JPH08294391A - Fused protein of dihydrofolic acid reductase-antigen polypeptide of chlamydia pneumoniae, dna coding for the same, recombinant vector containing the same dna, transformer containing the same recombinant vector and production of anti-chlamydia pneumoniae antibody - Google Patents

Abstract

PURPOSE: To obtain the subject protein for producing antibody and antigen for diagnosis, etc., of infection of Chlamydia pneumoniae by binding a dihyrofolic acid reductase having a specific amino acid sequence and an antigen peptide of Chlamydia pneumoniae. CONSTITUTION: This fused protein of dihydrofolic acid reductase-antigen polypeptide of Chlamydia pneumoniae comprises binding a polypeptide B containing contained at least five amino acid sequences into a polypeptide having an amino acid sequence expressed by formula II directly or through amino acid sequence expressed by formula I. The fused protein is useful for production, etc., of anti-Chlamydia pneumoniae antibody and antigen polpypeptides, etc., effectively utilized for producing a diagnostics for Chlamydia pneumoniae infectious diseases. The fused protein is obtained by binding DNA coding for dihydrofolic acid reductase to DNA coding for antigen polypeptide of Chlamydia pneumoniae and expressing the bound DNA in a host.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dihydrofolate reductase-Chlamydia pneumoniae antigenic polypeptide fusion protein, a DNA encoding the same, a recombinant vector containing the DNA, and a transformant containing the recombinant vector. And a method for producing an anti-Chlamydia pneumoniae antibody. INDUSTRIAL APPLICABILITY The present invention is effectively used in the pharmaceutical industry, particularly in the manufacture of a diagnostic agent for Chlamydia pneumoniae infection.

[0002]

2. Description of the Related Art As a bacterium belonging to the genus Chlamydia, species (Species) such as Chlamydia trachomatis, Chlamydia sittaci, Chlamydia pneumoniae are known. Chlamydia trachomatis is a causative bacterium that causes trachoma, sexually transmitted lymphogranuloma, genitourinary tract infection, inclusion body conjunctivitis, neonatal pneumonia, etc. Is a causative bacterium of respiratory infections, atypical pneumonia and the like.

The symptoms of respiratory tract infections caused by Chlamydia pneumoniae are similar to those of infections caused by Mycoplasma pneumoniae and influenza viruses, and are therefore easily misdiagnosed. Therefore, development of a simple diagnostic method for Chlamydia pneumoniae has been desired.

Diagnosis of infectious diseases is usually made definite by detecting the presence of causative bacteria at the site of infection or the like, or by detecting the presence of antibodies (against the causative bacteria) in serum or other body fluids. The former is called antigen test, the latter is called antibody test,
Both have important clinical significance, and as an antibody test for Chlamydia pneumoniae, a method of detecting the presence of an antibody using the basic bodies of Chlamydia pneumoniae is known.

On the other hand, as a plasmid capable of expressing a large amount of protein in E. coli, pBBK1
0MM is known (Japanese Patent Laid-Open No. 4-117284). This plasmid can express a fusion protein of dihydrofolate reductase (hereinafter abbreviated as DHFR) and an antiallergic peptide. Since the fusion protein obtained here also retains the enzymatic activity of DHFR, the fusion protein can be easily purified by utilizing the characteristics and activity of DHFR.

[0006]

[Problems to be Solved by the Invention] However, it is difficult to culture Chlamydia bacteria and it is not easy to obtain elementary bodies. An object of the present invention is to provide a technique for synthesizing a large amount of an antigen polypeptide for detecting Chlamydia bacteria.

[0007]

[Means for Solving the Problems] First, the present inventors extracted genomic DNA from Chlamydia pneumoniae and partially digested it with a restriction enzyme to obtain the phage vector λgt11.
Insert into DNA to create genomic DNA library,
This was infected with Escherichia coli Y1090r- strain, a colony of infected Escherichia coli expressing the Chlamydia pneumoniae antigenic polypeptide was screened using a Chlamydia pneumoniae-specific monoclonal antibody, and λ phage was extracted from the positively infected E. coli. Repeat the operation λ
The phage was purified and its DNA was obtained. Then, a recombinant plasmid vector pCPN6023 was constructed from the DNA of this λ phage and the above-mentioned plasmid pBBK10MM using a gene recombination technique, and the present invention was completed.

That is, the present invention provides the following (1) to (1
0). (1) Dihydrofolate reductase in which a polypeptide B containing at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 2 is bound to the polypeptide of SEQ ID NO: 1 directly or via an intervening amino acid sequence -Chlamydia
Pneumoniae antigen polypeptide fusion protein. (2) The fusion protein according to (1) above, wherein the polypeptide B is a polypeptide lacking 1 to 320 amino acids from the polypeptide of SEQ ID NO: 2. (3) The fusion protein according to (1) above, wherein the polypeptide B is a polypeptide in which 1 to 100 amino acids in the polypeptide of SEQ ID NO: 2 are substituted with other amino acids. (4) The fusion protein according to (1) above, which is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. (5) The fusion protein according to (1) above, which is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. (6) DNA encoding the fusion protein according to any one of (1) to (5) above or DN complementary thereto
A. (7) A recombinant vector containing the DNA of (6) above. (8) The recombinant vector according to (7) above, which is a pCPN6023 plasmid. (9) A transformant containing the recombinant vector according to (7) or (8) above. (10) A method for producing an anti-Chlamydia pneumoniae antibody, which comprises using the fusion protein according to any one of (1) to (5) above as an antigen.

The details of the present invention are as follows. Preparation of transformants, 2. 2. Method for producing fusion protein, 3. The resulting fusion protein, 4. DN encoding a fusion protein
A, and 5. The method for producing an anti-Chlamydia pneumoniae antibody using the fusion protein as an antigen will be described in this order.

[0010] 1. Preparation of transformants 1.1 Cultivation of Chlamydia pneumoniae A cell suspension was prepared from cultivated HL cells, the culture supernatant was removed, and then a suspension of Chlamydia pneumoniae was added to culture and centrifuge. To obtain HL cells infected with Chlamydia pneumoniae. Examples of Chlamydia pneumoniae include Chlamydia pneumoniae YK41
Stocks (Kanamoto et al .: Microbiological Immunology, 37
Volume, 495-498, 1993 (Y. Kanamoto et al., Microbio
l. Immunol., Vol.37, p.495-498, 1993)) can be used. 1.2 Purification of basic bodies of Chlamydia pneumoniae HL cells infected with Chlamydia pneumoniae are disrupted, centrifuged, and the supernatant is recovered. This supernatant is added to a continuous density gradient solution using urographin (made by Schering) and centrifuged to collect a band corresponding to the elementary bodies of Chlamydia pneumoniae.

1.3 Preparation of Chlamydia pneumoniae Genomic DNA The basic bodies of Chlamydia pneumoniae were treated with 1 mM ED.
10 mM Tris-HCl buffer (pH 8.0) containing TA
The suspension is suspended in (hereinafter referred to as TE buffer), 1% sodium dodecyl sulfate (SDS) aqueous solution and 1 mg / ml proteinase K aqueous solution are added and kept warm to dissolve the elementary bodies.
0.1 M Tris-hydrochloric acid buffer (pH 8.0) saturated phenol is added, and the mixture is stirred and centrifuged to collect the aqueous layer. Further, RNA degrading enzyme (RNase) treatment, phenol / chloroform / isoamyl alcohol treatment and ethanol precipitation treatment were performed, and Chlamydia pneumoniae genome D
Get NA.

