JP2000342272A - NEW POLYPEPTIDE, DNA CODING FOR THE POLYPEPTIDE, IMMUNOLOGICAL ASSAY OF ANTI-MYCOBACTERIUM TUBERCULOSIS 38kDa PROTEIN ANTIBODY AND ASSAYING REAGENT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new polypeptide composed of a polypeptide containing a specific amino acid sequence and useful for the immunoassay of anti- mycobacterium tuberculosis 38kDa protein antibody, etc., and the serodiagnosis of tuberculosis by the easy and quick detection of Mycobacterium tuberculosis. SOLUTION: This new polypeptide contains the amino acid sequence expressed by the formula. The polypeptide can be used for the accurate, quick and easy immunoassay of anti-mycobacterium tuberculosis 38kDa protein antibody and as a reagent for the immunoassay and is useful for the serodiagnosis of tuberculosis by the detection of Mycobacterium tuberculosis. The protein has been prepared by a gene recombination technique based on the finding that a polypeptide composed of the 285-374th amino acid sequence of the 38kDa protein of Mycobacterium tuberculosis has epitope on anti-mycobacterium tuberculosis 38kDa protein antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel polypeptide, a DNA encoding the same, a plasmid containing the DNA, a microorganism, a cultured cell, or an insect transformed with the plasmid, and an anti-M. Tuberculosis 38 kDa protein antibody. And a reagent for analysis.

[0002]

2. Description of the Related Art Mycobacterium
tuberculosis) is an infectious agent responsible for human tuberculosis disease. All of its genes have already been elucidated and are known to consist of 4,411,529 bases [Nature 393, pp. 537-544].
P. (1998)]. Most routes of transmission of Mycobacterium tuberculosis are through the respiratory tract due to droplets from evacuation patients. Tuberculosis in humans is the result of chronic inflammation caused by Mycobacterium tuberculosis. The pathogenesis is largely related to the host immune response. Tuberculosis is attracting attention as a "re-emerging infectious disease" in both developing and developed countries. Recently, outbreaks have been observed in schools and nursing homes in Japan, and the number of cases of massive eradication of M. tuberculosis has been increasing especially among the elderly, young people and foreigners, and about 130,000 people have been infected with TB. The diagnosis of tuberculosis is confirmed by detecting tuberculosis bacteria from a sample (mainly sputum). Effective treatments for tuberculosis include isoniazid, rifampicin, ethambutol, streptomycin, and the like. Early detection and treatment of tuberculosis patients can eliminate and cure tuberculosis bacteria in patients.

[0003] As a method for detecting Mycobacterium tuberculosis, for example, a culture method, a smear method, or a PCR method is used. However, in the culture method, the growth rate of Mycobacterium tuberculosis is low,
An 8-week culture period is required. In addition, the smear method has a drawback that it is not possible to distinguish between Mycobacterium tuberculosis and atypical acid-fast bacterium, although it is possible to rapidly detect Mycobacterium tuberculosis. Identification of Mycobacterium tuberculosis is performed by culturing Mycobacterium tuberculosis present in a test sample and determining whether the niacin test is positive or negative. Since the niacin test requires a large amount of cells, it is necessary to further increase the number of Mycobacterium tuberculosis bacteria after isolating them, which takes a considerable number of days. Also, some atypical mycobacteria have been reported to be positive for the niacin test. In the PCR method,
Skill is required in the operation of extracting a gene from a test sample or in the technique of the PCR method, and further, it is necessary to prevent DNA contamination (contamination). In this method, it takes 5 to 6 hours to obtain a result.

[0004] As a method for eliminating such drawbacks of the known detection method, and for accurately, quickly and easily detecting Mycobacterium tuberculosis, a serodiagnosis method using the 38 kDa protein of Mycobacterium tuberculosis as an antigen is known. In the serodiagnosis method, a result can be obtained simply and in a short time, similarly to a normal serodiagnosis method.

[0005]

However, in the above-mentioned serodiagnosis method, it is necessary to use a Mycobacterium tuberculosis 38 kDa protein as an antigen, and its purification has been difficult in that a large amount of Mycobacterium tuberculosis must be cultured.

[0006] The present inventors have prepared a polypeptide containing the amino acid sequence represented by SEQ ID NO: 15 in the Sequence Listing by using gene recombination technology, and the polypeptide is an anti-M. Tuberculosis 38 kDa protein antibody. And useful for serodiagnosis of Mycobacterium tuberculosis. The amino acid sequence represented by SEQ ID NO: 15 in the sequence listing consists of 91 amino acid residues,
The amino acid sequence from position 91 to position 91 is 38 kD of Mycobacterium tuberculosis.
28th of protein a (number of constituent amino acid residues = 374)
It is the same as the 5th to 374th amino acid sequences. The present invention is based on such findings.

