JPH10218895A - Modified protein, dna capable of coding for the same, recombinant vector containing the same dna, transformant containing the same recombinant vector, production of modified protein, measurement and measuring reagent for anti-triponema pallidum antibody, diagnostic agent for infection with syphilis and production of antitreponema pallidum antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new modified protein, comprising an amino acid sequence in which cysteine residue of a protein is converted into a lipid-nonbinding amino acid residue and useful as an antigen and a measuring reagent, etc., for an anti-Treponema pallidum antibody and for diagnosis, etc., of syphilis. SOLUTION: This new modified protein has a specific amino acid sequence in which cysteine residue of a protein is converted into a lipid-nonbinding amino acid residue such as alanine residue, glutamic acid residue or serine residue, etc., and is at least one of antigenic proteins having about 47kDa and about 17kDa molecular weight of a Treponema pallidum spirochete, etc., and useful as a measuring reagent for an anti-Treponema pallidum antibody, a diagnostic agent for syphilis and an antigen, etc., for producing the anti-Toriponema paridum antibody. The modified protein is obtained by extracting a DNA from the Treponema pallidum spirochete, amplifying the extracted DNA with a primer in which the cysteine residue of a gene of antigen having 47kDa molecular weight is substituted by alanine residue and expressing the resultant modified protein gene in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a modified protein,
DNA encoding the same, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a method for producing a modified protein, a method and a reagent for measuring an anti-Treponemal pallidum antibody, a diagnostic agent for syphilis infection, and an anti-treponema -It relates to a method for producing a palydam antibody.

[0002]

2. Description of the Related Art Living organisms contain various proteins. Among them, lipoprotein, which is a complex of lipid and protein, is known to remain on the cell membrane and function as an antigen protein in various pathogens. An antibody measurement method using a lipoprotein-containing fraction as an antigen is widely used. For example, Treponema pallidum is known as a causative agent of syphilis. In testing and diagnosing infection of this bacterium, an antibody measurement method using a cell membrane fraction obtained from the cells of Treponema pallidum as an antigen is currently used. Since Treponema pallidum can only grow in the cells or tissues of infected animals, in order to obtain the cell membrane fraction of Treponema pallidum, which is the antigen, to date, Treponema pallidum has been used in rabbit testes. A method has been used in which infection is obtained, Treponema pallidum is obtained from the testis, and a cell membrane fraction is obtained from the cells. However, in this method, a large amount of antigen could not be obtained.

The cell membrane fraction of Treponema pallidum contains various antigenic proteins, which are lipoproteins. Until now, molecular weight about 47K
It is known that there are antigenic proteins of daltons, about 17K daltons and about 15K daltons, and the amino acid sequences of these antigenic proteins and the nucleotide sequences encoding them have already been reported (El M. Weiger et al., Infection and Immunity, 60
Volume, 1568-1576 (1992) (LMWeigel et al., Infec
t. Immun., Vol. 60, p. 1568-1576 (1992)), D. Eirkins et al., Infection and Immunity, 61, 1202-1210 (1993) (DRAkins et.
al., Infect. Immun., Vol. 61, p. 1202-1210 (1993)),
BK Precell, et al., Molecular Microbiology, Volume 4, pp. 1371-1379 (1990)) (BK Purecell e
t al., Mol. Microbiol., Vol. 4, p. 1371-1379 (199
0)). However, since these antigenic proteins, which are lipoproteins, are hard to be released outside the cells, it has not been realized to easily produce these antigenic proteins in large quantities.

[0004]

The object of the present invention is to provide a modified protein which can be secreted from a microorganism. The invention of claim 2 provides a modified protein having a high signal peptide cleavage efficiency and a high secretion efficiency in addition to the effects of the first invention. The invention of claim 3 provides a modified protein having antigenicity as a Treponema pallidum antigen in addition to the effects of the invention of claim 1 or 2.
The invention of claim 4 provides a modified protein having antigenicity as a Treponema pallidum-specific antigen in addition to the effects of the invention of any of claims 1 to 3. The inventions described in claims 5 and 6 are claims 1 to
4. A modified protein having excellent antigenicity as an antigen specific to Treponema pallidum, in addition to the effects of the invention described in any one of 4) above.

[0005] The invention of claim 7 provides a DNA useful for producing a modified protein capable of being secreted from a microorganism. The invention of claim 8 provides a DNA useful for producing a modified protein having excellent antigenicity as a Treponema pallidum-specific antigen, in addition to the effects of the invention of claim 7. The invention described in claim 9 provides a recombinant vector useful for producing a modified protein that can be secreted from a microorganism. The invention described in claim 10 provides a transformant useful for producing a modified protein that can be secreted from a microorganism. The invention according to claim 11 is the invention according to claim 10
It is intended to provide a transformant which is useful for producing a modified protein having excellent antigenicity as a Treponema pallidum-specific antigen in addition to the effects of the invention described above.

[0006] The invention of claim 12 provides a method for producing a modified protein capable of being secreted from a microorganism. The invention of claim 13 provides a method for producing a modified protein having excellent antigenicity, in addition to the effects of the invention of claim 12. The invention according to claim 14 is a method for measuring an anti-Treponemal pallidum antibody useful for diagnosing syphilis infection, wherein a modified protein capable of being secreted from a microorganism and having antigenicity as a Treponema pallidum antigen is used as an antigen. Is provided. Claim 15
The described invention is capable of secretion from microorganisms, and
It is intended to provide a reagent for measuring an anti-Treponemal pallidum antibody useful for diagnosis of syphilis infection, comprising a modified protein having antigenicity as a Treponema pallidum antigen. The invention according to claim 16 provides a diagnostic agent for syphilis infection, which is useful for diagnosing syphilis infection, comprising a modified protein capable of secretion from microorganisms and having antigenicity as Treponema pallidum antigen as an active ingredient. Things. The invention of claim 17 provides a method for producing an anti-Treponemal pallidum antibody useful for diagnosing syphilis infection.

[0007]

Means for Solving the Problems The present invention provides the following (1) to
Regarding (17). (1) A modified protein having an amino acid sequence in which a cysteine residue of a protein is converted to a non-lipid-binding amino acid residue. (2) The modified protein according to (1), wherein the non-lipid-binding amino acid residue is selected from the group consisting of an alanine residue, a glutamic acid residue and a serine residue. (3) The modified protein according to (1) or (2), wherein the protein is derived from Treponema pallidum. (4) The modified protein according to any one of (1) to (3), wherein the protein is at least one kind of antigenic protein of Treponema pallidum having a molecular weight of about 47 K daltons and about 17 K daltons. (5) The modified protein according to any one of (1) to (4), which is a protein represented by SEQ ID NO: 1. (6) The modified protein according to any one of (1) to (4), which is a protein represented by SEQ ID NO: 2.

(7) A DNA encoding the modified protein according to any of (1) to (6) or a DNA complementary thereto. (8) The DNA according to the above (7), which has the nucleotide sequence represented by SEQ ID NO: 4 or 5. (9) A recombinant vector containing the DNA of (7) or (8). (10) A transformant containing the recombinant vector according to (9). (11) FERM BP-5641 or FERM BP
The transformant according to the above (10), which has been deposited as -5642.

(12) A transformant into which the gene encoding the modified protein according to any one of (1) to (6) has been introduced is cultured, and the modified protein is produced and accumulated in the culture. And a method for producing a modified protein. (13) The modification according to the above (12), wherein ammonium sulfate is added to the culture supernatant of the obtained culture, centrifugation is performed to obtain a precipitate, and the precipitate is dissolved in a surfactant-containing aqueous solution. A method for producing proteins. (14) A method for measuring an anti-Treponemal pallidum antibody, comprising using the modified protein according to any one of (3) to (6) as an antigen. (15) A reagent for measuring an anti-Treponemal pallidum antibody, comprising the modified protein according to any of (3) to (6) as an antigen. (16) A diagnostic agent for syphilis infection comprising the modified protein according to any one of (3) to (6) as an active ingredient. (17) A method for producing an anti-Treponemal pallidum antibody, comprising using the modified protein according to any one of (3) to (6) as an antigen.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the sequence list, the amino acid at the amino terminal is numbered 1 in the amino acid sequence, and the base at the 5 'end is numbered 1 in the base sequence.

Modified Protein The modified protein of the present invention has an amino acid sequence in which a cysteine residue of a protein has been converted to a non-lipid-binding amino acid residue (hereinafter, referred to as amino acid sequence (A)). The protein is not particularly limited, and includes, for example, proteins derived from organisms such as animals, plants, microorganisms, and viruses. From the viewpoint of extracellular secretion, examples of the protein include non-secretory proteins, and examples of the non-secretory protein include a membrane protein and a cytoplasmic protein. Examples of membrane proteins include various enzymes and various receptors present on cell membranes. The non-secretory proteins also include lipoproteins, which are proteins to which lipids are bound. Further, from the viewpoint of antigenicity, examples of the protein include various antigenic proteins present on cell membranes.

Examples of various antigenic proteins that are derived from microorganisms and are present on cell membranes include, for example,
Antigen proteins derived from Treponema pallidum, antigenic proteins derived from Chlamydia pneumoniae, antigenic proteins derived from Chlamydia trachomatis, and the like. Examples of antigenic proteins derived from Treponema pallidum include, for example, a molecular weight of about 47K daltons and about 17K daltons.
Kdalton and about 15K dalton antigen proteins and the like. Examples of Chlamydia pneumoniae-derived antigen proteins include, for example, antigen proteins with molecular weights of about 39.5K daltons, about 53K daltons and about 70K daltons, and the like. -As an antigen protein derived from Trachomatis, for example, a molecular weight of about 39.
Antigen proteins of 5K dalton, about 59.5K dalton and about 75K dalton, and the like.

In the amino acid sequence (A), the cysteine residue to be converted is a cysteine residue located on the amino-terminal side of the mature protein from the viewpoint of lipid binding. In addition, the number of cysteine residues to be converted is 2 as long as the obtained modified protein retains characteristics inherent in the original protein, for example, the antigenicity as Treponema pallidum antigen and can be secreted from microorganisms. It may be the above. The non-lipid-binding amino acid residue is not particularly limited as long as it has no or weak lipid-binding properties.However, from the viewpoint of high signal peptide cleavage efficiency and high secretion efficiency, alanine Residues, glutamic acid residues or serine residues are preferred.

Further, the properties of the resulting modified protein in the original protein, for example,
The amino acid sequence (A) may be selected from antigenic proteins derived from Treponema pallidum bacteria (for example, 1 to 5
Amino acid residues (for example, 1 to 20) from the region up to the 0th amino acid) may be deleted. In addition, as long as the obtained modified protein maintains the above-mentioned various properties and can be secreted from microorganisms, the modified protein of the present invention may be directly or intervened at the amino terminal or carboxyl terminal of the amino acid sequence (A). It may have a structure in which one or more amino acid sequence (A) or another amino acid sequence (B) is bonded via the amino acid sequence. Intervening amino acid sequences include, for example, isoleucine residues. Examples of the amino acid sequence (A) include those described above.