1.4 Preparation of Genomic DNA Expression Library Genomic DNA was digested with restriction enzymes AccI, HaeIII and Al.
Digested with uI, phenol / chloroform / isoamyl alcohol treatment and ethanol precipitation treatment, partial digestion D
Get NA. A linker, adenosine 5'-triphosphat, was added to the partially digested DNA.
e, hereinafter abbreviated as ATP) and T4 ligase to add a linker to the partially digested DNA. this,
10 mM containing 0.1 M NaCl and 1 mM EDTA
Chroma Spin 60 with Tris-HCl buffer as mobile phase
It is applied to a 00 (Chroma spin 6000) column, the eluate is collected, and a fraction containing a DNA fragment of 1 kbp to 7 kbp is collected. ATP and T4 polynucleotide kinase are added to the obtained fractions and reacted to phosphorylate the 5'end of the DNA fragment. The reaction solution was treated with phenol / chloroform / isoamyl alcohol and ethanol, and then 5 ′.
A DNA fragment whose ends are phosphorylated is obtained. This DNA
Λgt that had been previously cleaved with the restriction enzyme EcoRI into the fragment
11DNA, ATP and T4 ligase are added and reacted,
The recombinant λ obtained using a commercially available packaging kit
The gt11 DNA is packaged to create a genomic DNA expression library.

1.5 D encoding an antigenic polypeptide
Cloning of NA A culture solution of E. coli Y1090r- strain was infected with the genomic DNA expression library, cultured on an agar medium, and immersed in an isopropylthio-β-D-galactoside (IPTG) aqueous solution using a nitrocellulose filter. ,
The protein produced in the cells by the expression of the inserted DNA is attached to the nitrocellulose filter. The filter is blocked with bovine serum albumin, washed, and then the filter is reacted with anti-Chlamydia pneumoniae monoclonal antibody. Examples of anti-Chlamydia pneumoniae monoclonal antibodies include:
There is a monoclonal antibody produced by hybridoma 60A23. After the reaction, the filter is washed and reacted with an anti-mouse IgG antibody labeled with an enzyme such as peroxidase. After the reaction, the filter is washed and a color-developing substrate solution is added to react. As the color-developing substrate solution, for example, a solution containing an aqueous solution of hydrogen peroxide and a methanol solution of 4-chloro-1-naphthol can be used. After the reaction, the filter is washed and air dried.

A plaque on the agar medium corresponding to the color development spot of the filter is identified, and λ phage contained in the plaque is obtained. The above procedure is repeated until all the plaques have reacted with the above-mentioned monoclonal antibody, the DNA encoding the antigen polypeptide is cloned, and the anti-Chlamydia pneumoniae monoclonal antibody reactive Chlamydia pneumoniae antigen polypeptide is expressed by λ.
Get the phage.

1.6 Acquisition of DNA Encoding Chlamydia pneumoniae Antigen Polypeptide The obtained λ phage is infected with Escherichia coli Y1090r-strain and cultured to produce a large amount of λ phage. DNA is obtained and purified from λ phage using a commercially available kit. A primer, Taq Polymerase (Taq Poly
merase) and deoxynucleotides, heat,
The process of cooling and heat retention was repeated, and D inserted in λgt11
Amplify NA. Examples of the primer include λgt
11 forward primer (λgt11 forward pri
mer) and λgt11 reverse primer (λgt11 re
verse primer) (both manufactured by Takara Shuzo Co., Ltd.)
Examples of tack polymerases include AmpliTaq DNA Polymerase
There is. The general method of this DNA amplification method is known as the PCR method, and the details are described in "Samblock et al., Edited by Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory) (1989)" (J. Samblook).
et al., Molecular Cloning 2nd ed., Cold Spring Ha
rbor Laboratory Press (1989), hereinafter referred to as the document "Molecular Cloning".

The amplified DNA is obtained, and the base sequence is determined and analyzed. A commercially available kit can be used to obtain the DNA, for example, Wizard PCR Prep kit (Promega product).
Can be used. The nucleotide sequence can be determined by a fluorescent label terminator cycle sequence method using tack polymerase, and a kit sold by Perkin-Elmer Japan Co. can be used for this method. In the analysis, a commercially available machine, for example, a 373A type DNA sequencer (Applied Biosystems) can be used.

After the nucleotide sequence is determined, the obtained DNA nucleotide sequence is analyzed by gene sequence analysis software to edit, link, and estimate the amino acid translation region. As the gene sequence analysis software, "DNASIS" (Hitachi Software Engineering Co., Ltd.) can be used. As a result of the analysis, when a full-length gene has not been obtained, DNA before and after the already obtained DNA is obtained by genome walking. To perform genome walking, Takara Shuzo
It is possible to use a kit sold by Co., Ltd.

1.7 Preparation of DNA Encoding DHFR DNA encoding DHFR can be excised from a plasmid vector containing the DNA using a restriction enzyme, or can be a plasmid DNA containing the DNA.
Alternatively, genomic DNA is used as a template and PCR is performed using appropriate primers to amplify the DNA. In the former method, DNA encoding DHFR
As the plasmid vector containing, for example, the plasmid vector pBBK10MM or pCPN6023 which is the recombinant vector of the present invention can be used. pBB
E. coli containing K10MM has accession number FERM BP-2
Escherichia coli containing 394 and pCPN6023 under the deposit number FERM P-14908 are both deposited at the Institute of Biotechnology, Institute of Biotechnology. To obtain a plasmid from these Escherichia coli (plasmid-carrying bacteria), an ordinary method for obtaining plasmid DNA may be used, and this method is described in the document "Molecular Cloning". When the plasmid pBBK10MM is used, BamHI and XhoI are used as restriction enzymes, and a DNA fragment of about 4.6 Kbp may be excised.

In the latter method, for example, the above-mentioned pBBK10MM or pCPN6023 can be used as the plasmid DNA as it is, and the genomic DNA of Bacillus subtilis can be used as the genomic DNA. In order to obtain genomic DNA, a general method for obtaining genomic DNA may be followed, and this method is described in the document "Molecular Cloning". The primers used in the latter method were 5'end and 3'end of DNA encoding DHFR.
It can be designed and synthesized in consideration of the base sequence at the end. For example, an oligonucleotide having a sequence from 1st to 20th of the base sequence of SEQ ID NO: 5 and an oligonucleotide having a sequence complementary to the sequence from 461st to 480th can be used. These oligonucleotides can be chemically synthesized using a commercially available DNA synthesizer.

1.8 D encoding the DHFR-Chlamydia pneumoniae antigenic polypeptide fusion protein
Preparation of Recombinant Vector Containing NA and Preparation of Transformant Containing It The DNA encoding the Chlamydia pneumoniae antigenic polypeptide obtained in 1.6 above and the DNA encoding DHFR obtained in 1.7 above , Using a commercially available kit. At that time, a linker is used if necessary. As a commercially available kit, for example, a DNA ligation kit (Takara Shuzo) can be used. When the DNA obtained by ligation does not have a replication origin and does not function as a plasmid, this DNA is inserted into a new plasmid vector. As this new plasmid vector, for example, pBR322, pUC18 or the like can be used.

The reaction product of the above ligation is put into a host to prepare a transformant. When a plasmid derived from Escherichia coli is used, Escherichia coli can be used as a host, and for example, Escherichia coli HB101 strain can be used. This host is treated so as to become competent cells. E. coli HB
Competent cells obtained by processing 101 strains are sold by Takara Shuzo. A method for producing a transformant by adding the reaction product of the above ligation to a host is described in the document "Molecular Cloning". The resulting transformants were cultured to form colonies, and plasmid DN was used from each colony.
A is obtained, cleaved with the appropriate restriction enzymes and analyzed by agarose gel electrophoresis to select transformants with the desired recombinant plasmid. An example of the plasmid vector produced in this manner is pCPN6023 plasmid. The transformant thus prepared is the Escherichia coli HB101 strain containing the above-mentioned recombinant vector pCPN6023.

