JPH10212298A - Histidine tag chlamydia pneumoniae antigen polypeptide fusion protein, its production, dna coding for the same fusion protein, recombinant vector containing the dna, transformant containing the recombinant vector, recombinant baculovirus, its production and production of anti-chlamydia pneumoniae antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new fusion protein excellent in stability, useful for producing an antibody for diagnosing infection with Chlamydia pneumoniae, by bonding a polypeptide of a specific amino acid sequence to a peptide containing a plural number of continuous histidines. SOLUTION: This fusion protein is a new histidine tag Chlamydia pneumoniae polypeptide fusion protein obtained by bonding a polypeptide containing a sequence composed of at least five continuous amino acids in a peptide of the formula directly or through an amino acid sequence to a peptide containing at least 6 continuous histidines, stably maintains antigenicity as a Chlamydia pneumoniae antibody, is readily purified and is useful as an antigen for producing an antibody for a diagnostic for infection with Chlamydia pneumoniae. The fusion protein is obtained by infecting an insect with a baculovirus integrated with a histidine tag Chlamydia pneumoniae antigen fusion protein and expressing the gene in cells of the insect.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a histidine tag.
Chlamydia pneumoniae antigen polypeptide fusion protein, method for producing the same, D encoding the fusion protein
The present invention relates to NA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a recombinant baculovirus, a method for producing the same, and a method for producing an anti-Chlamydia pneumoniae antibody.

[0002]

2. Description of the Related Art Species such as Chlamydia trachomatis, Chlamydia shittashi and Chlamydia pneumoniae are known as bacteria of the genus Chlamydia. Chlamydia trachomatis is a bacterium that causes trachoma, genital lymphogranuloma, genitourinary tract infections, conjunctivitis conjunctivitis, neonatal pneumonia, etc.Chlamydia sictus is a causative bacterium for parrot disease, etc. Is a causative bacterium for respiratory infections, atypical pneumonia and the like.

[0003] The symptoms of respiratory infections caused by Chlamydia pneumoniae are often misdiagnosed because they are similar to those of infections caused by Mycoplasma pneumoniae and influenza virus. Therefore, development of a simple diagnostic method for Chlamydia pneumoniae has been desired.

[0004] Diagnosis of infectious diseases is usually made definitively by detecting the presence of a causative bacterium at an infected site or the like, or by detecting the presence of an antibody (to the causative bacterium) in serum or other body fluids. The former is called an antigen test, the latter is called an antibody test,
All of these have important clinical significance, and as an antibody test for Chlamydia pneumoniae, a method for detecting the presence of an antibody using basic bodies of Chlamydia pneumoniae is known.

The basic body of Chlamydia pneumoniae is
Includes antigens that are also commonly found in Chlamydia bacteria other than Chlamydia pneumoniae, ie, Chlamydia trachomatis or Chlamydia sp. Therefore, the method using the basic body reacts not only with an antibody against Chlamydia pneumoniae but also with an antibody against other species of Chlamydia, and has the disadvantage of lacking specificity. Many attempts have been made to search. For example, Matsumoto et al. Found an antigen polypeptide having a molecular weight of about 53 K daltons as an antigen polypeptide specific to Chlamydia pneumoniae, and determined the nucleotide sequence of the gene and the amino acid sequence deduced from the nucleotide sequence. Using the gene of dihydrofolate reductase of Escherichia coli, a system for expressing the antigen polypeptide as a fusion protein with this enzyme was constructed (International Patent Publication No. WO96 / 09320). However, in this system, the fusion protein is expressed using a prokaryotic E. coli. Therefore, the Chlamydia pneumoniae antigen polypeptide portion in the obtained fusion protein tends to be unstable and tend to have a slightly different shape from the shape in nature, so that the obtained fusion protein has an antigen as Chlamydia pneumoniae antigen. There is a problem that it is difficult to maintain the stability stably.

[0006]

The object of the present invention is to provide a fusion protein in which the antigenicity of the Chlamydia pneumoniae antigen is stably maintained and which is easily purified. The invention of claim 2 provides a fusion protein in which the antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained, in addition to the effects of the invention of claim 1. In the invention according to claim 3, the antigenicity of the Chlamydia pneumoniae antigen is stably maintained,
It is intended to provide a DNA useful for producing a fusion protein which can be easily purified. The invention according to claim 4 provides, in addition to the effects of the invention according to claim 3, a DNA useful for producing a fusion protein in which antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained. The fifth and sixth aspects of the present invention provide a recombinant vector useful for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and is easily purified.

The invention according to claim 7 is characterized in that the antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained,
PHLTC useful for production of easily purified fusion protein
PN53 plasmid is provided. The invention described in claim 8 provides a transformant which stably retains antigenicity as a Chlamydia pneumoniae antigen and is useful for producing a fusion protein which is easily purified. The invention described in claim 9 provides a transformant which is useful for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae-specific antigen and is easily purified. The tenth aspect of the present invention provides a recombinant baculovirus which is useful for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and is easily purified.

The invention according to claim 11 is a recombinant baculovirus which stably retains antigenicity as a Chlamydia pneumoniae antigen and is capable of efficiently producing a recombinant baculovirus useful for producing a fusion protein which can be easily purified. A method for producing a virus is provided. The invention according to claim 12 is a recombinant baculovirus which stably retains antigenicity as a Chlamydia pneumoniae-specific antigen and is capable of efficiently producing a recombinant baculovirus useful for producing a fusion protein which is easily purified. Is provided. The invention of claim 13 provides a method for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and efficiently produce a fusion protein which is easily purified. The invention of claim 14 provides a method for producing an anti-Chlamydia pneumoniae antibody suitable as an active ingredient of a diagnostic agent for Chlamydia pneumoniae infection.

[0009]

Means for Solving the Problems The present invention provides the following (1)-
(14). (1) SEQ ID NO: 1 is directly or via an amino acid sequence in a peptide containing at least 6 consecutive histidines.
A histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein comprising a polypeptide comprising a sequence comprising at least 5 consecutive amino acids in the peptide represented by the formula: (2) The fusion protein according to the above (1), wherein the amino acid sequence is a peptide represented by SEQ ID NO: 2. (3) DNA encoding the fusion protein according to (1) or (2) or a DNA complementary thereto. (4) The DNA according to the above (3), having the base sequence represented by SEQ ID NO: 3. (5) A recombinant vector containing the DNA according to (3) or (4). (6) (a) DNA encoding histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein
And (b) the DN of (a), which comprises a promoter sequence.
D containing DNA located upstream of A and capable of undergoing homologous recombination with baculovirus DNA
NA is defined as the downstream of the DNA of (a) and the DN of (b).
The recombinant vector according to the above (5), which is contained upstream of A.

(7) pHLTCPN53 plasmid. (8) A transformant containing the recombinant vector according to (5) or (6). (9) A transformant deposited under accession number FERM P-16036. (10) Baculovirus DNA comprising (a) a DNA encoding a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein and (b) a promoter sequence, wherein the DNA is located upstream of the DNA of (a).
A recombinant baculovirus comprising

(11) The recombinant vector and the baculovirus DNA described in the above (6) are introduced into insect cells, whereby homologous recombination is caused between the recombinant vector and the baculovirus DNA in insect cells. A method for producing a recombinant baculovirus. (12) A method for producing a recombinant baculovirus, which comprises introducing a pHLTCPN53 plasmid and a baculovirus DNA into an insect cell, thereby causing homologous recombination between the plasmid and the baculovirus DNA in the insect cell. (13) A histidine tag comprising infecting insect cells with the recombinant baculovirus according to (10), culturing the insect cells, and obtaining a cell lysate or cell lysate of the cultured insect cells. A method for producing a Chlamydia pneumoniae antigen polypeptide fusion protein. (14) A method for producing an anti-Chlamydia pneumoniae antibody, comprising using the fusion protein according to (1) or (2) as an antigen.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Chlamydia pneumoniae antigen polypeptide In the present invention, the Chlamydia pneumoniae antigen polypeptide is derived from at least five consecutive amino acids in the peptide represented by SEQ ID NO: 1 from the viewpoint of the minimum size of the peptide having antigenicity. (Hereinafter referred to as "polypeptide A"). Since the longer the amino acid sequence derived from the natural Chlamydia pneumoniae antigen polypeptide contained in the peptide chain of the polypeptide A, the higher the sensitivity of the antigen-antibody reaction can be expected, the polypeptide represented by SEQ ID NO: 1 is used as the polypeptide A. Is preferably a polypeptide comprising a sequence consisting of 20 or more amino acids, more preferably a polypeptide comprising a sequence consisting of 100 or more amino acids, and a sequence consisting of 250 or more amino acids More preferably, the polypeptide comprises

As long as it retains the antigenicity as a Chlamydia pneumoniae antigen, polypeptide A is composed of the peptide represented by SEQ ID NO.
0) may be missing. If the number of missing amino acids is too large, the antigenicity of polypeptide A as a Chlamydia pneumoniae antigen tends to be impaired. When the number of missing amino acids is large (for example, 5
Or more), the antigenicity of the Chlamydia pneumoniae antigen is likely to decrease, and in order to minimize the decrease, the amino acid missing from the peptide represented by SEQ ID NO: 1 is continuous (for example, 5 or more). Preferably, it is

As long as the polypeptide A has antigenicity as a Chlamydia pneumoniae antigen, the polypeptide A
Polypeptides containing other amino acid sequences in addition to the amino acid sequence derived from the naturally occurring Chlamydia pneumoniae antigen polypeptide can also be used. It is required that there be no or low antigenicity as a substance other than the Chlamydia pneumoniae antigen. Examples of such polypeptides include SEQ ID NO: 1
(For example, 1 to 100)
Amino acid (eg, 1) in the polypeptide shown in SEQ ID NO: 1
To 100) are inserted.

When the number of amino acids to be substituted or inserted is large (for example, 5 or more), the antigenicity of Chlamydia pneumoniae antigen tends to decrease. The amino acids to be substituted or inserted in the peptide to be substituted are preferably continuous (for example, 5 or more). The amino acids to be substituted preferably have similar properties, and include, for example, substitution of glycine for alanine. Specific examples of the polypeptide A include, for example, a peptide represented by SEQ ID NO: 1, a peptide in which alanine, which is the 488th amino acid of the peptide represented by SEQ ID NO: 1, is substituted with glycine, and the like.

Histidine Tag-Chlamydia pneumoniae Antigen Polypeptide Fusion Protein The histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein of the present invention can be directly or via an amino acid sequence to a peptide containing at least six consecutive histidines. And a polypeptide (polypeptide A) containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1. A peptide comprising at least six consecutive histidines is an amino acid sequence comprising a histidine tag. The histidine tag is an amino acid sequence in which histidine is continuous, and has a high affinity for a nickel atom. Therefore, a fusion protein of the peptide and the target peptide can be purified using the histidine tag. Specific examples of the amino acid sequence of a peptide containing at least six consecutive histidines include, for example, a sequence consisting of the 1st to 14th amino acids in the peptide represented by SEQ ID NO: 2.

The polypeptide containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 1 (polypeptide A) is the above-mentioned Chlamydia.
It is the Pneumoniae antigen polypeptide part. The intervening amino acid sequence is not particularly limited, and examples thereof include an amino acid sequence of leucine and leucine-methionine. Specific examples of the histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein of the present invention include:
For example, the peptide represented by SEQ ID NO: 2 can be mentioned.
Among the peptides represented by SEQ ID NO: 2, from the first to 14
The sequence consisting of the amino acids up to the ninth position is a histidine tag portion (a peptide containing six consecutive histidines) containing the genetic code for translation initiation, and the sequence consisting of the 15th to the 41st amino acids is the amino acid sequence portion And the sequence consisting of amino acids 42 to 529 is the amino acid sequence of the entire 53 KDa antigenic polypeptide of natural Chlamydia pneumoniae. The peptide represented by SEQ ID NO: 2 has antigenicity as a Chlamydia pneumoniae antigen.

A method for producing the histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein The method for producing the histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein of the present invention includes, for example, a gene recombination method. As a gene recombination method, for example, a DNA encoding the fusion protein is inserted into a vector to construct a recombinant vector, and this recombinant vector and baculovirus DNA
, A method of producing a recombinant baculovirus, infecting insect cells with the recombinant baculovirus, culturing the insect cells, and purifying the desired fusion protein. DN encoding the fusion protein
A will be described later. Examples of the vector include a plasmid, a phage, and a virus. Less than,
The method for producing the recombinant vector and the recombinant baculovirus will be described in detail.