The sequence of the amino acid sequence (B) is not particularly limited as long as the obtained modified protein maintains the above various properties and can be secreted from a microorganism.
From the viewpoint that the modified protein of interest can be purified from the culture supernatant of the microorganism, an amino acid sequence that is recognized as a signal sequence in a microorganism that produces the modified protein of interest is preferable.As such an amino acid sequence, for example, , Bacillus brevi
An amino acid sequence recognized as a signal sequence in s). When the amino acid sequence (B) is a signal sequence, the amino acid sequence (B) needs to be bonded to the amino group terminal of the amino acid sequence (A). Thus, when a microorganism that produces the desired modified protein is cultured, first, a fusion protein consisting of the amino acid sequence (B) and the amino acid sequence (A) is produced in the microorganism, and this fusion protein moves to the vicinity of the cell membrane. The amino acid sequence (A) is cleaved from this fusion protein by a signal peptidase in the cell membrane and secreted out of the microorganism.

[0016] Specific examples of the modified protein of the present invention include, for example, the proteins represented by SEQ ID NOs: 1 to 3. The protein represented by SEQ ID NO: 1 lacks the 1st to 19th amino acid residues of the antigenic protein derived from Treponema pallidum and having a molecular weight of about 47K daltons,
It has a structure in which a cysteine residue, which is the 20th amino acid residue, is substituted with an alanine residue. This protein has an antigenicity as Treponema pallidum antigen because it produces an antigen-antibody reaction with the serum of syphilis patients. The protein represented by SEQ ID NO: 2 lacks amino acid residues 1 to 21 of an antigenic protein derived from Treponema pallidum and has a molecular weight of about 17K daltons.
It has a structure in which a cysteine residue as the second amino acid residue is substituted with an alanine residue. This protein has an antigenicity as Treponema pallidum antigen because it produces an antigen-antibody reaction with the serum of syphilis patients.

The protein represented by SEQ ID NO: 3 lacks amino acids 1 to 17 of the antigenic protein derived from Treponema pallidum and has a molecular weight of about 15 K daltons, and has a cysteine residue as the 18th amino acid residue. It has a structure substituted with an alanine residue. This protein has an antigenicity as Treponema pallidum antigen because it produces an antigen-antibody reaction with the serum of syphilis patients.

Method for Producing Modified Protein As a method for producing the modified protein of the present invention, there are a chemical synthesis method and a gene recombination method. As a chemical synthesis method,
For example, map (Multiple Antigen Peptide, MAP)
The method is suitable for synthesizing a protein having an amino acid sequence of 30 or less amino acids, and can be synthesized using a commercially available peptide synthesizer. Proteins can be synthesized in a repetitive manner by the mapping method. As a gene recombination method, for example, a recombinant vector is prepared by inserting a DNA encoding the modified protein of the present invention into a vector,
A method of preparing a transformant by inserting it into a host, culturing the transformant, and using the transformant itself or a culture supernatant can be mentioned. The DNA encoding the modified protein of the present invention will be described later. As a vector,
For example, a plasmid vector, a phage vector and the like can be mentioned. As a host, for example, Bacillus brevis
(Bacillus brevis), Bacillus subtilis, Escherichia coli, yeast and the like. Hereinafter, a method for producing a recombinant vector, a method for producing a transformant, and a method for producing a modified protein using the transformant will be described in detail.

The recombinant vector can be prepared by inserting a DNA encoding the modified protein of the present invention (described later) into a plasmid vector or phage vector that can be replicated in a host cell according to a conventional method. At that time, a linker is used if necessary. As a plasmid vector, a drug such as an erythromycin resistance gene or a neomycin resistance gene can be easily screened for a transformant having the recombinant vector to be prepared, and the recombinant vector is stably maintained in host cells. Preferably, it has a resistance gene. In addition, as described below, the plasmid vector must have a promoter sequence, a Shine-Dalgarno sequence (hereinafter, referred to as an SD sequence) and a terminator sequence because the modified protein of the present invention can be produced. is required.

Specific examples of the plasmid vector include, for example, plasmids pUC118, pUB110, pNU
200, pNU210, pHT926, pNH300,
pNH400 and the like. pUC118, pUB1
10 can be purchased from Takara Shuzo Co., Ltd. pNU2
For 00, see “Shigezo Udaka, Journal of the Japanese Society of Agricultural Chemistry, Vol. 61, 66.
9 (1987) ". pNU210
For details, see `` Bioindustry, Vol. 9, pp. 100-107
(1992) (BIOINDUSTRY, Vol. 9, p. 100-107 (1992)) ". The pHT926 is described in detail in Japanese Patent Application Laid-Open No. 6-137882. pN
H300 will be described in detail in an embodiment described later. pN
Regarding H400, see "Journal of Bacteriology, Vol. 177, pp. 745-749 (1995) (J. Bacteriol., Vol.
177, p. 745-749 (1995)) ". Further, as the phage vector, for example, λgt11 phage, λgt10 phage and the like can be used. In each case, a recombinant vector corresponding to the parent vector used is obtained.

When the modified protein of the present invention is produced using a genetic recombination method, the recombinant plasmid to be prepared is
It is necessary that the plasmid encoding the modified protein of the present invention can express the DNA. Examples of a method for preparing such a plasmid include a method for inserting the DNA of the present invention near the promoter sequence and the SD sequence and downstream thereof, a DNA encoding the expressible amino acid sequence (B), and the like. The reading frame is aligned (ie, in frame) downstream of the amino acid sequence (A).
(Accordingly, the modified protein of the present invention obtained in the host is a fusion protein consisting of the amino acid sequence (B) and the amino acid sequence (A)). DN which is the recombinant plasmid of the present invention and encodes the modified protein of the present invention
Specific examples of plasmids capable of expressing A include, for example, plasmids pNH400TP47, pNH300T
P17 and the like. A general method for producing a recombinant plasmid is described in "Samblock et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory) (1989)" (J. Samblook et al., Molecul).
ar Cloning 2nd ed., Cold Spring Harbor Laboratory
Press (1989), hereafter referred to the document "Molecular
Cloning ").

The transformant of the present invention can be prepared by inserting the above-mentioned recombinant plasmid into a host. The host is not particularly limited as long as it expresses the DNA of the present invention in the recombinant plasmid, and examples thereof include Bacillus brevis, Bacillus subtilis (Bacillus subtilis), yeast, and Escherichia coli. For any of the microorganisms, it is preferable to use a non-proteolytic enzyme-producing strain, since the modified protein of the present invention produced in the transformant is not easily degraded. The plasmids pNH400TP47 and pNH300TP are used as recombinant plasmids.
17 is used, for example, Bacillus brevis 4
Seven strains, Bacillus brevis strain H102 (this strain is the same strain as Bacillus brevis strain HPD31) and the like can be used. As a method for inserting the recombinant plasmid into the host, for example, an electric pulse (Electroporatio
n) method, Protoplast method, calcium chloride method, Tris-PEG (Tris-PEG) method and the like, which are appropriately selected depending on the type of host.
When using Brevis, in terms of efficiency and simplicity,
It is preferable to use an electric pulse method.

A general method for preparing a transformant using a recombinant plasmid is described in the document “Molecular Cloning”, and is described in the aforementioned Bacillus brevis 47.
For example, a method for producing a transformant using Bacillus brevis H102 as a host is described in "Takahashi et al., Journal of
Bacteriology, 156, 1130-1134, 1983 (Takah
ashi et al., J. Bacteriol., Vol.156, p.1130-1134 (1
983))), `` Takagi et al., Agricultural Biological Chemistry, 53, 3099-3100, 1989 (Taka
gi et al., Agric. Biol. Chem., Vol. 53, p. 3099-3100
(1989)) ".

As a specific example of the transformant of the present invention, for example, Bacillus brevis strain HPD31 (Bacillus) into which the above-mentioned plasmid pNH400TP47 has been inserted.
brevis HPD31 / pNH400TP4
7), the Bacillus brevis HPD31 strain into which the plasmid pNH300TP17 was inserted (Bacillus b
revis HPD31 / pNH300TP17) and the like. Bacillus brevis HPD
31 / pNH400TP47 and Bacillus b
revis HPD31 / pNH300TP17 has been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology under the accession numbers FERM BP-5642 and FERM BP-5641.

For culturing the transformant, for example, a method in which the transformant is placed in a medium in which the transformant can grow and shaken or allowed to stand at an appropriate temperature can be used. As the medium, a medium containing a carbon source and a nitrogen source can be used. Examples of the carbon source include sugars and organic acids, and examples of the sugars include glucose, glycerol, starch, dextran, and molasses. Examples of the nitrogen source include an organic nitrogen source and an inorganic nitrogen source.Examples of the organic nitrogen source include casein, peptone, meat extract, yeast extract, casamino acid, and glycine. Examples thereof include ammonium sulfate and ammonium nitrate. When the transformant shows auxotrophy, nutrients necessary for its growth are added to the medium. Such nutrients include, for example, amino acids, vitamins, nucleic acids, salts and the like. As the medium, for example, a synthetic medium mainly composed of a sugar and an inorganic nitrogen source can be used. When a solid medium is used as the medium, agar is further added.

From the viewpoint of stably retaining the recombinant plasmid and suppressing the growth of a strain that does not have the recombinant plasmid, it is preferable to add an antibiotic to the medium. Examples of such antibiotics include penicillin, erythromycin, neomycin, chloramphenicol,
Bacitracin, D-cycloserine, ampicillin and the like. If necessary, soybean oil, lard oil, various surfactants and the like may be added to the medium as an antifoaming agent. The pH of the medium is usually 5 to 9, and preferably 6.5 to 7.5, from the viewpoint of the pH at which the transformant can grow. As a specific example of the medium, for example, a TMB medium (peptone 1%, meat extract 0.5%, yeast extract 0.2%, glucose 1%, MgSO ₄ 0.01
%, FeSO ₄ 0.01%, MnSO ₄ 0.001
%, ZnSO ₄ 0.0001% and erythromycin 10 μg / ml, pH 7.0).

For example, when Bacillus brevis is used as a host, the culture temperature is usually 15 to 42 ° C., preferably 24 to 37 ° C. The culture time is
Depending on the type of host, the physical state of the medium (solid or liquid), the culture device, the culture temperature, etc., Bacillus brevis is used as the host and TMB liquid medium is used as the culture medium, and the culture temperature is 15 to 42 ° C. In this case, the culture time is usually 16 to 166 hours, and preferably 24 to 96 hours.

As a method for producing the modified protein of the present invention by culturing the transformant, for example, the cultured transformant is crushed or dissolved to obtain a crushed solution or a lysate, and the crushed solution or the lysate is obtained. And a method for purifying the modified protein of the present invention. Further, when the modified protein of the present invention obtained in a host is a fusion protein of the signal sequence and the amino acid sequence (A), the signal sequence is removed from the fusion protein by signal peptidase of the transformant, Since the amino acid sequence (A) is secreted from the transformant, a method of obtaining a culture supernatant of the transformant and purifying the modified protein of the present invention as the amino acid sequence (A) from the culture supernatant is used. Can also.

Examples of the method for disrupting the transformant include a method for physically disrupting the transformant. For example, a method in which the transformant is suspended in a buffer and irradiated with ultrasonic waves, A method in which the transformant is kneaded with quartz sand and suspended in a buffer solution can be used. On the other hand, examples of a method for lysing a transformant include a method for lysing the transformant with an enzyme and a surfactant that lyse the cell wall of the transformant. Lysozyme can be used as this enzyme, and sodium dodecyl sulfate (SDS) can be used as a surfactant.

