle shown below.

f) The nine H-11 resin obtained from the F+noc group removal cycle was swollen by shaking twice with DMF 20 d.

g) Fmoc-Les+ (0,283ffi9
, 0.8 Z mol) of DMFm solution (20+++j) was added and shaken for 1 minute.

h) 3 ynl of dicyclohexyl carmediimir methylene chloride solution was added and shaken for 700 minutes.

1) Washed twice with DMF20mj.

j) Washed twice with Ingro-Mothernol 2Qysl.

Here, it was confirmed whether the condensation was completed by the Kaiser method, and if it was incomplete, the above condensation cycle was repeated. Moreover, if the condensation was completely completed, the product was again subjected to the Pm o e group removal cycle, and then subjected to the condensation cycle performed in step g) using Fmoa -Tyr (tBu ). Thereafter, the Fmoc group removal cycle and Fmoc amino acid condensation cycle are repeated in the same manner to obtain Fmo
c-Glu (otBu), Fmoc-Glu (o
tBu), Fm66-GlyFmoc-Asn (DOD
), Fmoe-Tyr(tBu), Fmoe-Lys(
Boa), Boc-Its t-condensed sequentially. Next, it was subjected to a desorption step from the resin.

That is, it was washed twice with methylene chloride 201117, suspended in a mixed solution of methylene chloride (10d)-anisole (2,0Qml)-thiophenol (0,66IIL), and then washed with trifluoroacetic acid (20,IIL/)-chloride. Methylene (2,324) was added and shaken for 1 hour. The resin was filtered, ether was added to the obtained filtrate, and the mixture was filtered to obtain a white precipitate. The obtained precipitate was subjected to reverse phase liquid chromatography, the desired pegutide fraction was collected, and the obtained eluted fraction km was condensed to dryness. Next, distilled water was added and the mixture was concentrated and dried several times, then dissolved in a small amount of distilled water and freeze-dried.

Thus purified 11*-Lys-Tyr-Asn-
Gly-Glu-Glu-'pyr-L@u-IIs
I got it.

It was confirmed by thin layer chromatography and reversed phase liquid chromatography that the product was synthesized with high purity.

Furthermore, the synthetic pegyptate obtained in this invention reacted with an anti-MPB57 protein polyclonal antibody.

<Example 3> ■ Cleavage at the BmH1 site in MPB 57 gene of plasmid pKKM 57 MPB 57 protein direct expression vector pKKM 57 p
To eliminate the BamHI site derived from KK233-2! 1
After digestion with the restriction enzymes EeoRI and 5alI, ligation was performed.

i.e. plasmids pKKM 572111 and 100
mM Tris-HCl buffer (p'7.5), 7mM
Magnesium chloride A, 50-point sodium chloride and restricted # element E
After reacting a mixture of 20 units of eoRI at 37°C for 2 hours, add appropriate amounts of 3M sodium chloride and water to chlorinate the mixture.) Adjust the concentration of IJ to 175 rnM, add 20 units of restriction enzyme 5aiI, and incubate for 2 hours at 37°C. Allowed time to react. After the reaction, extract once each with phenol and chloroform.

After that, it was precipitated with ethanol. After drying, add 7mM Nu-HCl buffer (p&(7,5), 20m
M sodium chloride, 7mM magnesium chloride, 0.1
After reacting with 4 units of Klenow fragment (DNA polymerase I, manufactured by Zenshuzo Co., Ltd.) in an mM EDTA bath solution at 16°C for 30 minutes, ethanol precipitation is performed. After drying, it was dissolved in 8 μl of solution A of Ligauge Incinkinoto (Takara Shuzo Juku), 1 μl of solution B was added, and the mixture was allowed to react at 16° C. for 30 minutes.

1 μl of the reaction mixture was added to E. colt JM109 strain competent cells (100 μl of Zenshuzo Xi cellulose) and incubated at 0° C. for 60 minutes. After this, the mixture was further heated at 42°C for 90 seconds and then at 0°C for 5 minutes. Add 300 μl of L-Pross medium to this mixture and incubate at 37° C. for 30 minutes. Next, centrifugation was performed at 5,000 rpm for 5 minutes, half of the supernatant was discarded, and the mixture was resuspended, spread on an L-agar medium containing 50 μl of ampicillin, and cultured at 37° C. for about 16 hours to obtain ampicillin-resistant clones. From the clone obtained here II
The MIM-X plasmid has a unique BamHI site derived from the MPB 57 gene.

Therefore, 1.0μI of this Grasmid, 10mM Tris-HCl buffer (pH 8,0), 7mM magnesium chloride, 100ml of sodium chloride, and restriction enzyme BJ were added.
A mixture of 10 units of LmH is reacted at 30°C for 2 hours.

