JP2000041678A - Soluble fused protein and gene encoding the same, and production of fused protein and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new fused protein consisting at least part of major outer membrane proteins of Clamydia trachomatis, a hydrophilic polypeptide having no immune response to human serum and the connecting portion thereof and useful e.g. for the diagnosis of infection with the above bacterium. SOLUTION: This new fused protein consists of at least part of major outer membrane proteins of Clamydia trachomatis, at least one of hydrophilic polypeptide having no immune response to human serum and the connecting portion thereof and maintain its soluble state in the absence of any protein solubilizer, and be usable for Clamydia trachomatis antibody tests and useful e.g. for the diagnosis of infection with Clamydia trachomatis. This fused protein is obtained by infecting cells with the basic unit of Clamydia trachomatis to propagate it, extracting chromosomal DNA, cloning it through PCR technique using a primer consisting of its partial sequence, binding the resultant gene to a gene encoding an artificially designed hydrophilic polypeptide, followed by expression in an expression system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antigen used for diagnosing Chlamydia trachomatis infection, a method for producing the same, and a method for testing an antibody using the same.
The present invention relates to a soluble fusion protein which can be used for Chlamydia trachomatis antibody testing, a gene encoding the same, a method for producing the fusion protein, an antibody testing method using the same, and an antibody testing kit.

[0002]

2. Description of the Related Art Chlamydia
Trachomatis) is a bacterium belonging to the genus Chlamydia and, unlike general bacteria, is an obligate intracellular parasitic pathogen that requires live host cells for growth. Infectious particles, small elementary bodies (abbreviated as EB; straight line 0.3 μm) are taken up into cells by phagocytosis and infected, and phagosomes (Phagosome)
Within a reticulate body (abbreviated as RB; diameter 0.5 to 2.
It is said to have a unique growth cycle in which it is converted to 0 μm), proliferates in two divisions, matures again into EB, is released extracellularly, and causes infection one after another.

Chlamydia trachomatis mainly infects the eyes and genitourinary tract, trachoma and inclusion body conjunctivitis, Sexually Transmitted Disease (STD), nongonococcal urethritis, cervicitis, neonatal pneumonia, etc. Cause In recent years, attention has been drawn to the spread of infection as a causative agent of STD especially in developed countries including Japan [Clinical testing, 40
(6), 655-658 (1996)].

[0004] Chlamydia trachomatis has 15 serotypes (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3). These are serologically divided into three groups, B-complex (B,
Ba, E, D, L1, L2), C-complex (C, J, H, I, A), intermediate type (G, F,
K, L3) [J. Infect.Dis., 152, 791-800 (198
Five)].

[0005] Test methods for Chlamydia trachomatis infection include an antigen test method for chlamydia cells in patient materials collected by abrasion and an antibody test method using serum as a sample. Although the antigen test method has been employed for some time, there have been problems such as pain in patients and difficulty in collecting antigen test materials. Recently, antibody testing methods that facilitate sample collection have been widely used. Antibody tests are also commonly used for screening or epidemiological investigation of persons at high risk of infection.

[0006] Five subclasses of antibodies are currently known, but IgG and IgG are important as indicators of bacterial infection.
IgM. A few months after infection, IgG reaches its maximum level and maintains its level for a long time, and is an indicator of persistent infection or a history of past infection. IgM is only produced early in infection and is an indicator of early infection. Furthermore, in the case of Chlamydia trachomatis infection, IgA is regarded as an important indicator of active infection [clinical examination, 40 (6), 693-698 (1
996)] Therefore, it is desirable that these three types of antibodies can be measured individually in the Chlamydia trachomatis antibody test.

[0007] Antigens used for the antibody test for Chlamydia trachomatis include purified EB of L2 strain or major outer membrane protein (Major) derived from L2 strain which is an antigen (species-specific antigen) specific to Chlamydia trachomatis. Outer Membrene Pr
otein, hereinafter abbreviated as MOMP, which uses about 60% of the outer membrane protein) is a partially purified product (outer membrane protein fraction). Cross-reaction occurs with antibodies from Chlamydia pneumoniae and Chlamydia sp. Therefore, in recent years, it has been attempted to produce MOMP by a genetic recombination method which can be obtained in large quantities and can reduce cross-reaction.

[0008] In recent years, antibody testing methods by enzyme immunoassay (EIA) have been developed. EIA has the advantage that multiple samples can be measured easily and quickly. In the EIA for measuring IgG and IgA, it is common to use a so-called antigen solid phase method in which an antigen immobilized on a microplate or the like is reacted with a test serum, and after washing, the antibody is reacted with a labeled antibody as a secondary antibody. is there. However, when the antigen solid phase method is used as it is for IgM detection, when a rheumatoid factor is present in serum, false positives may be generated by detecting IgG via the rheumatoid factor. Rheumatoid factor may be found not only in patients with autoimmune diseases but also in the sera of normal persons, which causes a problem that the specificity is greatly reduced. In addition, a decrease in sensitivity due to the presence of IgG also poses a problem. Therefore, a treatment has been carried out in which an IgG absorbent is added to the test serum in advance to remove the IgG.

However, it is difficult to completely remove IgG with an absorbent, and part of IgM is also absorbed and removed by the IgG absorbent, which causes a reduction in specificity and sensitivity. To solve this problem, an anti-IgM antibody is immobilized in advance,
It is effective to use a so-called antibody capture method in which only the antigen is allowed to bind, and then reacted with the antigen, and then reacted with a labeled antibody that binds to the antigen. By using this method, it is possible to detect IgM specifically and with high sensitivity.

[0010] MOMP is a highly hydrophobic protein. To solubilize it, a high concentration of a denaturant such as urea or guanidine hydrochloride, or sodium dodecyl sulfate (Sodium Dodecyl sulfate) is used.
A protein solubilizing agent represented by a strong ionic surfactant such as Sulfate (hereinafter abbreviated as SDS) is required. However, when MOMP is used as an antigen in the antibody capture method described above, it is normal if a high concentration of a protein solubilizer such as a denaturant or an ionic surfactant is contained in the antigen solution to maintain a solubilized state. No antigen-antibody reaction is established. If the antigen solution does not contain a sufficient concentration of a protein solubilizing agent such as a denaturant or ionic surfactant, MOMP aggregates and precipitates, or nonspecific adsorption to the solid surface Will occur.
In any case, there has been a problem that it becomes impossible to detect IgM, which is an important antibody as an indicator of initial infection.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to maintain solubility even in the absence of a protein solubilizing agent such as a denaturing agent or an ionic surfactant, and to have a natural antigenicity against Chlamydia trachomatis antibodies. Providing a means to measure Chlamydia trachomatis antibody with high sensitivity and specificity by producing a protein identical to type MOMP by genetic recombination and using it as an antigen for Chlamydia trachomatis antibody test .

[0012]

Means for Solving the Problems The present inventors have designed a DNA encoding a hydrophilic polypeptide not immunoreactive with human serum at the end or inside of a gene encoding at least a part of Chlamydia trachomatis MOMP. When a gene is prepared and introduced into a host such as Escherichia coli or insect cells and expressed, at least a portion of the expression product, Chlamydia trachomatis MOMP, and a hydrophilic polypeptide having no immunoreactivity with human serum It has been found that a fusion protein with the above maintains a solubilized state even in the absence of a protein solubilizing agent such as a denaturing agent or an ionic surfactant. Also,
Ig by antibody supplementation method using this fusion protein as antigen
As a result of performing EIA for M detection, we succeeded in improving sensitivity and specificity compared to EIA by the antigen solid phase method using an IgG absorbent. Furthermore, using this fusion protein as an antigen, an EIA for detecting IgG, an EIA for detecting IgA,
As a result of performing EIA for IgM detection, MOMP was used as the antigen.
The present inventors have confirmed that they have the same performance as EIA, and have completed the present invention.

That is, when the present invention is used as an antigen in a Chlamydia trachomatis antibody test utilizing an antigen-antibody reaction such as EIA, it is possible to detect Chlamydia trachomatis antibody with high sensitivity and specificity. A fusion protein of at least a portion of Chlamydia trachomatis MOMP and a hydrophilic polypeptide having no immunological cross-reactivity with human serum, a method for producing the fusion protein by genetic recombination,
And a method for testing Chlamydia trachomatis antibodies using the same as an antigen and a kit for testing Chlamydia trachomatis antibodies.

Hereinafter, the present invention will be described in more detail.

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the structure of a MOMP will be described. MOMP has four VD (Variable Domain) regions (having VDI-IV regions in order from the N-terminus) that differ greatly in amino acid sequence between Chlamydia trachomatis strains and are estimated to be exposed outside the cell membrane. There are five highly hydrophobic regions (N-terminal, C-terminal and
It consists of a region sandwiched between VD regions. For convenience, in this specification
(Common domain, hereinafter referred to as CD region, abbreviated as CDI to V region in order from the N-terminus). Each area is
CDI, VDI, CDII, VDII, CDIII, VDIII, CDIV, VDI from N-terminal
V and CDV are arranged in this order.

It is essential that a hydrophilic polypeptide to be fused with MOMP has no immunoreactivity with human serum. When a hydrophilic protein or a part thereof is selected as the hydrophilic polypeptide, an example of a protein satisfying this condition is bovine serum albumin (Bovine Serum Albumin,
However, the present invention is not limited to this. When a part of the hydrophilic protein is used, it is desirable to select a part having strong hydrophilicity. A hydropathy plot can be used as a measure of hydrophilicity. This involves assigning parameters according to the degree of hydrophilicity and hydrophobicity for each amino acid residue, averaging the parameters for several consecutive amino acid residues in the protein, and assigning residues from the amino terminal to the carboxy terminal. The plot is made while moving one by one. There are several parameter assignment methods. For example, the method of Doolite et al. [J. Mol. Biol., 15
7, 105-132 (1982)], the portion where the plot is negatively shifted is the hydrophilic region.

Even when a polypeptide having an artificially designed amino acid sequence is selected as the hydrophilic polypeptide, the above-mentioned parameters of Doolite et al. Can be used for the design. It is desirable to design a sequence containing a large number of amino acid residues having negative parameters, particularly asparagine, aspartic acid, glutamine, glutamic acid, histidine, arginine residues and the like. At this time, it is essential to make the sequence immune to human serum.

The MOMP that binds the hydrophilic polypeptide may be a fragment (part) as long as it has a necessary epitope,
Further, they may be connected. The binding position may be at the amino terminus, carboxy terminus or inside of MOMP, but is preferably at the CD region. In the case of internal binding, some amino acid sequences may be overlapped before and after the binding portion to prevent destruction of the epitope in MOMP. Further, the hydrophilic polypeptide may be arranged in only one place or may be dispersed in some places. When dispersed, polypeptides having the same sequence may be arranged, or polypeptides having different sequences may be arranged. Also, the size of the hydrophilic polypeptide to be bound is preferably such that the average value of the hydropathy parameters of Doolite et al. Of all amino acid residues in the fusion protein is -0.2 or less per amino acid residue, and -0.3 or less. More desirably.

In order to obtain the MOMP gene, EBs are infected to appropriate cells, for example, HeLa or L929 cells, and Chlamydia trachomatis is grown. After crushing the cells, a large amount of sucrose density gradient centrifugation is performed. Obtain EB and extract and purify chromosomal DNA by a conventional method. As a cloning method of the MOMP gene, there are λgt10, λgt11, PCR method and the like, and recently, the PCR method [Nature 324, 163 (1986)] is often used. What is PCR? DNA consisting of partial sequence of target gene
DNA consisting of a primer and a partial sequence complementary to the gene
The primer is annealed, and an amplification reaction is performed using Taq polymerase, a thermostable DNA polymerase. In the DNA primer, it is possible to add a partial sequence to the partial sequence of the target gene or its complementary sequence as necessary, or to mutate a part of the sequence, but in that case, carefully consider the reaction conditions There is a need to.

Then, the obtained amplified fragment containing the MOMP gene is inserted into a cloning vector such as pUC19 or pBR322, and a host such as Escherichia coli is transformed to select a clone having the desired DNA. gene
DNA is obtained. To obtain a gene encoding a hydrophilic protein or a part thereof, it is convenient to carry out cloning from a commercially available gene library by PCR. MOMP gene DNA is used as the DNA primer for PCR.
It is necessary to add a part of the sequence or introduce a mutation so that it has an appropriate restriction enzyme recognition site for binding to the inside.

Gene DNA encoding a polypeptide having an artificially designed amino acid sequence can be prepared by chemical synthesis. In order to bind to the MOMP gene, it is necessary to design the DNA to be synthesized so as to have an appropriate restriction enzyme recognition site. For methods of removing unnecessary parts from the gene DNA incorporated in the vector using restriction enzymes and binding other gene DNA, see [Mo
lecular Cloning, Cold Spring Harbar Laboratory Pres
s (1989)]. According to this method, it is possible to prepare a gene DNA encoding the designed fusion protein. The sequence of the obtained DNA can be determined by the dideoxy method or the like.

For production of a fusion protein, it is necessary to select a host-vector system that stably expresses the protein. For this purpose, Escherichia coli, yeast, insect cells or mammalian cells can be used as a host.

In particular, when an antigen used for antibody testing by EIA is prepared using insect cells, the protein derived from the insect cells contaminating the antigen does not have a nonspecific reaction with human serum, so that purification is easy. In addition, the cut-off value of EIA can be set lower. Therefore, the measurable range of the absorbance of EIA is widened. In addition, it is possible to reduce a determination error due to a difference in nonspecific reaction between samples.

An expression vector using E. coli as a host can be constructed as follows. As the promoter, a Tac promoter, a Trp promoter, a Lac promoter, a T7 promoter, a PL promoter and the like can be used. A DNA fragment of the fusion protein gene is inserted downstream of the promoter in the transcription direction. The method for introducing an expression vector into E. coli is described in the following document [Molecular Cloning, Cold Sprin
g Harbar Laboratory Press (1989)].
The transformed Escherichia coli can be cultured by a conventional method, subjected to an appropriate induction treatment, and allowed to express the desired fusion protein. Expression of the fusion protein was determined by Western blotting using an anti-Chlamydia trachomatis MOMP monoclonal antibody [Proc. Natl. Acad. Sci. USA., 76 (3), 3116-31.
20 (1979)].