As a method for purifying the modified protein of the present invention from the obtained crushed solution or lysate, for example, the crushed solution or lysate is centrifuged to remove cell debris, the supernatant is obtained, and streptomycin is obtained. A method of adding sulfate, stirring and centrifuging, thereby removing nucleic acids as a precipitate, obtaining a supernatant, adding ammonium sulfate to the supernatant, stirring, and centrifuging to obtain a precipitate. Can be used. In the final centrifugation, since the modified protein of the present invention may be contained in the supernatant, sampling is performed to confirm the presence or absence of the modified protein of the present invention in the supernatant and precipitate fractions. Preferably. On the other hand, as a method for purifying the modified protein of the present invention from the culture supernatant of the transformant, for example, ammonium sulfate is added to the culture supernatant (for example, 25%), the mixture is stirred, and the precipitate is obtained by centrifugation. The method can be used. At that time, in order to remove contaminants, a low-concentration ammonium sulfate is first added (for example, 10%), followed by stirring, centrifugation to obtain a supernatant, and further adding ammonium sulfate to the supernatant (for example, , Final concentration 25%),
It is preferable to stir and centrifuge to obtain a precipitate.

The obtained precipitate is preferably dissolved in a surfactant-containing aqueous solution from the viewpoint that a modified protein having excellent antigenicity can be obtained. As the aqueous solution, for example, buffers commonly used for protein purification, such as PBS and Tris-HCl buffer, can be used. As the surfactant, for example, SDS, Tween
20 and the like. The concentration of the surfactant in the aqueous solution is preferably 0.05 to 2%, more preferably 0.1 to 1%, and still more preferably 0.5 to 1%. When the concentration is less than 0.05% or more than 2%, the antigenicity of the obtained modified protein tends to be impaired. Especially,
When the protein is derived from Treponema pallidum, the obtained precipitate is dissolved in a surfactant-containing aqueous solution having the above concentration, since a modified protein having excellent antigenicity as Treponema pallidum can be obtained. Preferably. The modified protein of the present invention can also be purified by fractionating the culture supernatant of the transformant by cation column chromatography. Specific methods for removing cell debris, removing nucleic acids by adding streptomycin sulfate, and obtaining proteins by adding ammonium sulfate, etc., are described in the document "Molecular Cloning".

DNA encoding the modified protein In the present invention, the DNA encoding the modified protein is defined as a triplet code table in which the amino acid sequence of the modified protein is assigned to each amino acid. Selected from the group of DNA when read into the nucleotide sequence according to
Say. The modified proteins of the present invention include those described in the section of the modified proteins, and DNAs encoding the modified proteins also include those having nucleotide sequences corresponding to the amino acid sequences of these modified proteins. Specific examples of the DNA encoding the modified protein include, for example, DNAs having the nucleotide sequences shown in SEQ ID NOs: 4 to 6. The DNA having the nucleotide sequence of SEQ ID NO: 4 is a DNA encoding the protein of SEQ ID NO: 1. In this base sequence, the coding region is the sequence at positions 3 to 1276. The DNA having the base sequence of SEQ ID NO: 5 is a DNA encoding the protein of SEQ ID NO: 2. In this base sequence, the coding region is the sequence at positions 3 to 407.
The DNA having the nucleotide sequence represented by SEQ ID NO: 6 is a DNA encoding the protein represented by SEQ ID NO: 3, and the entire nucleotide sequence is a coding region.

The DNA encoding the modified protein can be prepared by a chemical synthesis method or a genetic recombination method. The chemical synthesis method includes, for example, a phosphoramidite method, which is suitable for synthesizing a DNA consisting of a base sequence having a total length of 100 bases or less, and can be synthesized using a commercially available DNA synthesizer. In order to prepare DNA having a total length of more than 100 bases, a gene recombination method described later can be used, but it can also be prepared as follows. That is, the base sequence is divided into less than 100 bases, DNA fragments consisting of each base sequence are chemically synthesized as described above, these DNA fragments are mixed, and these DNA fragments are separated using T4-DNA ligase. connect. At that time, a DNA fragment of a complementary strand is also synthesized, and when the DNA fragments are paired with each other so that a protruding end is generated, the protruding end serves as a sticky end, so that a target DNA is easily obtained.

The gene recombination method is designed, for example, by using the genomic DNA of an organism producing the original protein (cDNA if the organism is a eukaryote) as a template and the base sequence of the original protein. A method of performing a polymerase chain reaction (PCR) method using the prepared primer DNA can be used.
When the organism producing the original protein is Treponema pallidum, as Treponema pallidum,
For example, Treponema pallidum Nichols strain (Trepone
ma pallidum Nichols strain) can be used,
This strain is owned by Lee Laboratories
(US). The method of obtaining the genomic DNA or cDNA of the organism that produces the original protein is appropriately selected depending on the type of the organism, and is not particularly limited. For example, the method described in the document “Molecular cloning” can be used. Can be.

Genomic DNA of Treponema pallidum
Is, for example, 1 M of Treponema pallidum cells
Sodium chloride, 1N sodium hydroxide and 2% S
It can be obtained by suspending in a DS-containing aqueous solution, boiling, neutralizing by adding 0.5 M Tris (pH 7.0), extracting with phenol, performing an ethanol precipitation treatment, and drying the precipitate. The method of obtaining this genomic DNA is described in "M V. Norgard et al., Journal of Clinical Microbiology, Vol. 29, pp. 62-69 (1991)" (MVNor
gard et al., J. Clin. Microbiol., Vol. 29, p. 62-69
(1991)).

The nucleotide sequence of the antigenic protein derived from Treponema pallidum is described in the literature of Weiger et al., The literature of Eikens et al., The publication of Precel et al. Although the sequence is designed based on the base sequence of this antigen protein, the base sequence of this primer DNA should not be a base sequence corresponding to the amino acid sequence of the antigen protein, but should be in the amino terminal region of the antigen protein. It is necessary to design such that a certain cysteine residue is converted into a non-lipid-binding amino acid residue (an alanine residue, a glutamic acid residue, a serine residue, etc.). The primer DNA thus designed can be produced by a chemical synthesis method using a commercially available DNA synthesizer. A general method of replicating DNA using the PCR method is described in the document "Molecular Cloning". Once the DNA encoding the modified protein is obtained, the DNA can be replicated by using the genetic recombination method or the PCR method described above, so that the genomic DNA or the genomic DNA of the organism producing the original protein can be obtained. The operation of obtaining the cDNA again is unnecessary.

The modified protein of the present invention can be used as an active ingredient of various diagnostic agents. In particular, when the protein is derived from Treponema pallidum, the resulting modified protein is derived from Treponema pallidum. Since it has antigenicity, it can be used as an active ingredient of a diagnostic drug for syphilis infection.

The assay method and assay reagent for anti-Treponemal pallidum antibody using the modified protein as an antigen and the diagnostic method for syphilis infection diagnostic anti-Treponemal pallidum antibody containing the modified protein as an active ingredient are the modified protein of the present invention. There is no particular limitation as long as a protein obtained from a protein derived from Treponema pallidum is used as part or all of an antigen. As a method for measuring the anti-Treponemal pallidum antibody, for example, an immunoassay using various labels, a latex agglutination method using latex carrier particles, an immunoturbidimetric method, and the like can be used.

Hereinafter, the immunoassay using various labels will be described in detail. As a method for measuring an anti-Treponemal pallidum antibody using an immunoassay utilizing various labels using the modified protein, for example, immobilized antigen by physically or chemically immobilizing the modified protein on a carrier Is prepared, and the immobilized antigen is brought into contact with the sample sample and kept warm for a certain period of time, whereby, if an anti-Treponemal pallidum antibody is present in the sample sample, the antibody binds to the immobilized antigen and the antigen antibody Using a method in which a complex is formed on a carrier, washed once if necessary, and then contacted with a labeled antibody against the antibody in the specimen sample (in some cases, the specimen sample and the labeled antibody may be contacted simultaneously). Can be. When the antigen-antibody complex is formed, the labeled antibody is further bound to the complex, and the labeled antibody is also immobilized on the carrier. Thereafter, the amount of the label on the labeled antibody bound to the carrier or the label on the unbound label antibody is measured by a measuring method according to the label, and the presence or amount of the anti-Treponemal pallidum antibody in the sample is determined from the value. Can be. In the present invention,
"Measurement" means not only quantitative or semi-quantitative measurement but also qualitative measurement (detection, etc.).

The carrier is not particularly limited as long as it can immobilize the antigen. Examples thereof include plastic materials such as polystyrene and vinyl chloride, fiber materials such as cellulose, nitrocellulose and nylon, and glass. , Silica gel and other inorganic materials, erythrocytes, liposomes, polyvinylidene difluoride (PDVF), etc., and can be used in the form of microtiter plates, beads, magnetic beads, paper disks,
Although any shape such as a membrane and a thread can be used, it is preferable to use polystyrene beads or a microtiter plate from the viewpoint of simplicity, and a polystyrene microtiter plate is particularly preferable. As a method for physically immobilizing the modified protein on a carrier, for example, a method in which the modified protein-containing solution (antigen solution) is brought into contact with a carrier and left overnight at a low temperature (for example, 4 ° C.) is used. Can be. Further, as a method of chemically fixing the modified protein to a carrier, for example, a method of mixing the modified protein, a carrier having a carboxyl group on its surface, and carbodiimide, and allowing the mixture to stand can be used.

Examples of the sample include various liquid components of human, and human blood, human tears, human pharyngeal swab, human urine, etc. are preferable from the viewpoint of reflecting the clinical picture of the sample provider. Human blood is more preferred, and human serum is even more preferred. In order to prevent other antibodies and the like in the sample sample from non-specifically binding to the carrier, it is preferable to block the surface of the carrier with bovine serum albumin or the like before adding the sample sample. As the washing solution, for example, Tris containing a surfactant, a phosphate buffer, or the like can be used.

Examples of the labeled antibody include those obtained by labeling an antibody (eg, a human antibody) in a specimen sample with various labeling substances. Examples of the labeled antibody include, for example, anti-human IgG antibody, anti-human IgA antibody, anti-human Ig
M antibody and the like or partially degraded products thereof (F (ab ') ₂ , Fa
b) and the like, and these can be appropriately used depending on the type of the antibody to be measured. From the point that the clinical image of the sample provider is reflected, in the step of contacting a labeled antibody against the antibody in the sample sample, measurement is performed by separately reacting labeled antibodies with different types of antibodies to one sample sample. Is preferred. Examples of the labeled antibodies having different types of antibodies include a labeled anti-human IgG antibody, a labeled anti-human IgA antibody, a labeled anti-human IgM antibody, and the like.

As the labeling substance used for the labeled antibody, for example, an enzyme, a radioisotope, a fluorescent substance, a luminescent substance and the like can be used. Examples of the enzyme include malate dehydrogenase (enzyme number 1.1.1.37), glucose-6-phosphate dehydrogenase (enzyme number 1.1.1.49), glucose oxidase (enzyme number 1.1.3.4), and horseradish peroxidase (enzyme) No. 1.11.1.7), acetylcholinesterase (enzyme number 3.1.1.7), alkaline phosphatase (enzyme number 3.1.3.1), glucoamylase (enzyme number 3.2.1.
3), lysozyme (enzyme number 3.2.1.17), β-galactosidase (enzyme number 3.2.1.23) and the like. As the fluorescent substance, for example, fluorescine
Etc. are available. Among these labeling substances, it is preferable to use an enzyme in terms of sensitivity, safety, simplicity and the like, and the immunoassay using the enzyme as the labeling substance is usually performed by enzyme immunoassay (ELISA). Called. As an enzyme-labeled antibody, it is preferable to use an alkaline phosphatase-labeled antibody or a horseradish peroxidase-labeled antibody because simple and highly sensitive measurement is possible, and these enzyme-labeled antibodies are commercially available. In addition, in order to bind the antibody and the label, between the antibody and the label, biotin (Biotin), avidin (Avidin), streptavidin (Streptoavidin), digoxigenin (Digoxig
A chemical substance such as enin) may be interposed.