After the reaction, the mixture was extracted once with phenol and once with chloroform, and then precipitated with ethanol.

After drying, 50mM)
8,4) Dissolve in 194μ, add 1.5 units of alkaline phosphatase (E, colic75) and incubate at 65°C.
After 1 hour of reaction, 1.5 units of alkaline 7-osphatase was added, and after 1 hour of reaction, the mixture was extracted twice with phenol and twice with chloroform.

Ethanol was added to the aqueous layer to precipitate DNA. This precipitate was dissolved in 10 mM) Lys-HCl buffer (pH 8.0).
) Dissolve pKKM in 10 μl of 1 mM EDTA.
BamHI in the MPB 57 gene of the 577' lasmid
An in situ cleavage (PKKM57B) was prepared.

■ Chemically synthesized oligonucleotide y4 5', t! J Co-v-C):) GATCAAG
TACAACGGCGAG GAA TACCT Orico? - (Bottom) 5'GAT CAG GTA TrCC
TCGCCGTr GTA CTT" were synthesized. The specific synthesis operations are shown below. First, an automatic DN synthesis machine (Applied Biosystems, USA, 380A
After synthesizing the desired DN oligomer using (type), all groups other than the dimethoxytrityl group were removed and placed in a reversed phase. Pressure ram chromatography (conditions 20, using an 8-silica gel column, using a concentration gradient solution consisting of acetonitrile in ethylamine acetate buffer (P) (7,0), 100 mM as mobile phase)? I did my best.

After removing the somethoxytrityl group using 80% acetic acid in t and q, reverse phase HPLC (conditions: YMCPA
Using a CKAM-31400S column, 1
0° tin triethylamine acetate buffer (pH 7
, 0) A concentration gradient solution consisting of acetonitrile in medium is used.

) and lyophilized.

This synthetic oligomer (f), (bottom) is each l, 5
nmol each in the presence of ATP, lower left 4 polynucleotide kinase (E, aollA19) (manufactured by Zenshuzo)
10 units k was added and reacted at 37°C for 1 hour. After the reaction was completed, the mixture was treated with phenol and chloroform, and then heated at 65° C. for 5 minutes. After heating at 27oC, the desired oligomer as shown below was obtained by slow cooling.

”G ATCAAG TACAACGGCGAG
AA TACCT3'3, TTCATG TTGCCG
CTCCTT ATG GACTAG.

The oligomer prepared in this way has the property of being able to connect many units by repeating annealing and adding riders, with each unit having a size above K.
Furthermore, when amino fiK conversion is performed, 10 amino acids are linked back to ib so that no frame shift occurs. Therefore.

It is possible to easily obtain polymers (2-10) of antigen-determining regions.

Then, by applying 8% polyacrylamide (K) to a mixture having various units, a polymer of an appropriate size can be obtained.

■ Construction of pKKM 57 vector containing a polymer of antigen-determining region <Example 3> - Vector pKKM 57 B (obtained in Example 3)
BamHI cleavage) 0.1 μg, 3.3 pmol of the antigen region polymer of the BamHI fragment obtained in Example 3-■, and 12 μl of A bath solution and 3 μl of B bath solution of the DNA Ridersin kit (manufactured by Zenshuzo). The mixture was reacted for 30 minutes at 16°C.

This reaction mixture 10 Al f E, call JM
Add to 100 μl of IO9 strain competent cells (Takara Shuzo Rip).

Incubate for 60 minutes, then at 42℃ for 90 seconds, then at 0℃ for 2 seconds.
Transformation was carried out by allowing the cells to stand for a minute.

300 μl of L-pross medium was added to 1 ei of this reaction volume, and the mixture was incubated at 37° C. for 30 minutes.

Next, centrifuge at 5000 rpm for 5 minutes, and divide the supernatant by half.
Discarded and resuspended after death, L containing Ambicirif 50 μg/go
- Agar medium (add 1.3% agar to L-broth)
and cultured at 37°C for about 16 hours to obtain ampicillin-resistant clones. That is, in this way, the parent Baekme pKKM containing the antigen-determining region polymer (n=4)
57 prisoners were obtained (Figure 1).

■MPB containing polymer of antigen-determining region by E. coli
Production of 57 fusion protein pKKM 57 E containing CAI, eoll JMI
09 strain (E, callAJ12416.FERMP-
10445) and pKKM 57 (E, coil A
JI 2407, FERMP-10151) and E, co containing pKK233-2 as a control.
1, 1 JM109 strain in YT medium (Tryptone 16El/l, Yeast Extract 101/l)
, NaCt51! /l) 1. After culturing at 37° C. for 5 hours in OrnL (ambicilif 50 μm/ILI-containing solution), IPTG was added at a concentration of 200 μm/R1, and the cells were further cultured for 15 hours.