2. Method for producing fusion protein In the case of producing the fusion protein of the present invention, culture of the transformant, disruption of the transformant, treatment with streptomycin,
Ammonium sulfate precipitation and mesotrixate (hereinafter abbreviated as MTX)
Each step of binding affinity chromatography is performed. The transformant is cultured by shaking the incubator at an appropriate temperature until the fusion protein is sufficiently accumulated in the transformant in a medium in which the transformant can grow. When the E. coli HB101 strain containing the above-mentioned recombinant vector pCPN6023 is used as a transformant, L containing ampicillin is used.
The cells are cultivated with shaking in B medium at 37 ° C. overnight, and then this culture solution is inoculated into TB medium containing ampicillin and trimethoprim and further cultivated with shaking overnight at 37 ° C. For the preparation method of TB medium, refer to the document "Molecular Cloning".
It is described in. In order to disrupt the cultured transformants, the transformants are collected by centrifugation, suspended in a buffer and irradiated with ultrasonic waves. When the transformant is E. coli,
Lysozyme may be added to the above suspension, and the cells may be lysed by adding a buffer containing SDS. After disruption of the transformant, centrifugation is performed to remove cell debris and obtain a supernatant. Streptomycin sulfate was added to this supernatant,
By stirring for a while and centrifuging, the nucleic acid is removed as a precipitate, and the supernatant is obtained. This supernatant is MTX
-Run on an agarose column to adsorb the fusion protein to the column and then elute the fusion protein using an eluate containing folic acid.

3. Obtained Fusion Protein The fusion protein of the present invention comprises the polypeptide of SEQ ID NO: 1 directly or via an intervening amino acid sequence, SEQ ID NO: 2
Polypeptide B containing at least 5 contiguous amino acid sequences among the above polypeptides is bound. Among the fusion proteins, the polypeptide of SEQ ID NO: 1 is DH
It is the FR part. The polypeptide B containing at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 2 is preferably composed of 20 or more, more preferably 100 or more, and further preferably 200 or more amino acids. . As the polypeptide B, for example, the polypeptide of SEQ ID NO: 2 can be used, and among the polypeptide of SEQ ID NO: 2 lacking 1 to 320 amino acids or the polypeptide of SEQ ID NO: 2 It is also possible to use polypeptides in which 1 to 100 amino acids have been replaced by other amino acids. Examples of the polypeptide lacking 1 to 320 amino acids from the polypeptide of SEQ ID NO: 2 include those having the amino acid sequence of 203 to 439 of the polypeptide of SEQ ID NO: 2, for example.

As a polypeptide in which 1 to 100 amino acids in the polypeptide of SEQ ID NO: 2 are substituted with other amino acids, amino acids at arbitrary positions in the polypeptide of SEQ ID NO: 2 have similar properties. There is a polypeptide in which another amino acid is substituted, for example, a polypeptide in which tyrosine which is the 544th amino acid in the polypeptide of SEQ ID NO: 2 is substituted with phenylalanine.

As the polypeptide B, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 2 can be used. The polypeptide B also includes a polypeptide having at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 2 to which 1 to 20 amino acids are bound. Examples of such a polypeptide B include, for example, 203 to 43 of the polypeptide of SEQ ID NO: 2.
At the C-terminal of the polypeptide consisting of the 9th amino acid sequence, lysine-glycine-phenylalanine-glutamic acid-leucine-proline-tryptophan-glycine-
There is a polypeptide to which a polypeptide consisting of proline-leucine-isoleucine-asparagine has been added. The intervening amino acid sequence is not particularly limited, and examples thereof include an amino acid sequence consisting of leucine and leucine-methionine, glutamine-phenylalanine-glutamic acid-proline-
There is an amino acid sequence consisting of leucine-arginine-glutamic acid-glycine-valine-arginine and the like.

Specific examples of the fusion protein of the present invention include a polypeptide having the amino acid sequence of SEQ ID NO: 3 and a polypeptide having the amino acid sequence of SEQ ID NO: 4. Among the above fusion proteins, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 is desirable.

4. DNA encoding the fusion protein As the DNA encoding the fusion protein, various DNAs having a genetic code corresponding to the amino acid sequence of the fusion protein can be used.

5. Method for producing anti-Chlamydia pneumoniae antibody using fusion protein as an antigen To produce a Chlamydia pneumoniae antibody, a fusion protein of the present invention is used as an antigen to immunize a mouse, and its spleen cells are fused with a myeloma cell line to form a hybridoma. And make 53KD of Chlamydia pneumoniae
It can be obtained by selecting a hybridoma that recognizes the antigen polypeptide of a and culturing it. Examples of myeloma cell lines include P3X63Ag8.653
(ATCC CRL-1580) and P3 / NSI / 1-
Ag4-1 (ATCC TIB-18) can be used. An anti-Chlamydia pneumoniae antibody is produced according to a known general method of immunizing a mouse to obtain an antibody, except that the fusion protein of the present invention is used as an antigen.

[0029]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Example 1 Chlamydia pneumoniae 60
Preparation of DNA Encoding K Antigen Polypeptide (A) Culturing of Host Cell (HL Cell) HL cells grown in advance on the bottom surface of a plastic culture flask (75 cm ² ) were phosphate buffered saline (hereinafter , PBS) magnesium-free (-) liquid 5
PB containing 0.1% (w / v) trypsin after washing with ml
After adding 5 ml of S to the whole cell surface and discarding the liquid, it was incubated at 37 ° C for 10 minutes, 5 ml of Dulbecco's MEM medium containing 10% (v / v) fetal bovine serum was added, and HL cells were pipetted. Was peeled off to prepare a cell suspension.

When culturing in a 75 cm ² plastic culture flask, 1 ml of the above cell suspension and 15-20 ml of Dulbecco's MEM medium containing 10% (v / v) fetal bovine serum were added to the culture flask, and 6-well plastic was used. When culturing in a culture vessel, make 8 ml and 10 ml of the above cell suspension.
4 ml of a mixed solution with 292 ml of Dulbecco's MEM medium containing% fetal bovine serum was added to each well, and the cells were cultured in a 5% (v / v) carbon dioxide atmosphere.

(B) Chlamydia pneumoniae YK4
Cultivation of 1 The culture supernatant of HL cells grown in a 6-well plastic culture container (on the bottom) was removed with a pipette, and 1 liter of the suspension of the Chlamydia pneumoniae YK41 strain described above [Chlamydia pneumoniae YK41 stock solution was Dilute 12 to 24 times with an aqueous solution (hereinafter referred to as SPG) containing 75 g of sucrose, 0.52 g of monopotassium phosphate, 1.22 g of dipotassium phosphate and 0.72 g of glutamic acid, and treat with ultrasonic waves for 1 minute, 2 ml per well] of the supernatant obtained by centrifugation at 2,000 rpm for 3 minutes,
Centrifugal adsorption was performed at 2,000 rpm for 1 hour. After centrifugal adsorption, remove Chlamydia pneumoniae suspension, and 1 μg / ml
Add 4 ml of Dulbecco's MEM medium containing cycloheximide and 10% (v / v) fetal bovine serum to each well, and add 5%.
(V / v) Culture was carried out at 36 ° C. for 3 days in a carbon dioxide atmosphere. After culturing, the cells were peeled off with a sterilized silicon piece, and the cells were collected. This was centrifuged at 8,000 rpm for 30 minutes, the precipitate was resuspended in SPG, and stored at -70 ° C (hereinafter, referred to as Chlamydia pneumoniae YK41 stock solution).

(C) Chlamydia pneumoniae YK41
Purification of elementary bodies of Chlamydia pneumoniae Y stored at -70 ° C
The K41-infected frozen HL cell suspension was thawed and homogenized with a Teflon homogenizer. 10 at 2,500 rpm
Centrifuged for minutes and the supernatant was collected. The precipitate was suspended in SPG again and the same operation was performed to collect the supernatant. The same operation was performed twice more, and the obtained supernatants were collected and combined.