Preparation of Recombinant Vector The recombinant vector of the present invention contains a DNA encoding the fusion protein, and includes, for example, (a) a DNA encoding a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein and A) a DNA comprising a promoter sequence, comprising a DNA located upstream of the DNA of (a) and (c) capable of causing homologous recombination with baculovirus DNA;
And a recombinant vector comprising the DNA of (b) above and the DNA of (b) above.

The DNA of (a) will be described later.
The DNA of (b) has a function of expressing the fusion protein. This DNA preferably contains an SD sequence, and the base sequence between the promoter sequence and the DNA of (a) is preferably 200 bases or less. As the promoter sequence, for example,
The promoter sequence of the gene of the polyhedrin protein constituting the inclusion body of baculovirus is exemplified. As a specific example of this sequence, a sequence consisting of the 4030th to 4092th bases in the base sequence shown in SEQ ID NO: 10 can be mentioned. The nucleotide sequence represented by SEQ ID NO: 10 is the entire nucleotide sequence of the plasmid pVCPN53 used for preparing the recombinant vector of the present invention, and the plasmid pVCPN53 will be described later.

The DNA (c) is a DNA for incorporating the DNA (a) and the DNA (b) into a baculovirus DNA, and is a DNA capable of causing homologous recombination with the baculovirus DNA ( This DNA
Is sometimes referred to as a flanking sequence). In homologous recombination, a cross-reaction occurs between two sets of double-stranded DNAs having identical or highly homologous base sequences, and as a result, one double-stranded DNA is combined with the other double-stranded DNA from the middle. It is a changing phenomenon. When there are two sites where homologous recombination occurs, double homologous recombination occurs, and the phenomenon of rearrangement of double-stranded DNA occurs twice. As a result, in the two sets of double-stranded DNAs, the regions between the sites capable of causing homologous recombination are switched.

Examples of the baculovirus DNA include baculovirus genomic DNA, DNA having a base sequence deleted from the genomic DNA, DNA having another base sequence inserted into the genomic DNA, and base sequence in the genomic DNA. And DNAs substituted with the base sequence described above. The DNA having a base sequence deleted from the genomic DNA includes D, in which a region essential for the growth of baculovirus is deleted.
NA and DNA in which a region not necessarily required for the growth of baculovirus is deleted. Examples of the shape of the baculovirus DNA include a linear DNA and a completely circular DNA. These baculovirus DNAs are obtained from D.c.
NA or its DNA itself may be processed, DNA may be cloned from baculovirus and amplified by PCR or the like, or may be synthesized by chemical synthesis. Is also good.

As a specific example of the baculovirus DNA, BaculoGold Linearized Baculovirus DNA (BaculoGold Linearized Baculovirus DNA)
) (PharMingen (San Diego, Calif.) Trade name). This DN
A is a DNA obtained by digesting a baculovirus genomic DNA with a restriction enzyme to delete a region essential for the growth of the baculovirus from the genomic DNA and to make the DNA linear. The recombinant vector comprises a DNA capable of causing homologous recombination with a baculovirus DNA, downstream of the DNA of (a) and upstream of the DNA of (b). Therefore, homologous recombination occurs between the recombinant vector and the baculovirus DNA, and as a result, a recombinant baculovirus in which the DNA of (a) and the DNA of (b) are incorporated into the baculovirus DNA is formed.

The DNA capable of causing homologous recombination with the baculovirus DNA includes a DNA comprising an arbitrary part of the base sequence on the baculovirus DNA.
And a DNA having a base sequence that is the same as or has a high homology to the base sequence, and those having the same base sequence are preferable. As the base sequence of any part on the baculovirus DNA, only the recombinant baculovirus can grow, and thus, only the target recombinant baculovirus can be selected. The base sequence of DNA which is a certain region and which is complemented by homologous recombination between the recombinant vector and baculovirus DNA is preferable. The degree of homology is not particularly limited as long as the frequency of homologous recombination is not significantly reduced, but is preferably, for example, 90% or more. Also,
The length of DNA capable of undergoing homologous recombination with baculovirus DNA is not particularly limited as long as the frequency of homologous recombination is not significantly reduced.
For example, a 2-5 kilobase is preferred. DN capable of causing homologous recombination with baculovirus DNA
In A, the DNA downstream of the DNA (a) and the DNA upstream of the DNA (b) may be the same or different.

The DNA of (c) may be directly obtained from a baculovirus, or may be a DNA cloned from a baculovirus and amplified by a PCR method or the like.
Or a compound synthesized by a chemical synthesis method. As a specific example of the nucleotide sequence of the DNA (c), 7
A sequence consisting of the nucleotides from the 40th to the 4029th, 563
And a sequence consisting of the 5th to 8467th bases.

The recombinant vector of the present invention comprises the aforementioned (a)
A DNA encoding a histidine tag,
It can be obtained by inserting into a vector containing the DNA of (b) and the DNA of (c). This vector contains the D of (b)
Preferably, a DNA encoding a histidine tag immediately after NA, followed by a cloning site for foreign DNA is present. Examples of the vector containing the DNA encoding the histidine tag, the DNA of (b) and the DNA of (c) include, for example, plasmids pAcSG-His NT-A, pAcGHLT-B, pAcHLT-
C and the like. In addition, the recombinant vector of the present invention is prepared by inserting the DNA of (a) into a vector containing the DNA of (b) and the DNA of (c) to prepare a recombinant vector. It can also be obtained by cutting out the DNA containing the DNA (a) and inserting it into a vector containing the DNA encoding the histidine tag, the DNA (b) and the DNA (c). Examples of the vector containing the DNA of (b) and the DNA of (c) include, for example, plasmids pVL1393, pAcSG1, pAcSG1,
AcGP67, pAcJP1, pAcMP1, pAcUW51, pAcUW1, pAcUW41, and the like.These plasmids include Pharmingen (P
harMingen) (San Diego, CA, USA).

[0027] The recombinant vector of the present invention is preferably introduced into an appropriate host for storage and amplification. The host is appropriately selected depending on the type of the recombinant vector, but it is necessary to use a host in which the recombinant vector is stably replicated and no deletion occurs. When a plasmid vector is used as a vector, it is preferable to use a plasmid vector derived from Escherichia coli, which has been well studied historically. In this case, Escherichia coli is preferable as a host, and such Escherichia coli is, for example, Escherichia coli HB1
01 strains. When Escherichia coli is used as a host, the bacteria are treated so as to become competent cells. Competent cells obtained by treating Escherichia coli HB101 are sold by Takara Shuzo Co., Ltd. and others. A general method for preparing a transformant by introducing a recombinant vector into a host is described in "Samblock et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory) (1989)" (J. Samblook). et al., Molecul
ar Cloning 2nd ed., Cold Spring Harbor Laboratory
Press (1989), hereafter referred to the document "Molecular
Cloning ").

The transformant was cultured on a solid medium to form colonies, and plasmid DNA was obtained from each colony using an alkaline lysis method, cut with an appropriate restriction enzyme,
Analyze by agarose gel electrophoresis and select transformants having the desired recombinant vector. This transformant is
For example, the transformant is cultured by shaking overnight in an LB medium containing ampicillin at 37 ° C., and then inoculating this culture solution into a TB medium containing ampicillin, and further culturing with shaking at 37 ° C. overnight. Can be propagated, and accordingly, the recombinant vector can be amplified. The method for preparing the TB medium is described in the document "Molecular Cloning". The recombinant vector of the present invention includes, for example, a pHLTCPN53 plasmid, which has a base sequence represented by SEQ ID NO: 11. Further, as the transformant of the present invention,
For example, an Escherichia coli HB101 strain containing the pHLTCPN53 plasmid can be mentioned, and this strain has an accession number of FER.
It has been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology as MP-16036.

Method for Producing Recombinant Baculovirus Recombinant baculovirus is obtained by introducing the recombinant vector of the present invention and baculovirus DNA into insect cells, whereby the double homology between the recombinant vector and baculovirus DNA in insect cells is obtained. It can be produced by causing recombination. Examples of the baculovirus include, for example, Autograph California Nuclear Polyhedrosis Virus (Autograp
ha californica nuclear polyhedorosis virus). This virus has the American Type Culture Collection (ATCC) VR-1344 or V
Since it is registered as R-1345, it can be purchased there. Insect cells are not particularly limited as long as they can introduce the recombinant vector of the present invention and baculovirus DNA.
f9 cells, Sf21 cells, Tn5 cells, etc., and these insect cells can be purchased from Pharmingen. In addition, Sf9 cells are ATCC CRL1711.
Since it is registered as, you can also purchase from there.

The recombinant vector and the baculovirus DNA are introduced into the insect cells by mixing and culturing the recombinant vector and the baculovirus DNA with the suspension of the insect cells. By introducing the recombinant vector and baculovirus DNA into insect cells,
In insect cells, double homologous recombination occurs between the recombinant vector and the baculovirus DNA to form a recombinant baculovirus. Since the formed recombinant baculovirus is also released outside the cells, the recombinant baculovirus can be obtained from the culture supernatant.

Here, the method for producing the recombinant baculovirus of the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing an example of a process for producing a recombinant baculovirus of the present invention using a recombinant vector and baculovirus DNA. The recombinant vector comprises (a) DNA encoding a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein and (b) a promoter sequence, and DN is located upstream of the DNA of (a).
A, and (c) DNA capable of causing homologous recombination with baculovirus DNA
A downstream and upstream of the DNA of (b). Of the DNA of (c), the one located upstream of the DNA of (b) is (c1), and the one located downstream of the DNA of (a) is (c2). The nucleotide sequence of (c1) is shown in FIG.
DNA of the terminal portion of the baculovirus DNA (c)
The base sequence of (c2) has a high homology with the DNA (c2 ') at the other end of the baculovirus DNA of FIG.

Recombinant vector and baculovirus DN
When A is introduced into insect cells,
Homologous recombination occurs between (c1) and (c1 ') and between (c2) and (c2'), respectively, thereby forming a recombinant baculovirus. In the recombinant baculovirus, (c1 ") and (c2") are between (c1) and (c1 ') and (c2) and (c
DNA having a nucleotide sequence resulting from homologous recombination between 2 ′).

Next, a method for producing a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein will be described. The histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein is produced by infecting insect cells with a recombinant baculovirus, culturing the insect cells, and obtaining a cell lysate or cell lysate of the cultured insect cells. be able to. The above-mentioned cells can be used as insect cells. By mixing this insect cell with the recombinant baculovirus, it is possible to infect the insect cell with the recombinant baculovirus.

Insect cell culture is performed, for example, using EX-cell 400
Medium (JR Biosciences)
(JRH Biosciences), using a suitable medium such as TC100 medium containing 10% fetal bovine serum (Gibco BS), and keeping it at an appropriate temperature. As a method of disrupting cultured insect cells, for example, a method of collecting insect cells by centrifugation, suspending the suspension in a buffer solution to prepare a suspension, and applying a physical impact to the suspension is employed. can do. As the buffer, for example, a TE buffer, a phosphate buffer, PBS, or the like can be used. As a method of applying a physical impact to the suspension, for example, a method of irradiating the suspension with ultrasonic waves can be used.
As a method for lysing cultured insect cells, for example, SDS or Triton X100 (Triton
X100) can also be adopted. After crushing or lysing insect cells,
Centrifuge to remove cell debris and obtain supernatant.

As a method for purifying the fusion protein, for example, removal of nucleic acid by adding streptomycin sulfate and acquisition of protein by adding ammonium sulfate are performed on the supernatant obtained by disrupting or lysing insect cells and then centrifuging. Can be used. As a step of removing streptomycin sulfate by adding streptomycin sulfate, for example, adding streptomycin sulfate to the supernatant, stirring for a while, and centrifuging to remove the nucleic acid as a precipitate and obtain the supernatant Operations that can be used. As a step of adding ammonium sulfate to obtain a protein, for example, an operation of adding ammonium sulfate to the supernatant after removing the nucleic acid as a precipitate, stirring, and centrifuging can be used. Normal,
Although a precipitate is obtained, the target fusion protein may be contained in the supernatant. Therefore, it is preferable to sample and confirm the presence or absence of the target fusion protein.