As a method for measuring the anti-Treponemal pallidum antibody using the modified protein and the latex agglutination method, for example, the modified protein is physically or chemically immobilized on latex particles (carrier). In this method, an immobilized antigen is prepared, and the immobilized antigen is brought into contact with a sample sample, kept at a constant temperature for a certain period of time, and the turbidity of a mixed solution of the sample sample and the immobilized antigen is measured. By contacting the immobilized antigen with the specimen sample, if an anti-Treponemal pallidum antibody is present in the specimen sample, the antibody binds to the immobilized antigen to form an antigen-antibody complex.
At this time, since the antibody has two portions that bind to the antigen, a structure is formed in which the antigen-antibody complex crosslinks each other, and an agglutination reaction occurs. This reaction increases the turbidity of the mixed solution of the specimen sample and the immobilized antigen. The turbidity is measured visually or by using an absorption photometer. In addition,
As a modification of the latex agglutination method, a method of using various other fine particles such as red blood cells and liposomes instead of latex particles can also be used.

The modified protein of the present invention, obtained using a protein derived from Treponema pallidum, can be used as an antigen as a reagent for measuring an anti-Treponemal pallidum antibody. Although the components of the reagent vary depending on the measurement method, examples of the reagent for measuring the anti-Treponemal pallidum antibody using the immunoassay using the above-mentioned various labels include, for example, an immobilized antigen immobilized on a carrier and an immobilized antigen. And a labeled antibody that reacts with the antibody to be used. The reagent for measuring an anti-Treponemal pallidum antibody using the latex agglutination method is, for example, immobilized on latex particles (carrier). Immobilized antigen. Examples of the carrier and the labeled antibody include those described above. The labeled antibody can be dispersed in a buffer or the like.

In the above reagent, a labeled anti-human Ig antibody can be used as the labeled antibody. Examples of the labeled anti-human Ig antibody include a labeled anti-human IgG antibody, a labeled anti-human IgA antibody, and a labeled anti-human IgM antibody. The labeled antibody to be used may be any one of these antibodies for one sample sample, but from the point that the clinical picture of the sample provider is reflected, It is preferable to use these three types of labeled antibodies. These labeled antibodies may be prepared separately or in a mixture. Other components are combined with the reagent as needed. For example, in the case of a measurement reagent used in an immunoassay using the above-mentioned various labeled substances, as other components, for example, a negative control sample, a positive control sample, a washing solution, a reaction substrate when the labeling substance is an enzyme or the like, a dilution, Liquid, a sensitizer, a reaction stop solution, and the like.These may be used alone or in combination.Examples of the form of the reagent include, for example, a kit in which a necessary amount of the above components are enclosed, a bulk of these individual products, and the like. Can be

In the case of the measuring reagent used in the latex agglutination method, examples of other components include a negative control sample, a positive control sample, a washing solution, a sensitizer, and the like. The sensitizer includes, for example, bovine serum albumin and polyethylene glycol. The reagent for measuring anti-Treponemal pallidum antibody is effectively used for diagnosis of syphilis infection. As the syphilis infection diagnostic agent containing the modified protein as an active ingredient, for example, the above reagent can be used as it is.

Method for Producing Anti-Treponemal pallidum Antibody Using Modified Protein as Antigen The anti-Treponemal pallidum antibody is a modified protein of the present invention obtained by using a protein derived from Treponema pallidum. And obtained as an antiserum or an isolated anti-Treponemal pallidum antibody. As a method for producing antiserum, for example,
A method of immunizing an animal such as a rabbit or a mouse with the antigen and obtaining the serum thereof can be used. Also, anti-treponema
As a method for isolating and manufacturing a palydam antibody, for example, a rabbit or a mouse is immunized with the antigen, a spleen cell thereof is fused with a myeloma cell to prepare a hybridoma, and a hybridoma recognizing the antigen is prepared therefrom. A method of selecting, culturing this, and obtaining the culture supernatant can be used.
In order to enhance antigenicity, if necessary, an appropriate carrier protein may be bound to the antigen to form an immunogen.

Various adjuvants can be used at the time of immunization, but Freund's complete adjuvant (F
CA) and incomplete Freund's adjuvant (FIA) are preferred. As myeloma cells, for example, P3X63A
g8.653 (ATCC (American Type Culture Coll
section) CRL-1580) or P3 / NSI / 1-A
g4-1 (ATCC TIB-18) can be used. In addition, rabbits, such as rabbits,
Mice, rats, cows, sheep, goats, chickens and the like can be used, but are not limited to the animal species exemplified here. Except for using the modified protein of the present invention as an antigen, which is obtained using a protein derived from Treponema pallidum, according to a known general method for immunizing an animal to obtain an antibody, -Paridam antibodies can be produced. Antibodies include polyclonal and monoclonal antibodies.
In addition, these antisera and isolated and produced anti-Treponemal pallidum antibodies may be modified with various enzymes, colloids and the like. The obtained antiserum and the isolated and produced anti-Treponemal pallidum antibody are effectively used for diagnosis of syphilis infection.

[0050]

The present invention will be described below in detail with reference to examples. Example 1 Molecular weight of about 4 derived from Treponema pallidum
Production of modified protein of 7K dalton antigen protein, examination of antigenicity of modified protein by Western blotting, and measurement of anti-Treponemal pallidum antibody by ELISA (1) Molecular weight of about 47K derived from T. pallidum
Preparation of Recombinant Plasmid Expressing Dalton Antigen Protein Modified Protein A recombinant plasmid was prepared according to the procedure shown in FIG. As Treponema pallidum Nichols strain, Treponema pallidum Nichols strain
(Purchased from Lee Laboratories (USA), product code: SPROJ-TPURE) was used.

The genomic DNA of Treponema pallidum was obtained in the following manner according to the method described in M. Bu. 5 × 10 ⁷ cells of Treponema pallidum were suspended in an aqueous solution containing 1M sodium chloride, 1N sodium hydroxide and 2% SDS, and boiled for 1 minute. Next, 0.5 M Tris (pH 7.0) four times the amount of this aqueous solution was added for neutralization. Furthermore, phenol was added to obtain an aqueous layer, ethanol was added to the aqueous layer, and the mixture was centrifuged to obtain a precipitate, and the precipitate was dried. Finally, the precipitate is dissolved in sterilized water, and the genome DN of Treponema pallidum is dissolved.
Solution A was used. This genomic DNA solution is used for PC
Used as template DNA in the R method.

The aforementioned Weiger et al. Document describes the nucleotide sequence of a DNA encoding an antigenic protein derived from Treponema pallidum and having a molecular weight of about 47 K daltons (the nucleotide sequence represented by SEQ ID NO: 7). This molecular weight is about 4
The 7K dalton antigen protein is considered to be a cell membrane protein in Treponema pallidum by the following mechanism. First, it is synthesized as a precursor protein containing a signal sequence (amino acid sequence at positions 1 to 434 added to the nucleotide sequence shown in SEQ ID NO: 7) in Treponema pallidum cells, and then fatty acids present in the cells are synthesized. A thioester bond is formed with the cysteine residue at position 20 in this precursor protein. Then, the signal peptide peptidase present in the cells removes the amino acid sequence at positions 1 to 19 in the precursor protein as a signal sequence, and further fixes the bound fatty acid in the lipid bilayer of the cell membrane. Amino acid sequences after the 20th (mature protein) in the body protein are fixed as cell membrane proteins.

Since this fatty acid is considered to hinder the secretory expression of the antigen protein, in order to prevent the binding of the fatty acid, the cysteine residue at the amino terminus of the mature protein is changed to an alanine residue so as to be an alanine residue. Side primer DNA was designed. The base sequence of this primer DNA is shown by SEQ ID NO: 8. Of the base sequence represented by SEQ ID NO: 8, the fourth to ninth sequences are the restriction enzyme Pst
It is a recognition site of I, and the 6th to 17th sequences are base sequences corresponding to each amino acid residue of alanine, glycine, serine, and serine in order. This primer DNA is
It was chemically synthesized with a synthesizer. This is designated as primer 47-1. On the other hand, a primer DNA on the carboxyl group terminal side was designed so as to include a genetic code of the end of translation in the gene and to form a recognition site for the restriction enzyme BamHI immediately thereafter. The nucleotide sequence of this primer DNA is SEQ ID NO: 9
Indicated by In the base sequence represented by SEQ ID NO: 9, the fourth to ninth sequences are recognition sites for the restriction enzyme BamHI, and the tenth to twelfth sequences are the genetic code of the end of translation. This primer DNA was chemically synthesized using a DNA synthesizer. This is designated as primer 47-2.

Then, using the chromosomal DNA of Treponema pallidum as a template DNA, 62 μl of water,
1 μl of template DNA, Taq polymerase buffer 10
μl, 1.25 mM dNTP (dATP, dCTP,
An equimolar mixture of dGTP and dTTP) 16 μl, 2
0 pmole / μl aqueous solution of primer 47-1 5μ
1, 20 pmole / μl 5 μl of aqueous solution of primer 47-2 and 1 μl of Taq polymerase (5 uni
t) was mixed, heated at 96 ° C. for 0.5 minute, heated at 94 ° C. for 1 minute, cooled at 54 ° C. for 1 minute, and kept at 70 ° C. for 1 minute as one cycle. 2
The PCR operation was performed under the condition of repeating 5 cycles. A part of the obtained reaction solution was digested with restriction enzymes PstI and KpnI, and the rest was digested with restriction enzymes KpnI and BamHI, respectively, and a PstI-KpnI fragment (654 bp) and Kpn
An I-BamHI fragment (624 bp) was obtained.

The plasmid pNH400 [Journal of Bacteriology, 177, 745-749 (1995) (J.
Bacteriol., Vol. 177, p. 745-749 (1995))], digested with restriction enzymes PstI and BamHI, and digested with the PstI-KpnI fragment (654 bp) and K
The pnI-BamHI fragment (624 bp) was mixed, 20 μl of solution A and 5 μl of solution B contained in Takara DNA Ligation Kit (Takara Shuzo) were added, and the mixture was incubated at 16 ° C. for 30 minutes to carry out a ligation reaction. And the plasmid pNH40, which is approximately 5.4 kbp in size.
0TP47 was constructed.

(2) Preparation of Transformant Containing Plasmid pNH400TP47
The strain was introduced into Brevis HPD31 strain to obtain a transformant (Bacillus brevis HPD31 strain carrying plasmid pNH400TP47). This transformant has the accession number F
ERM BP-5642 has been deposited at the National Institute of Biotechnology and Industrial Technology.