Separately, 0.03 M Tris-hydrochloric acid buffer solution (pH 7.4) containing 50% (w / v) sucrose in a centrifuge tube, and then 3 volumes of urographine 76% (Schering Co.) and 0.03 M Tris. -A mixed solution with 7 volumes of hydrochloric acid buffer (pH 7.4) was overlaid, and the supernatant previously collected was carefully overlaid thereon and centrifuged at 8,000 rpm for 1 hour. Fifty
A 0.03 M Tris-hydrochloric acid buffer (pH 7.4) layer containing 0.1% (w / v) sucrose and a precipitate were collected, and SPG of the same volume was added to the collected liquid, followed by centrifugation at 10,000 rpm for 30 minutes. The supernatant was discarded and the precipitate was suspended in SPG. In a centrifuge tube, a continuous density gradient liquid of urographine 76% (made by Schering) and 0.03M Tris-HCl buffer (pH 7.4) from 35% to 50% (volume ratio of the former to the total amount) was prepared. Then, the suspension is layered on this, and
Centrifuge at m for 1 hour. Chlamydia pneumoniae Y
The band corresponding to the basic body of K41 was collected and was collected as S
It was diluted 2-fold with PG and centrifuged at 10,000 rpm for 30 minutes. The obtained precipitate was suspended in SPG, the protein concentration was measured (using a protein measurement kit manufactured by Bio-Rad, using bovine serum albumin as a standard), and then stored at -70 ° C.

(D) Chlamydia pneumoniae YK-4
Preparation of genomic DNA of 1 strain 300 μl of suspension of elementary bodies of the above purified Chlamydia pneumoniae YK-41 strain (protein concentration: 1.37 mg
/ ml) was centrifuged at 4 ° C. and 12,000 rpm for 5 minutes.
The precipitate was suspended by adding 500 μl of 10 mM Tris-hydrochloric acid buffer pH 8.0 (hereinafter referred to as TE buffer) containing 1 mM EDTA. Repeat the same centrifugation again to precipitate 3 times.
Suspended in 00 μl TE buffer. 1% SDS aqueous solution 3
0 μl and 30 μl of 1 mg / ml proteinase K aqueous solution were added and incubated at 56 ° C. for 30 minutes to dissolve the elementary bodies. 0.1 M Tris-HCl buffer (pH 8.0)
350 μl of saturated phenol was added, mixed well with a vortex mixer, and then centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to recover an aqueous layer (extraction of DNA). This extraction operation was repeated once again. Add 2 μl of 10 mg / ml RNase solution and incubate at 37 ℃ for 2 hours.
A was disassembled. 0.1 M Tris-HCl buffer (pH 8.
0) 25: 24: 1 (volume ratio) mixture of saturated phenol, chloroform and isoamyl alcohol (hereinafter referred to as P
300 μl of CI) was added, mixed well with a vortex mixer, and centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to collect an aqueous layer. This operation was repeated 5 times in total.

To the obtained solution, 1/10 volume of 10 M ammonium acetate aqueous solution and 2 volumes of ethanol were added and allowed to stand for 5 minutes to precipitate DNA, and then at 4 ° C. for 12,000.
Centrifuge at 0 rpm for 5 minutes. For precipitation, 600 μl of 70% ethanol aqueous solution was added and mixed, and the mixture was mixed at 4 ° C. for 12,000.
The washing with centrifugation at rpm for 5 minutes was repeated twice. The precipitate was left to stand for 15 minutes with the lid of the centrifuge tube open, to dry the precipitate, and 200 μl of TE was added to dissolve the precipitate, which was stored at -20 ° C.

(E) Preparation of genomic DNA expression library M-buffer 1 for restriction enzyme was added to 100 μl of genomic DNA solution.
0 μl, restriction enzyme mixture (AccI, HaeIII and 1
10 μl of 0.4 μl each of AluI diluted with 50/50 and 20 μl of TE were mixed), and reacted at 37 ° C. for 20 minutes. It should be noted that the reaction time of the above 20 minutes is 1 kbp to 7 k for DNA.
Pre-tested with a small amount of genomic DNA at the time it was degraded to partially digested DNA of bp size. 100 μl of PCI was added to the above reaction solution, mixed well with a vortex mixer, and centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to collect an aqueous layer. To this, 10μ of 3M sodium acetate solution
1 and 220 μl of ethanol were added and the mixture was allowed to stand at −80 ° C. for 15 minutes to precipitate a partially digested DNA. 4 ° C, 12,
After centrifuging at 000 rpm for 5 minutes and discarding the supernatant,
To the precipitate, 500 μl of 70% aqueous ethanol solution was added and mixed, and the mixture was again centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dried under reduced pressure.

20 μl of purified water was added to the partially digested DNA obtained.
19 μl of the linker (14 μl) of linker (20 pmole / μl) shown in the following chemical formula 1 and 10 mM AT
P 4.5 μl, 50 mM MgCl ₂ , 50 mM dithiothreitol and 500 μg / ml bovine serum albumin-containing 0.2 M Tris-HCl buffer (pH 7.6, hereinafter referred to as 10-fold concentration ligation buffer) 4.5 μl, purified water 2 μl and 1 μl of T4 ligase were added and reacted at 16 ° C. for 4 hours to add a linker.

Embedded image

The partially digested DNA to which a linker has been added,
It was applied to a Chroma spin 6000 column having a mobile phase of 10 mM Tris-HCl buffer containing 0.1 M NaCl and 1 mM EDTA. Two drops of the eluate were collected, and a part of each fraction was analyzed by 0.8% agarose gel electrophoresis to obtain 1 kbp.
A fraction containing a 7 kbp DNA fragment was collected from. To the obtained fraction 144 μl, purified water 13 μl, 10 mM A
TP 20 μl, 0.1 M MgCl ₂ , 50 mM dithiothreitol, 1 mM spermidine hydrochloride and 1 mM
0.5M Tris-HCl buffer containing EDTA (pH 7.6,
Hereinafter referred to as 10 times concentration phosphorylation reaction buffer) 20μ
1 and 3 μl of T4 polynucleotide kinase,
The reaction was carried out at 37 ° C for 30 minutes to phosphorylate the 5'end of the DNA fragment. After adding 200 μl of PCI and shaking well, the mixture was centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to collect the aqueous layer. 20 mg / ml glycogen aqueous solution 1 μl, 3
20 μl of M sodium acetate aqueous solution and ethanol 400
μl was added to precipitate nucleotides. 4 ° C, 12,
The mixture was centrifuged at 000 rpm for 10 minutes, the supernatant was discarded, 200 μl of 70% ethanol was added to the precipitate and mixed, the mixture was centrifuged again, the supernatant was discarded, the precipitate was air dried, and 1 μl of purified water was added to dissolve it.

0.6 μl of this solution was previously added with the restriction enzyme Eco.
Λgt11 DNA cleaved with RI (1 μg / μl, Stratagene) 1 μl, 10-fold concentration ligation buffer 0.5 μl, 10 mM ATP 0.5 μ
1, 0.4 μl of T4 ligase and 2 μl of purified water were added,
The reaction was carried out at 4 ° C overnight. Then, Gigapac
k) II Gold packaging kit (Stratagene)
The obtained recombinant λgt11 DNA was packaged using.

(F) DN encoding the antigen polypeptide
Cloning of A. One platinum loop of E. coli Y1090r- strain was treated with 10 mM MgS.
LB (containing 5 g of NaCl, 10 g of polypeptone and 5 g of yeast extract in 1 L of water) containing 3 ml of O ₄ , 0.2% maltose and 50 μg / ml ampicillin was inoculated into a medium,
After culturing with shaking at 37 ° C overnight, this was 2,000 rp.
Centrifuge at m for 10 minutes. 10 mM for precipitation (E. coli)
9 ml of an aqueous MgSO ₄ solution was added and mixed, 0.35 ml of this E. coli suspension was taken, 0.1 to 10 μl of a λgt11 (DNA library) suspension was added thereto, and the mixture was incubated at 37 ° C. for 20 minutes, E. coli was infected with λgt11. 2.5 ml of liquid LB agar medium pre-heated to 47 ℃
To the above, λgt11-infected E. coli was added, and this was immediately added to LB.
Spread on agar medium. 42 after the upper agar medium has solidified
Incubate at ℃ for 3-4 hours, and 1 when plaque is observed.
A nitrocellulose filter (φ82 mm) immersed in a 0 mM IPTG aqueous solution was placed on the upper layer agar medium and cultured at 37 ° C. for 12 hours. After asymmetrically puncturing the needle with black ink at three points to mark the filter, the filter was removed from the agar medium, and 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 150 mM NaCl and 0.1% Tween 20 was added. (Hereinafter, referred to as TTBS buffer solution) was washed 3 times. The agar medium was stored in the refrigerator.