The 285th to 374th amino acid sequence of the 38 kDa protein of Mycobacterium tuberculosis is
One of the regions where epitopes of known monoclonal antibodies specifically reacting with the 8 kDa protein are present [I
nfection and Immunity, 57, pp. 2481 to 2488 (1989)]. According to the literature, various C-terminal deletions of the Mycobacterium tuberculosis 38 kDa protein (that is, polypeptides in which the C-terminal region of the Mycobacterium tuberculosis 38 kDa protein is deleted) are prepared, and various known anti-M. Tuberculosis 38 kDa proteins are produced. It is disclosed that the epitope existence region of the known monoclonal antibody was determined by examining the reactivity with the monoclonal antibody. That is, the above-mentioned literature includes a known anti-tuberculosis bacterium 3
Among the 8 kDa protein monoclonal antibodies, monoclonal antibody HYT28, monoclonal antibody HAT2,
The epitopes recognized by the monoclonal antibodies HGT3 and TB72 are present in the C-terminal region (region consisting of the 285th to 374th amino acid sequence) of the Mycobacterium tuberculosis 38 kDa protein, while the monoclonal antibodies HBT12 and TB71
Is disclosed to be present in the central region (region consisting of the 117th to 284th amino acid sequences) of the Mycobacterium tuberculosis 38 kDa protein. However, the literature does not produce a polypeptide consisting of 90 amino acids 285 to 374, and discloses whether the polypeptide is effective in clinical tests using actual biological samples. Not. Therefore, it does not mention at all that the polypeptide comprising 91 amino acids found by the present inventor is effective in clinical tests using biological samples.

[0008]

Means for Solving the Problems The present invention relates to a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 15 in the sequence listing. The present invention also relates to a DNA encoding the polypeptide. Further, the present invention provides the DNA
And a plasmid comprising: The present invention also relates to a microorganism, a cultured cell, or an insect transformed by the plasmid. Further, the present invention provides a method for bringing a polypeptide into contact with a test sample, wherein the polypeptide and an anti-M. Tuberculosis 38
The present invention relates to an immunological analysis method for an anti-M. tuberculosis 38 kDa protein antibody, which comprises detecting a complex with a kDa protein antibody. Furthermore, the present invention relates to a reagent for immunological analysis of an anti-M. Tuberculosis 38 kDa protein antibody, which comprises the polypeptide.

[0009] The polypeptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 15 in the sequence listing. The amino acid sequence represented by SEQ ID NO: 15 in the sequence listing comprises 9
It consists of one amino acid residue, and its 2nd to 91st amino acid sequence (90 amino acids) has 38
It is identical to the 285th to 374th amino acid sequence of kDa protein (number of constituent amino acid residues = 374). The 285-th to 374-th amino acid sequence of the 38 kDa Mycobacterium tuberculosis protein does not contain a cysteine (SH group-containing amino acid) residue, nor does it have an amino acid sequence to which a sugar chain binds.

[0010] As described above in the section of the prior art, the 285th to 37th amino acids in the 38 kDa protein of M. tuberculosis.
The fourth amino acid sequence itself is a known anti-M. Tuberculosis 38 kDa protein monoclonal antibody, monoclonal antibody HYT28, monoclonal antibody HAT.
2. It is known as an epitope region of monoclonal antibody HGT3 and monoclonal antibody TB72 [I
nfunction and Immunity, 57,
Pages 2481 to 2488 (1989)], the polypeptide itself consisting of the amino acid sequence is novel without being described in the literature. Therefore, a polypeptide consisting of the “amino acid sequence represented by the sequence of SEQ ID NO: 15” in which methionine is further linked to the N-terminus of the amino acid sequence, A polypeptide comprising the `` amino acid sequence represented ''
Of course, it is new.

The polypeptide of the present invention is not particularly limited as long as it contains the amino acid sequence represented by SEQ ID NO: 15 in the Sequence Listing, and is not limited to the amino acid sequence represented by SEQ ID NO: 15 in the Sequence Listing. At the N-terminal and / or C-terminal of
It can be a polypeptide consisting of an amino acid sequence to which an arbitrary amino acid sequence is added. Examples of the amino acid sequence that can be added include, for example, all or a part of the amino acid sequence of glutathione S-transferase (GST) or β-galactosidase α peptide (LacZα).

The DNA of the present invention is not particularly limited as long as it encodes the polypeptide of the present invention. For example, a DNA comprising the base sequence represented by SEQ ID NO: 13 in the Sequence Listing may be mentioned. it can. The base sequence represented by SEQ ID NO: 13 in the sequence listing uses a codon frequently used in E. coli (Molecular B).
iology of the Gene, 4th Ed
ition, J. et al. D. Watson et al; Th
e Benjiam-in / Cummings Pub
lishing Company, Inc. , Cali
fornia), and therefore, is different from the above-mentioned nucleotide sequence represented by SEQ ID NO: 13 in the sequence listing, and the corresponding natural nucleotide sequence of the Mycobacterium tuberculosis 38 kDa protein.

The polypeptide of the present invention can be produced, for example, by a genetic recombination technique according to the procedure described below. First, based on the amino acid sequence of the polypeptide of the present invention, DN corresponding to those amino acid sequences
Select A codon and design nucleotide sequence. In designing a DNA codon, the properties of the host microorganism or cell in which the desired polypeptide is to be expressed are taken into account. For example, when expressing in E. coli, a codon advantageous for expression in E. coli is selected. Furthermore, a restriction enzyme cleavage site used for insertion into an expression vector, or an amino acid sequence that can be cleaved with a proteolytic enzyme (for example, thrombin or factor Xa) that restricts degradation when expressed as a fusion protein. May be appropriately introduced.

Next, it is divided into units suitable for DNA synthesis,
Sense DNA and antisense DNA (typically about 50 nucleotides or less) are individually chemically synthesized. After phosphorylation of the 5 'end with polynucleotide kinase, annealing is performed between the complementary DNAs to prepare a double-stranded DNA for each of the divided units. The plurality of double-stranded DNAs synthesized in this manner are sequentially linked using T4 DNA ligase, and an insertion DNA fragment consisting of the double-stranded synthetic DNAs (that is, DNG encoding the polypeptide of the present invention)
Create A). Subsequently, the DNA fragment for insertion is
It is incorporated into an expression vector cut with a restriction enzyme, and the obtained recombinant vector is introduced into a host microorganism or cell to form a transformant. After isolating the transformant, the transformant is cultured and gene expression is performed to prepare the polypeptide of the present invention.