(3) Production of Modified Protein A TMN agar plate medium (peptone 1%, meat extract 0.5%, yeast extract 0.2%, glucose 1%, MgSO ₄ 0.1%) autoclaved at 121 ° C. for 5 minutes.
01%, FeSO ₄ 0.01%, MnSO ₄ 0.00
1%, ZnSO ₄ 0.0001%, agar 1.5%, neomycin 50 μg / ml, pH 7.0 medium)
Bacillus harboring plasmid pNH400TP47
Brevis HPD31 strain was smeared and cultured at 30 ° C. for 48 hours to form colonies. The resulting colonies are
A 500 ml Erlenmeyer flask containing 100 ml of a liquid medium (medium obtained by removing agar from the TMN agar plate medium) was inoculated into a 500 ml Erlenmeyer flask, cultured at 30 ° C. for 2 days with shaking, and the culture was centrifuged.
The supernatant was obtained and used as a modified protein-containing fraction of an antigen protein having a molecular weight of about 47 K daltons derived from Treponema pallidum.

(4) SDS- of fraction containing modified protein
Polyacrylamide gel electrophoresis The culture supernatant obtained in the above (3) and the sample processing solution (0.
0625M Tris-HCl (pH 6.8), 2% SDS,
A mixture of 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue) was mixed well in an equal volume, and heated at 100 ° C. for 5 minutes to obtain a sample. Using a polyacrylamide gradient gel (manufactured by Daiichi Pure Chemicals, trade name: Multigel 10/20) having an acrylamide concentration of 10% to 20%,
10 μl of the sample is added to the gel, and the running buffer (250 mM
Using 25 mM Tris buffer (pH 8.3) containing glycine and 0.1% SDS at a current of 40 mA, SDS-
Polyacrylamide gel electrophoresis was performed. Two sample migration lanes were used, and a molecular weight marker was simultaneously migrated.

(5) Confirmation of Modified Protein After the completion of electrophoresis, a gel containing a molecular weight marker and one sample migration lane was cut out and stained with Coomassie brilliant blue (CBB). As a result, as shown in FIG. 2, a blue band was observed at a position of a molecular weight of about 47K dalton (lane C), and it was confirmed that a protein having a molecular weight of about 47K dalton was contained in the culture supernatant. Was.

(6) Examination of antigenicity of modified protein by Western blot method On the other hand, the remaining gel was immersed in a transfer buffer (aqueous solution containing 0.02 M Tris, 0.15 M glycine and 20% ethanol) for 15 minutes. Equilibrated. Subsequently, the gel was placed on a nitrocellulose membrane (manufactured by Bio-Rad) that had been equilibrated in the same manner, and both sides were similarly sandwiched between equilibrated filter papers.
The protein contained in the gel was transferred onto a nitrocellulose membrane by applying a constant voltage of V for 1 hour. After the transfer, the nitrocellulose membrane was immersed in a 1% bovine serum albumin (hereinafter abbreviated as BSA) solution (manufactured by KPL, trade name: 10-fold concentrated solution for BSA dilution / blocking) and allowed to stand at 4 ° C. overnight.

The obtained nitrocellulose membrane was washed with a washing buffer (0.015 M Tris, 0.2 M NaCl,
Washed with 0.05% Tween 20) for 10 minutes. At that time, the washing buffer was replaced with a new one every 2 minutes. The nitrocellulose membrane was cut into a book having a width of about 5 mm for each lane and placed in a grooved reaction vessel. 1 ml of syphilis patient serum diluted 100-fold with a serum dilution buffer (aqueous solution containing 0.015 M Tris, 0.2 M NaCl and 0.2% BSA) is placed in a reaction vessel, and reacted for 2 hours under shaking conditions. I let it. After the reaction, the nitrocellulose membrane was washed for 35 minutes using a washing buffer under shaking conditions. At that time, the washing buffer was replaced with a new one once after 15 minutes, and thereafter three times every 5 minutes thereafter. Subsequently, the nitrocellulose membrane was placed in a grooved reaction vessel, and allowed to react with 1 ml of a peroxidase-labeled anti-human IgG antibody (manufactured by KPL) diluted 500-fold with a serum dilution buffer under shaking conditions for 2 hours. . After the reaction, the nitrocellulose membrane was washed for 35 minutes using a washing buffer under shaking conditions. At this time, the washing buffer was first added once after 15 minutes, and then 5 times.
Replaced with a new one three times every minute. After washing, a diaminobenzidine solution (0.05 M Tris, 0.9% NaCl solution 20
ml, 32% of a 31% hydrogen peroxide solution, and 10 mg of diaminobenzidine (solution)) for 10 minutes. And
The nitrocellulose membrane was thoroughly washed with distilled water and dried.

As a result, as shown in FIG. 2, a brown band was observed at the position of a molecular weight of about 47 K daltons (lane W), and the serum of a syphilis patient was found to be an antigenic protein of a molecular weight of about 47 K daltons derived from Treponema pallidum. It was confirmed that it reacted with the modified protein. Incidentally, judging from the band intensity, the production amount of the modified protein was about 1 g /
(1 liter of culture). On the other hand, the same operation was performed using serum of a healthy person instead of serum of a syphilis patient, but no band was observed, confirming that the modified protein did not react with the serum of a healthy person.

From the above, it was shown that the modified protein of the antigen protein having a molecular weight of about 47 K daltons derived from Treponema pallidum obtained in the present invention specifically reacts with the serum of syphilis patients.

(7) Anti-treponema by ELISA
Measurement of paridum antibody While gently stirring the culture supernatant obtained in the above (3), ammonium sulfate was added so that the concentration became 25%.
Stir at 37 ° C overnight, further stir at 37 ° C for 1 hour,
The precipitate was obtained by centrifugation at 000 × g for 30 minutes. The resulting precipitate is washed with 50 mM ammonium sulfate containing 50%
The precipitate was washed by suspending in a Tris-HCl buffer (pH 8.6) and centrifuging under the above conditions to wash the precipitate, and this washing operation was performed twice. The resulting precipitate is washed with a small amount of buffer (5% containing 1% SDS).
0 mM Tris-HCl buffer (pH 8.6)) to obtain a protein solution. This protein solution is
Diluted with 0 mM Tris-HCl buffer (pH 8.6),
When the protein concentration is 0.95 μg / ml and the SDS concentration is 0.0
A 2% antigen solution was prepared, and 100 µl of the solution was poured into a well (recess) of a microtiter plate and allowed to stand at 4 ° C overnight.

The antigen solution was removed by suction, and the wells were
Washing solution (0.05% Tween 20 and 0.1% Na
Washed twice with N3-containing phosphate buffered saline (PBS)). Subsequently, 250 μl of a blocking solution (a solution obtained by diluting a 10-fold concentrated bovine serum albumin (BSA) solution manufactured by KPL Corporation with PBS 2 times) was poured into the wells,
After leaving still at 37 ° C. for 1 hour, the blocking solution was removed by suction. Then, 100 μl of serum of a syphilis patient diluted 200-fold with the washing solution was poured into the wells, and allowed to stand at 37 ° C. for 1 hour.
Washed 3 times with 1 of the washing solution. Further, a goat anti-human IgG monoclonal antibody (manufactured by KPL) labeled with alkaline phosphatase was diluted with a diluent (Tris 0.60).
6 g, MgCl ₂ .H ₂ O 20.3 mg, NaN ₃ 0.1
g, 5 g of BSA, 50 μl of Tween 20, 0.3 ml of goat serum and 10 ml of glycerin in a total volume of 100
100 μl of a solution (secondary antibody solution) diluted 1000-fold with 0.1 ml of an aqueous solution, adjusted to pH 8.0 with HCl) was poured into the wells, allowed to stand at 37 ° C. for 1 hour, and then the secondary antibody solution was removed by suction. The wells were washed three times with 250 μl of the washing solution.

Finally, a coloring reagent (MgCl ₂ .6H ₂ O)
0.1374 g and diethanolamine 141.9 ml
(A solution obtained by diluting 1 ml of a total of 270 ml of an aqueous solution containing distilled water with distilled water 5 times and dissolving 1 tablet of a substrate tablet (manufactured by KPL Co., Ltd., containing 10 mg of p-nitrophenyl phosphate))
After 100 μl was poured into the well and left at room temperature for 10 minutes to cause a color reaction, 25 μl of 3N NaOH was poured into the well to stop the color reaction, and the absorbance at 405 nm was measured with a microplate reader (manufactured by Tosoh Corporation). . The obtained absorbance was 0.425. Thereby, the molecular weight of about 4 derived from Treponema pallidum obtained in the present invention is obtained.
It was shown that an anti-Treponemal pallidum antibody in the serum of a syphilis patient can be measured by ELISA using a modified protein of the 7K dalton antigen protein as an antigen.

Example 2 Production of a modified protein of an antigenic protein derived from Treponema pallidum having a molecular weight of about 17 K daltons, examination of the antigenicity of the modified protein by Western blotting, and measurement of anti-Treponemal pallidum antibody by ELISA (1) Treponema・ Molecular weight approx. 17K derived from Pari dam
Preparation of Recombinant Vector Expressing Dalton Antigen Protein Modified Protein A recombinant vector was prepared according to the procedure shown in FIG. The aforementioned Akins et al. Document describes the nucleotide sequence of a DNA encoding an antigen protein having a molecular weight of about 17 K daltons derived from Treponema pallidum (SEQ ID NO: 10).
).

This antigenic protein having a molecular weight of about 17K daltons is considered to become a cell membrane protein in Treponema pallidum by the following mechanism. First, it is synthesized as a precursor protein containing a signal sequence (an amino acid sequence of positions 1 to 156 added to the base sequence shown in SEQ ID NO: 10) in Treponema pallidum cells,
Subsequently, the fatty acid present in the bacterium forms a thioester bond with the cysteine residue at position 22 in this precursor protein. Then, the amino acid sequence at positions 1 to 21 in this precursor protein is removed as a signal sequence by a signal peptidase present in the cells, and further,
The bound fatty acids are fixed in the lipid bilayer of the cell membrane,
Thereby, the amino acid sequence after the 22nd position (mature protein) in the precursor protein is fixed as a cell membrane protein.

Since this fatty acid is considered to hinder the secretory expression of the antigen protein, in order to prevent the binding of the fatty acid, the cysteine residue at the amino terminal of the mature protein is changed to an alanine residue so as to be an alanine residue. Side primer DNA was designed. The base sequence of this primer DNA is represented by SEQ ID NO: 11. In the base sequence represented by SEQ ID NO: 11, the fourth to eighth sequences are the restriction enzyme P
It is a recognition site of stI, and the 6th to 14th sequences are base sequences corresponding to amino acid residues of alanine, valine, and serine, respectively. This primer DNA was chemically synthesized using a DNA synthesizer. This is designated as primer 17-1. On the other hand, a primer DNA on the carboxyl group terminal side was designed so as to include the genetic code of the end of translation in the gene and to have a recognition site for the restriction enzyme HindIII immediately thereafter. The base sequence of this primer DNA is represented by SEQ ID NO: 12. In the base sequence represented by SEQ ID NO: 12, the 4th to 9th sequences are recognition sites for the restriction enzyme HindIII, and the 8th to 10th sequences are the genetic code for translation completion. This primer DNA was chemically synthesized using a DNA synthesizer. This is designated as primer 17-2.