A transfer vector using an insect cell as a host can be constructed as follows. First, as a promoter in insect cells, a polyhedrin promoter, a basic promoter and the like are used. A DNA fragment of the target fusion protein is inserted downstream of the promoter in the direction of transcription. The method for introducing a transfer vector into insect cells is described in the following literature [Baculovirus Expression Vectors, WH Freeman & Com.
pany (1992)]. The transformed insect cells can be subjected to suspension culture or adherent culture by a conventional method. As a medium, for example, TC-100 or Grace's medium can be used.
A medium supplemented with ~ 10% serum may be used. 3 to
After culturing for 4 days, the desired fusion protein can be expressed.

The expressed fusion protein can be purified by the following method. If solubilized in the cells, the cells are disrupted by sonication, centrifuged, and the supernatant is recovered.
The fusion protein can be purified to a higher purity than the supernatant by a general protein purification method such as salting out, preparative electrophoresis or column chromatography. When aggregates are formed in the cells, the cells are crushed using an enzyme and a nonionic surfactant, centrifuged, and the precipitate is collected. The precipitate is solubilized using a denaturing agent such as urea or guanidine hydrochloride or an ionic surfactant such as SDS. The solubilized fusion protein can be purified to high purity by a general protein purification method such as preparative electrophoresis or column chromatography. In either case, the purified fusion protein solution can be replaced with a buffer of the desired composition by means such as dialysis.

An EIA method for IgM detection by an antibody capture method using the fusion protein obtained by the above-described method is described below. Anti-human IgM antibody is applied to a polystyrene flat microplate at a concentration of 1 to 100 μg / ml, preferably 5 to 100 μg / ml.
When the solid phase is immobilized at a concentration of ５０50 μg / ml, an anti-human IgM antibody solid phase plate is formed. Appropriately diluted human serum is added to the plate and reacted at a constant temperature for a fixed time, and then the plate is washed to remove unreacted substances. By this operation, only IgM in the serum binds to the anti-human IgM antibody immobilized on the plate. Next, add 0.2 ~
100 μg / ml, preferably 0.5 to 50 μg / m
1 μl of the fusion protein solution is added, and the mixture is reacted at a fixed temperature for a fixed time. After that, the plate is washed to remove unreacted substances. By this operation, the fusion protein binds to anti-Chlamydia trachomatis IgM bound to the anti-human IgM antibody immobilized on the plate.

Next, the plate is reacted with a labeled antibody, preferably an enzyme-labeled anti-Chlamydia trachomatis MOMP antibody, and then the plate is washed to remove unreacted substances.
Here, alkaline phosphatase, horseradish peroxidase and the like are generally used as the enzyme used for labeling.
Finally, a substrate for the enzyme is added, and the degree of color development is measured with an absorptiometer to determine the presence or absence of antibodies, that is, the presence or absence of infection.

The EIA method by the solid phase antigen method using the fusion protein obtained by the above method is described below. The fusion protein is placed on a polystyrene flat microplate at a concentration of 0.1 to 10 μg / ml, preferably 0.2 to 10 μg / ml.
When the solid phase is immobilized at a concentration of 2 μg / ml, a fusion protein solid phase plate is formed. Add appropriately diluted human serum to the plate. If the measurement target is IgM, add an IgG absorbent to the serum in advance. After adding the serum, the plate is reacted at a fixed temperature for a fixed time, and then the plate is washed to remove unreacted substances. By this operation, only the anti-Chlamydia trachomatis antibody in the serum binds to the fusion protein immobilized on the plate.

Next, the plate is reacted with a labeled antibody, preferably an enzyme-labeled anti-human IgG, IgA or IgM, and then the plate is washed to remove unreacted substances. Here, alkaline phosphatase, horseradish peroxidase and the like are generally used as the enzyme used for labeling. Finally, a substrate for the enzyme is added, and the degree of color development is measured with an absorptiometer to determine the presence or absence of antibodies, that is, the presence or absence of infection.

[0030]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Cloning of L2 strain Chlamydia trachomatis MOMP gene Preparation of Chlamydia trachomatis genomic DNA (1) Culture of Chlamydia trachomatis and purification of EB Chlamydia trachomatis L2 strain (L2 / 434 / Bubo)
L929 cells grown in Eagle's MEM medium containing 0% fetal calf serum (FCS) were inoculated. 2-3 at 37 ° C
Cultured for one day. In addition, a part of the infected cells was collected, and the growth of Chlamydia was confirmed by a direct fluorescent antibody method. After completion of the culture, the culture solution was discarded, Hanks BSS was added, and the cells were scraped off with rubber policemen. Next, the infected cell suspension was sonicated (2.
After 0 KHz, 30 seconds), the supernatant was collected by centrifugation (500 × g, 4 ° C., 15 minutes). The supernatant was used in 35% Renogranfin solution (10 mM HE
PES, 0.15 M NaCl), 43,000 xg, 4 ° C,
Centrifuged for 1 hour. SPG (10 mM phosphate buffer containing 0.25 M sucrose and 5 mM glutamic acid (pH 7.
2)) was added and suspended. This was layered on the discontinuous density gradient (40% / 44% / 52%) of the Renogranfin solution, and 43,000 × g,
Centrifuged at 4 ° C for 1 hour. The band at the interface of 44% and 52% was collected, diluted with SPG, and then 30,000 ×
g, and centrifuged at 4 ° C. for 30 minutes to obtain purified EB.

(2) Chlamydia trachomatis genome D
Preparation of NA A 10-fold amount of Lysis Buffer (10 mM Tr) was added to the purified EB obtained above.
is-HCl (pH8.0), 5mM EDTA, 0.5% SDS, 100 μg / ml Protein
After adding ase K) and reacting at 55 ° C. for 90 minutes, phenol / chloroform extraction was performed to precipitate genomic DNA with ethanol. The obtained genomic DNA is transferred to TE Buffer (10 mM T
ris-HCl (pH7.5), 1mM EDTA)
/ Ml, and reacted at 60 ° C for 30 minutes. After completion of the reaction, phenol / chloroform extraction was performed to precipitate genomic DNA with ethanol. Genomic DNA was dissolved in an appropriate amount of TE Buffer.

2. Chlamydia trachomatis MOMP
Cloning of Gene Using 1 μl of the genomic DNA solution of Chlamydia trachomatis L2 strain obtained in Example 1-1, the method of Saiki et al. [Na
Nature, 324, 163 (1986)].
The gene region containing MOMP was amplified by a PCR method using it (manufactured by Takara Shuzo). That is, 10 mM Tris-HCl (pH 8.3), 50 mM
1 μl of genomic DNA solution of Chlamydia trachomatis L2 strain in a reaction solution containing M KCl, 5 mM MgCl ₂ and 0.1% gelatin
And SEQ ID NO: 13 synthesized using a DNA synthesizer
In addition, 20 pmoles and 5 units of Taq polymerase were added to each of the two types of oligonucleotide primers shown in FIGS.

This reaction solution was heated at 94 ° C., 3 × min. → [(94
° C, 45sec. → 55 ° C, 45sec. → 72 ° C, 2.5min.) × 30 cycles) → [(94 ° C, 45sec. → 55 ° C, 45sec. → 72 ° C, 10min.)
× 5 cycles]. As a result of fractionating the reaction completed solution by 1% agarose gel electrophoresis, a PCR amplification product of about 1.3 Kb was confirmed. This approximately 1.3 Kb PCR amplification product is
After recovery with clean II (BIO101), DNA Blun
Smoothing was performed using ting Kit (Takara Shuzo). Then pUC19
After digesting 2 μg with 4 units of restriction enzyme SmaI at 30 ° C. for 2 hours, the 5 ′ end was dephosphorylated with alkaline phosphatase (BRL). The PCR amplified fragment and pUC19 fragment were
gation Kit (manufactured by Takara Shuzo Co., Ltd.) and the CaCl ₂ method [Molecular Cloning, Cold Spring Harbar Laboratory
Press (1989)]. Appropriately selected 10 transformants were obtained, DNA was purified by the alkali-SDS method of Birnboim et al. [Nuc. Acids Res., 7, 1513 (1979)], and clones having PCR amplified fragments were obtained by restriction enzyme mapping. (named pMOMP-L2) (Fig. 1
reference).

Plasmid DNA was purified from the obtained clone, and its nucleotide sequence was determined using DNA sequencer 373A manufactured by PerkinElmer Japan. As a result, the known nucleotide sequence of the L2 strain [J. Bacteriol., 168 (3), 1277-1287 (1986)]
Exact match.

Embodiment 2 FIG. MOMP and BSA by E. coli
Expression of fusion protein of hydrophilic portion Preparation of MOMP expression vector 20 μg of the plasmid pMOMP-L2 obtained in Example 1 was digested with 20 units of BstYI restriction enzyme at 60 ° C. for 2 hours, followed by phenol extraction and ethanol precipitation. After dissolving the DNA,
1% after digestion with KpnI 20units at 37 ° C for 2 hours
Perform agarose gel electrophoresis.
ean II (BIO101). Next, JP-A-7-147986
Promoter expression vector pPLN-MCS 5
After digesting μg with 5 units of KpnI restriction enzyme at 37 ° C for 2 hours,
Phenol extraction-ethanol precipitation was performed. After dissolving the DNA, it was digested with 5 units of NdeI restriction enzyme at 37 ° C. for 2 hours, subjected to 1% agarose gel electrophoresis, and a 3.2 kb fragment was recovered with Geneclean II. Furthermore, the oligonucleotides shown in SEQ ID NOS: 15 and 16 were synthesized using a DNA synthesizer. Oligonucleotides to each ME
After phosphorylation of the 5 'end using GALABEL (manufactured by Takara Shuzo), the phosphorylated oligonucleotide was annealed. 0.
The 8 kb fragment, the 3.2 kb fragment and the annealed oligonucleotide were ligated and introduced into E. coli N99cI ⁺ . Appropriate 24 of the obtained transformants were selected, the DNA was purified by the alkali-SDS method, and the plasmid pCT33 was obtained by mapping with restriction enzymes NdeI and KpnI and determining the nucleotide sequence near the oligonucleotide (see FIG. 2).

2. Preparation of fusion protein expression vector of MOMP and BSA hydrophilic part (1) Cloning of BSA gene Gene Amp PCR Reagent Kit from Bovine Liver QUICK-Clone ^™ cDNA (CLONETECH) according to the method of Saiki et al.
The BSA gene region was amplified by a PCR method (manufactured by Takara Shuzo). That is, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM
Bovine Liver Q in a reaction solution containing MgCl ₂ and 0.1% gelatin
1 μl of UICK-Clone ^™ cDNA and 20 pmoles and 5 units of Taq polymerase, respectively, of the two types of primers of SEQ ID NOS: 17 and 18 synthesized using a DNA synthesizer were added to a final volume of 50 μl. This reaction solution was heated at 94 ° C for 3 mi.
n. → [(94 ℃, 45sec. → 55 ℃, 45sec. → 72 ℃, 2.5min.)
× 30 cycles) → [(94 ° C, 45sec. → 55 ° C, 45sec. → 72
C., 10 min.) × 5 cycles]. 1
% Agarose gel electrophoresis, about 1.8 k
The PCR amplification product of b was confirmed. This 1.8 kb P
After the CR amplification product was recovered with Geneclean II (manufactured by BIO101), the product was further blunted using a DNA Blunting Kit (manufactured by Takara Shuzo). Next, 2 μg of pUC19 was digested with 4 units of SmaI restriction enzyme at 30 ° C. for 2 hours, and then alkaline phosphatase (BRL) was digested.
5 'end was dephosphorylated. The PCR amplified fragment and the pUC19 fragment were ligated using a Ligation Kit (Takara Shuzo) and introduced into E. coli JM109 by the CaCl ₂ method. Appropriately obtained 10 transformants were selected, the DNA was purified by the alkali-SDS method, and a clone (designated pBSA) having a PCR amplified fragment was obtained by restriction enzyme mapping (see FIG. 3). Plasmid DN was obtained from the resulting clone.
A was purified, and its nucleotide sequence was determined using DNA sequencer 373A manufactured by PerkinElmer Japan. As a result, B
Although the amino acid sequence, which was partially different from the known base sequence of SA, was encoded by a known sequence [GeneBank accession No.
X58989].

(2) Preparation of Type A Expression Vector A fusion protein (SEQ ID NO: 1) in which a BSA hydrophilic portion was inserted into the CDIII region of MOMP and 12 amino acid sequences in the CDIII region overlapped before and after the junction. An expression vector (a vector having a coding sequence of the nucleotide sequence shown in SEQ ID NO: 7) pCT78 of a fusion protein having the amino acid sequence shown in Table 1 and designated as Type A was prepared by the following method. The gene encoding a particularly high hydrophilicity part in BSA was
PBSA for insertion into the gene encoding the CDIII region
And use Gene Amp PCR Reage as described above.
The BSA gene region was amplified by PCR using nt Kit (Takara Shuzo). That is, 10 mM Tris-HCl (pH 8.3), 50 mM
PBSA in a reaction solution containing M KCl, 5 mM MgCl ₂ , 0.1% gelatin
1 ng and the two types of mutagenic oligonucleotide primers shown in SEQ ID NOs: 19 and 20 synthesized using a DNA synthesizer were 20 pmoles and Taq polymerase, respectively.
5 units were added to a final volume of 50 μl. This reaction solution was heated at 94 ° C for 3 minutes → [(94 ° C, 45sec. → 50 ° C, 45sec. → 72
℃, 2.5min.) × 30 cycles] → [(94 ° C, 45sec. → 50
° C, 45 sec. → 72 ° C, 10 min.) X 5 cycles].
The reaction completion solution was fractionated by 1% agarose gel electrophoresis, and as a result, a PCR amplification product of about 250 b was confirmed. About 2
Gene Clean II (BIO101) for PCR amplification product of 50b
Collected using Plasmid obtained in Example 2-1
5 µg of pCT33 was added to 5 units of each of the restriction enzymes SacI and XmaI.
After digestion at 37 ° C for 2 hours, 1% agarose gel electrophoresis was performed, and a fragment of about 4.1 kb was recovered using Geneclean II. Plasmid pCT33 (20 μg) was digested with restriction enzymes Bst1107I and Xm
After digesting aI at 37 ° C for 2 hours at 5 units each, 1.2
% Agarose gel electrophoresis.
Collected with neclean II. Furthermore, the above PC of about 250b
R amplification product is 5 units each of restriction enzymes SacI and Bst1107I
After digestion at 37 ° C. for 2 hours, 1.5% agarose gel electrophoresis was performed, and a fragment of about 220 b was recovered using Geneclean II. 4.1 kb fragment, 740b fragment and 220b
Was ligated, and Escherichia coli N99cI ^{+ was} introduced. Appropriate 24 of the obtained transformants were selected, the DNA was purified by the alkali-SDS method, and the plasmid pCT78 was obtained by mapping with the restriction enzymes SacI and Bst1107I and determining the nucleotide sequence near the binding site and the PCR amplified fragment (FIG. 4).