As the host used in the above-mentioned polypeptide preparation step, a commonly used known microorganism such as Escherichia coli or yeast (Saccharomyces cerev) is used.
siae) or known cultured cells, for example, animal cells (for example, CHO cells or COS cells) or insect cells (for example, BmN4 cells). In addition, as the expression vector, a known plasmid can be appropriately selected depending on the host. For example, for E. coli, pUC, pTV, pGEX, pKK, or pT
rcHis; for yeast, pEMBLY or pY
ES2; pMAMneo for CHO cells; C
For OS cells, pcDNA3; for BmN4 cells, a vector having the polyhedrin promoter of silkworm nucleopolyhedrovirus (BmNPV) (for example, pB3).
K283) can be used. When the BmNPV expression system is used, the insect cells can be used, and by infecting a silkworm larva individual, it is possible to produce a larger amount of polypeptide (hundreds of times that in the case of using Escherichia coli). .

Further, the obtained polypeptide of the present invention can be isolated and purified by a known method, for example, affinity column chromatography or antibody column chromatography.

The polypeptide of the present invention analyzes anti-M. Tuberculosis 38 kDa protein antibodies present in the blood of M. tuberculosis infected patients ("detection" to determine the presence or absence of antibodies, (Including "measurement" to be quantitatively determined). As an analysis method using the polypeptide of the present invention as an antigen, conventionally known immunological analysis methods, for example, ELISA
A, the hemagglutination (PHA) method or the bead method can be used. As a test sample, antituberculosis bacterium 38k
The sample is not particularly limited as long as it is a sample that may contain the Da protein antibody, and particularly includes a biological liquid sample such as serum or saliva. In the immunological analysis method according to the present invention, the polypeptide of the present invention is brought into contact with a test sample, and an antigen-antibody reaction is carried out.
Incubation is performed under conditions capable of forming an antibody complex, and the formation of an antigen-antibody complex formed by binding of the polypeptide to the anti-M. Tuberculosis 38 kDa protein antibody is detected.

For example, when the ELISA method is used, the polypeptide of the present invention is immobilized on a suitable insoluble carrier (for example, an ELISA plate), a test sample is added to the insoluble carrier, and the antigen-antibody reaction is performed. Incubate under possible conditions (for example, at about 37 ° C. for about 30 minutes to 4 hours), wash the test sample, add an enzyme-labeled secondary antibody, and perform the same conditions under which an antigen-antibody reaction as described above is possible. , The second antibody is washed, the substrate for the above-mentioned labeled enzyme is added, the mixture is incubated under conditions that allow the enzyme reaction, and the signal from the enzyme reaction product is measured. The reagent for immunological analysis according to the present invention can be constituted using the same components as those of conventionally known immunological analytical reagents of the same type except that the polypeptide of the present invention is contained.

[0019]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << DN coding for 91AA polypeptide
Preparation of A >> DNA synthesis and ligation Sequence Nos. 1 to 1 in the sequence listing were obtained by a conventional method using an automatic type 381 DNA synthesizer (Applied Biosystems).
Single-stranded DNA 1 having a base sequence represented by each sequence of No. 2
Two types were synthesized. Hereinafter, the obtained DNAs are referred to as “DNA # 1” to “DNA # 12”, respectively. FIG. 1 shows the relationship between each base sequence and each DNA of the twelve types of DNAs # 1 to # 12 obtained by synthesis. Next, a kinase buffer solution containing 20 μg of each single-stranded DNA [composition: 5
0 mM-Tris-HCl (pH 8.0), 10 mM magnesium chloride, 5 mM dithiothreitol (DT
T), 1 mM-ATP], 0.1 ml of polynucleotide kinase (Toyobo), and adding each single-stranded DNA
Was phosphorylated at the 5 ′ end. FIG. 2 shows the phosphorylation step and each step to be performed below.

Phosphorylated DNA # 1 and DNA # 1
2 (10 μg each) in 0.1 ml of 50 mM Tris buffer
In addition, after heating at 95 ° C. for 5 minutes, the temperature is returned to room temperature over about 6 hours to carry out annealing to obtain a double-stranded D
NA [hereinafter referred to as DNA (I)] was prepared. In a similar manner, DNA # 2 and DNA # 11
(II), DNA # 3 and DNA # 10 were used to prepare DN.
A (III) was converted from DNA # 4 and DNA # 9 to DNA (I
V) from DNA # 5 and DNA # 8 to DNA (V)
And DNAs (V
I) was prepared respectively.

DNA # 1 contains CATGG at the 5 'end so that the start codon ATG in the base sequence of DNA (I) becomes a restriction enzyme NcoI cleavage sequence, while DNA # 1 contains
12 contains C at the 3 'end. Therefore, DNA # 1 and D
At the 5 ′ end of DNA (I) formed from NA # 12 (the 5 ′ end of DNA # 1), an end cut with the restriction enzyme NcoI is formed. DNA # 6 is 3 ′
It contains G at the end, while DNA # 7 has GA at the 5 'end.
Includes TCC. Therefore, the 3 ′ end (3 ′ of DNA # 6) of DNA (VI) formed from DNA # 6 and DNA # 7
(End), an end cut with the restriction enzyme BamHI is formed.