Subsequently, a step of obtaining a fraction containing the desired fusion protein is performed. In this step, for example, the precipitate is dissolved in a small amount of a buffer, or the supernatant is fractionated by liquid chromatography, and the fraction containing the fusion protein of interest is identified and obtained. A method can be used. Examples of a method for identifying the fraction containing the fusion protein of interest include a Western blotting method using a Chlamydia pneumoniae-specific monoclonal antibody (described below), the serum of a Chlamydia pneumoniae-infected patient, and the like. Specific methods for removing cell debris, removing DNA to which streptomycin sulfate is added, obtaining protein to which ammonium sulfate is added, and Western blotting are described in the document "Molecular Cloning". On the other hand, a method of obtaining a fraction containing the target fusion protein by utilizing the affinity of the histidine tag can also be used. This method includes, for example, crushing or dissolving the insect cells and further centrifuging the supernatant, etc., and flowing the mixture through a nickel affinity column, adsorbing the fusion protein to the column, and then using imidazole to fuse the fusion protein. Can be used. The purified fusion protein can be used as it is as a Chlamydia pneumoniae antigen,
It is preferable to remove the histidine tag portion using an appropriate proteolytic enzyme.

The fusion protein of the present invention comprises Chlamydia
Since it has antigenicity as a Pneumoniae antigen, it can be used as a reagent for measuring an anti-Chlamydia pneumoniae antibody or an active ingredient of a Chlamydia pneumoniae infection diagnostic agent. As a method for measuring the anti-Chlamydia pneumoniae antibody, for example, an immunoassay using various labels, a latex agglutination method using latex carrier particles, an immunoturbidimetric method and the like can be used. As a method for measuring an anti-Chlamydia pneumoniae antibody using an immunoassay utilizing various labels using the fusion protein, for example, the fusion protein may be physically or chemically used as a carrier such as a polystyrene microtiter plate. To prepare an immobilized antigen, and contact the immobilized antigen with a sample sample and keep it warm for a certain period of time, so that if an anti-Chlamydia pneumoniae antibody is present in the sample sample, the antibody is An antigen-antibody complex is formed on the carrier by binding to the antigen, washed once if necessary, and then brought into contact with a labeled antibody against the antibody in the sample sample (optionally, the sample sample and the labeled antibody can be simultaneously contacted. Good) method can be used. As the label, for example, an enzyme label such as peroxidase can be used.

DNA encoding the fusion protein In the present invention, the DNA encoding the fusion protein refers to the amino acid sequence of the fusion protein of the present invention as a triplet code table (1 to 6 nucleotide sequences for each amino acid). Has been assigned) and the amino acid is replaced with the nucleotide sequence according to the DNA
It is a DNA selected from the group. The DNA encoding the histidine tag portion in the fusion protein is a DNA containing a nucleotide sequence having six or more consecutive histidine-encoding nucleotide sequences, and includes those having a genetic code for translation initiation. The DNA encoding the Chlamydia pneumoniae antigen polypeptide is a DNA encoding the polypeptide A. Examples of the polypeptide A include those described in the section on the Chlamydia pneumoniae antigen polypeptide.

[0039] DN encoding the fusion protein of the present invention
Specific examples of A include, for example, a DNA having the base sequence of SEQ ID NO: 3. In the nucleotide sequence represented by SEQ ID NO: 3, the sequence consisting of the 1st to 42nd nucleotides is a nucleotide sequence encoding a histidine tag portion (a peptide containing 6 consecutive histidines) containing the genetic code for translation initiation. Wherein the sequence consisting of the 43rd to 123rd bases is a base sequence encoding the intervening amino acid sequence portion, and the sequence consisting of the 124th to 1587th bases is a natural Chlamydia pneumoniae 53KDa antigen polypeptide This is a nucleotide sequence encoding the entire amino acid sequence. In addition, a nucleotide sequence portion encoding glycine which is the 159th amino acid of this antigen polypeptide (a sequence consisting of nucleotides 598 to 600 in the nucleotide sequence shown in SEQ ID NO: 3)
Is not the “GGT” already reported by Matsumoto et al., But is mutated to “GGC”.

The DNA encoding the fusion protein can be prepared by a gene recombination method. As a gene recombination method, for example, as described above, a DNA encoding a Chlamydia pneumoniae antigen polypeptide is converted into a DNA encoding a histidine tag, a vector containing the DNA of (b) and the DNA of (c). There is a method of inserting. As the vector containing the DNA encoding the histidine tag, the above-mentioned commercially available products can be used.

Next, a method for cloning a DNA encoding a Chlamydia pneumoniae antigen polypeptide from Chlamydia pneumoniae will be described in detail. Culturing of Chlamydia pneumoniae Cultured HL cells and the like are infected with Chlamydia pneumoniae in advance and cultured to sufficiently form Chlamydia inclusion bodies, and then the cells are transformed into an SPG solution (75.0 g of sucrose, monopotassium phosphate). 0.52 g, dipotassium phosphate 1.22 g
And 0.72 g of glutamic acid dissolved in 1 liter of water, pH 7.4 to 7.6), and the animal cells were crushed or dissolved and centrifuged to separate the supernatant (a suspension of Chlamydia pneumoniae). get. As Chlamydia pneumoniae, for example, Chlamydia pneumoniae YK
41 strains (Kanemoto et al .: Microbiological Immunology, Vol. 37, pp. 495-498, 1993 (Y. Kanamoto et al., Mi
crobiol. Immunol., Vol. 37, p. 495-498, 1993)) and ATCC in the American Type Culture Collection.
The strain deposited as VR1310 can be used.

After removing the culture supernatant from the cultured HL cells and the like, the above-mentioned suspension of Chlamydia pneumoniae is added, and the mixture is centrifuged to promote the adsorption of Chlamydia pneumoniae to HL cells and the like. Chlamydia
Remove the Pneumoniae suspension and Dulbecco M containing 1 μg / ml cycloheximide and 10% (v / v) fetal calf serum.
An appropriate medium such as an EM medium is added, and the cells are cultured for 3 to 4 days. Thereby, Chlamydia pneumoniae-infected HL cells in which Chlamydia pneumoniae has proliferated in the cells are obtained.

Purification of basic bodies of Chlamydia pneumoniae Chlamydia pneumoniae-infected HL cells are disrupted, centrifuged, and the supernatant is recovered. This supernatant is added to a continuous density gradient solution using urografin (manufactured by Schering) and centrifuged. In a preliminary experiment, it was confirmed by an electron microscope that the basic body of Chlamydia pneumoniae was contained in a yellowish white band, so this band was collected.

Genomic DNA of Chlamydia pneumoniae
Preparation of the basic body of Chlamydia pneumoniae in lysis buffer (500 mM potassium chloride, 25 mM magnesium chloride,
10 mM Tris-HCl buffer (pH 8.5) containing 0.45% Triton X100 and 0.45% Nonidet P40, 5 μl of Pr
An oteinaseK aqueous solution (20 mg / ml) is added, and the mixture is kept at 56 ° C. for 1 hour to dissolve the basic bodies, and heated at 95 ° C. for 10 minutes to inactivate ProteinaseK. Then, 0.1 M Tris-HCl buffer (pH 8.0) saturated phenol is added, stirred, centrifuged, and the aqueous layer is collected. Further, the cells are subjected to an RNase (RNase) treatment, a phenol / chloroform / isoamyl alcohol treatment and an ethanol precipitation treatment to obtain genomic DNA of Chlamydia pneumoniae.

Cloning of DNA Encoding Chlamydia pneumoniae Antigen Polypeptide DNA Encoding Native Chlamydia pneumoniae Antigen Polypeptide
Has the nucleotide sequence of International Patent Publication No. WO 96/09320.
The base sequence (two types) serving as a primer is determined based on the base sequence, and a primer DNA having this base sequence is chemically synthesized using a commercially available DNA synthesizer. Examples of the base sequence of such a primer include the base sequences shown in SEQ ID NOs: 4 and 5.

Next, the above-mentioned primer DNA, Taq polymerase and deoxynucleotides are added to the genomic DNA of Chlamydia pneumoniae, and the steps of heating, cooling and keeping the temperature are repeated to obtain the Chlamydia pneumoniae antigen polypeptide. DNA encoding
Is amplified. As tack polymerase, for example,
Amplitak DNA Polymerase (AmpliTaq DNA)
Polymerase) (trade name of Takara Shuzo Co., Ltd.). This DN
A general method of the A amplification method is known as a PCR method, and is described in detail in the document "Molecular Cloning". After the obtained DNA is subjected to blunt-end treatment or appropriate restriction enzyme treatment, the DNA is inserted into an appropriate plasmid or phage vector or the like, whereby the DNA encoding the Chlamydia pneumoniae antigen polypeptide is obtained. Is cloned. In the PCR method, since the DNA to be amplified may have a mutation, it is preferable to confirm the nucleotide sequence of the cloned DNA.

Production of Anti-Chlamydia pneumoniae Antibody Using Fusion Protein as Antigen As a method for producing anti-Chlamydia pneumoniae antibody, for example, a mouse is immunized with the fusion protein of the present invention as an antigen, and its spleen cells are transformed into myeloma cells. A hybridoma that recognizes the Chlamydia pneumoniae 53 KDa antigenic polypeptide is selected from the hybridomas, cultured, and cultured to obtain an anti-Chlamydia pneumoniae antibody from the culture supernatant. Can be used. Examples of myeloma cell lines include P
3X63Ag8.653 (ATCC CRL-158
0) and P3 / NSI / 1-Ag4-1 (ATCC TI
B-18) can be used. Except for using the fusion protein of the present invention as an antigen, an anti-Chlamydia pneumoniae antibody is produced according to a known general method of immunizing a mouse to obtain an antibody.

[0048]

The present invention will be described below in detail with reference to examples, but the present invention is not limited thereto. Example 1 Preparation of DNA Encoding Chlamydia pneumoniae-Specific 53K Antigen Polypeptide (A) Culture of Host Cells (HL Cells) HL cells grown in advance on the entire bottom surface of a plastic culture flask (75 cm ² ) were phosphorylated. Washed with 5 ml of a magnesium-free (-) solution containing an acid-buffered saline (hereinafter referred to as PBS), and PB containing 0.1% (w / v) trypsin.
After adding 5 ml of S to the entire cell surface, discarding the solution, keeping the mixture at 37 ° C. for 10 minutes, adding 5 ml of Dulbecco's MEM medium containing 10% (v / v) fetal bovine serum, and pipetting HL cells. Was removed to prepare a cell suspension. 4 ml of a mixture of 8 ml of this cell suspension and 292 ml of Dulbecco's MEM medium containing 10% fetal bovine serum was added to each well of a 6-well plastic culture vessel, and 5% (v / v
v) The cells were cultured in a carbon dioxide atmosphere.

(B) Chlamydia pneumoniae YK-4
1. Culture of Chlamydia Chlamydia pneumoniae YK-4
1 share (Kanemoto et al .: Microbiol. Immunol., Vol. 37, P. 495-498,
1993). Chlamydia in advance to the obtained HL cells
After infecting and culturing P. pneumoniae strain YK-41 to sufficiently form Chlamydia inclusion bodies, the cells were transformed with SPG.
The suspension is placed in a polypropylene centrifuge tube, irradiated with ultrasonic waves at intervals of 1 second for 30 times, and the centrifuge tube is centrifuged at 1,500 × g for 3 minutes to obtain a supernatant, which is used for Chlamydia pneumoniae. A suspension was used. The culture supernatant of the HL cells grown in a 6-well plastic culture vessel (on the bottom) was removed with a pipette, and 2 ml of Chlamydia pneumoniae suspension was added to each well to give 2,000.
Centrifugal adsorption was performed at rpm for 1 hour. After centrifugal adsorption, the suspension of Chlamydia pneumoniae was removed, and Dulbecco M containing 1 μg / ml cycloheximide and 10% (v / v) fetal calf serum.
4 ml of EM medium was added per well, and the cells were cultured at 36 ° C. for 3 days in a 5% (v / v) carbon dioxide atmosphere. After the culture, the cells were peeled off with a piece of sterilized silicon, and the cells were collected. This is centrifuged at 8,000 rpm for 30 minutes to separate the precipitate by SPG.
And stored at -70 ° C.