Filter containing 150 mM NaCl 2
0 mM Tris-HCl buffer (pH 7.5) (hereinafter TBS
It was immersed in a 0.1% bovine serum albumin-containing solution (referred to as a buffer) and shaken at 37 ° C. for 1 hour to carry out a blocking reaction. Then, the filter was washed twice with TTBS buffer, and then immersed in a TTBS solution of 5 to 10 μg / ml of anti-Chlamydia pneumoniae monoclonal antibody (60A23) and shaken at 37 ° C. for 1 hour. Filter TTBS
After washing three times with buffer, the peroxidase-labeled (5
0ng / ml) anti-mouse IgG antibody solution (TTBS buffer)
Shake at 37 ° C for 1 hour. Filter TTBS
After washing three times with a buffer solution and three times with a TBS buffer solution, a chromogenic substrate solution (60 ml of a 30% hydrogen peroxide aqueous solution and 20 ml of a methanol solution of 0.3% 4-chloro-1-naphthol in 100 ml of the TBS buffer solution). In addition, it was dipped in (prepared) and left at room temperature for about 30 minutes. When the color was sufficiently developed, the filter was taken out, washed with purified water and air-dried.

Plaques on the agar medium corresponding to the color development spots on the filter were searched and identified, and the agar in this portion was pierced with a Pasteur pipette to recover the plaques. The recovered plaque was 0.1M with 1 drop of chloroform added.
It was taken in 50 mM Tris-hydrochloric acid buffer (pH 7.5) (hereinafter referred to as SM buffer) containing NaCl, 8 mM magnesium sulfate and 0.01% gelatin, and left overnight at 4 ° C. to extract λ phage in the plaque. . The above procedure was repeated until the plaques were all reacted with the above monoclonal antibody, and the DNA encoding the antigen polypeptide was cloned. Thus, a λ phage expressing an anti-Chlamydia pneumoniae monoclonal antibody-reactive Chlamydia pneumoniae antigen polypeptide was obtained, which was designated as 60-2Sλ phage.

(G) Cultivation of 60-2Sλ phage and DN
A Purification The plaques were formed in the same manner as described in (F) above, one plaque was collected, placed in 100 μl of SM buffer and left at 4 ° C. overnight to extract λ phage. LB
E. coli Y1090r-strain 250μ cultured overnight in culture medium
Add 5-10 μl of λ phage solution to 1 and add 20 at 37 ° C.
After allowing to stand for a minute, E. coli was infected with λ phage. 3 in advance
L containing 10 mM magnesium sulfate warmed to 7 ° C
B culture medium (50 ml) was inoculated and shake-cultured at 37 ° C. for 5 to 7 hours until lysis of Escherichia coli by λ phage occurred. Two
Add 50 μl of chloroform and add 10 at 3,000 rpm.
Centrifugation was performed to remove E. coli cell debris to obtain a λ phage suspension. The λ phage DNA was purified using the Wizard λ preps kit (Promega).

(H) Amplification of DNA encoding Chlamydia pneumoniae antigen polypeptide In a microtube for 600 μl, purified water (61.5 μm) was added.
1, 10-fold concentration reaction buffer (500 mM KCl,
Tris-HCl buffer containing 15 mM MgCl ₂ , 0.01% gelatin, pH 8.3) 10 μl, 20 mM dNT
P 1 μl, 53-3Sλ phage DNA solution 0.1 μ
1, 20 nM λgt11 forward primer (Takara Shuzo Co., Ltd.) 1 μl, 20 nM λgt11 reverse primer
(Takara Shuzo Co., Ltd.) 1 μl, AmpliTaq DNA Polymerase
0.5 μl was added and a few drops of mineral oil were overlaid. Incubation of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 73 ° C for 2 minutes was repeated 30 times,
Was amplified. After the reaction, perform 1.2% low temperature melting agarose gel electrophoresis and cut out the amplified DNA,
d Purified with PCR Prep Kit (Promega).

(I) DNA Nucleotide Sequence Analysis The DNA nucleotide sequence analysis is carried out by using a DNA amplified by PCR as a template and performing a sequencing reaction by a fluorescent labeled terminator cycle sequence method using Taq DNA polymerase to obtain a 373A type DNA sequencer (Applied Bio Systems). The obtained DNA base sequence was edited, ligated, and edited using gene sequence analysis software "DNASIS" (Hitachi Software Engineering Co., Ltd.).
The amino acid translation region was estimated.

Example 2 Construction of Recombinant Vector Containing DNA Encoding Fusion Protein of DHFR and Polypeptide Containing Part of Chlamydia pneumoniae Antigen Polypeptide and Construction of Transformant Containing it Plasmid pBBK10MM is restricted Enzymes BamHI and X
It was cleaved with hoI, subjected to 1.2% low temperature melting agarose gel electrophoresis, and a DNA fragment of about 4.6 Kbp was cut out and purified. In addition to this, DNAs of SEQ ID NO: 6 and SEQ ID NO: 7
The fragment was added to construct plasmid pADA431.
On the other hand, the 60-2Sλ phage DNA was cleaved with the restriction enzyme EcoRI, and the digested product of the restriction enzyme MunI of the plasmid pADA431 was added thereto and ligated to construct a recombinant plasmid vector pCPN6023. This plasmid vector expresses a fusion protein in which a polypeptide containing a part of the Chlamydia pneumoniae 60KDa antigen polypeptide (heat shock protein) is linked to the C-terminus of DHFR via an intervening sequence. The deduced amino acid sequence of this fusion protein was as shown in SEQ ID NO: 4.

Example 3 Confirmation of Fusion Protein of DHFR and Polypeptide Containing Part of Chlamydia pneumoniae 53KDa Antigen Polypeptide Escherichia coli HB10 carrying plasmid pCPN6023
One platinum loop of each strain was inoculated into 3 ml of LB medium containing 50 mg / l of ampicillin, and shake-cultured overnight at 37 ° C. 10 μl of a loading buffer solution (0.01% bromophenol blue, 10% mercaptoethanol, 20% glycerol, 0.156 M Tris-HCl buffer solution containing 5% SDS, pH 6.8) was added to 10 μl of the medium containing this E. coli, 8
After heating at 0 ° C. for 5 minutes, the reaction solution was subjected to 5-20% polyacrylamide gradient gel electrophoresis. 0.3M containing 10% methanol and 0.05% sodium dodecyl sulfate on the anode plate of the semi-dry blotting apparatus.
One piece of filter paper moistened with Tris solution, 10% methanol,
A piece of filter paper moistened with a 25 mM Tris aqueous solution containing 0.05% sodium dodecyl sulfate, 10% methanol, 0.1%.
1 sheet of nitrocellulose membrane moistened with a 25 mM Tris aqueous solution containing 05% sodium dodecyl sulfate and 40 mM aminocaproic acid, polyacrylamide gel after the above electrophoresis, and 2 sheets of filter paper moistened with a 25 mM Tris aqueous solution containing 40 mM aminocaproic acid. In this order, the cathode plates were set and a current was passed at a current density of ^2.5 mA / cm ² for 1 hour to transfer the protein in the polyacrylamide gel to the nitrocellulose membrane. Put this nitrocellulose membrane in TBS buffer containing 0.1% bovine serum albumin,
It was left standing for 1 hour or more at room temperature to perform blocking. The nitrocellulose membrane was washed twice with TTBS buffer and then at 37 ° C. in a monoclonal antibody solution produced by hybridoma 60A23 (in 5 to 10 μg / ml TTBS buffer) at 1 ° C.
Shake for time. The nitrocellulose membrane was washed 3 times with TTBS buffer, and then shaken in a peroxidase-labeled anti-mouse IgG antibody solution (in 50 ng / ml TTBS buffer) at 37 ° C. for 1 hour. Nitrocellulose membrane TTB
After washing 3 times with S buffer, chromogenic substrate solution (100 ml TB
20 ml of 60 μl 30% hydrogen peroxide solution in S buffer
4-chloro-1-naphthol methanol solution was mixed) and reacted at room temperature for 30 minutes. The nitrocellulose membrane was taken out, washed with purified water and air dried. As a result, a colored band was observed at a position corresponding to the size of the fusion protein, and Escherichia coli having the plasmid pCPN6023 expresses the 60 KDa antigen capable of reacting with the monoclonal antibody reactive with Chlamydia pneumoniae. Was shown.