Centrifuge this culture solution (12,000 rpm for 0 minutes)
After collecting the cells, the cells were suspended in sterilized water.

Ultrasonic treatment was performed. Then, collect the bacteria again and add 2 to the supernatant.
Double a degree sample & Kayei 1F! : Added an equal amount and boiled at 100°C for 3 minutes to obtain a bacterial cell extract. 5O8 extract of one body of 3a
- After polyacrylamide gel electrophoresis, proteins were transferred to a nitrocellulose filter and anti-FilPB 57
The reaction between the protein and the polyclonal antibody was investigated (Figure 2).

pKK producing MPB 57 protein as shown in Figure 2.
Large B box containing M57 vector (FE Belly P-1015
In the band reacting with the bacterial cell extract in step 1), pKKM 57 (large MW containing Al vector
(E, eo11AJ12416.FERMP-104
Large MJ as a band was observed in the bacterial cell extract of 45).
It was confirmed that the gene for the MPB 57 protein containing the antigen-determining region polymer was expressed in - and that the fused -eftide was produced. Analysis of the amino acid sequence of this fusion peptide revealed the following amino acid sequence.

Met-Aim Ly @ Val Asn Ile L
ys Pro Leu Glu AapLys II@
L@u Val Gin Ala Asn Glu A
la GluThr Thr Thr Thr Ala S@r
Gly Leu Val IIs Pr.

Asp Thr Ala Lys Glu Lys P
ro Gin Glu GlyThr Mal val
Alm Vat Gly Pro Gly Arg
TrpAsp Glu AJIpGly Glu Ly
e Arg-(IIs-Lyg-TyrAsn-Gly
-Glu-Glu-Tyr-Leu)4-11s Pr
o LeuAsp Val Ala Glu Gly
Asp Thr Mal IIs TyrS@r L
ye Tyr Gly Gly Thr G
lu Ile Lys TyrAsn Gly
Glu Glu Tyr Lsu Ile Leu
Sar AliArg Amp Val Mm
l Gly Arg Arg Ph@L
Yeing effect> The peptide containing the antigen-determining region of the BCG bacterium-derived MPB 57 protein provided by the present invention has an antigen-antibody reaction (EL).
ISA method) It can be used as a diagnostic agent for tuberculosis caused by K.

Furthermore, the gene encoding the antigen-determining region of the MPB57 protein of the BCG bacterium provided by the present invention, and the MPB 57 of BCG Komahirarai produced by genetic engineering techniques using the gene, are also available.
A method for producing a peptide containing the antigen-determining region of the protein is essential in order to provide a large amount and pure MPB 57 artificial protein useful as a diagnostic agent for tuberculosis.

Furthermore, DNA encoding the peptide discovered by the present invention
Select a preferred sequence from among the sequences and apply a DNA probe labeled with biotin or radioisotope to MA.
Diagnosis of tuberculosis is also possible using this method.

[Brief explanation of the drawing]

Figure 1 shows the structure of the expression vector pKKM57. Iil!
shows. Figure 2 shows the large intestine containing pKKM57 @JM109 and PK
Large intestine containing KM57(A) @JM109 and pKK233
This is a diagram obtained by analyzing a bacterial cell extract of colon $iJM109 containing -2'& by Western blotting. 3 E containing pKK233-2, coll JMI09 strain C control Figure 2

Claims (10)

[Claims] (1) A peptide containing the antigen-determining region of MPB57 protein derived from BCG bacteria. (2) The peptide according to claim (1), which is represented by the following structure (I). Structure (I): [There is a gene sequence] n = 1 to 15 (3) The peptide according to claim (1), which is represented by the following structure (II). Structure (II): [There is a gene sequence] n = 1 to 15 (4) The peptide according to claim (1), which is represented by the following structure (III). Structure (III): [There is a gene sequence] n = 1 to 15 (5) Claims that the product is manufactured by chemical synthesis (
Peptides described in 1) to (3). (6) The peptide according to claims (1) to (4), which is produced by a recombinant DNA method. (7) A synthetic DNA encoding the peptide according to claims (1) to (4). (8) A recombinant DNA containing the synthetic DNA according to claim (7). (9) A transformant transformed with the recombinant DNA according to claim (8). (10) A method for producing a target peptide, which comprises culturing the transformant according to claim (9) to produce a peptide containing at least the antigen-determining region of the target BCG bacterium-derived MPB57 protein.