(3) Preparation of Type B Expression Vector A BSA hydrophilic portion was inserted into the CDII and IV regions of MOMP, and 20 amino acid sequences before and after the junction in the CDII region, and before and after the junction in the CDIV region. The expression vector pCT80 of a fusion protein (fusion protein having the sequence shown in SEQ ID NO: 2; designated as Type B) in which 13 amino acid sequences overlap each other (a vector whose coding part is the base sequence shown in SEQ ID NO: 8) Was produced by the following method. In order to insert a gene encoding a particularly hydrophilic portion in BSA into a gene encoding the CDII region of MOMP, the gene described in Saiki et al.
The BSA gene region was amplified by PCR using Amp PCR Reagent Kit (Takara Shuzo). That is, 10 mM Tris-
In a reaction solution containing HCl (pH 8.3), 50 mM KCl, 5 mM MgCl ₂ and 0.1% gelatin, 20 pmoles of each of the two types of mutagenic oligonucleotide primers shown in SEQ ID NOS: 21 and 22 synthesized using 1 ng of pBSA and a DNA synthesizer And Ta
5 units of q polymerase was added to a final volume of 50 μl.
The reaction solution was heated at 94 ° C. for 3 min. → [(94 ° C., 45 sec. → 50
℃, 45sec. → 72 ℃, 2.5min.) × 30 cycles) → [(94
° C, 45 sec. → 50 ° C, 45 sec. → 72 ° C, 10 min.) X 5 cycles]. As a result of fractionating the reaction completed solution by 1% agarose gel electrophoresis, a PCR amplification product of about 150 b was confirmed. This about 150 b PCR amplification product is
It was recovered using II (manufactured by BIO101). 5 μg of the plasmid pCT33 obtained in Example 2-1 was mixed with restriction enzymes AflII and EagI.
After digesting each with 5 units at 37 ° C. for 2 hours, 1% agarose gel electrophoresis was performed, and a fragment of about 4.5 kb was
Collected by clean II. After digesting 20 μg of the plasmid pCT33 with 5 units of each of the restriction enzymes AflII and SspI at 37 ° C. for 2 hours, 1.2% agarose gel electrophoresis was performed.
The fragment of 0b was recovered by Gene clean II. Furthermore, the above-mentioned PCR amplification product of about 150 b was digested with restriction units EagI and SspI at 5 ° C. for 5 hours each at 37 ° C., and subjected to 1.5% agarose gel electrophoresis.
ean II. The 4.5 kb, 350b and 120b fragments were ligated and introduced into E. coli N99cI ⁺ . Appropriate 24 of the resulting transformants were selected, the DNA was purified by the alkali-SDS method, and plasmid pCT79 was obtained by mapping with restriction enzymes EagI and SspI and determining the base sequence near the binding site and PCR amplified fragment (FIG. 5). In order to insert a gene encoding a particularly highly hydrophilic portion in BSA into a gene encoding the CDIV region of MOMP, GeA was used according to Saiki et al.
The BSA gene region was amplified by a PCR method using ne Amp PCR Reagent Kit. That is, 10 mM Tris-HCl (pH 8.3), 5
PBSA in a reaction solution containing 0 mM KCl, 5 mM MgCl ₂ , 0.1% gelatin
1 ng and two kinds of mutagenic oligonucleotide primers shown in SEQ ID NOS: 23 and 24 synthesized using a DNA synthesizer were 20 pmoles and Taq polymerase 5 respectively.
units were added to a final volume of 50 μl. 9
4 ℃, 3min. → [(94 ℃, 45sec. → 50 ℃, 45sec. → 72
℃, 2.5min.) × 30 cycles] → [(94 ° C, 45sec. → 50
° C, 45 sec. → 72 ° C, 10 min.) X 5 cycles].
As a result of fractionating the reaction completed solution by 1% agarose gel electrophoresis, a PCR amplification product of about 140 b was confirmed. About 1
The 40b PCR amplification product was recovered using Geneclean II.

5 μg of the plasmid pCT79 was replaced with the restriction enzyme PvuII.
And XhoI were digested with 5 units at 37 ° C for 2 hours,
% Agarose gel electrophoresis was performed, and a fragment of about 4.8 kb was recovered using Geneclean II. Plasmid pCT79 20μg
With restriction enzymes PvuII and BlpI in 5 units each at 37 ° C,
After digestion for 2 hours, 1.2% agarose gel electrophoresis was performed, and a 220b fragment was recovered using Geneclean II. Further, the above-mentioned PCR amplification product of about 140b was digested with restriction enzymes XhoI and Bl.
After digesting the pI for 5 hours at 37 ° C for 2 hours, 1.5
% Agarose gel electrophoresis.
Collected by Geneclean II. 4.8 kb fragment, 220b
And the fragment of 110b were ligated, and Escherichia coli N99cI ⁺
Was introduced. Appropriately selected 24 transformants obtained,
Purify DNA by alkali-SDS method and use restriction enzyme XhoI
Plasmid pCT80 was obtained by mapping with BlpI and determining the nucleotide sequence near the binding site and the PCR amplified fragment (see FIG. 6).

3. Expression of fusion protein of MOMP and BSA hydrophilic moiety Plasmid pPLN-MCS, Plasmid pCT33 for MOMP expression
, Type A expression plasmid pCT78 and Type B
Escherichia coli N4830-1 for protein expression using the expression plasmid pCT80
Was transformed. 50 μg / m of each transformant
The cells were cultured at 30 ° C. for 16 hours in 1 ml of LB medium containing l Ampicillin. 1 ml of the preculture was added to 100 m of fresh LB medium.
1 was inoculated. After culturing at 30 ° C for about 3 hours (OD600 = 0.4-
0.5), the culture temperature was shifted to 42 ° C., and the culture was further performed for 3 hours. After the culture, the cells were centrifuged to collect the cells. After suspending some of the cells in Phosphate Buffered-Saline (PBS) and disrupting the cells with an ultrasonic disrupter, Laemmli's method [Nature, 22
7,680 (1970)] and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue (CBB). After electrophoresis, Western blotting was performed using a species-specific anti-Chlamydia trachomatis MOMP monoclonal antibody. as a result,
CBB staining shows that cells transformed with pCT33 have a molecular weight of about 4
The control pPLN-M was located at a molecular weight of about 51,000 in the cells transformed with pCT78 at the position of 000, and about 52,000 in the cells transformed with pCT80 at the position of 000.
A band not observed in the cells transformed with CS was confirmed (see FIG. 13). pCT33 molecular weight of about 40,000, pC
The molecular weight of T78 is about 51,000 and the molecular weight of pCT80 is about 5
The band at the 2,000 position was confirmed by Western blotting to react with the anti-MOMP antibody, and it was confirmed that the target protein was expressed (see FIG. 14). The cells disrupted by sonication were centrifuged at 5,500 × g for 10 minutes to separate a supernatant fraction and a precipitated fraction. As a result of examining each fraction by Western blotting, it was found that all of the expressed proteins were found in the precipitated fraction and formed aggregates.

4. Purification of fusion protein of MOMP and BSA hydrophilic portion One liter of each of E. coli expressing MOMP, Type A and Type B was cultured by the above-described culture method. After collecting cells by centrifugation, 8 ml of Lysis buffer (50 mM Tris-HCl (p
H8.0), 25% (W / V) sucrose, 1 mM EDTA, 4 mg / ml Lysozyme) and left on ice for 30 minutes. Then the final concentration is 10 mM
MgCl ₂ , 10mM MnCl ₂ , 10μg / ml DNaseI, 10μg / ml RNaseA
And left at 4 ° C. for 30 minutes.
Further, add 20 ml of Detergent buffer I (20 mM Tris-HCl (p
H7.5), 0.2M NaCl, 2mM EDTA, 1% Na-deoxycholate, 1.6% N
P-40) was added, and the mixture was further left for 30 minutes. 5,000 ×
The precipitate was collected by centrifugation at g for 15 minutes. Detergent buffer II (50 mM Tris-HCl
(pH 8.0), 50 mM NaCl, 10 mM EDTA, 0.5% Triton X-100), leave at 4 ° C for 1 hour, and
The precipitate was collected by centrifugation for minutes. Detergent buffer I
The washing of the precipitate with I was repeated three times. The resulting precipitate
10 ml of 2% sarcosine solution (2% Sodium N-Lauroyl Salcosi
nate, PBS containing 1.5 mM EDTA) using a Teflon homogenizer and shaken at 45 ° C. for 1 hour.
The suspension was centrifuged at 100,000 × g for 1 hour to collect the precipitate. This operation was repeated twice.

The obtained precipitate was mixed with 20 ml of 8M Urea, 1 mM.
It was sufficiently suspended in DTT, 10 mM Na-phosphate buffer (pH 6.0) using a Teflon homogenizer, and shaken at 45 ° C. for 1 hour. The suspension was centrifuged at 100,000 × g for 1 hour to recover the supernatant. This solution was converted to a hydroxyapatite resin Macro-Prep Ceramic HydroxyapatiteTypeI
Purification was carried out as follows using column chromatography (manufactured by Bio-Rad). 8M Urea, 1mM DTT, 10mM Na-phosphate buff
The solution was sent at 5 ml / min to a φ5 cm × 15 cm column equilibrated with er (pH 6.0), the concentration of Na-phosphate buffer was increased to 200 mM, and the solution was washed thoroughly.
The concentration of the buffer was increased to 300 mM, and the eluted fraction was collected. MOMP is 50mM containing 0.1% SDS
It was dialyzed against NaCl and 50 mM boric acid (pH 8.5), and insolubles were removed by centrifugation to obtain a purified product. Type A and Ty
PeB was dialyzed against 50 mM NaCl, 50 mM boric acid (pH 8.5), and insolubles were removed by centrifugation to obtain a purified product. Separated by SDS-PAGE to test protein purity of purified product,
After CBB staining, the protein was analyzed using an image master manufactured by Pharmacia, and as a result, it was found that the purity of MOMP, Type A and Type B proteins was almost 100%.

Embodiment 3 FIG. Expression of fusion protein of part of MOMP and BSA hydrophilic part by E. coli Preparation of fusion protein expression vector of a part of MOMP and BSA hydrophilic part (1) Preparation of Type C expression vector A part of the CDII region was removed from Type A, and a VDIV region having strong immunogenicity was inserted instead. VDIV
Expression vector (fusion vector having the nucleotide sequence shown in SEQ ID NO: 9) of a fusion protein having a region (fusion protein having the sequence shown in SEQ ID NO: 3; designated as Type C) pCT81
Was produced by the following method. 5 μg of the plasmid pCT78 obtained in Example 2-2 (2) was digested with 5 units of each of the restriction enzymes EagI and EcoRV at 37 ° C. for 2 hours, subjected to 1% agarose gel electrophoresis, and the approximately 5.0 kb fragment was subjected to Geneclean.
Collected by II (BIO101). Further, oligonucleotides shown in SEQ ID NOS: 25 and 26 were synthesized using a DNA synthesizer. Oligonucleotides to each ME
After phosphorylation of the 5 'end using GALABEL (manufactured by Takara Shuzo), the phosphorylated oligonucleotide was annealed. 5.
The 0 kb fragment and the annealed oligonucleotide were ligated and introduced into E. coli N99cI ⁺ . The obtained transformants were appropriately selected, the DNA was purified by the alkali-SDS method, and the plasmid pC was determined by mapping with the restriction enzymes EagI and EcoRV and determining the nucleotide sequence near the oligonucleotide.
T81 was obtained (see Fig. 7).

(2) Preparation of Type D Expression Vector The CDII region remaining in Type C and the inserted VDIV
A fusion protein having a VSA region and a BSA hydrophilic region inserted between the two regions (a fusion protein having the sequence shown in SEQ ID NO: 4; Type D;
The expression vector (the vector whose coding part is the nucleotide sequence shown in SEQ ID NO: 10) pCT82 was named by the following method. Plasmid pCT81 5 obtained in Example 3-1 (1)
After digesting μg with 5 units of restriction enzyme EagI at 37 ° C for 2 hours,
1% agarose gel electrophoresis was performed, a fragment of about 5.1 kb was recovered with Geneclean II (manufactured by BIO101), and the 5 ′ end was dephosphorylated with alkaline phosphatase (manufactured by BRL). 5 μm of plasmid pCT80 obtained in Example 2-2 (3)
g at 37 ° C for 2 hours with 5 units of restriction enzyme EagI.
A 1.2% agarose gel electrophoresis was performed, and a fragment of about 180 b was recovered using Geneclean II. 5.1 kb and 180
The fragment of b was ligated and introduced into E. coli N99cI ⁺ . Appropriately select 24 obtained transformants, alkaline-SDS
DNA was purified by the method, and plasmid pCT81 was obtained by mapping with the restriction enzyme EagI and determining the nucleotide sequence near the junction (see FIG. 8).

2. Expression of fusion protein of a part of MOMP and BSA hydrophilic part Plasmid pPLN-MCS, Plasmid pCT33 for MOMP expression
, Type C expression plasmid pCT81 and Type D
E. coli N4830-1 for protein expression using the expression plasmid pCT82
Was transformed. 50 μg / m of each transformant
The cells were cultured at 30 ° C. for 16 hours in 1 ml of LB medium containing l Ampicillin. 1 ml of the preculture was added to 100 m of fresh LB medium.
1 was inoculated. After culturing at 30 ° C for about 3 hours (OD600 = 0.4-
0.5), the culture temperature was shifted to 42 ° C., and the culture was further performed for 3 hours. After the culture, the cells were centrifuged to collect the cells. After suspending some of the cells in PBS and crushing the cells with an ultrasonic crusher,
SDS-PAGE was performed and CBB staining was performed. After electrophoresis, Western blotting was performed using a species-specific anti-Chlamydia trachomatis MOMP monoclonal antibody. As a result, the CBB staining showed that the cells transformed with pCT33 had a molecular weight of about 40,000, the cells transformed with pCT81 had a molecular weight of about 50,000, and the cells transformed with pCT82 had a molecular weight of about 50,000. Control at 52,000 positions
A band not observed in the cells transformed with pPLN-MCS was confirmed (see FIG. 13). The molecular weight of pCT33 is about 40,00
0, the bands at the molecular weight of about 50,000 for pCT81 and about 52,000 for pCT82 were colored by Western blotting, confirming that the target protein was expressed (see FIG. 14). The cells disrupted by sonication were centrifuged at 5,500 × g for 10 minutes to separate a supernatant fraction and a precipitated fraction. As a result of examining each fraction by Western blotting, it was found that all of the expressed proteins were found in the precipitated fraction and formed aggregates.