Using the chromosomal DNA obtained in Example 1 (1) as the template DNA and the primers 17-1 and 17-2 as the primer DNA, the PCR operation was performed in the same manner as in Example 1 (1). went. The obtained PCR reaction solution was digested with restriction enzymes HindIII and PstI to obtain a HindIII-PstI fragment (405 bp) (this fragment is referred to as an HP fragment).

Plasmid pNU210 [Hideo Yamagata et al., Protein Nucleic Acid Enzyme, Vol. 37, pp. 258-268 (1992)] was used as a template DNA, and the Bacillus sp.
Brevis cell wall protein (MWP) gene [Yamagata et al., Journal of Bacteriology, 169, 124]
5-1289 (1987) (Yamagata, H., et al, J. Bacterio
l., Vol. 169, p. 1245-1289 (1987))] so that the amino acid-terminal primer DNA represented by SEQ ID NO: 13 is amplified so that the MWP signal sequence and the multiple cloning site are amplified. And designing a carboxyl group terminal side primer DNA represented by SEQ ID NO: 14,
These primer DNAs were synthesized with a DNA synthesizer.

Next, the plasmid p
NU210 1 μl, water 62 μl, Taq polymerase buffer 10 μl, 1.25 mM dNTP (dAT
An equimolar mixture of P, dCTP, dGTP and dTTP)
16 μl, 20 pmole / μl Aqueous solution of amino-terminal primer DNA 5 μl, 20 pmole / μl
l Aqueous solution of primer DNA at the carboxyl group end
5 μl and 1 μl of Taq polymerase (5 units)
And heating at 96 ° C. for 0.5 minute, heating at 94 ° C. for 1 minute, cooling at 54 ° C. for 1 minute, and keeping at 70 ° C. for 1 minute as one cycle, and this step is 25 cycles Perform PCR operation under repeated conditions and obtain the PC
The R reaction solution was digested with restriction enzymes SmaI and EcoRI.

On the other hand, plasmid pUB110 (manufactured by Takara Shuzo Co., Ltd.) is digested with restriction enzymes EcoRI and PvuII.
20 μl of solution A and 5 μl of solution B contained in (Takara Shuzo Co., Ltd.)
Was added and incubated at 16 ° C. for 30 minutes to perform a ligation reaction to obtain a plasmid pNH300.
This plasmid pNH300 was replaced with the restriction enzyme HindIII.
And PstI. This digest was mixed with the HP fragment, and a ligation reaction was carried out in the same manner using the kit.
300TP17 was constructed.

(2) Preparation of Transformant Containing Plasmid pNH300TP17 The plasmid constructed by the electric pulse method was
A transformant (Bacillus brevis HP containing plasmid pNH300TP17) was introduced into Brevis HPD31 strain.
D31 strain). This transformant has the accession number FERM
BP-5641 has been deposited with the National Institute of Advanced Industrial Science and Technology.

(3) Production of Modified Protein The above plasmid pNH300TP17 was used as a transformant.
In the same manner as in Example 1 (3), except that Bacillus brevis HPD31 strain containing Bacillus brevis HPD31 was used, a modified protein-containing fraction of an antigenic protein derived from Treponema pallidum and having a molecular weight of about 17 K daltons was obtained.

(4) SDS- of fraction containing modified protein
Polyacrylamide gel electrophoresis The same operation as in Example 1 (4) was performed except that the culture supernatant obtained in Example 2 (2) was used.

(5) Confirmation of Modified Protein After completion of electrophoresis, a gel containing a molecular weight marker and one sample migration lane was cut out in the same manner as in Example 1 (5).
The staining operation was performed with Coomassie Brilliant Blue (CBB). As a result, as shown in FIG.
A blue band was observed at the position of 7K dalton (lane C), confirming that the culture supernatant contained a protein having a molecular weight of about 17K dalton.

(6) Examination of antigenicity of modified protein by Western blotting Western blot was performed in the same manner as in Example 1 (6). As a result, as shown in FIG.
A brown band was observed at the position of the dalton (lane W), confirming that the serum of the syphilis patient reacted with the modified protein of the antigenic protein derived from Treponema pallidum and having a molecular weight of about 17 K daltons. Judging from the band density, the production amount of the modified protein is considered to be about 1 g / (1 liter of culture solution). On the other hand, the same operation was performed using serum of a healthy person instead of serum of a syphilis patient, but no band was observed, confirming that the modified protein did not react with the serum of a healthy person.

From the above, it was shown that the modified protein of the antigen protein having a molecular weight of about 17 K daltons derived from Treponema pallidum obtained in the present invention specifically reacts with the serum of syphilis patients.

(7) Anti-Treponema by ELISA
Measurement of paridum antibody The same operation as in Example 1 (7) was performed except that the culture supernatant obtained in Example 2 (3) was used as the culture supernatant. Here, in order to match the number of moles of the protein contained in the antigen solution with the case of the antigen solution used in Example 1 (7), a 50 mM Tris-HCl buffer (pH 8.6) and a small amount of SDS were used. An antigen solution having a protein concentration of 0.30 μg / ml and an SDS concentration of 0.02% was prepared. Then, the absorbance at 405 nm of the reaction solution after the color reaction was measured in the same manner as in Example 1 (7). The resulting absorbance is 0.8
28. Thus, ELI using the modified protein of the antigenic protein derived from Treponema pallidum fungi obtained in the present invention and having a molecular weight of about 17K daltons as an antigen
It was shown that SA can measure anti-Treponemal pallidum antibodies in the serum of syphilis patients.

Example 3 Production of anti-Treponemal pallidum antibody using modified protein as antigen Anti-Treponemal pallidum antibody is produced using the modified protein of the present invention as an antigen, for example, as follows. (A) Culture and passage of myeloma cell line Myeloma cell line was P3X63Ag8.653 (ATCC
(CRL-1580) was cultured in RPMI1640 medium containing 10% (v / v) fetal calf serum, and was cultured at 37 ° C., 5% (v / v).
v) Passage in the presence of CO2. Two weeks before subjecting to cell fusion, 0.13 mM 8-azaguanine, 0.5 μg / ml
MC-210 (Mycoplasma remover, Dainippon Pharmaceutical Co., Ltd.)
RPM containing 10% (v / v) fetal calf serum
The cells are cultured in the I1640 medium for one week, and then in the normal medium for the next one week.

(B) Mouse immunization 200 μl of a suspension of the modified protein of the present invention having a protein concentration of 270 μg / ml was centrifuged at 12,000 rpm for 10 minutes, and 200 μl of PBS was added to the precipitate, followed by resuspension. To this, 200 μl of Freund's complete adjuvant is added to form an emulsion, and 150 μl of the emulsion is injected subcutaneously on the back of the mouse (this day is defined as day 0). On days 14, 34 and 49, a suspension 1 of the modified protein having a protein concentration of 270 μg / ml
On day 69, 50 μl of a suspension of the modified protein having a protein concentration of 800 μg / ml was injected intraperitoneally, and on day 92, 100 μl of the suspension was injected intraperitoneally. On day 95, the spleen is removed and subjected to cell fusion.

(C) Cell fusion To 10 ⁸ spleen cells obtained from the spleen, 10 ⁷ myeloma cells were placed in a round-bottom glass tube and mixed well.
Centrifuge at 1400 rpm for 5 minutes, remove the supernatant and mix the cells better. 30% pre-heated to 37 ° C
(W / v) RPMI16 containing polyethylene glycol
Add 0.4 ml of 40 medium and leave for 30 seconds. 700rp
After centrifugation at 6 m for 6 minutes, RPMI 1640 medium 10 m
and slowly rotate the glass tube so that the polyethylene glycol is well mixed.
After centrifugation for 5 minutes, the supernatant was completely removed, and 5 ml of H was added to the precipitate.
Add AT medium and leave for 5 minutes. Further, 10 to 20 ml of HAT medium was added and left for 30 minutes, HAT medium was added so that the myeloma cell concentration was 3.3 × 10 ⁵ / ml, and the cells were suspended. Dispense two drops into wells of a plastic culture vessel. 5
% (V / v) carbon dioxide atmosphere at 36 ° C., and after 1 day, 7 days, and 14 days, 1-2 wells of HAT medium were added to the wells.
Add drops.

(D) Screening for antibody-producing cells
And suspended in 0.05M sodium bicarbonate buffer (pH 9.6) containing 0.02% (w / v) sodium azide so that
A dialysate was dialyzed against a 0.05 M sodium bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, and then diluted to a protein concentration of 1 to 10 μg / ml, and then used as a 96-well EIA made of vinyl chloride. 50μl in the well of the plate
The mixture is left at 4 ° C. overnight to adsorb the antigen. The supernatant was removed and 0.02% (w / v) Tween 20 was added to the wells.
Is added and left for 3 minutes, followed by removal and washing. After one more washing operation, the wells contained 1% (v / v) PBS10 containing bovine serum albumin.
Add 0 μl and leave at 4 ° C. overnight for blocking. After removing PBS containing bovine serum albumin,
After similarly washing twice with PBS containing 0.02% (w / v) Tween 20, 50 μl of the culture supernatant of the fused cells was added to the wells.
In addition, leave at room temperature for 2 hours. After similarly washing three times with PBS containing 0.02% (w / v) Tween 20, 25 ng / ml peroxidase-labeled goat anti-mouse Ig was added to the wells.
Add 50 μl of G antibody and leave at room temperature for 2 hours. 0.0
After similarly washing three times with PBS containing 2% (w / v) Tween 20, 50 μl of an ABTS solution (manufactured by KPL) was added to the wells.
l, and left at room temperature for 15 minutes to 1 hour to cause a color reaction,
The absorbance at 405 nm is measured with a 96-well EIA plate photometer. Then, the cells in the positive wells are collected with a Pasteur pipette, transferred to a 24-well plastic culture vessel, added with 1-2 ml of HAT medium, and cultured similarly.

(E) Cloning by limiting dilution method The cell concentration of the two fused cells grown in a 24-well plastic culture vessel was measured, and each was diluted with HT medium so that the number of cells became 20 cells / ml. . Separately, 4 to 6-week-old mouse thymocytes suspended in HT medium are placed in a 96-well plastic culture vessel at 1 to 2 × 10 ⁵ cells / well,
The above fused cells (cell concentration: 20 cells / ml) were added to 50
Add 1 μl / well, and culture at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere, and add 1 to 2 drops / well of HT medium after 1 day, 7 days and 14 days. 50 μl of the culture supernatant in the well in which cell proliferation was observed was collected, and the above (D)
Antibody production is confirmed in the same manner as in “Screening of antibody-producing cells”. Cells that have only a single cell colony in the well, produce antibodies that react with the modified protein of the present invention, and that proliferate rapidly are collected from the well and subsequently grown in a 24-well plastic culture vessel. Further, the same cloning operation is repeated to obtain a hybridoma that produces an anti-Treponemal pallidum antibody. This is cultured, and an anti-Treponemal pallidum antibody is produced from the culture supernatant.

[0086]

The modified protein according to claim 1 can be secreted from a microorganism. The modified protein according to claim 2 has the effect of the modified protein according to claim 1,
Further, the signal peptide has high cleavage efficiency and high secretion efficiency. The modified protein according to claim 3 has the effect of the modified protein according to claim 1 or 2, and further has antigenicity as Treponema pallidum antigen. The modified protein according to claim 4 has the effect of the modified protein according to any one of claims 1 to 3, and further has antigenicity as a Treponema pallidum-specific antigen. The modified protein according to claims 5 and 6 has the effect of the modified protein according to any one of claims 1 to 4, and has excellent antigenicity as a Treponema pallidum-specific antigen.