Next, using the DNA ligation kit (Takara Shuzo), the DNA (I) and DNA (II)
(2 μg each) was added to 10 μl of the A reaction solution.
Add 10 μl of B enzyme solution containing NA ligase, and add
For 30 minutes to perform ligation to obtain double-stranded DNA [hereinafter referred to as DNA (VII)].
Was prepared. In a similar manner, DNA (III) and D
Double stranded DNA (VIII) from NA (IV) and DN
Double-stranded DNA (IX) from A (V) and DNA (VI)
Each was produced. Further, DNA is prepared in the same manner as described above.
(VII), DNA (VIII), and DNA (IX) (each 2μ)
g) was added to 10 μl of the A reaction solution, and 10 μl of the B enzyme solution containing T4 DNA ligase was further added.
Ligation was performed by reacting for 0 minutes.

2. Purification of 280 bp DNA The obtained reaction product was subjected to electrophoresis on a 3.5% new sieve gel, and φX174 used as a marker was used.
A band corresponding to the 280 bp band of / HaeIII Digest (Toyobo) was cut out. Then, DNA P
The DNA was purified using rep (Dayatron) to obtain a double-stranded DNA (X) shown in FIG. The nucleotide sequence of the obtained DNA (X) is, by design, a nucleotide sequence represented by SEQ ID NO: 13 in the Sequence Listing, and the present invention comprises an amino acid sequence represented by SEQ ID NO: 15 in the Sequence Listing. (Hereinafter referred to as 91AA polypeptide). The actual nucleotide sequence was confirmed in Example 5 described later.

[0024]

Example 2 << Preparation of recombinant plasmid containing DNA encoding 91AA polypeptide / LacZα fusion protein >> Vector digestion and ligation with restriction enzymes BamHI and NcoI In this example, a recombinant plasmid containing a DNA encoding the 91AA polypeptide / LacZα fusion protein (20 kDa) shown in (1) of FIG. 6 is shown in FIG. It was prepared according to the procedure.

As an expression vector, pTV119N (Takara Shuzo) capable of binding to the LacZα gene and producing a fusion protein was used. The vector pTV119N was constructed using restriction enzymes NcoI and NcoI.
After performing an enzyme reaction at 37 ° C. for 4 hours using the buffer solution for I, phenol / chloroform extraction was performed, and ethanol precipitation was performed. This precipitate is subjected to Speed Vac (Amco).
After drying in a TE buffer [composition: 10 mM-Tris
-HCl (pH 7.4), 1 mM-EDTA (pH 8.
0)], a restriction enzyme BamHI and a buffer for BamHI were added, and the enzyme reaction was carried out at 37 ° C. for 2 hours to obtain a linear vector having NcoI cohesive ends and BamHI cohesive ends at both ends. The linear vector and the double-stranded DNA obtained in Step 2 of Example 1
(X) was ligated with T4 DNA ligase.

2. Transformation Using the reaction product obtained in the above step 1, transformation was carried out by a conventional method. That is, 50 mM-CaCl ₂
Competent cells (E. coli MV118)
(4 strains).

3. Confirmation of DNA Encoding Fusion Protein The transformant colony obtained in the above step 2 was inoculated into L-broth containing ampicillin (50 μg / ml) and cultured at 37 ° C. overnight. 1.5 m of this culture
was placed in an Eppendorf tube, cells were collected by centrifugation, and plasmid DNA was isolated by alkaline lysis (Molecular Cloning, A Labo).
rattro Manual 2nd Edition
n, J. et al. Sambrook, E .; F. Fritsch,
T. Maniatis; ColdSpring Har
Bor Laboratory, New York).
This plasmid was digested with restriction enzymes BamHI and NcoI, and subjected to electrophoresis using a 3.5% new sieve gel. The presence of a DNA band of about 280 bp was confirmed by a UV lamp in the presence of ethidium bromide.

[0028]

Example 3 << Preparation of Recombinant Plasmid Containing DNA Encoding Glutathione S-Transferase (GST) / 91AA Polypeptide Fusion Protein >> In this example, glutathione S-transferase (GST) shown in FIG. A) / 91AA polypeptide fusion protein (molecular weight = 37 kDa) was prepared in accordance with the procedure shown in FIG. In this example, as a vector containing a glutathione S-transferase (GST) gene, pGEX- capable of binding to the glutathione S-transferase gene and producing a fusion protein
Using 2T (Pharmacia), the BamHI site was used to prepare the title plasmid.

1. Synthesis of Primers Two types of primers (Primer A and Primer B) were synthesized by a conventional method using an automatic type 381 DNA synthesizer (Applied Biosystems). Each of the primers is composed of 30 bases and has the following base sequence including a BamHI cleavage site. Primer A: TGCAAGGATC CATGGCCGCT GGTTTCGCTT Primer B: GAACTGGATC CTAAGAAGAG ATAGTAGCGA

2. D encoding the 91 AA polypeptide
Amplification of NA 280 bp DNA purified in step 2 of Example 1
Using the two types of primers synthesized in step 1 of the present example, Polymerase ChainReac
The DNA was amplified by the Tion (PCR) method.
One cycle includes: (1) DNA denaturation step: 1 minute at 94 ° C. (2) Annealing step between single-stranded DNA and primer: 1 minute at 60 ° C. (3) DNA synthesis step by DNA polymerase: 72
The cycle was repeated 35 times, consisting of 1 minute at ° C.