3. Purification of fusion protein of a part of MOMP and BSA hydrophilic part One liter of each of E. coli expressing TypeC and TypeD was cultured by the above-mentioned culture method. After collecting cells by centrifugation, 8 ml of Lysis buffer (50 mM Tris-HCl (pH 8.0), 25% (W /
V) After suspending in Sucrose, 1 mM EDTA, 4 mg / ml Lysozyme), the suspension was left on ice for 30 minutes. Next, a final concentration of 10 mM MgCl ₂ , 10 mM
MnCl ₂ , 10 μg / ml DNaseI, and 10 μg / ml RNaseA were added thereto, respectively, and left at 4 ° C. for 30 minutes. 20m more
l of Detergent buffer I (20 mM Tris-HCl (pH 7.5), 0.2 MN
aCl, 2 mM EDTA, 1% Na-deoxycholate, 1.6% NP-40) was added, and the mixture was further left for 30 minutes. The precipitate was collected by centrifugation at 5,000 xg for 15 minutes. 10 to this precipitate
Double volume of Detergent buffer II (50 mM Tris-HCl (pH 8.0), 50m
M NaCl, 10 mM EDTA, 0.5% Triton X-100), left at 44 ° C. for 1 hour, and centrifuged at 5,000 × g for 10 minutes to collect a precipitate. Washing of the precipitate with Detergent buffer II was repeated three times. The resulting precipitate is
ml of 2% sarcosine solution (2% Sodium N-Lauroyl Salco)
sinate, PBS (pH 6.8) containing 1.5 mM EDTA) using a Teflon homogenizer, and shaken at 45 ° C. for 1 hour. The suspension was centrifuged at 100,000 × g for 1 hour to collect the precipitate. This operation was repeated twice.

The obtained precipitate was mixed with 20 ml of 8M Urea, 1 mM DTT, 10 ml.
The suspension was sufficiently suspended in mM Na-phosphate buffer (pH 6.0) using a Teflon homogenizer, and shaken at 45 ° C. for 1 hour. The suspension was centrifuged at 100,000 × g for 1 hour to recover the supernatant. This solution was purified as follows using a hydroxyapatite resin Macro-Prep Ceramic Hydroxyapatite Type I (manufactured by Bio-Rad) column chromatography. 8M Urea, 1mM DTT, 10mM Na-phosphate buffer (pH6.
5ml in a φ5cm x 15cm column equilibrated in 0)
/ Min, and adjust the concentration of Na-phosphate buffer to 20
After washing to 0 mM and washing well, Na-phosphate buffe
The fraction eluted by increasing the concentration of r to 300 mM was collected. This fraction was dialyzed against 50 mM NaCl and 50 mM boric acid (pH 8.5), and insolubles were removed by centrifugation to obtain a purified product.
The purified product was separated by SDS-PAGE in order to examine the protein purity, stained with CBB, and analyzed with an image master manufactured by Pharmacia. As a result, it was found that the purity of Type C and Type D proteins was almost 100%.

Embodiment 4 FIG. Expression of a fusion protein of a part of MOMP and a hydrophilic polypeptide by Escherichia coli Preparation of Expression Vector for Fusion Protein of Part of MOMP and Hydrophilic Polypeptide (1) Preparation of Type E Expression Vector A part of the CDIII region of MOMP was removed and replaced with 20.
Expression vector (fusion protein having the sequence shown in SEQ ID NO: 5; named as TypeE) into which a hydrophilic polypeptide containing two consecutive asparagine residues has been inserted (the coding portion is the nucleotide sequence shown in SEQ ID NO: 11) (Vector) pCT
51 was prepared by the following method. 5 μg of the plasmid pCT33 obtained in Example 2-1 was ligated with the restriction enzymes Bst1107I and PvuI.
After digesting each I at 5 units at 37 ° C. for 2 hours, 1% agarose gel electrophoresis was carried out, and a fragment of about 4.7 kb was converted into Ge.
The product was recovered with neclean II (manufactured by BIO101) and dephosphorylated at the 5 'end with alkaline phosphatase (manufactured by BRL). Further, using a DNA synthesizer, SEQ ID NOS: 27 and 2
The oligonucleotide shown in No. 8 was synthesized. Oligonucleotides were 5 'each using MEGALABEL (Takara Shuzo)
After phosphorylation of the termini, the phosphorylated oligonucleotide was annealed. The 4.7 kb fragment and the annealed oligonucleotide were ligated and introduced into E. coli N99cI ⁺ . Appropriate 24 of the obtained transformants were selected, the DNA was purified by the alkali-SDS method, and the base sequence near the oligonucleotide was determined to obtain the plasmid pCT51 (see FIG. 9).

(2) Preparation of TypeF Expression Vector A fusion protein (SEQ ID NO: 1) obtained by removing a part of the CDIII and CDIV regions and the VDIII region of MOMP and inserting 20 consecutive hydrophilic polypeptides containing aspartic acid instead Fusion protein having the sequence shown in No. 6; named TypeF)
An expression vector pCT52 (a vector whose coding portion is the nucleotide sequence shown in SEQ ID NO: 12) was prepared by the following method. 5 μg of the plasmid pCT33 obtained in Example 2-1 was ligated with the restriction enzymes Bst1107I and BlpI in 5 units each at 37 ° C.
After digestion for 2 hours, 1% agarose gel electrophoresis was performed, and a fragment of about 4.5 kb was recovered using Geneclean II (manufactured by BIO101). Furthermore, oligonucleotides shown in SEQ ID NOS: 29 and 30 were synthesized using a DNA synthesizer. Each oligonucleotide was phosphorylated at the 5 'end using MEGALABEL (manufactured by Takara Shuzo), and then the phosphorylated oligonucleotide was annealed. The 4.5 kb fragment and the annealed oligonucleotide were ligated and E. coli N99cI ⁺
Was introduced. Appropriately selected 24 transformants obtained,
DNA was purified by the alkali-SDS method, and plasmid pCT52 was obtained by mapping with the restriction enzyme BlpI and determining the nucleotide sequence near the oligonucleotide (see FIG. 9).

2. Expression of fusion protein of a part of MOMP and hydrophilic polypeptide Plasmid pPLN-MCS, Plasmid pCT33 for MOMP expression, Ty
Escherichia coli N4830-1 for protein expression was transformed with plasmid pCT51 for peE expression and plasmid pCT52 for TypeF expression. Each transformant was treated with 50 μg / ml Ampicilli
The cells were cultured in 1 ml of LB medium containing n at 30 ° C. for 16 hours. 1 ml of the preculture was inoculated into 100 ml of fresh LB medium.
After culturing at 30 ° C. for about 3 hours (OD600 = 0.4-0.5), the culturing temperature was shifted to 42 ° C., and culturing was further performed for 3 hours. After culture
The cells were collected by centrifugation. After suspending some of the cells in PBS and crushing the cells with an ultrasonic crusher, SDS-PAGE was performed,
CBB staining was performed. After electrophoresis, Western blotting was performed using a species-specific anti-Chlamydia trachomatis MOMP monoclonal antibody. As a result, CBB
For staining, cells transformed with pCT33 had a molecular weight of about 40,0
At position 00, the bacterial cell transformed with pCT51 has a molecular weight of about 3
At 7,000 positions, a band not observed in the cells transformed with pPLN-MCS as a control was observed in the cells transformed with pCT52 at the molecular weight of about 31,000 (see FIG. 13). The molecular weight of pCT33 is about 40,000, the molecular weight of pCT51 is about 37,000, and the molecular weight of pCT52 is about 3,100.
Color development was observed in the band at position 0 by Western blotting, and it was confirmed that the target protein was expressed (see FIG. 14). In addition, the cells sonicated
The mixture was centrifuged at 500 × g for 10 minutes to separate a supernatant fraction and a precipitate fraction. As a result of examining each fraction by Western blotting, it was found that all of the expressed proteins were found in the precipitated fraction and formed aggregates.

3. Purification of fusion protein of a part of MOMP and hydrophilic polypeptide One liter of each of E. coli expressing TypeE and TypeF was cultured by the above-mentioned culture method. After collecting cells by centrifugation, 8 ml of Lysis buffer (50 mM Tris-HCl (pH 8.0), 25% (W /
V) After suspending in Sucrose, 1 mM EDTA, 4 mg / ml Lysozyme), the suspension was left on ice for 30 minutes. Next, a final concentration of 10 mM MgCl ₂ , 10 mM
MnCl ₂ , 10 μg / ml DNaseI, and 10 μg / ml RNaseA were added thereto, respectively, and left at 4 ° C. for 30 minutes. Another 20ml
Detergent buffer I (20 mM Tris-HCl (pH 7.5), 0.2 M NaCl,
2mM EDTA, 1% Na-deoxycholate, 1.6% NP-40)
Left for 0 minutes. The precipitate was collected by centrifugation at 5,000 xg for 15 minutes. 10 times the amount of Deterg
ent buffer II (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM
The precipitate was sufficiently suspended in EDTA, 0.5% Triton X-100), left at 4 ° C. for 1 hour, and centrifuged at 5,000 × g for 10 minutes to collect a precipitate. This washing of the precipitate with Detergent buffer II was repeated three times. The obtained precipitate was added to 10 ml of a 2% sarcosine solution (2% sodium N-Lauroyl Salcosinate, 1.5 mM).
The suspension was sufficiently suspended in a PBS containing EDTA) using a Teflon homogenizer, and shaken at 45 ° C. for 1 hour. 1 suspension
The precipitate was collected by centrifugation at 00000 × g for 1 hour. This operation was repeated twice.

The obtained precipitate was mixed with 20 ml of 8 M Urea, 1 mM DTT, 10
The suspension was sufficiently suspended in mM Na-phosphate buffer (pH 6.0) using a Teflon homogenizer, and shaken at 45 ° C. for 1 hour. The suspension was centrifuged at 100,000 × g for 1 hour to recover the supernatant. This solution was purified using a hydroxyapatite resin Macro-Prep Ceramic Hydroxyapatite Type I (manufactured by Bio-Rad) column chromatography as follows. 8M Urea, 1mM DTT, 10mM Na-phosphate buffer (pH
5m on a φ5cm x 15cm column equilibrated in 6.0)
1 / min and adjust the concentration of Na-phosphate buffer to 2
After increasing to 00 mM and washing well, Na-phosphate buf
The eluted fraction was collected by increasing the concentration of fer to 300 mM. This fraction was dialyzed against 50 mM NaCl and 50 mM boric acid (pH 8.5), and insolubles were removed by centrifugation to obtain a purified product.
The purified product was separated by SDS-PAGE to test the protein purity, stained with CBB, and analyzed with an image master manufactured by Pharmacia. As a result, it was found that the purity of Type E and Type F proteins was almost 100%.

Embodiment 5 FIG. Detection of anti-Chlamydia trachomatis antibody using fusion protein expressed in Escherichia coli Preparation of Test Plate (1) Preparation of Anti-Human IgM Antibody Solid Plate for Antibody Capture Method EIA Goat anti-human IgM monoclonal antibody (Miles)
Was diluted with a 50 mM carbonate buffer (pH 9.5) to a concentration of 15 μg / ml, and a polystyrene flat microplate (manufactured by Nunc Corporation) was used.
Was dispensed at 100 μl / well and left at 4 ° C. overnight. Allow the microplate to stand for 18 hours or more
Tris-HCl (pH7.6) containing 0.05% Tween20 300μl / w
After washing twice with a cell, a final concentration of 0.5% BSA and 0.05% Tween2
200 μl / well of Tris-HCl (pH 7.6) containing 0 was added and allowed to stand at 4 ° C. overnight to prepare an anti-human IgM monoclonal antibody solid phase microplate.

(2) Antigen solid phase method Preparation of antigen solid phase plate for EIA The purified MOMP and Type A to F proteins described in Examples 2 to 4 were each 1 μg / m 2 in 50 mM carbonate buffer (pH 9.5).
and dispensed at 100 μl / well into a polystyrene flat microplate (manufactured by Nunc) at 4 ° C.
And left overnight. The microplate that had been allowed to stand for 18 hours or more was treated with 200 μl of PBS (pH 7.0) containing 0.05% Tween20 at a final concentration.
After washing 3 times with l / well, final concentration 0.5% BSA and 0.05%
Add 200μl / well of PBS containing Tween20 and add 4 ℃
The mixture was allowed to stand overnight to prepare an antigen solid phase microplate.

2. Anti-Chlamydia trachomatis IgM
(1) Detection by antibody supplementation method EIA After removing the plate preservation solution in the anti-human IgM monoclonal antibody solid phase microplate wells described in Example 5-1 (1), antigen positive was diagnosed as neonatal Chlamydia pneumonia. 0.5% BSA from 30 infant sera and 50 normal sera
And 200-fold diluted with PBS (pH 7.0) containing 0.05% Tween20, 100 μl was added to the wells of the solid-phase microplate, and reacted at room temperature (15 ° C. to 25 ° C.) for about 1 hour. After the reaction, PBS containing 0.05% Tween20 (pH 7.0) 200 μl / we
Washed three times with 1 l. Then, the antigen lysate was mixed with 1% BSA and 0.
Using 50 mM NaCl containing 50% Tween20, 50 mM boric acid (pH 8.5), the purified products described in Example 2-4 were added at 10 μg / ml and 1 μm for MOMP, Type A and Type B, respectively.
At 0 μg / ml and 5 μg / ml, TypeC and Ty
In the case of peD, the purified product described in Example 3-3 was used for 2 each.
In the case of Type E and Type F, the purified products described in Example 4-3 were each diluted to 5 μg / ml. After adding the antigen lysis solution to the wells of the solid phase microplate at 100 μl / well, room temperature (15 ° C. to 25 ° C.)
C) for about 1 hour. After the reaction, the wells were washed three times with 200 μl / well of PBS containing 0.05% Tween20.