(C) Chlamydia pneumoniae YK-4
Purification of basic body 1 Chlamydia pneumoniae Y stored at -70 ° C
The K-41 infected frozen HL cell suspension was thawed and homogenized with a Teflon homogenizer. 1 at 2,500 rpm
After centrifugation for 0 minutes, the supernatant was collected. Precipitation is again SPG
, And the same operation was performed to recover the supernatant. The same operation was further performed twice, and the obtained supernatants were collected and combined.

Separately, a 0.03 M Tris-HCl buffer (pH 7.4) containing 50% (w / v) sucrose was placed in a centrifuge tube, and then
A mixed solution of 3 volumes of urografin 76% (manufactured by Schering) and 7 volumes of 0.03 M Tris-HCl buffer (pH 7.4) was overlaid, and the previously collected supernatant was carefully overlaid thereon. Centrifuged at 2,000 rpm for 1 hour. 50%
(W / v) 0.03 M Tris-HCl buffer containing sucrose (pH
7.4) Collect the layer and the precipitate, and add the same volume of S
The PG solution was added and centrifuged at 10,000 rpm for 30 minutes. The supernatant was discarded, and the precipitate was suspended in the SPG solution. In a centrifuge tube, a continuous density gradient solution of 35% to 50% (volume ratio of the former to the total amount) of 76% urografin (manufactured by Schering) and 0.03M Tris-HCl buffer (pH 7.4) is prepared. Then, the suspension was layered on this, and 8000 rp
Centrifugation at 1 m for 1 hour. Chlamydia pneumonia Y
A yellowish cloudy band corresponding to the basic body of K41 was collected, and this was diluted 2-fold with SPG solution, and collected.
Centrifuged at 00 rpm for 30 minutes. SP obtained
It was suspended in solution G, and the protein concentration was measured (using a protein measurement kit from 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 one strain 200 μl (1.3 × 10 ⁹ / ml) of a suspension of the basic body of the purified Chlamydia pneumoniae strain YK-41 was added to 4
The mixture was centrifuged at 12,000 rpm for 10 minutes at 1 ° C. to obtain Chlamydia pneumoniae basic bodies as a precipitate. 200 μl of lysis buffer (500 mM potassium chloride, 25 mM magnesium chloride, 0.45% Triton X1
10 mM Tris containing 00 and 0.45% Nonidet P40
Hydrochloric acid buffer (pH 8.5) and 5 μl of an aqueous solution of Proteinase K (20 mg / ml) were added, and the mixture was incubated at 56 ° C. for 1 hour to dissolve the basic bodies, and then heated at 95 ° C. for 10 minutes.
K was deactivated. Then, 350 μl of 0.1 M Tris-HCl buffer (pH 8.0) saturated phenol was added, and the mixture was mixed well by a vortex mixer.
For 5 minutes, and an aqueous layer was collected (DNA extraction). This extraction operation was repeated once. 10mg / ml
Of RNase solution was added and incubated at 37 ° C. for 2 hours to degrade RNA. 300 μl of a 25: 24: 1 (volume ratio) mixture of 0.1 M Tris-HCl buffer (pH 8.0) saturated phenol, chloroform and isoamyl alcohol (volume ratio) was added, and mixed well with a vortex mixer. 4 ° C, 12,000 rp
After centrifugation at 5 m for 5 minutes, the aqueous layer was collected. This operation was repeated five times in total.

A 1/10 volume of a 10 M ammonium acetate aqueous solution and 2 volumes of ethanol were added to the obtained solution, and the mixture was left for 5 minutes to precipitate DNA.
Centrifuged at rpm for 5 minutes. For precipitation, add 600 μl of a 70% aqueous ethanol solution, mix, and mix at 4 ° C., 12,000 rp.
Washing by centrifugation at m for 5 minutes was repeated twice. Leave the centrifuge tube lid open for 15 minutes to dry the precipitate,
200 μl of 10 mM Tris-HCl buffer (pH 8.0)
1 was added to dissolve and stored at -20 ° C.

(E) Cloning of DNA Encoding Chlamydia pneumoniae Antigen Polypeptide The nucleotide sequence of the DNA encoding the natural Chlamydia pneumoniae 53 kDa antigen polypeptide and the nucleotide sequences before and after it are shown in SEQ ID NO: 6. So, Chlamydia
The primer DNA on the amino-terminal side was designed so as to include the genetic code for translation initiation of DNA encoding the Pneumoniae 53 kDa antigen polypeptide. The base sequence of this primer DNA is shown in SEQ ID NO: 4. In the base sequence represented by SEQ ID NO: 4, the sequence consisting of the first to seventh bases is for adding a DNA fragment that is cleaved by the restriction enzyme BamHI, and the bases at the eighth to 28th bases are added. The sequence consisting of natural chlamydia
It is a sequence that hybridizes to the vicinity of the genetic code at the start of translation of the DNA encoding the Pneumoniae 53 kDa antigen polypeptide and functions as a primer for a PCR reaction. This primer DNA was chemically synthesized using a DNA synthesizer. This is designated as primer DNA1.

On the other hand, the primer DN on the carboxyl terminal side is included so that the genetic code of the end of translation in the gene is included.
A was designed. The nucleotide sequence of this primer DNA is shown in SEQ ID NO: 5. In the base sequence represented by SEQ ID NO: 5, the sequence consisting of the first to eighth bases is for adding a DNA fragment that is cleaved by the restriction enzyme NheI, and the ninth to 28th bases are added. Is a sequence that hybridizes to the vicinity of the translation-completed genetic code of the DNA encoding the natural Chlamydia pneumoniae 53 kDa antigen polypeptide and functions as a primer for a PCR reaction. This primer DNA is
It was chemically synthesized with a synthesizer. This is designated as primer DNA2. The obtained primer DNA1 and primer DNA
2 was an aqueous solution.

Then, 35.7 μl of purified water, a 10-fold concentration PCR buffer (100 mM containing 500 mM potassium chloride)
M Tris-HCl buffer pH 8.3) 5μl, 2.5mM dNTP
Aqueous solution 4μl, 25mM magnesium chloride aqueous solution 3
μl, 100μM primer DNA1 aqueous solution 0.5μ
l, 100 μM primer DNA2 aqueous solution 0.5 μl,
1 μl of Taq DNA polymerase (5 U / μl), a genomic DNA solution of the Chlamydia pneumoniae strain YK-41
After mixing 1 μl and overlaying several drops of mineral oil, 9
Heating at 4 ° C. for 4 minutes, followed by heating at 94 ° C. for 30 seconds,
A cycle consisting of cooling at 50 ° C. for 30 seconds and incubation at 73 ° C. for 1 minute was repeated 40 times to obtain D encoding the native Chlamydia pneumoniae 53 kDa antigen polypeptide.
NA was amplified. After that, except for mineral oil, 0.
10 μl of a 30% glycerin aqueous solution containing 1% bromophenol blue was added, and 1% agarose gel electrophoresis was performed. After the electrophoresis, the gel was immersed in a 0.5 μg / ml aqueous ethidium bromide solution for 30 minutes, observed under ultraviolet irradiation, cut out of a 1.5 kbp band, and then subjected to a QIAQ Quick Gel Extraction Kit. (QIA
The DNA was purified using a quick gel extraction kit (QIAGEN), and the DNA was added to 100 μl of TE buffer (10 mM containing 1 mM ethylenediaminetetraacetic acid).
M Tris-HCl buffer (pH 8.0). This DN
The above-mentioned PCI (100 μl) was added to the solution A, mixed well, and then centrifuged at 12,000 rpm for 2 minutes to collect an aqueous layer (phenol extraction). To this, 10 μl of a 3M sodium acetate aqueous solution and 220 μl of ethanol were added, mixed, and kept on ice for 30 minutes.
After centrifugation at 15 m for 15 minutes, the DNA was obtained as a precipitate. After adding 500 μl of a 70% ethanol aqueous solution to the precipitate, the mixture was centrifuged at 12,000 rpm for 5 minutes at 4 ° C., and the supernatant was removed. (Ethanol precipitation). This PCR
Chlamydia pneumoniae 53 amplified by the reaction
The DNA encoding the kDa antigen polypeptide has a restriction enzyme Ba at the 5 'end of the open reading frame region.
A nucleotide sequence that is cleaved by mHI and a nucleotide sequence that is cleaved by the restriction enzyme NheI are added to the 3 ′ end.

Example 2 Preparation of Recombinant Vector Containing DNA Encoding Chlamydia pneumoniae Antigen Polypeptide 45 μl of purified water was added to the obtained DNA encoding the Chlamydia pneumoniae 53 kDa antigen polypeptide, and the DNA was dissolved. 5 μl of double concentration K buffer for restriction enzyme (200 mM Tris-HCl buffer containing 100 mM magnesium chloride, 10 mM dithiothreitol, 1000 mM potassium chloride, pH 8.5), 2 μm restriction enzyme BamHI
l (8 U / μl) was added and the mixture was kept at 30 ° C. for 2 hours. Thereafter, the same phenol extraction and ethanol precipitation as described above were performed. To the obtained DNA, 45 μl of purified water was added and dissolved, and a 10-fold concentration of M buffer for restriction enzyme (100
mM magnesium chloride, 10 mM dithiothreitol, 5
5 μl of 100 mM Tris-HCl buffer containing 00 mM sodium chloride (pH 7.5), 0.5 μl of 1% Triton X-100 aqueous solution, and 2 μl (8 U / μl) of restriction enzyme NheI were added, and the mixture was kept at 37 ° C. for 2 hours. Thereafter, agarose gel electrophoresis and DNA purification were performed in the same manner as above, and phenol extraction and ethanol precipitation were performed in the same manner as described above.
The obtained DNA was dissolved in 50 μl of TE buffer to obtain a DNA encoding a Chlamydia pneumoniae 53 kDa antigen polypeptide treated with a restriction enzyme.

On the other hand, 4 μl of plasmid pVL1393 solution (Pharmingen) (0.5 μg / μl) was added to purified water 41
μl, 10 μl concentration of K buffer for restriction enzyme 5 μl, restriction enzyme
2 μl (8 U / μl) of BamHI was added, and the mixture was kept at 30 ° C. for 2 hours. Thereafter, phenol extraction and ethanol precipitation were performed in the same manner as described above. To the obtained DNA, 40 μl of purified water was added to dissolve the DNA, and 5 μl of a 10-fold concentration M buffer solution for restriction enzyme, 5 μl of 1% bovine serum albumin aqueous solution, and 2 μl (8 U / μl) of restriction enzyme XbaI were added. Incubated at 2 ° C. for 2 hours. Thereafter, agarose gel electrophoresis and DNA purification were carried out in the same manner as described above, and phenol extraction and ethanol precipitation were carried out in the same manner as described above.
The plasmid pVL1393 dissolved in μl of TE buffer and treated with a restriction enzyme was obtained.

D coding for the Chlamydia pneumoniae 53 kDa antigen polypeptide treated with the restriction enzyme
5 μl of the NA solution and 1 μl of the plasmid pVL1393 solution treated with the restriction enzyme were mixed, and 10 μl of purified water was added thereto.
l, 10-fold concentration ligation buffer (66 mM magnesium chloride, 100 mM dithiothreitol, 1 mM AT
660 mM Tris-HCl buffer containing P, pH 7.6) 2
100 μl of T4 DNA ligase was added thereto, and the mixture was incubated at 16 ° C. for 4 hours to ligate DNA. This reaction solution 3
Add 100 μl of E. coli HB101 competent cells (Takara Shuzo Co., Ltd.) and add 3 μl at 42 ° C. for 30 minutes in ice.
After incubating for 2 seconds in ice and for 2 minutes in ice, 1 ml of LB medium (1% tryptone, 0.5%
% Yeast extract, 1% aqueous sodium chloride solution)
Shaking culture was performed at 37 ° C. for 1 hour. This E. coli is
20 ml of LB medium containing μg / ml ampicillin and 1.5% agar was applied to three solidified 90 mmφ dishes, and cultured overnight at 37 ° C. to obtain colonies of Escherichia coli into which the recombinant plasmid had been introduced. Was formed on a Petri dish.
Some of the obtained colonies of Escherichia coli were picked up, inoculated in 2 ml of LB medium containing 100 μg / ml of ampicillin, and cultured with shaking at 37 ° C. overnight. From this Escherichia coli, plasmid DN
A was extracted and dissolved in 50 μl of TE buffer. To 1 μl of each plasmid DNA solution, 8 μl of purified water, 1 μl of 10-fold concentration M buffer for restriction enzyme, 0.2 μl of restriction enzyme PvuII
(12 U / μl) and incubated at 37 ° C. for 1 hour.
The cleavage pattern was observed by performing 0.7% agarose gel electrophoresis, and 3705, 3104, 2364, 1399,
The presence or absence of the 431 and 96 bp bands was examined, and the plasmid in which these bands were observed was selected as a plasmid into which the DNA encoding the Chlamydia pneumoniae 53 kDa antigen polypeptide had been correctly integrated, and this plasmid was designated as p.
It was named VCPN53 plasmid.