3. Digestion of PCR product with restriction enzyme BamHI Next, the PCR product obtained in the above step 2 was treated with 3.5%
The gel corresponding to the 280 bp band of φX174 / HaeIII Digest (Toyobo) used as a marker was cut out by gel electrophoresis of New Sheave, and the DNA was removed.
DNA was purified using Prep (Dayatron). This DNA was dissolved in TE buffer, and the restriction enzyme Ba was
mHI and BamHI buffer were added to perform the enzyme reaction.
The reaction was performed at 7 ° C. for 2 hours to obtain a DNA fragment having BamHI cohesive ends at both ends.

4. Digestion and Ligation of Expression Vector with Restriction Enzyme BamHI After digestion of the expression vector pGEX-2T with the restriction enzyme BamHI, phenol / chloroform extraction was performed, followed by ethanol precipitation. The precipitate was dried with a Speed Vac (Amco) to obtain a linear vector having BamHI cohesive ends at both ends. This linear vector and the DNA fragment obtained in the step 3 were subjected to T4DN
Ligation was performed using A ligase.

5. Transformation Using the reaction product obtained in the above step 4, transformation was carried out by a conventional method. That is, 50 mM-CaCl ₂
Competent cells prepared by treatment (Escherichia coli DH5 strain)
Was introduced with plasmid DNA.

6. Confirmation of DNA Encoding Fusion Protein The colony of the transformant obtained in the above step 5 was inoculated into L-broth containing ampicillin (50 μg / ml) and cultured at 37 ° C. overnight. 1.5 m of this culture
was placed in an Eppendorf tube, the cells were collected by centrifugation, and the plasmid DNA was isolated by the alkaline lysis method. This plasmid is cut with the restriction enzyme BamHI,
Electrophoresis was performed using a 3.5% NewSieve gel, and the presence of a DNA band of about 280 bp was confirmed with a UV lamp in the presence of ethidium bromide.

[0035]

Example 4 << DN encoding 91AA polypeptide
Preparation of Recombinant Plasmid for Confirmation of Base Sequence of A >> In this example, the procedure shown in FIG. 5 was used in order to confirm the base sequence of “DNA encoding 91AA polypeptide” prepared in Example 1 above. Using the BamHI site contained in the polycloning site of pUC18 (Takara Shuzo), the recombinant plasmid of the title was prepared.

1. Digestion of Expression Vector with Restriction Enzyme BamHI and Ligation The vector pUC18 (Takara Shuzo) was digested with the restriction enzyme BamHI, followed by phenol / chloroform extraction and ethanol precipitation. The precipitate was dried with a Speed Vac (Amco) to obtain a linear vector having BamHI cohesive ends at both ends. This linear vector and the DNA fragment obtained in Step 3 of Example 3 were ligated using T4 DNA ligase.

2. Transformation Using the reaction product obtained in the above step 1, transformation was carried out by a conventional method. That is, 50 mM-CaCl ₂
Competent cells prepared by treatment (Escherichia coli DH5 strain)
Was introduced with plasmid DNA.

3. D encoding the 91AA polypeptide
Confirmation of NA The colony as a transformant obtained in the above step 2 was inoculated into L-broth containing ampicillin (50 μg / ml) and cultured at 37 ° C. overnight. 1.5 m of this culture
was placed in an Eppendorf tube, the cells were collected by centrifugation, and the plasmid DNA was isolated by the alkaline lysis method. This plasmid is cut with the restriction enzyme BamHI,
Electrophoresis was performed using a 3.5% NewSieve gel, and the presence of a DNA band of about 280 bp was confirmed with a UV lamp in the presence of ethidium bromide.

[0039]

Example 5 << DN encoding 91AA polypeptide
Confirmation of base sequence of A >> Isolation of plasmid The transformant obtained in Step 2 of Example 4 was inoculated into 50 ml of L-broth containing ampicillin (50 µg / ml), and cultured at 37 ° C overnight. This culture solution is
A plasmid Midi kit (Qiagen) was obtained from cells obtained by centrifugation at 000 rpm × 10 minutes.
Inc. ) Was used to purify the plasmid DNA.

2. Determination of DNA base sequence The DNA obtained in the above step 1 was subjected to Taq Dye De
oxy Terminator Cycle Sequ
Amplification was performed with a PCR device (Takara Shuzo) using an Encoding Kit (Applied Biosystems). Unreacted low-molecular-weight substrate is converted into a spin column (Boehringer, Qic
k Spin 50), followed by DNA Sequ
When the nucleotide sequence was determined using an encoder 373A apparatus, it was consistent with the nucleotide sequence represented by SEQ ID NO: 13 in the sequence listing.

[0041]

Example 6 << Confirmation of fusion protein >> Expression of fusion protein Each transformant in which the presence of a DNA band of about 280 bp was confirmed in step 3 of Example 2 and step 6 of Example 3 (transformant 5 confirmed in step 3 of Example 2)
The clone and the transformant (3 clones confirmed in step 6 of Example 3) were inoculated into L-broth containing ampicillin (50 μg / ml), and cultured at 37 ° C. overnight. Subsequently, induction protein isopropylthiogalactoside (IPTG) was added to express the fusion protein.