Next, 2 times with PBS containing 0.05% Tween20.
100 μl / well of a goat anti-MOMP peroxidase-labeled monoclonal antibody diluted to μg / ml was added,
The reaction was carried out at room temperature for about 1 hour. After the reaction, the wells were washed five times with 200 μl / well of PBS containing 0.05% Tween20. After washing, 3.3 mg /
To a 0.1 M citrate-phosphate buffer (pH 5.0) containing mlo-phenylenediamine, a substrate solution containing 0.02% aqueous hydrogen peroxide was added at 100 μl / well, and reacted at room temperature for about 30 minutes. After the reaction, 100 μl / well of 1.5N sulfuric acid was added to stop the reaction, and the O.D. of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm using a microplate colorimeter. D. The value was measured. The cutoff value was a value obtained by adding a value obtained by multiplying the average of the absorbance of normal human serum by three times the standard deviation. Table 1 shows the results. From Table 1, in order to evaluate the specificity to Chlamydia other than Chlamydia trachomatis, nine cases of Chlamydia pneumoniae antigen-positive human sera and four cases of Chlamydia shitashi antigen-positive human sera were used. The capture method EIA was performed, but none of the cases showed a positive result.

(2) Detection by antigen solid phase method EIA After removing the plate preservation solution in the antigen solid phase microplate well described in Example 5-1 (2),
mg / ml rabbit anti-human IgG antibody, 0.5% BSA and 0.05
The mixture was diluted 20-fold with PBS (pH 7.0) containing% Tween20 and left for 1 hour. Next, 100 μl of this serum diluent was added to the wells of the solid phase microplate, and reacted at room temperature (15 ° C. to 25 ° C.) for about 1 hour. After the reaction, P containing 0.05% Tween20
Washing was performed three times with 200 μl / well of BS. Then, 0.
Goat anti-human IgG peroxidase-labeled antibody (MBL) diluted approximately 40,000-fold with PBS containing 05% Tween20
Was added at 100 μl / well, and reacted at room temperature for about 1 hour. After the reaction, 200 μl / w of PBS containing 0.05% Tween20
Washed 3 to 4 times with ell. After washing, 3.3 mg / ml
100 μl / well of a substrate solution obtained by adding 0.02% aqueous hydrogen peroxide to 0.1 M citrate-phosphate buffer (pH 5.0) containing ο-phenylenediamine was added, and reacted at room temperature for about 30 minutes. . After the reaction, add 1.5 N sulfuric acid to 100 μl / we
Then, the reaction was stopped by adding a colorimeter for microplate, and the O.D. D. The value was measured. The cutoff value was a value obtained by adding a value obtained by multiplying the average of the absorbance of normal human serum by three times the standard deviation. Table 1 shows the results. From Table 1, in order to evaluate the specificity to Chlamydia other than Chlamydia trachomatis, nine cases of Chlamydia pneumoniae antigen-positive human sera and four cases of Chlamydia shitashi antigen-positive human sera were used. , The antigen solid phase method EIA was performed, but none of the cases showed a positive result.

[0058]

[Table 1]

3. Anti-Chlamydia trachomatis IgG
After removing the plate preservation solution from the antigen-solid phase microplate wells described above, 50 serum samples of Chlamydia trachomatis-infected patients and 50 normal serum samples determined by the FA method were used.
After diluting 200-fold with PBS containing 0.5% BSA and 0.05% Tween20, 100 µl of the diluted solution was added to the wells of a solid-phase microplate, and reacted at room temperature (15 ° C to 25 ° C) for about 1 hour. After the reaction, the wells were washed three times with 200 μl / well of PBS containing 0.05% Tween20. Then, about 4% with PBS containing 0.05% Tween20.
Goat anti-human IgG peroxidase-labeled antibody (MBL) diluted 0000-fold was added at 100 µl / well, and the mixture was reacted at room temperature for about 1 hour. After the reaction, the wells were washed 3 to 4 times with 200 μl / well of PBS containing 0.05% Tween20. After washing, 3.
To a 0.1 M citrate-phosphate buffer (pH 5.0) containing 3 mg / ml o-phenylenediamine, a substrate solution obtained by adding 0.02% aqueous hydrogen peroxide was added at 100 μl / well, and the mixture was added at room temperature for about 30 minutes.
Allowed to react for minutes. After the reaction, 1.5 N sulfuric acid was added to 100 μl /
The reaction was stopped by adding a well, and the O.D. of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm using a colorimeter for microplate. D. The value was measured. The cutoff value was a value obtained by adding a value obtained by multiplying the average of the absorbance of normal human serum by three times the standard deviation. Table 2 shows the results.

[0060]

[Table 2]

From Table 2, in order to evaluate the specificity to Chlamydia other than Chlamydia trachomatis, the antigen-positive human serum 9 of Chlamydia pneumoniae was evaluated.
For example, the antigen solid phase method EIA was carried out by the above method using four cases of Chlamydia shiitake antigen-positive human sera, but none of the cases showed a positive result.

From the above results, anti-Chlamydia trachomatis IgM can be detected with higher sensitivity and specificity by the antibody capture method EIA using various fusion proteins as antigens than the antigen solid phase method EIA using MOMP as antigen. Various fusion proteins can also be used as antigens in the antigen solid phase EIA.

Embodiment 6 FIG. Expression of fusion protein gene using insect cells Preparation of recombinant transfer vector and recombinant virus (1) Preparation of recombinant transfer vector pCT33, pCT78, pCT80, pCT81 obtained in Examples 2 to 4
20 μg of each of pCT82, pCT51 and pCT52 was replaced with restriction enzyme SmaI2
Digest at 30 ° C for 2 hours with 0 units, and restriction enzyme BsaBI
After digestion at 65 ° C for 2 hours, about 1.6 Kb, 1.9 Kb, 2.0 kb, 1.9 Kb, 2.1 kb and 1.6 kb were sequentially analyzed by 1% agarose gel electrophoresis.
The DNA fragments of kb and 1.4 kb were fractionated, and Geneclean II (BIO1
The DNA was collected from the same company. Next, transfer vector pAcYM
1 [J. Gen. Virol., 68, 1233 (1987)]
After digestion at 30 ° C for 2 hours, alkaline phosphatase (B
RL) was used to dephosphorylate the 5 'end. After linking the above six types of DNA fragments to pAcYM1, Escherichia coli HB101 was infected. Appropriately selected 20 transformants were obtained, DNA was purified by the alkali-SDS method, and the target plasmids pAcMOMP, pAcT
ypeA, pAcTypeB, pAcTypeC, pAcTypeD, pAcTypeE and
pAcTypeF was obtained (see FIGS. 11 and 12).

(2) Preparation of Recombinant Virus Using a φ35 mm dish (manufactured by Sumitomo Bakelite),
Night moth larva-derived cell line Sf9 (Spodopterd frugiperda) cells were cultured in a medium (TC-100 or Grace's medium) containing 10% fetal calf serum (FCS) in a night moth larva-derived cell line: Tn5 (Trich
oplusia ni) Cells should be in serum-free medium (TC-100 or Grac
e's medium) for 3 to 4 days. Then, φ35mmD
1 × 10 ⁶ / Di using ish (Sumitomo Bakelite)
sh, the cultured cells described above were adjusted to a medium volume of about 1.5 ml. After allowing to stand in this state for 30 to 40 minutes, the medium was aseptically removed and 1.5 ml of a serum-free medium (EX-CELL 400
Or Grace's medium). Here, AcNPV (kinuraba, Autographa californica nuclear polyh
edorosis virus) 1 μl of about 20 ng / μl DNA, 1 μl of 2-3 μg / μl recombinant transfer vector, 6 μl of sterile distilled water, 8 μl in total and 5 μl of lipofectin (BRL)
After gently mixing 8 μl of 3 μl of sterilized distilled water in an Eppendorf tube, the mixture was gently dropped dropwise into the serum-free medium (co-transfection). And put it in the moisture chamber,
Cultured at ２６26.5 ° C. for 3 days.

After the co-transfection, about 1 ml of the culture supernatant on the third day was transferred to a tube, diluted with 10 μl to 10 ⁻¹ to 10 ⁻³ using 20 μl of each, and 100 μl of them was diluted to 1 × 10 ⁶ / Dish ( Inoculate cells adjusted to φ35mm)
It was allowed to stand for a period of time for adsorption. After that, discard the virus solution.
2 ml of low-melting point agar (Sea Plaque: manufactured by Takara Shuzo Co., Ltd.), and after coagulation, 1 ml of medium (10% for Sf9 cells)
Use TC-100 or Grace's medium containing FCS. For Tn5 cells, use TC-100 or Grace's serum-free medium. ) And cultured at 26-26.5 ° C for 3-4 days.
1 ml of PBS (pH 7.0) containing 01% (W / V) Nutral Red was added, and the plaque was stained to select a transparent recombinant.

The plaque, which was considered to be a recombinant obtained by the plaque assay, was collected together with the gel using a Pasteur pipette, suspended in 400 μl of a cell culture medium, and thoroughly stirred. Agarose gel was precipitated by centrifugation for 10 minutes, and the supernatant was diluted with each medium to 10 ^-1 to 10 ^-3 as in the plaque assay.
Plaque assay was similarly performed using low melting point agar to purify the recombinant virus.

The recombinant titers obtained by plaque pulify were increased by infecting cells. Also at this time, about 1 × 10
⁶ / Dish Sf9 cells and Tn5 cells are prepared and left for about 30 minutes. After discarding most of the medium, about 100 μl of the obtained recombinant is added, and the mixture is gently shaken and mixed at about 15 minute intervals. This operation was performed four times in total for about one hour. Next, in the case of Sf9 cells, a medium containing 10% FCS was added, and in the case of Tn5 cells, 1.5 ml of a serum-free medium was added, followed by culturing for 3 to 4 days. Using the supernatant obtained at this time, cells were prepared in the same manner using two 75 cm ² culture bottles, and 1 ml of the recombinant virus solution already titrated once was used.
And after culturing for 3 to 4 days, the resulting supernatant was used as a high-titer recombinant for expression of the structural gene.

2. Expression of fusion protein The number of cells was adjusted at about 1 × 10 ⁶ / Dish to φ35 mm dish, most of the medium was aspirated, and the high titer recombinant (about 1
× 10 ⁷ pfu / ml) was inoculated to M.O.I.5 to 10 and this operation was continued for about 1 hour at a time of about 15 minutes with gentle shaking for a total of 4 times. Then, the medium (10% FCS for Sf9 cells)
Use Tc-100 or Grace's medium containing Tc5. For Tn5 cells, use a serum-free medium of Tc-100 or Grace's. ) Was added and cultured at 26 to 26.5 ° C. for 3 to 4 days to express various fusion proteins.

After culturing for 4 days, the cells were collected by centrifugation. A part of the cells was suspended in PBS, and the cells were crushed with an ultrasonic crusher, followed by SDS-PAGE. After electrophoresis, Western blotting showed that cells infected with the virus recombined with pAcMOMP developed color at a molecular weight of 40,000, and cells infected with the virus recombined with pAcTypeA showed a molecular weight of about 51,000. Coloring is seen at 000,
In cells infected with the virus recombined with pAcTypeB, color development was observed at a position of about 52,000 molecular weight, and in cells infected with the virus recombined with pAcTypeC about 50,000 molecular weight.
A color was observed at position 00, a color was observed at a molecular weight of about 52,000 in cells infected with the virus recombined with pAcTypeD, and a molecular weight of about 37,7 was observed in cells infected with the virus recombined with pAcTypeE. Color development was observed at the position of 000, and in cells infected with the virus recombined with pAcTypeF, color development was observed at the position of the molecular weight of about 31,000, and the expression of each protein was confirmed.

Further, the cells subjected to the ultrasonic crushing treatment were
The mixture was centrifuged at 0 × g for 10 minutes to separate a supernatant fraction and a precipitate fraction. As a result of examining each fraction by Western blotting, it was found that MOMP was found in the precipitated fraction and formed an insoluble substance. On the other hand, all the fusion proteins were found to be soluble in the supernatant fraction.

3. Purification of fusion protein MOMP was purified as follows. In the culture method described above, S
One liter of the f9 cells was cultured. After collecting cells by centrifugation, 8 ml of Lysis buffer II (50 mM Tris-HCl (pH 8.0), 25
% (W / V) Sucrose, 1 mM EDTA), followed by sonication. Then the final concentration is 10 mM MgCl ₂ , 10 mM MnCl ₂ , 10 μg / ml
DNAseI and 10 μg / ml RNaseA
It was left at 4 ° C. for 30 minutes. Another 20 ml Detergent bu
ffer I (20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 2 mM EDTA, 1%
Na-deoxy-cholate, 1.6% NP-40) was added, and the mixture was further left for 30 minutes. The precipitate was collected by centrifugation at 5,000 xg for 15 minutes. 10 times the volume of Detergent for this precipitate
buffer II (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM EDT
A, 0.5% Triton X-100), left at 4 ° C. for 5 minutes, and centrifuged at 5,000 × g for 10 minutes to collect a precipitate. Washing of the precipitate with Detergentbuffer II was repeated three times. The precipitate was dissolved in PBS containing 2% SDS, and the insoluble fraction was removed by centrifugation.
It was dialyzed against PBS containing DS to obtain a purified product.

On the other hand, various fusion proteins were purified as follows. One liter of Sf9 cells was cultured by the above-described culture method. After collecting cells by centrifugation, 8 ml of Lysis buffer II
(50 mM Tris-HCl (pH 8.0), 25% (W / V) sucrose, 1 mM EDTA), followed by sonication. After removing insolubles by centrifugation, 20% saturated ammonium sulfate was added, and the mixture was stirred at 4 ° C. for 15 hours. The precipitate was collected by centrifugation at 5,000 × g for 10 minutes, dissolved in 20 ml of 8 M Urea, 20 mM HEPES (pH 8.0), and insolubles were removed by centrifugation. PB
After dialysis against S, insolubles were removed by centrifugation to obtain a purified product.

Embodiment 7 FIG. Detection of anti-Chlamydia trachomatis antibody using fusion protein expressed in insect cells Preparation of Test Plate (1) Preparation of Anti-Human IgM Antibody Solid-Plate for Antibody Capture Method EIA Using the purified products of MOMP and various fusion proteins described in Example 6-3, proceed to Example 5-1 (1). It was prepared according to the described method.

(2) Antigen solid phase method Preparation of antigen solid phase plate for EIA Using the purified product of MOMP and various fusion proteins described in Example 6-3, according to the method described in Example 5-1 (2) Produced.