The DNA according to claim 7 is useful for producing a modified protein that can be secreted from a microorganism. The DNA according to the eighth aspect has the effects of the DNA according to the seventh aspect, and is useful for producing a modified protein having excellent antigenicity as a Treponema pallidum-specific antigen. The recombinant vector according to claim 9 is useful for producing a modified protein that can be secreted from a microorganism. The transformant according to claim 10 is useful for producing a modified protein that can be secreted from a microorganism. The transformant according to claim 11 has the effects of the transformant according to claim 10 and is useful for producing a modified protein having excellent antigenicity as a Treponema pallidum-specific antigen.

The method for producing a modified protein according to claim 12 is useful for producing a modified protein that can be secreted from a microorganism. The method for producing a modified protein according to claim 13 has the effects of the method for producing a modified protein according to claim 12, and is useful for producing a modified protein having excellent antigenicity. The method for measuring an anti-Treponemal pallidum antibody according to claim 14 is useful for diagnosis of syphilis infection. The reagent for measuring an anti-Treponemal pallidum antibody according to claim 15 is useful for diagnosis of syphilis infection. The diagnostic agent for syphilis infection according to claim 16 is useful for diagnosis of syphilis infection. The method for producing an anti-Treponemal pallidum antibody according to claim 17 is useful for diagnosis of syphilis infection.

[0089]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 415 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Ala Gly Ser Ser His His Glu Thr His Tyr Gly Tyr Ala Thr Leu Ser 1 5 10 15 Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln Ser Arg Asp Val Leu 20 25 30 Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly Asp Leu Asp Ala Gly 35 40 45 Met Phe Asp Ala Val Ser Arg Ala Thr His Gly His Gly Ala Phe Arg 50 55 60 Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly Glu Lys Val Leu Ser 65 70 75 80 Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys Lys Trp Glu Tyr Glu 85 90 95 Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala Ser Ala Ser Phe Gln 100 105 110 Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu Ala Val Glu Gly Ala 115 120 125 Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp Ser Glu Glu 130 135 140 Tyr Lys Ile Thr Asn Val Lys Val His Gly Met Lys Phe Val Pro Val 145 150 155 160 Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys Glu Lys Phe His Phe 165 170 175 Val Glu Asp Ser Arg Val Thr Glu Asn Th r Asn Gly Leu Lys Thr Met 180 185 190 Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser Met Glu Ser 195 200 205 Pro His Asp Leu Val Val Asp Thr Val Gly Thr Val Tyr His Ser Arg 210 215 220 Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu Lys Arg Ala Asp Gly 225 230 235 240 Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met Asn Phe Asn 245 250 255 Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu 260 265 270 Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp Ser Trp Trp Lys Thr 275 280 285 Gly Arg Val Pro Arg Ile Ser Cys Gly Ile Asn Tyr Gly Phe Asp Arg 290 295 300 Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn 305 310 315 320 Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu 325 330 335 Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val Leu Thr Ile Gly Gly 340 345 350 Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val Asp Val Phe Ala Asp 355 360 365 Gly Gln Pro Lys Leu Val Ser Asp Gln Ala Val Ser Leu Gly Gln Asn 370 375 380 Val Leu Ser Ala Asp Phe Thr Pro Gly Thr Gl u Tyr Thr Val Glu Val 385 390 395 400 Arg Phe Lys Glu Phe Gly Ser Val Arg Ala Lys Val Val Ala Gln 405 410 415

SEQ ID NO: 2 Sequence length: 135 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Ala Val Ser Cys Thr Thr Val Cys Pro His Ala Gly Lys Ala Lys Ala 1 5 10 15 Glu Lys Val Glu Cys Ala Leu Lys Gly Gly Ile Phe Arg Gly Thr Leu 20 25 30 Pro Ala Ala Asp Cys Pro Gly Ile Asp Thr Thr Val Thr Phe Asn Ala 35 40 45 Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu Lys Lys Ser Ala 50 55 60 Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg Glu Asp Gly 65 70 75 80 Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys Ala Pro His 85 90 95 Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn Ser Val Arg Tyr Met 100 105 110 Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu Met Ala Pro Phe Tyr 115 120 125 Val Leu Lys Lys Thr Lys Lys 130 135

SEQ ID NO: 3 Sequence length: 124 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Ala Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln 1 5 10 15 Asp Phe Asp Glu Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp 20 25 30 Gly Gly Lys Met Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly 35 40 45 Arg Phe Lys Ser Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser 50 55 60 Ser Gly Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu 65 70 75 80 Leu Glu Lys Gly Asn Pro Glu Met Val Asp Val Val Thr Gly Ala Thr 85 90 95 Val Ser Ser Gln Ser Phe Arg Arg Leu Gly Arg Ala Leu Leu Gln Ser 100 105 110 Ala Arg Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg 115 120 124

SEQ ID NO: 4 Sequence length: 1286 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTGCAGGCTC GTCTCATCAT GAGACGCACT ATGGCTATGC GACGCTAAGC TATGCGGACT 60 ACTGGGCCGG GGAGTTGGGG CAGAGTAGGG ACGTGCTTTT GGCGGGTAAT GCCGAGGCGG 120 ACCGCGCGGG GGATCTCGAC GCAGGCATGT TCGATGCAGT TTCTCGCGCA ACCCACGGGC 180 ATGGCGCGTT CCGTCAGCAA TTTCAGTACG CGGTTGAGGT ATTGGGCGAA AAGGTTCTCT 240 CGAAGCAGGA GACCGAAGAC AGCAGGGGAA GAAAAAAGTG GGAGTACGAG ACTGACCCAA 300 GCGTTACTAA GATGGTGCGT GCCTCTGCGT CATTTCAGGA TTTGGGAGAG GACGGGGAGA 360 TTAAGTTTGA AGCAGTCGAG GGTGCAGTAG CGTTGGCGGA TCGCGCGAGT TCCTTCATGG 420 TTGACAGCGA GGAATACAAG ATTACGAACG TAAAGGTTCA CGGTATGAAG TTTGTCCCAG 480 TTGCGGTTCC TCATGAATTA AAAGGGATTG CAAAGGAGAA GTTTCACTTC GTGGAAGACT 540 CCCGCGTTAC GGAGAATACC AACGGCCTTA AGACAATGCT CACTGAGGAT AGTTTTTCTG 600 CACGTAAGGT AAGCAGCATG GAGAGCCCGC ACGACCTTGT GGTAGACACG GTGGGTACCG 660 TCTACCACAG CCGTTTTGGT TCGGACGCAGGCGTGT GCTGAAA AGGGCTGATG 720 GCTCTGAGCT GTCGCACCGT GAGTTCATCG ACTATGTGAT GAACTTCAAC ACGGTCCGCT 780 ACGACTACTA CGGTGATGAC GCGAGCTACA CCAATCTGAT GGCGAGTTAT GGCACCAAGC 840 ACTCTGCTGA CTCCTGGTGG AAGACAGGAA GAGTGCCCCG CATTTCGTGT GGTATCAACT 900 ATGGGTTCGA TCGGTTTAAA GGTTCAGGGC CGGGATACTA CAGGCTGACT TTGATTGCGA 960 ACGGGTATAG GGACGTAGTT GCTGATGTGC GCTTCCTTCC CAAGTACGAG GGGAACATCG 1020 ATATTGGGTT GAAGGGGAAG GTGCTGACCA TAGGGGGCGC GGACGCGGAG ACTCTGATGG 1080 ATGCTGCAGT TGACGTGTTT GCCGATGGAC AGCCTAAGCT TGTCAGCGAT CAAGCGGTGA 1140 GCTTGGGGCA GAATGTCCTC TCTGCGCGGATT TCACTCCCGG CACTGAGTAC ACGGTTGAGG 1200 TTAGGTTCAA GGAATTCGGT TCTGTGCGTG CGAAGGTAGT GGCCCAGTAG AAGAGGGGTG 1260 TCCTATCCCG TGTGTCTTAA GGATCC 1286

SEQ ID NO: 5 Sequence length: 416 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTGCAGTCTC GTGCACAACC GTGTGTCCGC ACGCCGGGAA GGCCAAAGCG GAAAAGGTAG 60 AGTGCGCGTT GAAGGGAGGT ATCTTTCGGG GTACGCTACC TGCGGCCGAT TGCCCGGGAA 120 TCGATACGAC TGTGACGTTC AACGCGGATG GCACTGCGCA AAAGGTAGAG CTTGCCCTTG 180 AGAAGAAGTC GGCACCTTCT CCTCTTACCT ATCGCGGTAC GTGGATGGTA CGTGAAGACG 240 GAATTGTCGA ACTCTCGCTT GTGTCCTCGG AGCAATCGAA GGCACCGCAC GAGAAAGAGC 300 TGTACGAGCT GATAGACAGT AACTCCGTTC GCTACATGGG CGCTCCCGGC GCAGGAAAGC 360 CTTCAAAGGA GATGGCGCCG TTTTACGTGC TCAAAAAAAC AAAGAAATAG AAGCTT 416

SEQ ID NO: 6 Sequence length: 372 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCATCATTTA GTTCTATCCC GAATGGCACG TACCGGGCGA CGTATCAGGA TTTTGATGAG 60 AATGGTTGGA AGGACTTTCT CGAGGTTACT TTTGATGGTG GCAAGATGGT GCAGGTGGTT 120 TACGATTATC AGCATAAAGA AGGGCGGTTT AAGTCCCAGG ACGCTGACTA CCATCGGGTC 180 ATGTATGCAT CCTCGGGCAT AGGTCCTGAA AAGGCCTTCA GAGAGCTCGC CGATGCTTTG 240 CTTGAAAAGG GTAATCCCGA GATGGTGGAT GTGGTCACCG GTGCAACTGT TTCTTCCCAG 300 AGTTTCAGGA GGTTGGGTCG TGCGCTTCTG CAGAGTGCGC GGCGCGGCGA GAAGGAAGCC 360 ATTATTAGCA GG 372