2. Confirmation of fusion protein by Western blot 1.0 μl of the culture solution obtained in the above step 1 was placed in an Eppendorf tube, centrifuged, and then 100 μl of cells
The cells were suspended in phosphate buffered saline (PBS). 10 μl of the suspension and a sample buffer [composition: 1% sodium dodecyl sulfate (SDS), 1% mercaptoethanol,
0.01% bromophenol blue (BPB), 50m
After electrophoresis of 10 μl of M-Tris-glycine (pH 8.3, 20% glycerol) on a 12.5% polyacrylamide gel (presence of 0.4% SDS), a blotting buffer [25 mM-Tris-HCl (pH 8.3)] was used.
3), 192 mM glycine, and 20% methyl alcohol] (50 V;
70 minutes). This nitrocellulose membrane was washed with 10 mM Tris-HCl (pH 7.4) / 15 containing 10% FCS.
After blocking with 0 mM-NaCl, M. tuberculosis antibody-positive serum was used as the first antibody, and reacted for 1 hour at room temperature on a nitrocellulose membrane. The M. tuberculosis antibody-positive serum was used as a M. tuberculosis antibody test reagent (Anda Biol).
human sera that was positive in an ELISA method using E. coli (M.Ogicals).

Subsequently, a phosphatase-labeled goat anti-human IgG antibody was reacted as a second antibody at room temperature for 1 hour. Next, as a substrate, nitro blue tetrazolium (NBT)
And activity staining of alkaline phosphatase was performed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Note that the buffer solution contains 0.1 M-Tr
is-HCl (pH 9.5) /0.1 M NaCl / 5
Using mM-MgCl _2.

As a result of the Western blot,
About 5 clones of the transformant confirmed in step 3 of
In all the clones, a band appeared at the position of 20 kDa. With respect to the three clones of the transformant confirmed in step 6 of Example 3, a band appeared at a position of 37 kDa in all the clones.

[0045]

Example 7 << Detection of Mycobacterium tuberculosis Antibody in Human Serum Using Fusion Protein >> Preparation of Nitrocellulose Membrane for Western Blot The transformant obtained in step 5 of Example 3 was cultured overnight at 37 ° C. in L-broth containing ampicillin (100 μg / ml), and then fresh ampicillin (100 μg / ml) was added.
ml) of L-broth. IPTG (final concentration: 5 mM) when 4 hours have passed after culturing at 37 ° C.
Was added, and culturing was further performed for 4 hours. This culture was centrifuged (3,000 rpm; 10 minutes), the obtained cells were mixed with a sample buffer, and electrophoresed on a 12.5% polyacrylamide gel (with 0.4% SDS). Blotting buffer [25 mM Tris-HCl (pH
8.3), 192 mM glycine, and 20% methyl alcohol].
V; 70 minutes). This nitrocellulose membrane is 10% FC
10 mM Tris-HCl (pH 7.4) containing S /
After blocking with 150 mM NaCl, it was cut and divided into 22 equal parts. One of them was used for staining with amide black to confirm whether the transfer was successful.

2. Western Blot Positive sera include Mycobacterium tuberculosis antibody test reagent (Anda B)
11 human sera that were positive in the ELISA method using the same (Iologicals) were used. As a control, 10 normal human serum samples were used. Using the above-mentioned positive serum or normal human serum as the first antibody, the reaction was carried out at room temperature for 1 hour on the nitrocellulose membrane for western blot prepared in the above-mentioned step 1. Subsequently, a phosphatase-labeled goat anti-human IgG antibody was reacted at room temperature for 1 hour as a second antibody. NBT and BCIP as substrates
Was used for phosphatase activity staining to detect M. tuberculosis antibody present in the serum.

Table 1 shows the results. In Table 1, "++
"+" Is strong positive, "++" is medium positive, "+"
Means weakly positive. Step 5 of the third embodiment
The GST / 91AA polypeptide fusion protein (molecular weight = 37 kd) produced by the transformant obtained in the above was detected in 9 of 11 cases of M. tuberculosis antibody-positive samples,
No detectable in control normal human serum. Therefore, G contained in E. coli expressed by induction of IPTG
The ST / 91AA polypeptide fusion protein was confirmed to be effective in detecting a Mycobacterium tuberculosis 38 kDa protein antibody formed after infection with Mycobacterium tuberculosis.

<< Table 1 >> Western Blot (37 kDa) Sample Number of Samples ++++++++ Total (%) Mycobacterium tuberculosis antibody positive serum 1 1 4 3 2 9 81.8 Normal serum 1 000 000

[0049]

Example 8 << Purification of GST / 91AA polypeptide fusion protein >> Culture The transformant obtained in step 5 of Example 3 was inoculated into 100 ml of L-broth containing ampicillin (50 μg / ml), and cultured at 37 ° C. overnight. This culture solution 1
00 ml of L-containing ampicillin (50 μg / ml).
Spinner flask containing 3 liters of broth (capacity = 3
Liter) and cultured with stirring at 37 ° C. for 4 hours. Next, IPTG (700 mg) was added, and the cells were further cultured for 4 hours. The culture was returned to room temperature and stirring was continued overnight.

2. Preparation of GST / 91AA polypeptide fusion protein crude fraction The culture was centrifuged at 4 ° C (6,000 rpm; 1
5 minutes) [performed twice using 6 500 ml centrifuge tubes], and the precipitated cells were washed with 20 mM Tris-HC.
1 (pH 8.0) / 1% Triton X-100 about 4
0 ml, and centrifuged again at 4 ° C. (12,000
rpm × 10 minutes). The cells obtained here are called L
ysis Buffer [50 mM Tris-HCl
(PH 8.0) / 1 mM-EDTA / 100 mM-Na
Cl] (9 ml), 50 mM of 5 mM phenylmethanesulfonyl fluoride (PMSF) and 240 µl of lysozyme (10 mg / ml) were added, and the cells were lysed on ice for about 20 minutes. When the viscosity appears, DNase I (3
The DNA was degraded by adding 20 μl (mg / ml). PBS / 1% Triton X-100 was added to make the total volume about 40 ml, and freezing and thawing were repeated twice. This is centrifuged (12,000; 10 minutes)
The supernatant was obtained.