2. Detection of anti-Chlamydia trachomatis IgM (1) Detection by antibody supplementation method EIA Using the test plate described in Example 7-1 (1), the detection was performed according to the method described in Example 5-2 (1). Table 3 shows the results of comparison with Example 5-2 (1).

[0076]

[Table 3]

In order to evaluate the specificity to Chlamydia other than Chlamydia trachomatis, nine cases of Chlamydia pneumoniae antigen-positive human sera and four cases of Chlamydia shitashi antigen-positive human sera were used. The capture EIA was performed, but none of the cases showed a positive result.

(2) Detection by antigen solid phase method EIA Using the test plate described in Example 7-1 (2), the detection was performed according to the method described in Example 6-2 (2). Table 4 shows the results of comparison with Example 5-2 (2).

[0079]

[Table 4]

From Table 4, in order to evaluate the specificity against Chlamydia other than Chlamydia trachomatis, antigen-positive human sera of Chlamydia pneumoniae 9
For example, the antigen solid phase method EIA was carried out by the above method using four cases of Chlamydia shiitake antigen-positive human sera, but none of the cases showed a positive result.

3. Anti-Chlamydia trachomatis IgG
The detection was performed according to the method described in Example 6-3 using the test plate described in Example 7-1 (2). Example 5
Table 5 shows the results of comparison with -3.

[0082]

[Table 5]

From Table 5, in order to evaluate the specificity to Chlamydia other than Chlamydia trachomatis, antigen-positive human serum 9 of Chlamydia pneumoniae was used.
For example, the antigen solid phase method EIA was carried out by the above method using four cases of Chlamydia shiitake antigen-positive human sera, but none of the cases showed a positive result.

From the above results, the test plate using various fusion proteins produced by insect cells can be used with high sensitivity and specificity for the anti-Chlamydia trachomatis antibody, as in the case of using various fusion proteins produced by Escherichia coli. Can be detected. Further, since the cut-off value can be set lower than that in the case where E. coli is produced, the measurable range of the absorbance of EIA is widened. Further, it is possible to further reduce a determination error due to a difference in non-specific reaction between samples.

[0085]

The soluble fusion protein of the present invention is produced by a gene recombination method and used as an antigen for detecting Chlamydia trachomatis antibodies, thereby enabling more sensitive and specific diagnosis of Chlamydia trachomatis infection than before. It has become possible.

[0086]

[Sequence list] SEQ ID NO: 1 Sequence length: 457 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence characteristics: 1-177,250-457 Chlamydia trachomatis L2 strain MOMP region 178 Joining portion 179 -249 BSA region Sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu 85 90 95 Met Phe Thr Asn Ala Ala Tyr Met Ala Leu Asn Ile Trp Asp Arg Phe 100 105 110 Asp Val Phe Cys Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn 115 120 125 Ser Ala Ser Phe Asn Leu Val Gly Leu Phe Gly Asp Asn Glu Asn His 130 135 140 Ala Thr Val Ser Asp Ser Lys L eu Val Pro Asn Met Ser Leu Asp Gln 145 150 155 160 Ser Val Val Glu Leu Tyr Thr Asp Thr Thr Phe Ala Trp Ser Ala Gly 165 170 175 Ala Gln Leu Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met 180 185 190 Ala Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu 195 200 205 Ser His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro 210 215 220 Asn Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly 225 230 235 240 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg Tyr Thr Asp Thr Thr Phe Ala 245 250 255 Trp Ser Ala Gly Ala Arg Ala Ala Leu Trp Glu Cys Gly Cys Ala Thr 260 265 270 Leu Gly Ala Ser Phe Gln Tyr Ala Gln Ser Lys Pro Lys Val Glu Glu 275 280 285 Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys 290 295 300 Gly Tyr Val Gly Gln Glu Phe Pro Leu Asp Leu Lys Ala Gly Thr Asp 305 310 315 320 Gly Val Thr Gly Thr Lys Asp Ala Ser Ile Asp Tyr His Glu Trp Gln 325 330 335 Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr Ile 340 345 350 Gly Val Lys Trp Ser Arg Ala S er Phe Asp Ala Asp Thr Ile Arg Ile 355 360 365 Ala Gln Pro Lys Ser Ala Thr Thr Val Val Phe Asp Val Thr Thr Leu Asn 370 375 380 Pro Thr Ile Ala Gly Ala Gly Asp Val Lys Ala Ser Ala Glu Gly Gln 385 390 395 400 Leu Gly Asp Thr Met Gln Ile Val Ser Leu Gln Leu Asn Lys Met Lys 405 410 415 Ser Arg Lys Ser Cys Gly Ile Ala Val Gly Thr Thr Ile Val Asp Ala 420 425 430 Asp Lys Tyr Ala Val Thr Val Glu Thr Arg Leu Ile Asp Glu Arg Ala 435 440 445 Ala His Val Asn Ala Gln Phe Arg Phe 450 455

SEQ ID NO: 2 Sequence length: 484 Sequence type: amino acid Topology: linear Sequence type: protein Sequence characteristics: 1-108,149-348,385-484 Chlamydia trachomatis L2 strain MOMP region 384 junction 109-148,349-383 BSA region sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu 85 90 95 Met Phe Thr Asn Ala Ala Tyr Met Ala Leu Asn Ile Ala Ser Leu Arg 100 105 110 Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gln Glu Pro Glu 115 120 125 Arg Asn Glu Cys Phe Leu Ser His Lys Asp Asp Ser Pro Asp Leu Pro 130 135 140 Lys Leu Ly s Pro Gly Arg His Met Gln Asp Ala Glu Met Phe Thr Asn 145 150 155 160 Ala Ala Tyr Met Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys 165 170 175 Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn Ser Ala Ser Phe 180 185 190 Asn Leu Val Gly Leu Phe Gly Asp Asn Glu Asn His Ala Thr Val Ser 195 200 205 Asp Ser Lys Leu Val Pro Asn Met Ser Leu Asp Gln Ser Val Val Glu 210 215 220 Leu Tyr Thr Asp Thr Thr Phe Ala Trp Ser Ala Gly Ala Gly Ala Arg 225 230 235 240 Ala Ala Leu Trp Glu Cys Gly Cys Ala Thr Leu Gly Ala Ser Phe Gln 245 250 255 Tyr Ala Gln Ser Lys Pro Lys Val Glu Glu Leu Asn Val Leu Cys Asn 260 265 270 Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly Gln Glu 275 280 285 Phe Pro Leu Asp Leu Lys Ala Gly Thr Asp Gly Val Thr Gly Thr Lys 290 295 300 Asp Ala Ser Ile Asp Tyr His Glu Trp Gln Ala Ser Leu Ala Leu Ser 305 310 315 320 Tyr Arg Leu Asn Met Phe Thr Pro Tyr Ile Gly Val Lys Trp Ser Arg 325 330 335 Ala Ser Phe Asp Ala Asp Thr Ile Arg Ile Ala Gln Arg Glu Thr Tyr 340 345 350 Gly Asp Me t Ala Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu 355 360 365 Cys Phe Leu Ser His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Thr 370 375 380 Arg Ala Ser Phe Asp Ala Asp Thr Ile Arg Ile Ala Gln Pro Lys Ser 385 390 395 400 Ala Thr Thr Val Phe Asp Val Thr Thr Leu Asn Pro Thr Ile Ala Gly 405 410 415 Ala Gly Asp Val Lys Ala Ser Ala Glu Gly Gln Leu Gly Asp Thr Met 420 425 430 Gln Ile Val Ser Leu Gln Leu Asn Lys Met Lys Ser Arg Lys Ser Cys 435 440 445 Gly Ile Ala Val Gly Thr Thr Ile Val Asp Ala Asp Lys Tyr Ala Val 450 455 460 Thr Val Glu Thr Arg Leu Ile Asp Glu Arg Ala Ala His Val Asn Ala 465 470 475 480 Gln Phe Arg Phe

SEQ ID NO: 3 Sequence length: 454 Sequence type: amino acid Topology: linear Sequence type: protein Sequence features: 1-90,121-174,247-454 Chlamydia trachomatis L2 strain MOMP region 91-120 Chlamydia trachomatis L2 strain MOMP VDIV region insertion part 175 junction part 176-246 BSA region Sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg Ser Ala Thr Thr Val Phe 85 90 95 Asp Val Thr Thr Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys 100 105 110 Ala Ser Ala Glu Gly Gln Leu Gly Tyr Leu Lys Gly Asn Ser Ala Ser 115 120 1 25 Phe Asn Leu Val Gly Leu Phe Gly Asp Asn Glu Asn His Ala Thr Val 130 135 140 Ser Asp Ser Lys Leu Val Pro Asn Met Ser Leu Asp Gln Ser Val Val 145 150 155 160 Glu Leu Tyr Thr Asp Thr Thr Phe Ala Trp Ser Ala Gly Ala Gln Leu 165 170 175 Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala Asp Cys 180 185 190 Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser His Lys 195 200 205 Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn Thr Leu 210 215 220 Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys Tyr Leu 225 230 235 240 Tyr Glu Ile Ala Arg Arg Tyr Thr Asp Thr Thr Phe Ala Trp Ser Ala 245 250 255 Gly Ala Arg Ala Ala Leu Trp Glu Cys Gly Cys Ala Thr Leu Gly Ala 260 265 270 Ser Phe Gln Tyr Ala Gln Ser Lys Pro Lys Val Glu Glu Leu Asn Val 275 280 285 285 Leu Cys Asn Ala Ala Glu Phe Thr Ile Asn Lys Pro Lys Gly Tyr Val 290 295 300 300 Gly Gln Glu Phe Pro Leu Asp Leu Lys Ala Gly Thr Asp Gly Val Thr 305 310 315 320 Gly Thr Lys Asp Ala Ser Ile Asp Tyr His Glu Trp Gln Ala Ser Leu 325 330 Three 35 Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr Ile Gly Val Lys 340 345 350 Trp Ser Arg Ala Ser Phe Asp Ala Asp Thr Ile Arg Ile Ala Gln Pro 355 360 365 Lys Ser Ala Thr Thr Val Val Phe Asp Val Thr Thr Leu Asn Pro Thr Ile 370 375 380 Ala Gly Ala Gly Asp Val Lys Ala Ser Ala Glu Gly Gln Leu Gly Asp 385 390 395 400 400 Thr Met Gln Ile Val Ser Leu Gln Leu Asn Lys Met Lys Ser Arg Lys 405 410 415 Ser Cys Gly Ile Ala Val Gly Thr Thr Ile Val Asp Ala Asp Lys Tyr 420 425 430 Ala Val Thr Val Glu Thr Arg Leu Ile Asp Glu Arg Ala Ala His Val 435 440 445 Asn Ala Gln Phe Arg Phe 450

SEQ ID NO: 4 Sequence length: 514 Sequence type: amino acid Topology: linear Sequence type: protein Sequence characteristics: 1-108,149-150,181-234,307-514 Chlamydia trachomatis L2 strain MOMP region 151 -180 VDIV region insertion part of Chlamydia trachomatis L2 strain MOMP 235 junction 109-148,236-306 BSA region Sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu 85 90 95 Met Phe Thr Asn Ala Ala Tyr Met Ala Leu Asn Ile Ala Ser Leu Arg 100 105 110 Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gln Gl u Pro Glu 115 120 125 Arg Asn Glu Cys Phe Leu Ser His Lys Asp Asp Ser Pro Asp Leu Pro 130 135 140 Lys Leu Lys Pro Gly Arg Ser Ala Thr Thr Val Val Phe Asp Val Thr Thr 145 150 155 160 Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys Ala Ser Ala Glu 165 170 175 Gly Gln Leu Gly Tyr Leu Lys Gly Asn Ser Ala Ser Phe Asn Leu Val 180 185 190 Gly Leu Phe Gly Asp Asn Glu Asn His Ala Thr Val Ser Asp Ser Lys 195 200 205 Leu Val Pro Asn Met Ser Leu Asp Gln Ser Val Val Glu Leu Tyr Thr 210 215 220 Asp Thr Thr Phe Ala Trp Ser Ala Gly Ala Gln Leu Cys Lys Val Ala 225 230 235 240 Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gln 245 250 255 Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser His Lys Asp Asp Ser Pro 260 265 270 Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn Thr Leu Cys Asp Glu Phe 275 280 285 Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys Tyr Leu Tyr Glu Ile Ala 290 295 300 300 Arg Arg Tyr Thr Asp Thr Thr Phe Ala Trp Ser Ala Gly Ala Arg Ala 305 310 315 320 Ala Leu Trp Glu Cys Gly Cys Ala Thr Leu Gly Ala Ser Ph e Gln Tyr 325 330 335 Ala Gln Ser Lys Pro Lys Val Glu Glu Leu Asn Val Leu Cys Asn Ala 340 345 350 Ala Glu Phe Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly Gln Glu Phe 355 360 365 365 Pro Leu Asp Leu Lys Ala Gly Thr Asp Gly Val Thr Gly Thr Lys Asp 370 375 380 Ala Ser Ile Asp Tyr His Glu Trp Gln Ala Ser Leu Ala Leu Ser Tyr 385 390 395 400 Arg Leu Asn Met Phe Thr Pro Tyr Ile Gly Val Lys Trp Ser Arg Ala 405 410 415 Ser Phe Asp Ala Asp Thr Ile Arg Ile Ala Gln Pro Lys Ser Ala Thr 420 425 430 Thr Val Phe Asp Val Thr Thr Leu Asn Pro Thr Ile Ala Gly Ala Gly 435 440 445 Asp Val Lys Ala Ser Ala Glu Gly Gln Leu Gly Asp Thr Met Gln Ile 450 455 460 Val Ser Leu Gln Leu Asn Lys Met Lys Ser Arg Lys Ser Cys Gly Ile 465 470 475 480 480 Ala Val Gly Thr Thr Ile Val Asp Ala Asp Lys Tyr Ala Val Thr Val 485 490 490 495 Glu Thr Arg Leu Ile Asp Glu Arg Ala Ala His Val Asn Ala Gln Phe 500 505 510 Arg Phe