The DN encoding the Chlamydia pneumoniae 53 kDa antigen polypeptide in the resulting plasmid
The nucleotide sequence of A was confirmed as follows. First, DNA
Using a synthesizer, chemically synthesizing a DNA having the nucleotide sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 8,
Primer DNA3 and Primer DNA4, respectively
And each was dissolved in water. Then, 0.5 μl of the obtained plasmid solution, 36.25 μl of purified water, 5 μl of a 10-fold concentration buffer for PCR, 4 μl of a 2.5 mM dNTP aqueous solution,
3 μl of 25 mM magnesium chloride aqueous solution, 0.25 μl of 100 μM aqueous primer DNA3 solution, 0.25 μl of 100 μM aqueous primer-DNA4 solution and 0.25 μl of Taq polymerase (5 units / μl) were mixed, and several drops of mineral oil were layered. Heating at ℃ for 30 seconds,
A cycle consisting of cooling at 55 ° C. for 30 seconds and incubation at 72 ° C. for 1 minute was repeated 30 times to amplify the DNA encoding the Chlamydia pneumoniae 53 kDa antigen polypeptide incorporated into the plasmid. Amplified DNA
Base sequence of ABI PRISM Dye Terminator Cycle Sequencing Kit (ABI PRISM Dye Terminator Cycle Sequencing Kit)
(Perkin Elmer) and A
The test was carried out using an ABI PRISM 310 DNA Sequencer (Perkin Elmer).

As a result, the amplified DNA was represented by SEQ ID NO: 9.
It is confirmed that the translation completion genetic code exists immediately after this nucleotide sequence, and the amino acid sequence of the peptide obtained by translating these nucleotide sequences is represented by SEQ ID NO: 1. Was done. The entire base sequence of the obtained pVCPN53 plasmid is shown as SEQ ID NO: 10. 4029 from the seventh position in the base sequence represented by SEQ ID NO: 10
Sequence consisting of bases up to the 5th and 846
The sequences consisting of the bases up to the 7th base are all DNA sequences derived from the baculovirus, and the sequences consisting of the bases from the 4030th base to the 4092th base are the sequences of the polyhedrin protein gene constituting the inclusion body of the baculovirus. The promoter sequence, from position 4135 to 5601
The sequence consisting of bases up to the second is the DNA encoding the Chlamydia pneumoniae 53 kDa antigen polypeptide. The sequence consisting of the bases 5635 to 8467 is a sequence present on the 3 ′ side of the sequence consisting of the bases 7 to 4029 in the genomic DNA of baculovirus. In addition, this plasmid contains the ampicillin resistance gene and DNA sequences necessary for replication in E. coli. Note that pVCPN5
The Escherichia coli HB101 strain containing the three plasmids has the accession number F
It has been deposited as ERM P-16037 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

Example 3 Histidine tag-Chlamydia
Preparation of Recombinant Vector Containing DNA Encoding Pneumonier Antigen Polypeptide Fusion Protein 100 ng / μl of the obtained pVCPN53 plasmid aqueous solution
Into each were 43 μl of purified water, 5 μl of a 10-fold concentration of an H buffer for a restriction enzyme (500 mM Tris-HCl buffer containing 100 mM magnesium chloride, 10 mM dithiothreitol, and 1 M sodium chloride, pH 7.5) and a restriction enzyme EcoRI (20 U / μ
l) 1 μl was added, and the mixture was kept at 37 ° C. for 1 hour. Subsequently, phenol extraction and ethanol precipitation were performed in the same manner as described above, and the obtained DNA was dissolved in 45 μl of purified water, and 5 μl of a 10-fold concentration K buffer for restriction enzyme and BamHI (8 U / μl) were used.
l) 1 μl was added, and the mixture was kept at 37 ° C. for 1 hour. Then 1
% Agarose gel electrophoresis was performed, a 1.5 kbp band was cut out, purified in the same manner as above, and ethanol precipitated. The obtained DNA was dissolved in 8 μl of purified water, and DN
Add 1 μl of a 10-fold concentration buffer in A Blunting Kit (DNA Blunting Kit, manufactured by Takara Shuzo Co., Ltd.), and add
And then kept at 37 ° C. for 10 minutes. Further, 1 μl of T4 DNA polymerase in this kit was added, and 3
After keeping the temperature at 7 ° C. for 5 minutes, 40 μl of purified water was added, and the reaction solution was vigorously stirred to inactivate T4 DNA polymerase. Thereafter, phenol extraction and ethanol precipitation were performed in the same manner as described above, and the obtained DNA was further subjected to DN
The DNA solution was dissolved in 5 μl of A dilution buffer to obtain a blunt-ended DNA solution encoding a Chlamydia pneumoniae 53 kDa antigen polypeptide.

On the other hand, 1 μg / μl of plasmid pAcHLT-C
(Pharmingen) 2 μl, purified water 46 μl, 10 μl
5 μl of H buffer solution for double concentration of restriction enzyme and restriction enzyme XhoI
(12 U / μl) 1 μl was added and the mixture was kept at 37 ° C. for 1 hour.
Subsequently, phenol extraction and ethanol precipitation were performed in the same manner as described above. The obtained DNA was dissolved in 8 μl of purified water, 1 μl of a 10-fold concentration buffer in the DNA branding kit was added, and the mixture was added at 70 ° C. for 5 minutes. After keeping the temperature,
It was kept warm for 0 minutes. Further, 1 μl of T4 DNA polymerase in this kit was added, and the mixture was incubated at 37 ° C. for 5 minutes.
μl of purified water was added and the reaction was stirred vigorously to inactivate T4 DNA polymerase. Subsequently, phenol extraction and ethanol precipitation were performed in the same manner as described above, and the obtained DNA was
Dissolve in 3 μl of purified water and add 1M Tris-HCl buffer pH
8.0 15 μl and 2 μl of alkaline phosphatase (0.6 U / μl) derived from Escherichia coli were added, and the mixture was kept at 65 ° C. for 30 minutes. And phenol extraction (2 times) as above,
Ethanol precipitation was performed, and the obtained DNA was dissolved in 10 μl of a 10-fold concentration buffer in the DNA branding kit to obtain a blunt-ended plasmid solution.

D coding for the resulting blunt-ended Chlamydia pneumoniae 53 kDa antigen polypeptide
2 μl of the NA solution and 1 μl of the blunt-ended plasmid solution were mixed, and the DNA ligation kit version 1 (Takara Shuzo Co., Ltd.)
After adding 12 μl of solution A and 3 μl of solution B, and keeping the mixture at 16 ° C. for 1 hour, the DNA encoding the blunt-ended Chlamydia pneumoniae 53 kDa antigen polypeptide is added.
And the blunt-ended plasmid were ligated. Then 3 μl
Was added to 100 μl of E. coli HB101 competent cells (Takara Shuzo Co., Ltd.), and the mixture was incubated for 30 minutes in ice, at 30 ° C. for 30 seconds, and in ice for 2 minutes.
It was added to 1 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% aqueous sodium chloride solution) kept at 37 ° C, and cultured with shaking at 37 ° C for 1 hour. This Escherichia coli was mixed with 20 ml of an LB medium containing 100 μg / ml ampicillin and 1.5% agar and solidified by 90 mmφ.
Was applied to three Petri dishes, and cultured overnight at 37 ° C. to form colonies of Escherichia coli into which the recombinant plasmid had been introduced. Some of the obtained colonies of Escherichia coli were picked up, inoculated into 2 ml of LB medium containing 100 μg / ml of ampicillin, and cultured with shaking at 37 ° C. overnight. Plasmid DNA was extracted from the Escherichia coli by the alkaline lysis method and dissolved in 50 μl of TE buffer. To 1 μl of each plasmid solution, 8 μl of purified water, 1
1 μl of M buffer for 0-fold concentration of restriction enzyme, PvuII restriction enzyme
After adding 0.2 μl (12 U / μl), keeping the mixture at 37 ° C. for 1 hour, 0.7% agarose gel electrophoresis was performed to observe the cleavage pattern, and 3388, 2840, 2693, 57
The presence or absence of bands of 8 and 96 bp was examined, and a plasmid in which these bands were observed was selected as a plasmid in which DNA encoding the histidine tag-Chlamydia pneumoniae 53 kDa antigen polypeptide fusion protein was correctly integrated, and this was selected as pHLTCP53. It was named plasmid.

The entire base sequence of the resulting pHLTCPN53 plasmid is shown as SEQ ID NO: 11. Of the base sequence represented by SEQ ID NO: 11, the sequence consisting of the first to 2128th bases and the sequence consisting of the 3868th to 6480th bases are both sequences of DNA derived from baculovirus, and 2129 The sequence consisting of the 2nd to 2191st bases is the promoter sequence of the polyhedrin protein gene constituting the inclusion body of baculovirus, and the sequence consisting of the 2206th to 3795th bases is the D sequence encoding the fusion protein.
NA. The sequence consisting of the bases 3868 to 6480 is a sequence present on the 3 ′ side of the sequence consisting of the bases 1 to 2128 in the genomic DNA of baculovirus. Besides that,
This plasmid also contains the ampicillin resistance gene and the DNA sequences necessary for replication in E. coli. In addition, pH
The Escherichia coli HB101 strain containing the LTCPN53 plasmid has been deposited with the National Institute of Advanced Industrial Science and Technology as a deposit number FERM P-16036.

Example 4 Production of Recombinant Baculovirus and Preparation of Histidine Tag-Chlamydia pneumoniae Antigen Polypeptide Fusion Protein The pHLTCPN53 plasmid was replaced with baculovirus DN.
When introduced into insect cells together with A, two DNs derived from baculovirus contained in the pHLTCPN53 plasmid
A respectively undergoes homologous recombination with the baculovirus DNA to form a recombinant baculovirus. In addition,
PHLTCPN5 for the formation of recombinant baculovirus
3 The whole plasmid is not essential,
Recombinant baculovirus can also be formed by using a DNA having a sequence consisting of the 1st to 6480th bases in the base sequence represented by SEQ ID NO: 11 instead of the CPN53 plasmid. Therefore, a recombinant baculovirus was formed as follows to produce a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein.

2 μl of an aqueous solution (1 μg / μl) of the obtained pHLTCPN53 plasmid, 100 ng / μl of baculogold linearized baculovirus DNA
0.5 μl of (BaculoGold Linearized Baculovirus DNA) (Pharmingen), 5.5 μl of purified water, and 8 μl of lipofectin (GIBCO BRL) diluted 2-fold with purified water were mixed at room temperature for 15 minutes. After standing for minutes, 700,000 Sf9 cells (A
Add TCC CRL 1711) and add 1.2ml EX-cell
35 mmφ with 400 medium (JRH Biosciences)
Was added to a Petri dish. This petri dish was heated at 26.5 ° C for 2 hours.
After culturing for one day, Sf9 cells were detached from the Petri dish by pipetting to prepare a cell suspension,
It was equally divided and placed in a mmφ petri dish. To each petri dish, 1 ml of TC100 medium (manufactured by Gibco BS) containing 10% fetal bovine serum was added, and the cell suspension was cultured at 26.5 ° C for 3 days. Further, the Sf9 cells were detached from the Petri dish by pipetting to prepare a cell suspension, and the suspension was prepared at 2,000 rpm.
For 10 minutes to separate into a supernatant containing the recombinant baculovirus and a precipitate consisting of Sf9 cells infected with the recombinant baculovirus.

Next, the recombinant baculovirus containing the recombinant baculovirus was placed in a 25 cm 2 cell culture flask to which 1,700,000 Sf9 cells had been adhered in advance and 3 ml of TC100 medium containing 10% fetal bovine serum had been added. Clear solution 0.
3 ml was added, and the cells were cultured at 26.5 ° C. for 3 days. A cell suspension was prepared by detaching the Sf9 cells from the flask by pipetting, centrifuged at 2,000 rpm for 10 minutes, and again a supernatant solution containing the recombinant baculovirus and a precipitate comprising the recombinant baculovirus-infected Sf9 cells. Separated.