3. Separation and Purification by Glutathione-Sepharose 4B Column The fraction obtained in the step 2 was applied to a glutathione-Sepharose 4B column (2.5 × 4 cm), and PB
S / 1% Triton X-100 (10ml) and P
After washing with about 20 ml of BS, 5 mM glutathione / 5
Elution was carried out with 50 ml of 0 mM Tris-HCl (pH 8.0) and 20 ml of 3M NaCl. GST / 91AA
The eluted fraction of the polypeptide fusion protein was 12.5%
After electrophoresis on a polyacrylamide gel (in the presence of 0.4% SDS), it was confirmed by performing Coomassie staining.

4. Separation and purification using a butyl-Sepharose 4B column About 80 ml of the fraction obtained in the above step 3 was subjected to 2M- (NH
₄ ) Dialysis was performed overnight in a low-temperature room against ₂ SO ₄ (500 ml), and the liquid volume was about 50 ml. This was applied to a butyl-Sepharose 4B column (2.5 × 3 cm), washed with about 20 ml of a 2M aqueous solution of ammonium sulfate, and then washed with 30 ml of a 1M aqueous solution of ammonium sulfate.
Elution was carried out with 60 ml of 4M ammonium sulfate aqueous solution and 20 ml of 50 mM phosphate buffer (PB) (pH 6.5). The eluted fraction of the GST / 91AA polypeptide fusion protein was a 12.5% polyacrylamide gel (0.4
(In the presence of% SDS), and confirmed by Coomassie staining.

5. Separation and purification by Sephaacryl S-200 column 10 ml of the peak fraction of the GST / 91AA polypeptide fusion protein obtained in the above step 4 was separated by Sephaacryl S.
Apply to a -200 column (2.5 x 95 cm),
0.2M-Tris-HCl (pH 9.5) /0.2M
Eluted with -NaCl. The eluted fraction of the GST / 91AA polypeptide fusion protein was confirmed by performing Coomassie staining after electrophoresis on a 12.5% polyacrylamide gel (in the presence of 0.4% SDS).

[0054]

Example 9 << Separation and Purification of 91AA Polypeptide >> After dissolving 0.5 mg of the GST / 91AA polypeptide fusion protein purified in Example 8 in PBS, 50 μg of thrombin (Boehringer Mannheim; derived from human plasma) was added. At 37 ° C. for 2 hours. This reaction solution was applied to a glutathione-Sepharose 4B column (2.5 × 4 cm), and about 20 ml of PBS was applied.
And the fractions collected were passed through. 16. eluted fractions
Tricine-SDS using 5% polyacrylamide gel
After performing electrophoresis, Coomassie staining was performed to confirm that the polypeptide was a 91AA polypeptide. That is, a protein band appeared at the position of 9 kDa.

[0055]

Example 10 << Detection of Mycobacterium tuberculosis 38 kDa antibody in human blood by ELISA using 91AA polypeptide >> The 91AA polypeptide obtained in Example 9 was
It was dissolved in a 0.1 M bicarbonate buffer (pH 9.5) to a concentration of 1 μg / ml. Each well of a microtiter plate was coated with 100 μl of the lysate. Each well contains 0.01% SDS / 0.05% Twe
en 20 / PBS after washing with 1%
Blocking was performed with 0.1 M bicarbonate buffer (pH 9.5) containing bovine serum albumin (BSA).

The human serum used in Example 7 was reacted with the 91AA polypeptide coated on the plate at 37 ° C. for 2 hours. After washing the plate, an appropriately diluted goat anti-human IgG (H chain and L chain) alkaline phosphatase conjugated antibody [Kirkegaard & Perr
y Laboratories, Inc. (KPL)]
100 μl was added. The plate was reacted at 37 ° C. for 2 hours. After the last washing, the coloring reagent (AB
TS Peroxidase Substrate; K
PL), and the mixture was allowed to react at room temperature for 10 to 30 minutes. Thereafter, 100 μl of 1% SDS was added to stop the reaction. When the absorbance at 405 nm was measured, 9
It was confirmed that the 1AA polypeptide reacted with the Mycobacterium tuberculosis 38 kDa protein antibody. This result is shown in Example 7
Western blot (Table 1).

[0057]

According to the polypeptide of the present invention, it is possible to provide an accurate, rapid, and simple method for immunological analysis of anti-M. Tuberculosis 38 kDa protein antibody.

[0058]

[Sequence listing free text] Each base sequence described in the sequence of SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing corresponds to SEQ ID NO: 1 in the sequence listing.
3 is a partial sequence of the nucleotide sequence described in Sequence 3. Each base sequence described in the sequence of SEQ ID NO: 7 to SEQ ID NO: 12 in the sequence listing is a partial sequence of the base sequence described in the sequence of SEQ ID NO: 14 in the sequence listing. In addition, the base sequence described in SEQ ID NO: 13 in the sequence listing includes a base sequence encoding the amino acid sequence described in SEQ ID NO: 15 in the sequence listing. Also,
The base sequence described in SEQ ID NO: 14 in the sequence listing is a complementary base sequence to the base sequence described in SEQ ID NO: 13 in the sequence listing. Furthermore, the 2nd to 91st amino acid sequences in the amino acid sequence described in SEQ ID NO: 15 in the sequence listing are amino acid sequences derived from Mycobacterium tuberculosis.