SEQ ID NO: 5 Sequence length: 349 Sequence type: amino acid Topology: linear Sequence type: protein Sequence characteristics: 1-166,188-349 Chlamydia trachomatis L2 strain MOMP region 168-187 polyasparagine Region 167 Junction sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu 85 90 95 Met Phe Thr Asn Ala Ala Tyr Met Ala Leu Asn Ile Trp Asp Arg Phe 100 105 110 Asp Val Phe Cys Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn 115 120 125 Ser Ala Ser Phe Asn Leu Val Gly Leu Phe Gly Asp Asn Glu Asn His 130 135 140 Ala Thr Val Ser Asp Ser Lys Leu Val Pro Asn Met Ser Leu Asp Gln 145 150 155 160 Ser Val Val Glu Leu Tyr Val Asn Asn Asn Asn Asn Asn Asn Asn Asn 165 170 175 Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Ala Glu Phe Thr Ile 180 185 190 Asn Lys Pro Lys Gly Tyr Val Gly Gln Glu Phe Pro Leu Asp Leu Lys 195 200 205 Ala Gly Thr Asp Gly Val Thr Gly Thr Lys Asp Ala Ser Ile Asp Tyr 210 215 220 His Glu Trp Gln Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe 225 230 235 240 Thr Pro Tyr Ile Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp 245 250 255 Thr Ile Arg Ile Ala Gln Pro Lys Ser Ala Thr Thr Val Phe Asp Val 260 265 270 Thr Thr Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys Ala Ser 275 280 285 Ala Glu Gly Gln Leu Gly Asp Thr Met Gln Ile Val Ser Leu Gln Leu 290 295 300 Asn Lys Met Lys Ser Arg Lys Ser Cys Gly Ile Ala Val Gly Thr Thr 305 310 315 320 Ile Val Asp Ala Asp Lys Tyr Ala Val Thr Val Glu Thr Arg Leu Ile 325 330 335 Asp Glu Arg Ala Ala His Val Asn Ala Gln Phe Arg Phe 340 345

SEQ ID NO: 6 Sequence length: 277 Sequence type: amino acid Topology: linear Sequence type: protein Sequence characteristics: 1-166,188-277 Chlamydia trachomatis L2 strain MOMP region 168-187 polyasparagine Acid region 167 junction sequence Met Leu Pro Val Gly Asn Pro Ala Glu Pro Ser Leu Met Ile Asp Gly 1 5 10 15 Ile Leu Trp Glu Gly Phe Gly Gly Asp Pro Cys Asp Pro Cys Thr Thr 20 25 30 Trp Cys Asp Ala Ile Ser Met Arg Met Gly Tyr Tyr Gly Asp Phe Val 35 40 45 Phe Asp Arg Val Leu Gln Thr Asp Val Asn Lys Glu Phe Gln Met Gly 50 55 60 Ala Lys Pro Thr Thr Ala Thr Gly Asn Ala Ala Ala Pro Ser Thr Cys 65 70 75 80 Thr Ala Arg Glu Asn Pro Ala Tyr Gly Arg His Met Gln Asp Ala Glu 85 90 95 Met Phe Thr Asn Ala Ala Tyr Met Ala Leu Asn Ile Trp Asp Arg Phe 100 105 110 Asp Val Phe Cys Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn 115 120 125 Ser Ala Ser Phe Asn Leu Val Gly Leu Phe Gly Asp Asn Glu Asn His 130 135 140 Ala Thr Val Ser Asp Ser Lys Leu Val Pro Asn Met Ser Leu Asp Gln 145 150 155 160 Ser Val Val Glu Leu Tyr Val Asp Asp Asp Asp Asp Asp Asp Asp Asp 165 170 175 Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Ile Ala Gln Pro Lys 180 185 190 Ser Ala Thr Thr Val Phe Asp Val Thr Thr Leu Asn Pro Thr Ile Ala 195 200 205 Gly Ala Gly Asp Val Lys Ala Ser Ala Glu Gly Gln Leu Gly Asp Thr 210 215 220 Met Gln Ile Val Ser Leu Gln Leu Asn Lys Met Lys Ser Arg Lys Ser 225 230 235 240 Val Thr Val Glu Thr Arg Leu Ile Asp Glu Arg Ala Ala His Val Asn 260 265 270 270 Ala Gln Phe Arg Phe 275

SEQ ID NO: 7 Sequence length: 1371 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA (MOMP region code of Chlamydia trachomatis L2 strain) cDNA to mRNA (BSA region coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1.1371 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAACCA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCTCTGTG CGATCTGTGCTCGTC ACCACTTGTGCTCGTC ACCACTTGTGCT TG AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 300 GCTGCTTACA TGGCATTGAA TATTTGGGAT CGTTTTGATG TATTCTGTAC ATTAGGAGCC 360 ACCAGTGGAT ATCTTAAAGG AAATTCAGCA TCTTTCAACT TAGTTGGCTT ATTCGGAGAT 420 AATGAGAACC ATGCTACAGT TTCAGATAGT AAGCTTGTAC CAAATATGAG CTTAGATCAA 480 TCTGTTGTTG AGTTGTATAC AGA TACTACT TTTGCTTGGA GTGCTGGAGC TCAATTGTGT 540 AAAGTTGCAT CCCTTCGTGA AACCTATGGT GACATGGCTG ACTGCTGTGA GAAACAAGAG 600 CCTGAAAGAA ATGAATGCTT CCTGAGCCAC AAAGATGATA GCCCAGACCT CCCTAAATTG 660 AAACCAGACC CCAATACTTT GTGTGATGAG TTTAAGGCAG ATGAAAAGAA GTTTTGGGGA 720 AAATACCTAT ACGAAATTGC TAGAAGGTAT ACAGATACTA CTTTTGCTTG GAGTGCTGGA 780 GCTCGTGCAG CTTTGTGGGA ATGTGGATGC GCGACTTTAG GCGCTTCTTT CCAATACGCT 840 CAATCCAAGC CTAAAGTCGA AGAATTAAAC GTTCTCTGTA ACGCAGCTGA GTTTACTATC 900 AATAAGCCTA AAGGATATGT AGGGCAAGAA TTCCCTCTTG ATCTTAAAGC AGGAACAGAT 960 GGTGTGACAG GAACTAAGGA TGCCTCTATT GATTACCATG AATGGCAAGC AAGTTTAGCT 1020 CTCTCTTACA GACTGAATAT GTTCACTCCC TACATTGGAG TTAAATGGTC TCGAGCAAGT 1080 TTTGATGCAG ACACGATTCG TATTGCTCAG CCGAAGTCAG CTACAACTGT CTTTGATGTT 1140 ACCACTCTGA ACCCAACTAT TGCTGGAGCT GGCGATGTGA AAGCTAGCGC AGAGGGTCAG 1200 CTCGGAGATA CCATGCAAAT CGTTTCCTTG CAATTGAACA AGATGAAATC TAGAAAATCT 1260 TGCGGTATTG CAGTAGGAAC AACTATTGTG GATGCAGACA AATACGCAGT TACAGTTGAG 1320 ACTCGCTTGA TCGATGAGAG AGCTGCTCAC GTAAAT GCAC AATTCCGCTT C 1371

SEQ ID NO: 8 Sequence length: 1452 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA (MOMP region code of Chlamydia trachomatis L2 strain) cDNA to mRNA (BSA region coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1.1452 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAACCA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCTCTGTG CGATCTGTGCTCGTC ACCACTTGTGCTCT CG AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 300 GCTGCTTACA TGGCATTGAA TATTGCATCC CTTCGTGAAA CCTATGGTGA CATGGCTGAC 360 TGCTGTGAGA AACAAGAGCC TGAAAGAAAT GAATGCTTCC TGAGCCACAA AGATGATAGC 420 CCAGACCTCC CTAAATTGAA ACCCGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 480 GCTGCTTACA TGGCATTGAA TAT TTGGGAT CGTTTTGATG TATTCTGTAC ATTAGGAGCC 540 ACCAGTGGAT ATCTTAAAGG AAATTCAGCA TCTTTCAACT TAGTTGGCTT ATTCGGAGAT 600 AATGAGAACC ATGCTACAGT TTCAGATAGT AAGCTTGTAC CAAATATGAG CTTAGATCAA 660 TCTGTTGTTG AGTTGTATAC AGATACTACT TTTGCTTGGA GTGCTGGAGC TGGAGCTCGT 720 GCAGCTTTGT GGGAATGTGG ATGCGCGACT TTAGGCGCTT CTTTCCAATA CGCTCAATCC 780 AAGCCTAAAG TCGAAGAATT AAACGTTCTC TGTAACGCAG CTGAGTTTAC TATCAATAAG 840 CCTAAAGGAT ATGTAGGGCA AGAATTCCCT CTTGATCTTA AAGCAGGAAC AGATGGTGTG 900 ACAGGAACTA AGGATGCCTC TATTGATTAC CATGAATGGC AAGCAAGTTT AGCTCTCTCT 960 TACAGACTGA ATATGTTCAC TCCCTACATT GGAGTTAAAT GGTCTCGAGC AAGTTTTGAT 1020 GCAGACACGA TTCGTATTGC TCAGCGTGAA ACCTATGGTG ACATGGCTGA CTGCTGTGAG 1080 AAACAAGAGC CTGAAAGAAA TGAATGCTTC CTGAGCCACA AAGATGATAG CCCAGACCTC 1140 CCTAAATTGA CTCGAGCAAG TTTTGATGCA GACACGATTC GTATTGCTCA GCCGAAGTCA 1200 GCTACAACTG TCTTTGATGT TACCACTCTG AACCCAACTA TTGCTGGAGC TGGCGATGTG 1260 AAAGCTAGCG CAGAGGGTCA GCTCGGAGAT ACCATGCAAA TCGTTTCCTT GCAATTGAAC 1320 AAGATGAAAT CTAGAAAATC TTGCGGTATT GCAGTA GGAA CAACTATTGT GGATGCAGAC 1380 AAATACGCAG TTACAGTTGA GACTCGCTTG ATCGATGAGA GAGCTGCTCA CGTAAATGCA 1440 CAATTCCGCT TC 1452

SEQ ID NO: 9 Sequence length: 1362 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA (Chlamydia trachomatis L2 strain MOMP region code portion) cDNA to mRNA (BSA region coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1..1362 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAGACAA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCTCTGTG CGATCTGTGCT CGACTTCTGTGCT ACT AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGT TCTGCTACCA CTGTCTTTGA TGTTACCACT 300 CTGAACCCAA CTATTGCTGG AGCTGGCGAT GTGAAAGCAA GCGCTGAGGG TCAGCTGGGA 360 TATCTTAAAG GAAATTCAGC ATCTTTCAAC TTAGTTGGCT TATTCGGAGA TAATGAGAAC 420 CATGCTACAG TTTCAGATAG TAAGCTTGTA CCAAATATGA GCTTAGATCA ATCTGTTGTT 480 GAGTTGTATA CAGATACTAC TTT TGCTTGG AGTGCTGGAG CTCAATTGTG TAAAGTTGCA 540 TCCCTTCGTG AAACCTATGG TGACATGGCT GACTGCTGTG AGAAACAAGA GCCTGAAAGA 600 AATGAATGCT TCCTGAGCCA CAAAGATGAT AGCCCAGACC TCCCTAAATT GAAACCAGAC 660 CCCAATACTT TGTGTGATGA GTTTAAGGCA GATGAAAAGA AGTTTTGGGG AAAATACCTA 720 TACGAAATTG CTAGAAGGTA TACAGATACT ACTTTTGCTT GGAGTGCTGG AGCTCGTGCA 780 GCTTTGTGGG AATGTGGATG CGCGACTTTA GGCGCTTCTT TCCAATACGC TCAATCCAAG 840 CCTAAAGTCG AAGAATTAAA CGTTCTCTGT AACGCAGCTG AGTTTACTAT CAATAAGCCT 900 AAAGGATATG TAGGGCAAGA ATTCCCTCTT GATCTTAAAG CAGGAACAGA TGGTGTGACA 960 GGAACTAAGG ATGCCTCTAT TGATTACCAT GAATGGCAAG CAAGTTTAGC TCTCTCTTAC 1020 AGACTGAATA TGTTCACTCC CTACATTGGA GTTAAATGGT CTCGAGCAAG TTTTGATGCA 1080 GACACGATTC GTATTGCTCA GCCGAAGTCA GCTACAACTG TCTTTGATGT TACCACTCTG 1140 AACCCAACTA TTGCTGGAGC TGGCGATGTG AAAGCTAGCG CAGAGGGTCA GCTCGGAGAT 1200 ACCATGCAAA TCGTTTCCTT GCAATTGAAC AAGATGAAAT CTAGAAAATC TTGCGGTATT 1260 GCAGTAGGAA CAACTATTGT GGATGCAGAC AAATACGCAG TTACAGTTGA GACTCGCTTG 1320 ATCGATGAGA GAGCTGCTCA CGTAAATGCA CAATTC CGCT TC 1362

SEQ ID NO: 10 Sequence length: 1542 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA (MOMP region coding portion of Chlamydia trachomatis L2 strain) cDNA to mRNA (BSA region coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1..1542 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAACCA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCCTTG CGATCTGAG CGACTTGTGCT TG CGTGTTTTGC AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 300 GCTGCTTACA TGGCATTGAA TATTGCATCC CTTCGTGAAA CCTATGGTGA CATGGCTGAC 360 TGCTGTGAGA AACAAGAGCC TGAAAGAAAT GAATGCTTCC TGAGCCACAA AGATGATAGC 420 CCAGACCTCC CTAAATTGAA ACCCGGCCGT TCTGCTACCA CTGTCTTTGA TGTTACCACT 480 CTGAACCCAA CTATTGCTGG AGCTGGCGAT GTGAAAGCAA GCGCTGAGGG TCAGCTGGGA 540 TATCTTAAAG GAAATTCAGC ATCTTTCAAC TTAGTTGGCT TATTCGGAGA TAATGAGAAC 600 CATGCTACAG TTTCAGATAG TAAGCTTGTA CCAAATATGA GCTTAGATCA ATCTGTTGTT 660 GAGTTGTATA CAGATACTAC TTTTGCTTGG AGTGCTGGAG CTCAATTGTG TAAAGTTGCA 720 TCCCTTCGTG AAACCTATGG TGACATGGCT GACTGCTGTG AGAAACAAGA GCCTGAAAGA 780 AATGAATGCT TCCTGAGCCA CAAAGATGAT AGCCCAGACC TCCCTAAATT GAAACCAGAC 840 CCCAATACTT TGTGTGATGA GTTTAAGGCA GATGAAAAGA AGTTTTGGGG AAAATACCTA 900 TACGAAATTG CTAGAAGGTA TACAGATACT ACTTTTGCTT GGAGTGCTGG AGCTCGTGCA 960 GCTTTGTGGG AATGTGGATG CGCGACTTTA GGCGCTTCTT TCCAATACGC TCAATCCAAG 1020 CCTAAAGTCG AAGAATTAAA CGTTCTCTGT AACGCAGCTG AGTTTACTAT CAATAAGCCT 1080 AAAGGATATG TAGGGCAAGA ATTCCCTCTT GATCTTAAAG CAGGAACAGA TGGTGTGACA 1140 GGAACTAAGG ATGCCTCTAT TGATTACCAT GAATGGCAAG CAAGTTTAGC TCTCTCTTAC 1200 AGACTGAATA TGTTCACTCC CTACATTGGA GTTAAATGGT CTCGAGCAAG TTTTGATGCA 1260 GACACGATTC GTATTGCTCA GCCGAAGTCA GCTACAACTG TCTTTGATGT TACCACTCTG 1320 AACCCAACTA TTGCTGGAGC TGGCGATGTG AAA GCTAGCG CAGAGGGTCA GCTCGGAGAT 1380 ACCATGCAAA TCGTTTCCTT GCAATTGAAC AAGATGAAAT CTAGAAAATC TTGCGGTATT 1440 GCAGTAGGAA CAACTATTGT GGATGCAGAC AAATACGCAG TTACAGTTGA GACTCGCTTG 1500 ATCGATGAGA GAGCTGCTCA CGTAAATGCAATTCC