SEQ ID NO: 7 Sequence length: 1302 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA sequence GTG AAA GTG AAA TAC GCA CTA CTT TCT GCC GGA GCG CTG CAG TTG TTG 48 Val Lys Val Lys Tyr Ala Leu Leu Ser Ala Gly Ala Leu Gln Leu Leu 1 5 10 15 GTT GTA GGC TGT GGC TCG TCT CAT CAT GAG ACG CAC TAT GGC TAT GCG 96 Val Val Gly Cys Gly Ser Ser His His Glu Thr His Tyr Gly Tyr Ala 20 25 30 ACG CTA AGC TAT GCG GAC TAC TGG GCC GGG GAG TTG GGG CAG AGT AGG 144 Thr Leu Ser Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln Ser Arg 35 40 45 GAC GTG CTT TTG GCG GGT AAT GCC GAG GCG GAC CGC GCG GGG GAT CTC 192 Asp Val Leu Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly Asp Leu 50 55 60 GAC GCA GGC ATG TTC GAT GCA GTT TCT CGC GCA ACC CAC GGG CAT GGC 240 Asp Ala Gly Met Phe Asp Ala Val Ser Arg Ala Thr His Gly His Gly 65 70 75 80 GCG TTC CGT CAG CAA TTT CAG TAC GCG GTT GAG GTA TTG GGC GAA AAG 288 Ala Phe Arg Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly Glu Lys 85 90 95 GTT CTC TCG AAG CAG GAG ACC GAA GAC AGC AGG GGA AGA AAA AAG TGG 336 Val Leu Ser Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys Lys Trp 100 105 110 GAG TAC GAG ACT GAC CCA AGC GTT ACT AAG ATG GTG CGT GCC TCT GCG 384 Glu Tyr Glu Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala Ser Ala 115 120 125 TCA TTT CAG GAT TTG GGA GAG GAC GGG GAG ATT AAG TTT GAA GCA GTC 432 Ser Phe Gln Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu Ala Val 130 135 140 GAG GGT GCA GTA GCG TTG GCG GAT CGC GCG AGT TCC TTC ATG GTT GAC 480 Glu Gly Ala Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp 145 150 155 160 AGC GAG GAA TAC AAG ATT ACG AAC GTA AAG GTT CAC GGT ATG AAG TTT 528 Ser Glu Glu Tyr Lys Ile Thr Asn Val Lys Val His Gly Met Lys Phe 165 170 175 GTC CCA GTT GCG GTT CCT CAT GAA TTA AAA GGG ATT GCA AAG GAG AAG 576 Val Pro Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys Glu Lys 180 185 190 TTT CAC TTC GTG GAA GAC TCC CGC GTT ACG GAG AAT ACC AAC GGC CTT 624 Phe His Phe Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn Gly Leu 195 200 205 AAG ACA ATG CTC ACT GAG GAT AGT TTT TCT GCA CGT AAG GTA AGC AGC 672 Lys Thr Met Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser 210 215 220 ATG GAG AGC CCG CAC GAC CTT GTG GTA GAC ACG GTG GGT ACC GTC TAC 720 Met Glu Ser Pro His Asp Leu Val Val Asp Thr Val Gly Thr Val Tyr 225 230 235 240 CAC AGC CGC CGT TTT GGT TCG GAC GCA GAG GCT TCT GTG ATG CTG AAA AGG 768 His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu Lys Arg 245 250 255 GCT GAT GGC TCT GAG CTG TCG CAC CGT GAG TTC ATC GAC TAT GTG ATG 816 Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met 260 265 270 AAC TTC AAC ACG GTC CGC TAC GAC TAC TAC GGT GAT GAC GCG AGC TAC 864 Asn Phe Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr 275 280 285 ACC ACC AAT CTG ATG GCG AGT TAT GGC ACC AAG CAC TCT GCT GAC TCC TGG 912 Thr Asn Leu Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp Ser Trp 290 295 300 TGG AAG ACA GGA AGA GTG CCC CGC ATT TCG TGT GGT ATC AAC TAT GGG 960 Trp Lys Thr Gly Arg Val Pro Arg Ile Ser Cys Gly Ile Asn Tyr Gly 305 310 315 320 TTC GAT CGG TTT AAA GGT TCA GGG CCG GGA TAC TAC AGG CTG ACT TTG 1008 Phe Asp Arg Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu Thr Leu 325 330 335 ATT GCG AAC GGG TAT AGG GAC GTA GTT GCT GAT GTG CGC TTC CTT CCC 1056 Ile Ala Asn Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe Leu Pro 340 345 350 AAG TAC GAG GGG AAC ATC GAT ATT GGG TTG AAG GGG AAG GTG CTG ACC 1104 Lys Tyr Glu Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val Leu Thr 355 360 365 ATA GGG GGC GCG GAC GCG GAG ACT CTG ATG GAT GCT GCA GTT GAC GTG 1152 Ile Gly Gly Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val Asp Val 370 375 380 TTT GCC GAT GGA CAG CCT AAG CTT GTC AGC GAT CAA GCG GTG AGC TTG 1200 Phe Ala Asp Gly Gln Pro Lys Leu Val Ser Asp Gln Ala Val Ser Leu 385 390 395 395 400 GGG CAG AAT GTC CTC TCT GCG GAT TTC ACT CCC GGC ACT GAG TAC ACG 1248 Gly Gln Asn Val Leu Ser Ala Asp Phe Thr Pro Gly Thr Glu Tyr Thr 405 410 415 GTT GAG GTT AGG TTC AAG GAA TTC GGT TCT GTG CGT GCG AAG GTA GTG 1296 Val Glu Val Arg Phe Lys Glu Phe Gly Ser Val Arg Ala Lys Val Val 420 425 430 GCC CAG TAG AAG AGG GGT GTC CTA TCC CGT GTG TCT 1302 Ala Gln 434

SEQ ID NO: 8 Sequence length: 36 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TAGCTGCAGG CTCGTCTCAT CATGAGACGC ACTATG 36

SEQ ID NO: 9 Sequence length: 36 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AAAGGATCCT TAAGACACAC GGGATAGGAC ACCCCT 36

SEQ ID NO: 10 Sequence length: 468 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: Genomic DNA sequence ATG AAA GGA TCT GTC CGC GCG CTG TGC GCG TTC CTT GGT GTT GGA GCG 48 Met Lys Gly Ser Val Arg Ala Leu Cys Ala Phe Leu Gly Val Gly Ala 1 5 10 15 CTC GGT AGC GCT TTG TGT GTC TCG TGC ACA ACC GTG TGT CCG CAC GCC 96 Leu Gly Ser Ala Leu Cys Val Ser Cys Thr Thr Val Cys Pro His Ala 20 25 30 GGG AAG GCC AAA GCG GAA AAG GTA GAG TGC GCG TTG AAG GGA GGT ATC 144 Gly Lys Ala Lys Ala Glu Lys Val Glu Cys Ala Leu Lys Gly Gly Ile 35 40 45 TTT CGG GGT ACG CTA CCT GCG GCC GAT TGC CCG GGA ATC GAT ACG ACT 192 Phe Arg Gly Thr Leu Pro Ala Ala Asp Cys Pro Gly Ile Asp Thr Thr 50 55 60 GTG ACG TTC AAC GCG GAT GGC ACT GCG CAA AAG GTA GAG CTT GCC CTT 240 Val Thr Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu 65 70 75 80 GAG AAG AAG TCG GCA CCT TCT CCT CTT ACC TAT CGC GGT ACG TGG ATG 288 Glu Lys Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met 85 90 95 GTA CGT GAA GAC GGA ATT GTC GAA CTC TCG CTT GTG TCC TCG GAG CAA 336 Val Arg Glu Asp Gly Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln 100 105 110 TCG AAG GCA CCG CAC GAG AAA GAG CTG TAC GAG CTG ATA GAC AGT AAC 384 Ser Lys Ala Pro His Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn 115 120 125 TCC GTT CGC TAC ATG GGC GCT CCC GGC GCA GGA AAG CCT TCA AAG GAG 432 Ser Val Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu 130 135 140 ATG GCG CCG TTT TAC GTG CTC AAA AAA ACA AAG AAA 468 Met Ala Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys 145 150 155 156

SEQ ID NO: 11 Sequence length: 32 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AAACTGCAGT CTCGTGCACA ACCGTGTGTC CG 32

SEQ ID NO: 12 Sequence length: 32 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAAAAGCTTA TTTCTTTGTT TTTTTGAGCA CG
32

SEQ ID NO: 13 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAACCCGGGA ATATACTAGA GATTTTTAAC 30

SEQ ID NO: 14 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAAGAATTCA AGCTTGAGCT CCTCGAG 27

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction of plasmid pNH400TP47.

FIG. 2 is a diagram of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis diagram and a Western blot of the modified antigen protein obtained in Example 1.

FIG. 3 is a construction diagram of plasmid pNH300TP17.

FIG. 4 is a diagram of a sodium dodecyl sulfate-polyacrylamide gel electrophoretogram and a Western blot of the modified antigen protein obtained in Example 2.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 15/09 ZNA C12P 21/02 C C12P 21/02 21/08 21/08 G01N 33/53 D G01N 33/53 33/571 33/571 33/577 B 33/577 C12N 15/00 ZNAA // (C12N 1/21 C12R 1:08) (C12N 15/09 ZNA C12R 1:01) (C12P 21/02 C12R 1:08) (72) Inventor Hiromi Iijima 4-3-1 Higashi-cho, Hitachi City, Ibaraki Prefecture Inside the Pharmaceutical Research Laboratory, Hitachi Chemical Co., Ltd. (72) Inventor Yoshiki Nakao 4-3-1-1, Higashi-machi, Hitachi City, Ibaraki Prefecture, Japan 72) Inventor Ken Sawazaki 4-3-1 Higashicho, Hitachi City, Ibaraki Prefecture, Hitachi Chemical Co., Ltd., Pharmaceutical Research Laboratories (72) Inventor Makoto Mizukami 2-8 Chuo-cho, Choshi City, Chiba Prefecture ) Inventor Yukihiro Nishikawa 2-8 Chuo-cho, Choshi-shi, Chiba Higeta Shoyu Co., Ltd. Research Institute (54) [Title of Invention] Modified protein, DNA encoding the same, recombinant vector containing the DNA, transformant containing the recombinant vector, method for producing modified protein, method for measuring anti-Treponemal pallidum antibody and reagent for measurement, diagnosis of syphilis infection For the production of drugs and anti-Treponemal pallidum antibodies

Claims (17)

[Claims] 1. A modified protein having an amino acid sequence in which a cysteine residue of a protein has been converted to a non-lipid-binding amino acid residue. 2. The modified protein according to claim 1, wherein the non-lipid-binding amino acid residue is selected from the group consisting of an alanine residue, a glutamic acid residue and a serine residue. 3. The modified protein according to claim 1, wherein the protein is derived from Treponema pallidum. 4. The modified protein according to any one of claims 1 to 3, wherein the protein is at least one antigen protein having a molecular weight of about 47 K dalton and about 17 K dalton of Treponema pallidum. 5. The modified protein according to claim 1, which is a protein represented by SEQ ID NO: 1. 6. The modified protein according to claim 1, which is a protein represented by SEQ ID NO: 2. 7. A DNA encoding the modified protein according to any one of claims 1 to 6, or D complementary thereto.
NA. 8. The DNA according to claim 7, which has the nucleotide sequence of SEQ ID NO: 4 or 5. 9. A recombinant vector comprising the DNA according to claim 7 or 8. 10. A transformant comprising the recombinant vector according to claim 9. 11. FERM BP-5641 or FER
The transformant according to claim 10, which has been deposited as MBP-5642. 12. A method comprising culturing a transformant into which a gene encoding the modified protein according to any one of claims 1 to 6 has been introduced, producing and accumulating the modified protein in a culture, and obtaining the culture. A method for producing a modified protein, characterized in that: 13. The method according to claim 12, wherein ammonium sulfate is added to the culture supernatant of the obtained culture, and the precipitate is obtained by centrifugation, and the precipitate is dissolved in an aqueous solution containing a surfactant. A method for producing a modified protein. 14. A method for measuring an anti-Treponemal pallidum antibody, comprising using the modified protein according to claim 3 as an antigen. 15. An anti-Treponemal comprising the modified protein according to claim 3 as an antigen.
Reagent for measuring Paridam antibody. 16. A diagnostic agent for syphilis infection comprising the modified protein according to any one of claims 3 to 6 as an active ingredient. 17. A method for producing an anti-Treponemal pallidum antibody, comprising using the modified protein according to claim 3 as an antigen.