A cell suspension was prepared by adding 0.25 ml of PBS to the precipitate, and 5 μl of the cell suspension was added to 5 μl of purified water and sodium lauryl sulfate-polyacrylamide gel containing 10% 2-mercaptoethanol. Sample buffer for electrophoresis (SDS-PAGE) (0.25 M Tris-HCl buffer containing 2% sodium lauryl sulfate, 30% glycerin, 0.01% bromophenol blue (p
H6.8)) 10 μl was added and mixed, followed by 5-20% polyacrylamide gradient gel electrophoresis.

Subsequently, three filter papers moistened with a blotting buffer (0.05% sodium lauryl sulfate, 20% methanol, 25 mM Tris, 192 mM glycine) were placed on the cathode plate of the semi-dry blotting apparatus. After that, a polyacrylamide gradient gel on which the above-mentioned electrophoresis was completed, a nitrocellulose membrane moistened with a blotting buffer, and three filter papers moistened with a blotting buffer were stacked, and the anode plate was placed thereon. , 12 V, and transfer was performed for 1 hour. After the nitrocellulose membrane after transfer was immersed in PBS containing 1% bovine serum albumin overnight, the membrane was washed with a washing solution for blotting (0.5 M sodium chloride, 0.1 M
7. 20 mM Tris-HCl buffer containing 20% Tween20, pH8.
After washing 3 times with 0), Chlamydia pneumoniae 53
SCP53, a monoclonal antibody specific to the kDa antigen polypeptide (Journal of Clinical Microbiology, 132, 583-588 (1994)) (J. Clin. Mi
crobiol., Vol. 132, p. 583-588, 1994)) It was immersed in a washing solution for blotting containing 3 μg / ml and left at room temperature for 2 hours. Further, after the membrane was washed three times with the washing solution for blotting, 0.3 μg / ml of peroxidase-labeled anti-mouse Ig was washed.
G antibody (Kirkegarrd & Perry Laboratories, Inc.)
(Manufactured by Co., Ltd.), and left for 1 hour at room temperature. Wash membrane 3 times with blotting washes, P
After washing twice with BS, the membrane was immersed in a TMB substrate solution (manufactured by Kirkeguard & Perry Laboratories, Inc.) for 5 minutes, washed with purified water, and a color band was observed on the membrane. The results are shown in FIG.

As is apparent from FIG. 2, when Sf9 cells infected with the recombinant baculovirus were used as a sample, a colored band was observed on the electrophoresis lane (lane W), and the position was determined by the chlamydia as the sample. When the cells of the Pseudomonas pneumoniae strain YK-41 were used, the position of the colored band observed on the electrophoresis lane (lane Y) was about 4 kDa higher than the position of the band. From these results, it was found that a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein was produced in Sf9 cells infected with the recombinant baculovirus, and that this fusion protein had antigenicity as a Chlamydia pneumoniae 53KDa antigen polypeptide. It has been shown. In addition,
The fusion protein of the present invention can be purified, for example, as follows.

A 75 cm 2 cell culture flask 5 to which 5,000,000 Sf9 cells had been previously attached and 10 ml of TC100 medium containing 10% FCS had been added.
To each of these, 0.5 ml of the supernatant containing the above-mentioned recombinant baculovirus is added, and cultured at 26.5 ° C. for 3 days.
The Sf9 cells are detached from the flask by pipetting to prepare a cell suspension, and centrifuged at 2,000 rpm for 10 minutes to remove the supernatant. 2 ml of cell lysate (16 μg / ml benzamidine HCl, 10 μg / ml phen
anthroline, 10 μg / ml aprotinin, 10 μg / ml leupe
ptin, 10μg / ml pepstatin A, 1mM phenylmethylsul
fonyl fluoride, 130 mM sodium chloride, 1% Triton X100, 10 mM sodium fluoride, 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM sodium phosphate, add, stir, place on ice for 1 hour to lyse cells I do. Centrifuge at 40,000 g for 30 minutes to remove insolubles. 400 μl of the Ni-NTA agarose gel suspension (Curegen) is centrifuged at 500 g for 3 minutes, and the supernatant is removed. Add 1.2 ml of washing buffer (300 mM sodium chloride, 50 mM sodium phosphate buffer containing 10% glycerin, pH 8.0) and resuspend.
Centrifuge for 1 minute, look at the supernatant, and wash the gel. The above-mentioned cell lysate is added to the gel, and the mixture is kept at 4 ° C. for 1 hour with stirring every 5 minutes, and then centrifuged at 500 g for 3 minutes to remove the supernatant. One ml of a washing buffer containing 20 mM imidazole is added and resuspended, and centrifuged similarly to remove the supernatant and wash the gel. The same washing is performed four more times. One milliliter of an eluate containing 300 mM imidazole (300 mM sodium chloride, 50 mM sodium phosphate buffer pH 6.0 containing 10% glycerin) was added, and the mixture was resuspended. The mixture was allowed to stand at room temperature for 5 minutes. Collect the fresh liquid. Further, the same elution operation is performed with an eluate containing 400 mM imidazole and an eluate containing 500 mM imidazole, and each supernatant is collected.

Example 5 Histidine tag-Chlamydia
Production of Anti-Chlamydia pneumoniae Antibody Using Pneumonie Antigen Polypeptide Fusion Protein as Antigen Anti-Chlamydia pneumoniae antibody is produced using the fusion protein of the present invention as an antigen, for example, as follows. (A) Culture and passage of myeloma cell line Myeloma cell line was P3X63Ag8.653 (ATCC
(CRL-1580) was cultured in RPMI1640 medium containing 10% (v / v) fetal calf serum, at 37 ° C., 5% (v / v).
Passage in the presence of CO ₂ . Two weeks prior to cell fusion, 0.13 mM 8-azaguanine, 0.5 μg / ml M
C-210 (Mycoplasma remover, Dainippon Pharmaceutical Co., Ltd.)
RPMI16 containing 10% (v / v) fetal bovine serum
The cells are cultured in a 40-medium for one week, and then cultured in a normal medium for the next one week.

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

(C) Cell fusion To 10 ⁸ spleen cells obtained from the above spleen, 10 ⁷ myeloma cells were placed in a round-bottom glass tube and mixed well.
Centrifuge at 1400 rpm for 5 minutes, remove the supernatant and mix the cells better. 30% pre-heated to 37 ° C
(W / v) RPMI164 containing polyethylene glycol
0 ml of medium 0 is added and left for 30 seconds. 700rpm
After centrifugation for 6 minutes in RPMI1640 medium 10 ml
And slowly spin the glass tube so that the polyethylene glycol is well mixed, centrifuge at 1400 rpm for 5 minutes, completely remove the supernatant, and add 5 ml of HA to the precipitate.
Add T medium and let stand for 5 minutes. 10-20 ml of H
AT medium was added, the mixture was left for 30 minutes, HAT medium was added so that the myeloma cell concentration was 3.3 × 10 ⁵ cells / ml, and the cells were suspended. Dispense 2 drops into wells of the container.
After culturing at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere, HAT medium was added to the wells after 1 day, 7 days, and 14 days.
Add drops.

(D) Screening of antibody-producing cells The above fusion protein was prepared at a protein concentration of 1 to 10 μg / ml.
Containing 0.02% (w / v) sodium azide so that
Suspended in 05M sodium bicarbonate buffer (pH 9.6),
A dialysate was dialyzed against 0.05 M sodium bicarbonate buffer (pH 9.6) containing 02% sodium azide, and then diluted to a protein concentration of 1 to 10 μg / ml. Take 50 μl per well and leave at 4 ° C. overnight to adsorb the fusion protein. The supernatant is removed, 150 μl of PBS containing 0.02% (w / v) Tween 20 is added to the wells, the mixture is left for 3 minutes, and then removed and washed. After one further washing operation, the wells contained 1% (v / v) bovine serum albumin containing P
Blocking is performed by adding 100 μl of BS and leaving the mixture at 4 ° C. overnight or more. After removing PBS containing bovine serum albumin and washing twice with PBS containing 0.02% (w / v) Tween 20, the culture supernatant of the fused cells was added to the wells.
Add μl and leave at room temperature for 2 hours. 0.02% (w / v)
After similarly washing three times with PBS containing Tween 20, 25 ng / ml peroxidase-labeled goat anti-mouse I was added to the wells.
Add 50 μl of gG antibody and leave at room temperature for 2 hours. 0.
After washing three times with PBS containing 02% (w / v) Tween 20 in the same manner, 50 μl of an ABTS solution (manufactured by KPL) was added to the wells.
l, and left at room temperature for 15 minutes to 1 hour to cause a color reaction,
The absorbance at 405 nm is measured with a 96-well EIA plate photometer. Then, the cells in the positive wells are collected with a Pasteur pipette, transferred to a 24-well plastic culture vessel, added with 1-2 ml of HAT medium, and cultured similarly.

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

[0078]

According to the present invention, the fusion protein of the present invention stably retains its antigenicity as a Chlamydia pneumoniae antigen and is easily purified. The fusion protein according to claim 2 has the effect of the fusion protein according to claim 1, and furthermore, the antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained. The DNA according to claim 3 is useful for production of a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and is easily purified. The DNA according to claim 4 has the effect of the DNA according to claim 3, and is useful for producing a fusion protein in which antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained. The recombinant vector according to claims 5 and 6 is useful for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and is easily purified.

The pHLTCPN53 plasmid according to claim 7 is useful for producing a fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae-specific antigen and is easily purified. The transformant according to claim 8 is useful for producing a suitable fusion protein which can stably maintain antigenicity as a Chlamydia pneumoniae antigen and is easily purified. The transformant according to claim 9 is a Chlamydia transformant.
The antigenicity as a pneumoniae-specific antigen is stably maintained, and is useful for producing a suitable fusion protein that is easily purified. The recombinant baculovirus according to claim 10,
The antigenicity as a Chlamydia pneumoniae antigen is stably maintained, and is useful for producing a fusion protein that can be easily purified.

In the method for producing a recombinant baculovirus according to the eleventh aspect, a recombinant baculovirus useful for producing a fusion protein which is easily purified while maintaining stable antigenicity as a Chlamydia pneumoniae antigen can be efficiently produced. be able to. The method for producing a recombinant baculovirus according to claim 12, wherein the antigenicity as a Chlamydia pneumoniae-specific antigen is stably maintained and a recombinant baculovirus useful for producing a fusion protein that is easily purified is efficiently produced. Can be. According to the method for producing a fusion protein according to the thirteenth aspect, an antigenicity as a Chlamydia pneumoniae antigen is stably maintained, and a fusion protein that is easily purified can be efficiently produced. The method for producing an anti-Chlamydia pneumoniae antibody according to claim 14 is suitable for producing an anti-Chlamydia pneumoniae antibody useful as an active ingredient of a diagnostic agent for Chlamydia pneumoniae infection.