SEQ ID NO: 11 Sequence length: 1047 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA (Chlamydia trachomatis L2 strain MOMP region code portion) Other Nucleic acid Synthetic DNA (polyasparagine region coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1..1047 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAACCA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCTGCT TG CGATCTGTCTGTCGATCTGCTCGTCGATCTGCTCGTCGATCTGCT ATGGTGACTT TGTTTTCGAC CGTGTTTTGC AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 300 GCTGCTTACA TGGCATTGAA TATTTGGGAT CGTTTTGATG TATTCTGTAC ATTAGGAGCC 360 ACCAGTGGAT ATCTTAAAGG AAATTCAGCA TCTTTCAACT TAGTTGGCTT ATTCGGAGAT 420 AATGAGAACC ATGCTACAGT TTCAGATAGT AAGCTTGTAC CAAATATGAG CTTAGATCAA 480 TCTGTTGTTG AGTTGTATGT TAACAATAAC AATAACAATA ACAATAACAA TAACAATAAC 540 AATAACAATA ACAATAACAA TGCTGAGTTT ACTATCAATA AGCCTAAAGG ATATGTAGGG 600 CAAGAATTCC CTCTTGATCT TAAAGCAGGA ACAGATGGTG TGACAGGAAC TAAGGATGCC 660 TCTATTGATT ACCATGAATG GCAAGCAAGT TTAGCTCTCT CTTACAGACT GAATATGTTC 720 ACTCCCTACA TTGGAGTTAA ATGGTCTCGA GCAAGTTTTG ATGCAGACAC GATTCGTATT 780 GCTCAGCCGA AGTCAGCTAC AACTGTCTTT GATGTTACCA CTCTGAACCC AACTATTGCT 840 GGAGCTGGCG ATGTGAAAGC TAGCGCAGAG GGTCAGCTCG GAGATACCAT GCAAATCGTT 900 TCCTTGCAAT TGAACAAGAT GAAATCTAGA AAATCTTGCG GTATTGCAGT AGGAACAACT 960 ATTGTGGATG CAGACAAATA CGCAGTTACA GTTGAGACTC GCTTGATCGA TGAGAGAGCT 1020 GCTCACGTAA ATGCACAATT CCGCTTC 1047

SEQ ID NO: 12 Sequence length: 831 Sequence type: nucleic acid Number of strands: double stranded Topology: linear Sequence type: Genomic DNA (Chlamydia trachomatis L2 strain MOMP region coding part) Others Nucleic acid of synthetic DNA (polyaspartic acid domain coding portion) Sequence characteristics: Symbol indicating characteristics: CDS Location: 1..831 Method used to determine characteristics: E sequence ATGCTGCCTG TGGGTAACCC TGCTGAACCA AGCCTTATGA TCGACGGGAT CCTATGGGAA 60 GGTTTCGGCG GAGATCCTTG CGACGCTGCTGTCGATCTGCT 120 ATGGGTTACT ATGGTGACTT TGTTTTCGAC CGTGTTTTGC AAACAGATGT GAATAAAGAA 180 TTCCAAATGG GTGCCAAGCC TACAACTGCT ACAGGCAATG CTGCAGCTCC ATCCACTTGT 240 ACAGCAAGAG AGAATCCTGC TTACGGCCGA CATATGCAGG ATGCTGAGAT GTTTACAAAT 300 GCTGCTTACA TGGCATTGAA TATTTGGGAT CGTTTTGATG TATTCTGTAC ATTAGGAGCC 360 ACCAGTGGAT ATCTTAAAGG AAATTCAGCA TCTTTCAACT TAGTTGGCTT ATTCGGAGAT 420 AATGAGAACC ATGCTACAGT TTCAGATAGT AAGCTTGTAC CAAATATGAG C TTAGATCAA 480 TCTGTTGTTG AGTTGTATGT CGACGATGAC GATGACGATG ACGATGACGA TGACGATGAC 540 GATGACGATG ACGATGACGA TATCGCTCAG CCGAAGTCAG CTACAACTGT CTTTGATGTT 600 ACCACTCTGA ACCCAACTAT TGCTGGAGCT GGCGATGTGA AAGCTAGCGC AGAGGGTCAG 660 CTCGGAGATA CCATGCAAAT CGTTTCCTTG CAATTGAACA AGATGAAATC TAGAAAATCT 720 TGCGGTATTG CAGTAGGAAC AACTATTGTG GATGCAGACA AATACGCAGT TACAGTTGAG 780 ACTCGCTTGA TCGATGAGAG AGCTGCTCAC GTAAATGCAC AATTCCGCTT C 831

SEQ ID NO: 13 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence CCAGAAAAAG ATAGCGAGCA CA

SEQ ID NO: 14 Sequence length: 22 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: Yes sequence AAAAAAACTG GACCCGACCG AA

SEQ ID NO: 15 Sequence length: 73 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TATGCTGCCT GTGGGTAACC CTGCTGAACC AAGCCTTATG ATCGACGGGA TCCTATGGGA AGGTTTCGGC GGA

SEQ ID NO: 16 Sequence length: 75 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Antisense: Yes Sequence GATCTCCGCC GAAACCTTCC CATAGGATCC CGTCGATCAT AAGGCTTGGT TCAGCAGGGT TACCCACAGG CAGCA

SEQ ID NO: 17 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGCACAATGA AGTGGGTGAC TTTTATTTCT

SEQ ID NO: 18 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: Yes sequence TCGTGTTTAG GCTAAGGCTG TTTGAGTTGA

SEQ ID NO: 19 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTTCACACTC TCTTTGGAGC TCAATTGTGT

SEQ ID NO: 20 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: Yes sequence ATAAAAGTAG GTATACCTTC TAGCAATTTC

SEQ ID NO: 21 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTGTGTAATA TTGCATCCCT TCGTGAAACC

SEQ ID NO: 22 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: Yes sequence ACACAAAGTA TTGCGGCCGG GTTTCAATTT

SEQ ID NO: 23 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTGTGTAAAG TTGCAGCTCA GCGTGAAACC

SEQ ID NO: 24 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA Antisense: Yes sequence AGTATTGGGG TCTCGAGTCA ATTTAGGGAG

SEQ ID NO: 25 Sequence length: 97 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGCCGTTCTG CTACCACTGT CTTTGATGTT ACCACTCTGA ACCCGACTAT TGCTGGTGCT GGCGAT GTGA AAGCAAGCGC TGAGGGTCAG CTGGGAT

SEQ ID NO: 26 Sequence length: 93 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence ATCCCAGCTG ACCCTCAGCG CTTGCTTTCA CATCGCCAGC ACCAGCAATA GTCGGGTTCA GAGTGG TAAC ATCAAAGACA GTGGTAGCAG AAC

SEQ ID NO: 27 Sequence length: 65 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TGTTAACAAT AACAATAACA ATAACAATAA CAATAACAAT AACAATAACA ATAACAATAA CAATG

SEQ ID NO: 28 Sequence length: 65 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Antisense: Yes Sequence CATTGTTATT GTTATTGTTA TTGTTATTGT TATTGTTATT GTTATTGTTA TTGTTATTGT TAACA

SEQ ID NO: 29 Sequence length: 69 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TGTCGACGAT GACGATGACG ATGACGATGA CGATGACGAT GACGATGACG ATGACGATGA CGATAT CGC

SEQ ID NO: 30 Sequence length: 72 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA Antisense: Yes sequence TGAGCGATAT CGTCATCGTC ATCGTCATCG TCATCGTCAT CGTCATCGTC ATCGTCATCG TCATCG TCGA CA

[Brief description of the drawings]

FIG. 1 shows the construction of a vector containing a major outer membrane protein gene of L2 strain.

FIG. 2 is a diagram showing the construction of an expression vector pCT33.

FIG. 3 shows the construction of a vector containing a BSA gene.

FIG. 4 is a view showing the construction of an expression vector pCT78.

FIG. 5 is a view showing the construction of an expression vector pCT79.

FIG. 6 is a diagram showing the construction of an expression vector pCT80.

FIG. 7 shows the construction of the expression vector pCT81.

FIG. 8 is a diagram showing the construction of an expression vector pCT82.

FIG. 9 is a diagram showing the construction of an expression vector pCT51.

FIG. 10 is a view showing the construction of an expression vector pCT52.

FIG. 11. Transfer vectors pAcMOMP, pAcTypeA, and pAcT
It is a figure showing construction of ypeB.

FIG. 12: Transfer vectors pAcTypeC, pAcTypeD, pAcType
It is a figure showing construction of E and pAcTypeF.

FIG. 13: Escherichia coli N483 transformed with various expression vectors
It is a figure which shows SDS-PAGEG of the crushed cell body of 0-1.

FIG. 14: Escherichia coli N483 transformed with various expression vectors
It is a figure which shows the western blotting of the cell crushed product of 0-1.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 5/10 C12P 21/02 C C12P 21/02 G01N 33/53 D G01N 33/53 33/569 F 33/569 33 / 571 33/571 C12N 5/00 B // (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1 : 91) (72) Inventor Yoshiyuki Ishii 3-5-1 Asahicho, Machida-shi, Tokyo F-term (reference) in Denki Kagaku Kogyo Co., Ltd. 4B024 AA13 BA80 CA01 DA02 DA06 EA04 GA11 HA08 HA09 HA11 4B064 AG01 CA02 CA10 CA19 CC24 DA15 4B065 AA01X AA01Y AA26X AA90X AB01 BA02 CA24 CA46 4H045 AA10 AA20 AA30 BA10 BA41 CA11 CA15 CA42 DA76 DA86 EA52 EA54 FA72 FA74 GA24 GA31 HA05 HA06 HA31

Claims (31)

[Claims] 1. A protein comprising at least a portion of a Chlamydia trachomatis major outer membrane protein, at least one hydrophilic polypeptide having no immunoreactivity with human serum, and a junction thereof. A fusion protein that maintains a solubilized state in the absence of a solubilizing agent. 2. The fusion protein according to claim 1, wherein at least one of the hydrophilic polypeptides is a polypeptide having the same amino acid sequence as at least a part of bovine serum albumin. 3. The fusion protein according to claim 1, wherein at least one of the hydrophilic polypeptides is a polypeptide in which two or more asparagine residues are continuously bonded. 4. The fusion according to claim 1, wherein at least one of the hydrophilic polypeptides is a polypeptide in which two or more aspartic acid residues are continuously bonded. protein. 5. The fusion protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 6. The fusion protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 2 in the sequence listing. 7. The fusion protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 3 in the sequence listing. 8. The fusion protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 4 in the sequence listing. 9. The fusion protein according to claim 3, which has the amino acid sequence of SEQ ID NO: 5 in the sequence listing. 10. The fusion protein according to claim 4, which has the amino acid sequence of SEQ ID NO: 6 in the sequence listing. 11. A DNA having a base sequence encoding the fusion protein according to claim 5. 12. The DNA according to claim 11, which has the nucleotide sequence of SEQ ID NO: 7 in the sequence listing, or the DNA having a nucleotide sequence complementary to the sequence. 13. A DNA comprising a base sequence encoding the fusion protein according to claim 6. 14. The DNA according to claim 13, which has the nucleotide sequence of SEQ ID NO: 8 in the sequence listing, or the DNA having a nucleotide sequence complementary to the sequence. 15. A DNA comprising a base sequence encoding the fusion protein according to claim 7. 16. The DNA according to claim 15, which has the nucleotide sequence of SEQ ID NO: 9 in the sequence listing, or the DNA having a nucleotide sequence complementary to the sequence. 17. A DNA having a base sequence encoding the fusion protein according to claim 8. 18. The DNA according to claim 17, which has the nucleotide sequence of SEQ ID NO: 10 in the sequence listing, or a DNA having a nucleotide sequence complementary to the sequence. 19. A DNA having a base sequence encoding the fusion protein according to claim 9. 20. The DNA according to claim 19, which has the nucleotide sequence of SEQ ID NO: 11 in the sequence listing, or a DNA having a nucleotide sequence complementary to the sequence. 21. A DNA having a base sequence encoding the fusion protein according to claim 10. 22. The DNA according to claim 21, which has the nucleotide sequence of SEQ ID NO: 12 in the sequence listing, or the DNA having a nucleotide sequence complementary to the sequence. A vector comprising the DNA according to any one of claims 11 to 22. 24. The vector according to claim 23, which is an expression vector capable of expressing the fusion protein according to claim 1 in a host. 25. A transformant obtained by transforming a host cell with the vector according to claim 23 or 24. 26. The transformant according to claim 25, wherein the host cell is a eukaryotic cell or a prokaryotic cell. 27. The transformant according to claim 26, wherein the host cell is Escherichia coli. 28. The transformant according to claim 26, wherein the host cell is an insect cell. 29. The transformant according to any one of claims 25 to 28, wherein the transformant is cultured under conditions in which the protein is expressed, and an expression product is collected from the cultured cell and cross-reacted with a Chlamydia bacterium. The method for producing a fusion protein according to any one of claims 1 to 10, wherein the fusion protein is substantially free of another protein showing 30. A method for testing a Chlamydia trachomatis antibody, comprising using the protein according to any one of claims 1 to 10 as an antigen. 31. A kit for testing a Chlamydia trachomatis antibody, comprising the method according to claim 30.