Example 4 Chlamydia pneumoniae 6
Acquisition of DNA encoding the entire 0KDa antigen polypeptide Plasmid pCPN6023 and DNA of λgt11 described above
Genome walking is performed using the library to obtain the DNA encoding the entire 60 KDa antigen polypeptide of Chlamydia pneumoniae. The amino acid sequence encoded by this DNA is as shown in Sequence No. 2.

Example 5 Construction of recombinant vector containing DNA encoding fusion protein of DHFR and whole Chlamydia pneumoniae 60 KDa antigen polypeptide,
And the production of a transformant containing the same. Using the plasmid pBBK10MM and the DNA encoding the entire 60 KDa antigen polypeptide of Chlamydia pneumoniae, a fusion protein of DHFR and the entire 60 KDa antigen polypeptide of Chlamydia pneumoniae was prepared in the same manner as in Example 2. A recombinant vector containing a DNA encoding the above and a transformant containing the recombinant vector are prepared. The amino acid sequence of this fusion protein is shown in SEQ ID NO: 3.

Example 6 Production of anti-Chlamydia pneumoniae antibody using fusion protein as antigen (A) Culture and passage of myeloma cell line Myeloma cell line was P3X63Ag8.653 (ATCC
CRL-1580) is cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum and subcultured. Two weeks before cell fusion, 0.13 mM 8-azaguanine, 0.5 μg / ml MC-210 (mycoplasma remover, Dainippon Pharmaceutical Co., Ltd.) and 10% (v / v) fetal bovine serum were used. The cells are cultured for 1 week in RPMI1640 medium containing the above, and then for 1 week thereafter in a normal medium.

(B) Immunization of Mouse 200 μl of the above-mentioned fusion protein suspension having a protein concentration of 270 μg / ml was centrifuged at 12000 rpm for 10 minutes, and 200 μl of PBS was added to the precipitate to resuspend it. To this, 200 μl of Freund's complete adjuvant is added to make an emulsion, and 150 μl of the emulsion is subcutaneously injected into the back of the mouse (this day is designated as day 0). Suspension 1 of the above fusion protein having a protein concentration of 270 μg / ml on the 14th, 34th and 49th days
00 μl was intraperitoneally injected into a mouse, and further, on day 69, 50 μl of a suspension of the above fusion protein having a protein concentration of 800 μg / ml, and on day 92, 100 μl of the same suspension was intraperitoneally injected into a mouse. On day 95, the spleen is removed and subjected to cell fusion.

[0053] The myeloma cells 10 ⁷ taken in a round bottom glass tube, mixed well (C) with respect to splenocytes ⁸ obtained from cell fusion the spleen,
Centrifuge at 1400 rpm for 5 minutes, remove the supernatant and mix the cells better. 30% pre-insulated at 37 ℃
(W / v) RPMI16 containing polyethylene glycol
Add 0.4 ml of 40 medium and let stand for 30 seconds. 700rp
After centrifugation at m for 6 minutes, RPMI1640 medium 10m
Add 1 l, slowly rotate the glass tube so that the polyethylene glycol is mixed well, and spin at 1400 rpm for 5
Centrifuge for minutes, remove the supernatant completely, and add 5 ml of H to the precipitate.
Add AT medium and let stand for 5 minutes. Add 10 to 20 ml of HAT medium, leave it for 30 minutes, add HAT medium to a myeloma cell concentration of 3.3 × 10 ⁵ / ml, suspend the cells, and use a Pasteur pipette to 96 wells. Dispense 2 drops into each well of a plastic culture vessel. 5%
(V / v) Culturing at 36 ° C. in a carbon dioxide atmosphere, and after 1 day, 7 days, and 14 days, 1 to 2 HAT medium was added to the wells.
Add drops.

(D) Screening of antibody-producing cells The above fusion protein has a protein concentration of 1 to 10 μg / ml.
Suspended in 0.05M sodium bicarbonate buffer (pH 9.6) containing 0.02% (w / v) sodium azide,
The solution was dialyzed against 0.05M sodium bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, and then diluted to a protein concentration of 1 to 10 μg / ml. 50μ in the well of the plate
Take l and let stand overnight at 4 ° C. to adsorb the antigen. Supernatant was removed and 0.02% (w / v) Tween 20 was added to the wells.
PBS (150 μl) is added and left for 3 minutes, then removed and washed. After further washing once, PBS10 containing 1% (v / v) bovine serum albumin in the well
Add 0 μl and leave at 4 ° C. or more overnight to perform blocking. After removing the PBS containing bovine serum albumin,
After washing twice with PBS containing 0.02% (w / v) Tween 20, 50 μl of the culture supernatant of the fused cells was added to the wells.
In addition, leave at room temperature for 2 hours. Similarly, after washing three times with PBS containing 0.02% (w / v) Tween 20, 25 ng / ml of peroxidase-labeled goat anti-mouse Ig was added to the wells.
Add 50 μl of G antibody and leave at room temperature for 2 hours. 0.0
Similarly, after washing 3 times with PBS containing 2% (w / v) Tween 20, 50 μl of ABTS solution (KPL) was added to the wells.
1 and left at room temperature for 15 minutes to 1 hour to allow color reaction
The absorbance at 405 nm is measured with a photometer for 96-well EIA plate. Then, the cells in the positive wells are collected with a Pasteur pipette, transferred to a 24-well plastic culture container, added with 1 to 2 ml of HAT medium, and cultured in the same manner.

(E) Cloning by limiting dilution method The cell concentration of the two fused cells grown in a 24-well plastic culture vessel was measured, and each was diluted with HT medium so that the number of cells was 20 cells / ml. . Separately, 4 to 6-week-old mouse thymocytes suspended in HT medium were taken in a 96-well plastic culture container at 1-2 × 10 ⁵ cells / well,
Add 50 of the above fused cells (cell concentration is 20 cells / ml) to this.
Add 1 μl / well each, incubate at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere, and add 1 to 2 drops / well of HT medium 1 day, 7 days, and 14 days thereafter. 50 μl of the culture supernatant of the well in which cell proliferation was observed was collected and
Confirm antibody production in the same manner as in "Screening of antibody-producing cells". Cells in which there are only single cell colonies in the well, which produce antibodies that react with the elementary bodies and which grow fast, are harvested from the wells and subsequently grown in 24-well plastic culture vessels. Further, the same cloning operation is repeated to obtain a hybridoma that produces an anti-Chlamydia pneumoniae antibody. This is cultured, and an anti-Chlamydia pneumoniae antibody is produced from the culture supernatant.