[0081]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 488 Sequence type: Amino acid Sequence type: Peptide sequence Met Ser Ile Ser Ser Ser Gly Pro Asp Asn Gln Lys Asn Ile Met 1 5 10 15 Ser Gln Val Leu Thr Ser Thr Pro Gln Gly Val Pro Gln Gln Asp Lys 20 25 30 Leu Ser Gly Asn Glu Thr Lys Gln Ile Gln Gln Thr Arg Gln Gly Lys 35 40 45 Asn Thr Glu Met Glu Ser Asp Ala Thr Ile Ala Gly Ala Ser Gly Lys 50 55 60 Asp Lys Thr Ser Ser Thr Thr Lys Thr Glu Thr Ala Pro Gln Gln Gly 65 70 75 80 Val Ala Ala Gly Lys Glu Ser Ser Glu Ser Gln Lys Ala Gly Ala Asp 85 90 95 Thr Gly Val Ser Gly Ala Ala Ala Thr Thr Ala Ser Asn Thr Ala Thr 100 105 110 Lys Ile Ala Met Gln Thr Ser Ile Glu Glu Ala Ser Lys Ser Met Glu 115 120 125 Ser Thr Leu Glu Ser Leu Gln Ser Leu Ser Ala Ala Gln Met Lys Glu 130 135 140 Val Glu Ala Val Val Val Ala Ala Leu Ser Gly Lys Ser Ser Gly Ser 145 150 155 160 Ala Lys Leu Glu Thr Pro Glu Leu Pro Lys Pro Gly Val Thr Pro Arg 165 170 175 Ser Glu Val Ile Glu Ile Gly Leu Ala Leu Ala Lys Ala Ile Gln Thr 180 1 85 190 Leu Gly Glu Ala Thr Lys Ser Ala Leu Ser Asn Tyr Ala Ser Thr Gln 195 200 205 Ala Gln Ala Asp Gln Thr Asn Lys Leu Gly Leu Glu Lys Gln Ala Ile 210 215 220 Lys Ile Asp Lys Glu Arg Glu Glu Tyr Gln Glu Met Lys Ala Ala Glu 225 230 235 240 Gln Lys Ser Lys Asp Leu Glu Gly Thr Met Asp Thr Val Asn Thr Val 245 250 255 Met Ile Ala Val Ser Val Ala Ile Thr Val Ile Ser Ile Val Ala Ala 260 265 270 Ile Phe Thr Cys Gly Ala Gly Leu Ala Gly Leu Ala Ala Gly Ala Ala 275 280 285 Val Gly Ala Ala Ala Ala Gly Gly Ala Ala Gly Ala Ala Ala Ala Thr 290 295 300 Thr Val Ala Thr Gln Ile Thr Val Gln Ala Val Val Gln Ala Val Lys 305 310 315 320 Gln Ala Val Ile Thr Ala Val Arg Gln Ala Ile Thr Ala Ala Ile Lys 325 330 335 Ala Ala Val Lys Ser Gly Ile Lys Ala Phe Ile Lys Thr Leu Val Lys 340 345 350 Ala Ile Ala Lys Ala Ile Ser Lys Gly Ile Ser Lys Val Phe Ala Lys 355 360 365 Gly Thr Gln Met Ile Ala Lys Asn Phe Pro Lys Leu Ser Lys Val Ile 370 375 380 Ser Ser Leu Thr Ser Lys Trp Val Thr Val Val Gly Val Gly Val Val Val 385 390 Three 95 400 Ala Ala Pro Ala Leu Gly Lys Gly Ile Met Gln Met Gln Leu Ser Glu 405 410 415 Met Gln Gln Asn Val Ala Gln Phe Gln Lys Glu Val Gly Lys Leu Gln 420 425 430 Ala Ala Ala Ala Asp Met Ile Ser Met Phe Thr Gln Phe Trp Gln Gln Ala 435 440 445 Ser Lys Ile Ala Ser Lys Gln Thr Gly Glu Ser Asn Glu Met Thr Gln 450 455 460 Lys Ala Thr Lys Leu Gly Ala Gln Ile Leu Lys Ala Tyr Ala Ala Ile 465 470 475 475 480 Ser Gly Ala Ile Ala Gly Ala Ala 485 488

SEQ ID NO: 2 Sequence length: 529 Sequence type: amino acid Sequence type: peptide sequence Met Ser Pro Ile Asp Pro Met Gly His His His His His Gly Arg 1 5 10 15 Arg Arg Ala Ser Val Ala Ala Gly Ile Leu Val Pro Arg Gly Ser Pro 20 25 30 Gly Leu Asp Gly Ile Cys Ser Arg Ser Met Ser Ile Ser Ser Ser Ser 35 40 45 Gly Pro Asp Asn Gln Lys Asn Ile Met Ser Gln Val Leu Thr Ser Thr 50 55 60 Pro Gln Gly Val Pro Gln Gln Asp Lys Leu Ser Gly Asn Glu Thr Lys 65 70 75 80 Gln Ile Gln Gln Thr Arg Gln Gly Lys Asn Thr Glu Met Glu Ser Asp 85 90 95 Ala Thr Ile Ala Gly Ala Ser Gly Lys Asp Lys Thr Ser Ser Thr Thr 100 105 110 Lys Thr Glu Thr Ala Pro Gln Gln Gly Val Ala Ala Gly Lys Glu Ser 115 120 125 Ser Glu Ser Gln Lys Ala Gly Ala Asp Thr Gly Val Ser Gly Ala Ala 130 135 140 Ala Thr Thr Ala Ser Asn Thr Ala Thr Lys Ile Ala Met Gln Thr Ser 145 150 155 160 Ile Glu Glu Ala Ser Lys Ser Met Glu Ser Thr Leu Glu Ser Leu Gln 165 170 175 Ser Leu Ser Ala Ala Gln Met Lys Glu Val Glu Ala Val Val Val Ala 180 185 190 Ala Leu Ser Gly Lys Ser Ser Gly Ser Ala Lys Leu Glu Thr Pro Glu 195 200 205 Leu Pro Lys Pro Gly Val Thr Pro Arg Ser Glu Val Ile Glu Ile Gly 210 215 220 Leu Ala Leu Ala Lys Ala Ile Gln Thr Leu Gly Glu Ala Thr Lys Ser 225 230 235 240 Ala Leu Ser Asn Tyr Ala Ser Thr Gln Ala Gln Ala Asp Gln Thr Asn 245 250 255 Lys Leu Gly Leu Glu Lys Gln Ala Ile Lys Ile Asp Lys Glu Arg Glu 260 265 270 Glu Tyr Gln Glu Met Lys Ala Ala Glu Gln Lys Ser Lys Asp Leu Glu 275 280 285 Gly Thr Met Asp Thr Val Asn Thr Val Met Ile Ala Val Ser Val Ala 290 295 300 300 Ile Thr Val Ile Ser Ile Val Ala Ala Ile Phe Thr Cys Gly Ala Gly 305 310 315 320 Leu Ala Gly Leu Ala Ala Gly Ala Ala Val Gly Ala Ala Ala Ala Gly 325 330 335 Gly Ala Ala Gly Ala Ala Ala Ala Thr Thr Val Ala Thr Gln Ile Thr 340 345 350 Val Gln Ala Val Val Gln Ala Val Lys Gln Ala Val Ile Thr Ala Val 355 360 365 Arg Gln Ala Ile Thr Ala Ala Ile Lys Ala Ala Val Lys Ser Gly Ile 370 375 380 Lys Ala Phe Ile Lys Thr Leu Val Lys Ala Ile Ala Lys Ala Ile Ser 385 390 395 400 Lys Gly Ile Ser Lys Val Phe Ala Lys Gly Thr Gln Met Ile Ala Lys 405 410 415 Asn Phe Pro Lys Leu Ser Lys Val Ile Ser Ser Leu Thr Ser Lys Trp 420 425 430 Val Thr Val Gly Val Gly Val Val Val Ala Ala Pro Ala Leu Gly Lys 435 440 445 445 Gly Ile Met Gln Met Gln Leu Ser Glu Met Gln Gln Asn Val Ala Gln 450 455 460 Phe Gln Lys Glu Val Gly Lys Leu Gln Ala Ala Ala Asp Met Ile Ser 465 470 475 480 Met Phe Thr Gln Phe Trp Gln Gln Ala Ser Lys Ile Ala Ser Lys Gln 485 490 495 Thr Gly Glu Ser Asn Glu Met Thr Gln Lys Ala Thr Lys Leu Gly Ala 500 505 510 Gln Ile Leu Lys Ala Tyr Ala Ala Ile Ser Gly Ala Ile Ala Gly Ala 515 520 525 Ala 529

SEQ ID NO: 3 Sequence length: 1587 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: Other nucleic acid Synthetic DNA sequence ATGTCCCCTA TAGATCCGAT GGGACATCAT CATCATCATC ACGGAAGGAG AAGGGCCAGT 60 GTTGCGGCGG GAATTTTGGT CCCTCGTCTCGAG ATCCCAGGTCTCGATGGA TTTCATCTTC TTCAGGACCT GACAATCAAA AAAATATCAT GTCTCAAGTT 180 CTGACATCGA CACCCCAGGG CGTGCCCCAA CAAGATAAGC TGTCTGGCAA CGAAACGAAG 240 CAAATACAGC AAACACGTCA GGGTAAAAAC ACTGAGATGG AAAGCGATGC CACTATTGCT 300 GGTGCTTCTG GAAAAGACAA AACTTCCTCG ACTACAAAAA CAGAAACAGC TCCACAACAG 360 GGAGTTGCTG CTGGGAAAGA ATCCTCAGAA AGTCAAAAGG CAGGTGCTGA TACTGGAGTA 420 TCAGGAGCGG CTGCTACTAC AGCATCAAAT ACTGCAACAA AAATTGCTAT GCAGACCTCT 480 ATTGAAGAGG CGAGCAAAAG TATGGAGTCT ACCTTAGAGT CACTTCAAAG CCTCAGTGCC 540 GCGCAAATGA AAGAAGTCGA AGCGGTTGTT GTTGCTGCCC TCTCAGGGAA AAGTTCGGGC 600 TCCGCAAAAT TGGAAACACC TGAGCTCCCC AAGCCCGGGG TGACACCAAG ATCAGAGGTT 660 ATCGAAATCG GACTCGCGCT TGCTAAAGCA ATTCAGACAT TGGGAGAAGC CACAAAATCT 720 GCCTT ATCTA ACTATGCAAG TACACAAGCA CAAGCAGACC AAACAAATAA ACTAGGTCTA 780 GAAAAGCAAG CGATAAAAAT CGATAAAGAA CGAGAAGAAT ACCAAGAGAT GAAGGCTGCC 840 GAACAGAAGT CTAAAGATCT CGAAGGAACA ATGGATACTG TCAATACTGT GATGATCGCG 900 GTTTCTGTTG CCATTACAGT TATTTCTATT GTTGCTGCTA TTTTTACATG CGGAGCTGGA 960 CTCGCTGGAC TCGCTGCGGG AGCTGCTGTA GGTGCAGCGG CAGCTGGAGG TGCAGCAGGA 1020 GCTGCTGCCG CAACCACGGT AGCAACACAA ATTACAGTTC AAGCTGTTGT CCAAGCGGTG 1080 AAACAAGCTG TTATCACAGC TGTCAGACAA GCGATCACCG CGGCTATAAA AGCGGCTGTC 1140 AAATCTGGAA TAAAAGCATT TATCAAAACT TTAGTCAAAG CGATTGCCAA AGCCATTTCT 1200 AAAGGAATCT CTAAGGTTTT CGCTAAGGGA ACTCAAATGA TTGCGAAGAA CTTCCCCAAG 1260 CTCTCGAAAG TCATCTCGTC TCTTACCAGT AAATGGGTCA CGGTTGGGGT TGGGGTTGTA 1320 GTTGCGGCGC CTGCTCTCGG TAAAGGGATT ATGCAAATGC AGCTCTCGGA GATGCAACAA 1380 AACGTCGCTC AATTTCAGAA AGAAGTCGGA AAACTGCAGG CTGCGGCTGA TATGATTTCT 1440 ATGTTCACTC AATTTTGGCA ACAGGCAAGT AAAATTGCCT CAAAACAAAC AGGCGAGTCT 1500 AATGAAATGA CTCAAAAAGC TACCAAGCTG GGCGCTCAAA TCCTTAAAGC GTATGCCGCA 1560 ATCAGCGGAG CCAT CGCTGG CGCAGCA 1587

SEQ ID NO: 4 Sequence length: 28 Sequence type: Number of nucleic acid strands: Single-stranded Sequence type: Other nucleic acid Synthetic DNA sequence GCGGATCCAT GTCTATTTCA TCTTCTTC 28

SEQ ID NO: 5 Sequence length: 28 Sequence type: number of nucleic acid chains: single-stranded Sequence type: other nucleic acid Synthetic DNA sequence ATGCTAGCTT TTATGCTGCG CCAGCGAT 28