[0059]

[Sequence List] <110> Iatron Laboratories, Inc <120> Novel Polypeptide, DNA Coding Therefor, and Method and Reagent for Immunologically Assaying Anti-Mycobaterium tuberculosis 38 kDa Protein Antibody <130> IAT99008P <160> 15 <210> 1 <211 > 46 <212> DNA <213> Artificial Sequence <220> <223> This is a partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. <400> 1 catggccgct ggtttcgctt ctaaaactcc tgctaaccag gctatc 46 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. is there. <400> 2 tctatgatcg acggtccggc tccggacggt tacccgatca tcaacta 47 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. is there. <400> 3 cgaatacgct atcgttaaca accgtcagaa agacgctgct actgctca 48 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. is there. <400> 4 gactctgcag gctttcctgc actgggctat cactgacggt aacaaggctt 50 <210> 5 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. is there. <400> 5 cgttcctgga ccaggttcac ttccagccgc tgccgccggc tgttgttaa 49 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 13 in the sequence listing. is there. <400> 6 actgtctgac gctctgatcg ctactatctc ttcttag 37 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> This is a partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. . <400> 7 gatcctaaga agagatagta gcgatcagag cgtcagacag tttaacaaca 50 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. is there. <400> 8 gccggcggca gcggctggaa gtgaacctgg tccaggaacg aagccttgt 49 <210> 9 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. is there. <400> 9 taccgtcagt gatagcccag tgcaggaaag cctgcagagt ctgagcagta 50 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. is there. <400> 10 gcagcgtctt tctgacggtt gttaacgata gcgtattcgt agttgatg 48 <210> 11 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. is there. <400> 11 atcgggtaac cgtccggagc cggaccgtcg atcatagaga tagcctg 47 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> A partial sequence of the base sequence described in SEQ ID NO: 14 in the sequence listing. is there. <400> 12 gttagcagga gttttagaag cgaaaccagc ggc 33 <210> 13 <211> 277 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 15 in Sequence Listing including. <400> 13 catggccgct ggtttcgctt ctaaaactcc tgctaaccag gctatctcta tgatcgacgg 60 tccggctccg gacggttacc cgatcatcaa ctacgaatac gctatcgtta acaaccgtca 120 gaaagacgct gctactgctc agactctgca ggctttcctg cactgggcta tcactgacgg 180 taacaaggct tcgttcctgg accaggttca cttccagccg ctgccgccgg ctgttgttaa 240 actgtctgac gctctgatcg ctactatctc ttcttag 277 <210> 14 <211> 277 <212> DNA < 213> Artificial Sequence <220> <223> This is a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO: 13 in the sequence listing. <400> 14 gatcctaaga agagatagta gcgatcagag cgtcagacag tttaacaaca gccggcggca 60 gcggctggaa gtgaacctgg tccaggaacg aagccttgtt accgtcagtg atagcccagt 120 gcaggaaagc ctgcagagtc tgagcagtag cagcgtcttt ctgacggttg ttaacgatag 180 cgtattcgta gttgatgatc gggtaaccgt ccggagccgg accgtcgatc atagagatag 240 cctggttagc aggagtttta gaagcgaaac cagcggc 277 <210> 15 <211> 91 <212> PRT < 213> Artificial Sequence <220> <222> (2) .. (91) <223> This is an amino acid sequence derived from Mycobacterium tuberculosis. <400> 15 Met Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn Gln Ala ILe 1 5 10 15 Ser Met ILe Asp Gly Pro Ala Pro Asp Gly Tyr Pro ILe ILe Asn 20 25 30 Tyr Glu Tyr Ala ILe Val Asn Asn Arg Gln Lys Asp Ala Ala Thr 35 40 45 Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala ILe Thr Asp Gly 50 55 60 Asn Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro Leu Pro 65 70 75 Pro Ala Val Val Lys Leu Ser Asp Ala Leu ILe Ala Thr Ile Ser 80 85 90 Ser

[Brief description of the drawings]

FIG. 1. D encoding the designed polypeptide of the present invention.
FIG. 2 is an explanatory diagram showing a base sequence of NA.

FIG. 2 is an explanatory view showing a process for producing the DNA shown in FIG.

FIG. 3 is an explanatory diagram showing a process for preparing plasmid pTV119.Pep1.

FIG. 4 is an explanatory diagram showing a step of preparing a plasmid pGEX-2T · Pep1.

FIG. 5 is an explanatory diagram showing a process for preparing plasmid pUC18 · Pep1.

FIG. 6 is an explanatory diagram showing the structure of a fusion protein.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Hidenobu Nogi 1-11-4 Higashikanda, Chiyoda-ku, Tokyo F-term in Yatron Co., Ltd. (Reference) 4B024 AA13 BA80 CA03 DA06 EA04 GA11 4B065 AA01Y AA26X AB01 AC14 BA02 CA24 CA46 4H045 AA10 BA21 CA11 DA86 EA50 FA72 FA74

Claims (6)

[Claims] 1. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 15 in the sequence listing. 2. A DNA encoding the polypeptide according to claim 1. 3. A plasmid containing the DNA according to claim 2. 4. A microorganism, cultured cell, or insect transformed by the plasmid of claim 3. 5. The polypeptide according to claim 1, which is brought into contact with a test sample, and the polypeptide and the anti-tuberculosis bacterium 38k are contacted.
An immunological analysis method for an anti-M. Tuberculosis 38 kDa protein antibody, which comprises detecting a complex with a Da protein antibody. 6. A reagent for immunological analysis of an anti-M. Tuberculosis 38 kDa protein antibody, which comprises the polypeptide according to claim 1.