[0056]

EFFECT OF THE INVENTION The fusion protein according to claim 1 can be used for antibody testing of Chlamydia pneumoniae. The fusion protein according to claim 2 exhibits the effect of the fusion protein according to claim 1, and further, since the length of the amino acid sequence is short, the number of antigen peptides that can be immobilized on a carrier or the like can be increased and the sensitivity can be increased. It can be used for the production of high diagnostic agents. The fusion protein according to claim 3 exerts the effect of the fusion protein according to claim 1, and since it can form a structure that is less susceptible to degradation by proteolytic enzymes, it has excellent stability and is stable as an antigen. Excel.
The fusion protein according to claim 4 exhibits the effect of the fusion protein according to claim 1, and further has the entire antigen polypeptide of Chlamydia pneumoniae, and therefore is extremely suitable for antibody tests and accurate diagnosis of Chlamydia pneumoniae infection. is there. The fusion protein according to claim 5 exerts the effect of the fusion protein according to claim 1, and further has an antigen portion of the antigen polypeptide of Chlamydia pneumoniae, and therefore is extremely useful for antibody tests and accurate diagnosis of Chlamydia pneumoniae infection. Appropriate. DNA according to claim 6.
Can be used for producing the fusion protein according to any one of claims 1 to 5, which is suitable for an antibody test for Chlamydia pneumoniae and a diagnosis for infection with Chlamydia pneumoniae. Claim 7
The recombinant vector described is suitable for producing a fusion protein suitable for antibody testing for Chlamydia pneumoniae, diagnosis of Chlamydia pneumoniae infection, and the like. The recombinant vector according to claim 8 exhibits the effects of the recombinant vector according to claim 7, and can express a fusion protein having an antigenic portion of the antigenic polypeptide of Chlamydia pneumoniae. Is very suitable for manufacturing. The transformant according to claim 9 is suitable for producing a fusion protein suitable for antibody tests for Chlamydia pneumoniae, diagnosis of Chlamydia pneumoniae infection, and the like. The method for producing an anti-Chlamydia pneumoniae antibody according to claim 10 is suitable for producing a diagnostic agent for Chlamydia pneumoniae infection.

[0057]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 160 Sequence type: Amino acid Sequence type: Peptide sequence Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Glu Phe Glu Ile Leu Glu Arg Arg Ile 145 150 155 160

SEQ ID NO: 2 Sequence length: 544 Sequence type: Amino acid Sequence type: Peptide sequence Met Ala Ala Lys Asn Ile Lys Tyr Asn Glu Glu Ala Arg Lys Lys Ile 1 5 10 15 His Lys Gly Val Lys Thr Leu Ala Gle Ala Val Lys Val Thr Leu Gly 20 25 30 Pro Lys Gly Arg His Val Val Ile Asp Lys Ser Phe Gly Ser Pro Gln 35 40 45 Val Thr Lys Asp Gly Val Thr Val Ala Lys Glu Ile Glu Leu Glu Asp 50 55 60 Lys His Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 65 70 75 80 Thr Ala Asp Lys Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 85 90 95 Glu Ala Ile Tyr Ser Glu Gly Leu Arg Asn Val Thr Ala Gly Ala Asn 100 105 110 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Lys Val Val Val 115 120 125 Asp Glu Leu Lys Lys Ile Ser Lys Pro Val Gln His His Lys Glu Ile 130 135 140 Ala Gln Val Ala Thr Ile Ser Ala Asn Asn Asp Ser Glu Ile Gly Asn 145 150 155 160 Leu Ile Ala Glu Ala Met Glu Lys Val Gly Lys Asn Gly Ser Ile Thr 165 170 175 Val Glu Glu Ala Lys Gly Phe Glu Thr Val Leu Asp Val Val Glu Gly 180 185 190 Met Asn Phe Asn Arg Gly Tyr Leu Ser Ser Tyr Phe Ser Thr Asn Pro 195 200 205 Glu Thr Gln Glu Cys Val Leu Glu Asp Ala Leu Ile Leu Ile Tyr Asp 210 215 220 Lys Lys Ile Ser Gly Ile Lys Asp Phe Leu Pro Val Leu Gln Gln Val 225 230 235 240 Ala Glu Ser Gly Arg Pro Leu Leu Ile Ile Ala Glu Glu Ile Glu Gly 245 250 255 Glu Ala Leu Ala Thr Leu Val Val Asn Arg Leu Arg Ala Gly Phe Arg 260 265 270 Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280 285 Leu Glu Asp Ile Ala Ile Leu Thr Gly Gly Gln Leu Val Ser Glu Glu 290 295 300 Leu Gly Met Lys Leu Glu Asn Thr Thr Leu Ala Met Leu Gly Lys Ala 305 310 315 320 Lys Lys Val Ile Val Thr Lys Glu Asp Thr Thr Ile Val Glu Gly Leu 325 330 335 Gly Asn Lys Pro Asp Ile Gln Ala Arg Cys Asp Asn Ile Lys Lys Gln 340 345 350 Ile Glu Asp Ser Thr Ser Asp Tyr Asp Lys Glu Lys Leu Gln Glu Arg 355 360 365 Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Ala Ala 370 375 380 Thr Glu Ile Glu Met Lys Glu Lys Lys Asp Arg val Asp Asp Ala Gln 385 390 395 400 His Ala Thr Ile Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly 405 410 415 Thr Ala Leu Val Arg Cys Ile Pro Thr Leu Glu Ala Phe Leu Pro Met 420 425 430 Leu Ala Asn Glu Asp Glu Ala Ile Gly Thr Arg Ile Ile Leu Lys Ala 435 440 445 Leu Thr Ala Pro Leu Lys Gln Ile Ala Ser Asn Ala Gly Lys Glu Gly 450 455 460 Ala Ile Ile Cys Gln Gln Val leu Ala Arg Ser Ala Asn Glu Gly Tyr 465 470 475 480 Asp Ala Leu Arg Asp Ala Tyr Thr Asp Met Ile Asp Ala Gly Ile Leu 485 490 495 Asp Pro Thr Lys Val Thr Arg Ser Ala Leu Glu Ser Ala Arg Ser Ile 500 505 510 Ala Gly Leu Leu Leu Thr Thr Glu Ala Leu Ile Ala Asp Ile Pro Glu 515 520 525 Glu Lys Ser Ser Ser Ala Pro Ala Met Pro Ser Ala Gly Met Asp Tyr 530 535 540 544

SEQ ID NO: 3 Sequence length: 704 Sequence type: Amino acid Sequence type: Peptide sequence Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Glu Phe Glu Ile Leu Glu Arg Arg Ile 145 150 155 160 Met Ala Ala Lys Asn Ile Lys Tyr Asn Glu Glu Ala Arg Lys Lys Ile 165 170 175 His Lys Gly Val Lys Thr Leu Ala Gle Ala Val Lys Val Thr Leu Gly 180 185 190 Pro Lys Gly Arg His Val Val Ile Asp Lys Ser Phe Gly Ser Pro Gln 195 200 205 Val Thr Lys Asp Gly Val Thr Val Ala Lys Glu Ile Glu Leu Glu Asp 210 215 220 Lys His Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 225 230 235 240 Thr Ala Asp Lys Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 245 250 255 Glu Ala Ile Tyr Ser Glu Gly Leu Arg Asn Val Thr Ala Gly Ala Asn 260 265 270 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Lys Val Val Val 275 280 285 Asp Glu Leu Lys Lys Ile Ser Lys Pro Val Gln His His Lys Glu Ile 290 295 300 Ala Gln Val Ala Thr Ile Ser Ala Asn Asn Asp Ser Glu Ile Gly Asn 305 310 315 320 Leu Ile Ala Glu Ala Met Glu Lys Val Gly Lys Asn Gly Ser Ile Thr 325 330 335 Val Glu Glu Ala Lys Gly Phe Glu Thr Val Leu Asp Val Val Glu Gly 340 345 350 Met Asn Phe Asn Arg Gly Tyr Leu Ser Ser Tyr Phe Ser Thr Asn Pro 355 360 365 Glu Thr Gln Glu Cys Val Leu Glu Asp Ala Leu Ile Leu Ile Tyr Asp 370 375 380 Lys Lys Ile Ser Gly Ile Lys Asp Phe Leu Pro Val LeuGln Gln Val 385 390 395 400 Ala Glu Ser Gly Arg Pro Leu Leu Ile Ile Ala Glu Glu Ile Glu Gly 405 410 415 Glu Ala Leu Ala Thr Leu Val Val Asn Arg Leu Arg Ala Gly Phe Arg 420 425 430 Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 435 440 445 Leu Glu Asp Ile Ala Ile Leu Thr Gly Gly Gln Leu Val Ser Glu Glu 450 455 460 Leu Gly Met Lys Leu Glu Asn Thr Thr Leu Ala Met Leu Gly Lys Ala 465 470 475 480 Lys Lys Val Ile Val Thr Lys Glu Asp Thr Thr Ile Val Glu Gly Leu 485 490 495 Gly Asn Lys Pro Asp Ile Gln Ala Arg Cys Asp Asn Ile Lys Lys Gln 500 505 510 Ile Glu Asp Ser Thr Ser Asp Tyr Asp Lys Glu Lys Leu Gln Glu Arg 515 520 525 Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Ala Ala 530 535 540 Thr Glu Ile Glu Met Lys Glu Lys Lys Asp Arg Val Asp Asp Ala Gln 545 550 555 560 His Ala Thr Ile Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly 565 570 575 Thr Ala Leu Val Arg Cys Ile Pro Thr Leu Glu Ala Phe Leu Pro Met 580 585 590 Leu Ala Asn Glu Asp Glu Ala Ile Gly Thr Arg Ile IleLeu Lys Ala 595 600 605 Leu Thr Ala Pro Leu Lys Gln Ile Ala Ser Asn Ala Gly Lys Glu Gly 610 615 620 Ala Ile Ile Cys Gln Gln Val leu Ala Arg Ser Ala Asn Glu Gly Tyr 625 630 635 640 Asp Ala Leu Arg Asp Ala Tyr Thr Asp Met Ile Asp Ala Gly Ile Leu 645 650 655 Asp Pro Thr Lys Val Thr Arg Ser Ala Leu Glu Ser Ala Arg Ser Ile 660 665 670 Ala Gly Leu Leu Leu Thr Thr Glu Ala Leu Ile Ala Asp Ile Pro Glu 675 680 685 Glu Lys Ser Ser Ser Ala Pro Ala Met Pro Ser Ala Gly Met Asp Tyr 690 695 700 704

SEQ ID NO: 4 Sequence length: 419 Sequence type: Amino acid Sequence type: Peptide sequence Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 Ala Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 Ala Gln Asn Ser His Ser Tyr Glu Phe Glu Ile Leu Glu Arg Arg Ile 145 150 155 160 Gln Phe Glu Pro leu Arg Glu Gly Val Arg Tyr Phe Ser Thr Asn Pro 165 170 175 Glu Thr Gln Glu Cys Val Leu Glu Asp Ala Leu Ile Leu Ile Tyr Asp 180 185 190 Lys Lys Ile Ser Gly Ile Lys Asp Phe Leu Pro Val Leu Gln Gln Val 195 200 205 Ala Glu Ser Gly Arg Pro Leu Leu Ile Ile Ala Glu Glu Ile Glu Gly 210 215 220 Glu Ala Leu Ala Thr Leu Val Val Asn Arg Leu Arg Ala Gly Phe Arg 225 230 235 240 Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 245 250 255 Leu Glu Asp Ile Ala Ile Leu Thr Gly Gly Gln Leu Val Ser Glu Glu 260 265 270 Leu Gly Met Lys Leu Glu Asn Thr Thr Leu Ala Met Leu Gly Lys Ala 275 280 285 Lys Lys Val Ile Val Thr Lys Glu Asp Thr Thr Ile Val Glu Gly Leu 290 295 300 Gly Asn Lys Pro Asp Ile Gln Ala Arg Cys Asp Asn Ile Lys Lys Gln 305 310 315 320 Ile Glu Asp Ser Thr Ser Asp Tyr Asp Lys Glu Lys Leu Gln Glu Arg 325 330 335 Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Ala Ala 340 345 350 Thr Glu Ile Glu Met Lys Glu Lys Lys Asp Arg val Asp Asp Ala Gln 355 360 365 His Ala Thr Ile Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly 370 375 380 Thr Ala Leu Val Arg Cys Ile Pro Thr Leu Glu Ala Phe Leu Pro Met 385 390 395 400 Leu Ala Asn Glu Asp Glu Ala Lys Gly Phe Glu Leu Pro Trp Gly Pro 405 410 415 Leu Ile Asn 419

SEQ ID NO: 5 Sequence length: 480 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: Other nucleic acid Genomic DNA sequence ATG ATC AGT CTG ATT GCG GCG TTA GCG GTA GAT CGC GTT ATC GGC ATG 48 Met Ile Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met 1 5 10 15 GAA AAC GCC ATG CCG TGG AAC CTG CCT GCC GAT CTC GCC TGG TTT AAA 96 Glu Asn Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys 20 25 30 CGC AAC ACC TTA AAT AAA CCC GTG ATT ATG GGC CGC CAT ACC TGG GAA 144 Arg Asn Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu 35 40 45 TCA ATC GGT CGT CCG TTG CCA GGA CGC AAA AAT ATT ATC CTC AGC AGT 192 Ser Ile Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser 50 55 60 CAA CCG GGT ACG GAC GAT CGC GTA ACG TGG GTG AAG TCG GTG GAT GAA 240 Gln Pro Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu 65 70 75 80 GCC ATC GCG GCG TGT GGT GAC GTA CCA GAA ATC ATG GTG ATT GGC GGC 288 Ala Ile Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly 85 90 95 G GT CGC GTT TAT GAA CAG TTC TTG CCA AAA GCG CAA AAA CTG TAT CTG 336 Gly Arg Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu 100 105 110 ACG CAT ATC GAC GCA GAA GTG GAA GGC GAC ACC CAT TTC CCG GAT TAC 384 Thr His Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr 115 120 125 GAG CCG GAT GAC TGG GAA TCG GTA TTC AGC GAA TTC CAC GAT GCT GAT 432 Glu Pro Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp 130 135 140 GCG CAG AAC TCT CAC AGC TAT GAG TTC GAA ATT CTG GAG CGG CGG ATC 480 Ala Gln Asn Ser His Ser Tyr Glu Phe Glu Ile Leu Glu Arg Arg Ile 145 150 155 160

SEQ ID NO: 6 Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single stranded Sequence type: Other nucleic acid Synthetic DNA Sequence GATCCAATTG CCATGGGGGC CCTTAATTAA TTAAC 35

SEQ ID NO: 7 Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Other nucleic acid Synthetic DNA Sequence TCGAGTTAAT TAATTAAGGG CCCCCATGGC AATTG 3
5

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 21/02 C12P 21/02 C 21/04 21/04 G01N 33/53 G01N 33/53 D 33 / 569 33/569 F 33/571 33/571 // A61K 49/00 A61K 49/00 A C07K 14/295 8517-4H C07K 14/295 C12N 9/02 C12N 9/02 (C12N 15/09 ZNA C12R 1 : 01) (C12P 21/02 C12R 1:19) (54) Title of the Invention Dihydrofolate reductase-Chlamydia pneumoniae antigen-polypeptide fusion protein, DNA encoding the same, recombinant vector containing the DNA, Transformant containing the recombinant vector, and method for producing anti-Chlamydia pneumoniae antibody

Claims (10)

[Claims] 1. A dihydrofolic acid wherein a polypeptide B comprising at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 2 is bound to the polypeptide of SEQ ID NO: 1 directly or via an intervening amino acid sequence. Reductase-Chlamydia pneumoniae antigenic polypeptide fusion protein. 2. The fusion protein according to claim 1, wherein the polypeptide B is a polypeptide lacking 1 to 320 amino acids from the polypeptide of SEQ ID NO: 2. 3. The fusion protein according to claim 1, wherein the polypeptide B is a polypeptide in which 1 to 100 amino acids in the polypeptide of SEQ ID NO: 2 are replaced with other amino acids. 4. The fusion protein according to claim 1, wherein the fusion protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. 5. The fusion protein according to claim 1, wherein the fusion protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. 6. A DNA encoding the fusion protein according to claim 1 or D complementary thereto.
NA. 7. A recombinant vector containing the DNA of claim 6. 8. The recombinant vector according to claim 7, which is the pCPN6023 plasmid. 9. A transformant containing the recombinant vector according to claim 7. 10. A method for producing an anti-Chlamydia pneumoniae antibody, which comprises using the fusion protein according to claim 1 as an antigen.