SEQ ID NO: 6 Sequence length: 1939 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: C. pneumoniae Strain name: YK-41 Direct Origin of clone: 53-3S Sequence characteristics Characteristic symbol: CDS Location: 236..1012 Characteristic determination method: P sequence TCAGTATCGG CGGAATTCGA ACCCCTTCGC GGCTCTTTCT GGAACTCTAG AATCTTTACA 60 TCTCGAAGAG TTAACTCAAG GATTATTCAC ACTCAGATCATGAC ACTCACGATCACTCACAG ATAAACAAAT 180 GGTTAAAGGC GCTGAGTAAA GCCCTTTGCA GAATCAAACC CCTTAGGATA CAAAC ATG 238 Met 1 TCT ATT TCA TCT TCT TCA GGA CCT GAC AAT CAA AAA AAT ATC ATG TCT 286 Ser Ile Ser Ser Ser Ser Gly Pro Asp Asn 15 Gle Lys Asn CTG ACA TCG ACA CCC CAG GGC GTG CCC CAA CAA GAT AAG CTG 334 Gln Val Leu Thr Ser Thr Pro Gln Gly Val Pro Gln Gln Asp Lys Leu 20 25 30 TCT GGC AAC GAA ACG AAG CAA ATA CAG CAA ACA CGT CAG GGT AAA AAC 382 Ser Gl y Asn Glu Thr Lys Gln Ile Gln Gln Thr Arg Gln Gly Lys Asn 35 40 45 ACT GAG ATG GAA AGC GAT GCC ACT ATT GCT GGT GCT TCT GGA AAA GAC 430 Thr Glu Met Glu Ser Asp Ala Thr Ile Ala Gly Ala Ser Gly Lys Asp 50 55 60 65 AAA ACT TCC TCG ACT ACA AAA ACA GAA ACA GCT CCA CAA CAG GGA GTT 478 Lys Thr Ser Ser Thr Thr Lys Thr Glu Thr Ala Pro Gln Gln Gly Val 70 75 80 GCT GCT GGG AAA GAA TCC TCA GAA AGT CAA AAG GCA GGT GCT GAT ACT 526 Ala Ala Gly Lys Glu Ser Ser Glu Ser Gln Lys Ala Gly Ala Asp Thr 85 90 95 GGA GTA TCA GGA GCG GCT GCT ACT ACA GCA TCA AAT ACT GCA ACA AAA 574 Gly Val Ser Gly Ala Ala Ala Thr Thr Ala Ser Asn Thr Ala Thr Lys 100 105 110 ATT GCT ATG CAG ACC TCT ATT GAA GAG GCG AGC AAA AGT ATG GAG TCT 622 Ile Ala Met Gln Thr Ser Ile Glu Glu Ala Ser Lys Ser Met Glu Ser 115 120 125 ACC TTA GAG TCA CTT CAA AGC CTC AGT GCC GCG CAA ATG AAA GAA GTC 670 Thr Leu Glu Ser Leu Gln Ser Leu Ser Ala Ala Gln Met Lys Glu Val 130 135 140 145 GAA GCG GTT GTT GTT GCT GCC CTC TCA GGG AAA AGT TCG GGT TCC GCA 718 Glu Ala Val Val Val Ala Ala Leu Ser Gly Lys Ser Ser Gly Ser Ala 150 155 160 AAA TTG GAA ACA CCT GAG CTC CCC AAG CCC GGG GTG ACA CCA AGA TCA 766 Lys Leu Glu Thr Pro Glu Leu Pro Lys Pro Gly Val Thr Pro Arg Ser 165 170 175 GAG GTT ATC GAA ATC GGA CTC GCG CTT GCT AAA GCA ATT CAG ACA TTG 814 Glu Val Ile Glu Ile Gly Leu Ala Leu Ala Lys Ala Ile Gln Thr Leu 180 185 190 GGA GAA GCC ACA AAA TCT GCC TTA TCT AAC TAT GCA AGT ACA CAA GCA 862 Gly Glu Ala Thr Lys Ser Ala Leu Ser Asn Tyr Ala Ser Thr Gln Ala 195 200 205 CAA GCA GAC CAA ACA AAT AAA CTA GGT CTA GAA AAG CAA GCG ATA AAA 910 Gln Ala Asp Gln Thr Asn Lys Leu Gly Leu Glu Lys Gln Ala Ile Lys 210 215 220 225 ATC GAT AAA GAA CGA GAA GAA TAC CAA GAG ATG AAG GCT GCC GAA CAG 958 Ile Asp Lys Glu Arg Glu Glu Tyr Gln Glu Met Lys Ala Ala Glu Gln 230 235 240 AAG TCT AAA GAT CTC GAA GGA ACA ATG GAT ACT GTC AAT ACT GTG ATG 1006 Lys Ser Lys Asp Leu Glu Gly Thr Met Asp Thr Val Asn Thr Val Met 245 250 255 ATC GCG GTT TCT GTT GCC ATT ACA GTT ATT TCT ATT GTT GCT GC T ATT 1054 Ile Ala Val Ser Val Ala Ile Thr Val Ile Ser Ile Val Ala Ala Ile 260 265 270 TTT ACA TGC GGA GCT GGA CTC GCT GGA CTC GCT GCG GGA GCT GCT GTA 1102 Phe Thr Cys Gly Ala Gly Leu Ala Gly Leu Ala Ala Gly Ala Ala Val 275 280 285 GGT GCA GCG GCA GCT GGA GGT GCA GCA GGA GCT GCT GCC GCA ACC ACG 1150 Gly Ala Ala Ala Ala Gly Gly Ala Ala Gly Ala Ala Ala Ala Thr Thr 290 295 295 300 305 GTA GCA ACA CAA ATT ACA GTT CAA GCT GTT GTC CAA GCG GTG AAA CAA 1198 Val Ala Thr Gln Ile Thr Val Gln Ala Val Val Gln Ala Val Lys Gln 310 315 320 GCT GTT ATC ACA GCT GTC AGA CAA GCG ATC ACC GCG GCT ATA AAA GCG 1246 Ala Val Ile Thr Ala Val Arg Gln Ala Ile Thr Ala Ala Ile Lys Ala 325 330 335 GCT GTC AAA TCT GGA ATA AAA GCA TTT ATC AAA ACT TTA GTC AAA GCG 1294 Ala Val Lys Ser Gly Ile Lys Ala Phe Ile Lys Thr Leu Val Lys Ala 340 345 350 ATT GCC AAA GCC ATT TCT AAA GGA ATC TCT AAG GTT TTC GCT AAG GGA 1342 Ile Ala Lys Ala Ile Ser Lys Gly Ile Ser Lys Val Phe Ala Lys Gly 355 360 365 ACT CAA ATG ATT GCG AAG AAC TTC CCC AAG CTC TCG AAA GTC ATC TCG 1390 Thr Gln Met Ile Ala Lys Asn Phe Pro Lys Leu Ser Lys Val Ile Ser 370 375 380 385 TCT CTT ACC AGT AAA TGG GTC ACG GTT GGG GTT GGG GTT GTA GTT GCG 1438 Ser Leu Thr Ser Lys Trp Val Thr Val Gly Val Gly Val Val Val Ala 390 395 400 GCG CCT GCT CTC GGT AAA GGG ATT ATG CAA ATG CAG CTC TCG GAG ATG 1486 Ala Pro Ala Leu Gly Lys Gly Ile Met Gln Met Gln Leu Ser Glu Met 405 410 415 CAA CAA AAC GTC GCT CAA TTT CAG AAA GAA GTC GGA AAA CTG CAG GCT 1534 Gln Gln Asn Val Ala Gln Phe Gln Lys Glu Val Gly Lys Leu Gln Ala 420 425 430 GCG GCT GAT ATG ATT TCT ATG TTC ACT CAA TTT TGG CAA CAG GCA AGT 1582 Ala Ala Asp Met Ile Ser Met Phe Thr Gln Phe Trp Gln Gln Ala Ser 435 440 445 AAA ATT GCC TCA AAA CAA ACA GGC GAG TCT AAT GAA ATG ACT CAA AAA 1630 Lys Ile Ala Ser Lys Gln Thr Gly Glu Ser Asn Glu Met Thr Gln Lys 450 455 460 465 GCT ACC AAG CTG GGC GCT CAA ATC CTT AAA GCG TAT GCC GCA ATC AGC 1678 Ala Thr Lys Leu Gly Ala Gln Ile Leu Lys Ala Tyr Ala Ala Ile Ser 470 475 480 GGA GCC ATC GCT GGC GCA GCA TAAAACCAAT AATTTTTAAA GGAGCATGTA 1729 Gly Ala Ile Ala Gly Ala Ala 485 488 TTGCTATGAC ATCAGGAGTT AGTGGAAGTT CAAGTCAGGA TCCCACATTG GCTGCGCAAT 1789 TGGCACAATC CTCTCAAAAA GCAGGAAATG CTCAGAGCGG TCACGATACT AAGAATGTAA 1849 CAAAGCAAGG GGCTCAAGCA GAAGTCGCTG CTGGAGGCTT TGAGGATCTC ATTCAAGACG 1909 CTTCAGCGCA AAGTACAGGA AAAAAAGAAG 1939

SEQ ID NO: 7 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Sequence type: other nucleic acid Synthetic DNA sequence ATTCCGGATT ATTCATACCG 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Sequence type: other nucleic acid Synthetic DNA sequence CAGGAAAGGA TCAGATCTGC 20

SEQ ID NO: 9 Sequence length: 1464 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: Other nucleic acid Synthetic DNA sequence ATGTCTATTT CATCTTCTTC AGGACCTGAC AATCAAAAAA ATATCATGTC TCAAGTTCTG 60 ACATCGACAC CCCAGGGCGT GCCCCAACAATCATAGACA CACGTCAGGG TAAAAACACT GAGATGGAAA GCGATGCCAC TATTGCTGGT 180 GCTTCTGGAA AAGACAAAAC TTCCTCGACT ACAAAAACAG AAACAGCTCC ACAACAGGGA 240 GTTGCTGCTG GGAAAGAATC CTCAGAAAGT CAAAAGGCAG GTGCTGATAC TGGAGTATCA 300 GGAGCGGCTG CTACTACAGC ATCAAATACT GCAACAAAAA TTGCTATGCA GACCTCTATT 360 GAAGAGGCGA GCAAAAGTAT GGAGTCTACC TTAGAGTCAC TTCAAAGCCT CAGTGCCGCG 420 CAAATGAAAG AAGTCGAAGC GGTTGTTGTT GCTGCCCTCT CAGGGAAAAG TTCGGGCTCC 480 GCAAAATTGG AAACACCTGA GCTCCCCAAG CCCGGGGTGA CACCAAGATC AGAGGTTATC 540 GAAATCGGAC TCGCGCTTGC TAAAGCAATT CAGACATTGG GAGAAGCCAC AAAATCTGCC 600 TTATCTAACT ATGCAAGTAC ACAAGCACAA GCAGACCAAA CAAATAAACT AGGTCTAGAA 660 AAGCAAGCGA TAAAAATCGA TAAAGAACGA GAAGAATACC AAGAGATGAA GGCTGCCGAA 720 CAGAA GTCTA AAGATCTCGA AGGAACAATG GATACTGTCA ATACTGTGAT GATCGCGGTT 780 TCTGTTGCCA TTACAGTTAT TTCTATTGTT GCTGCTATTT TTACATGCGG AGCTGGACTC 840 GCTGGACTCG CTGCGGGAGC TGCTGTAGGT GCAGCGGCAG CTGGAGGTGC AGCAGGAGCT 900 GCTGCCGCAA CCACGGTAGC AACACAAATT ACAGTTCAAG CTGTTGTCCA AGCGGTGAAA 960 CAAGCTGTTA TCACAGCTGT CAGACAAGCG ATCACCGCGG CTATAAAAGC GGCTGTCAAA 1020 TCTGGAATAA AAGCATTTAT CAAAACTTTA GTCAAAGCGA TTGCCAAAGC CATTTCTAAA 1080 GGAATCTCTA AGGTTTTCGC TAAGGGAACT CAAATGATTG CGAAGAACTT CCCCAAGCTC 1140 TCGAAAGTCA TCTCGTCTCT TACCAGTAAA TGGGTCACGG TTGGGGTTGG GGTTGTAGTT 1200 GCGGCGCCTG CTCTCGGTAA AGGGATTATG CAAATGCAGC TCTCGGAGAT GCAACAAAAC 1260 GTCGCTCAAT TTCAGAAAGA AGTCGGAAAA CTGCAGGCTG CGGCTGATAT GATTTCTATG 1320 TTCACTCAAT TTTGGCAACA GGCAAGTAAA ATTGCCTCAA AACAAACAGG CGAGTCTAAT 1380 GAAATGACTC AAAAAGCTAC CAAGCTGGGC GCTCAAATCC TTAAAGCGTA TGCCGCAATC 1440 AGCGGAGCCA TCGCTGGCGC AGCA 1464

SEQ ID NO: 10 Sequence length: 11099 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: Other nucleic acid Plasmid sequence AAGCTTTACT CGTAAAGCGA GTTGAAGGAT CATATTTAGT TGCGTTTATG AGATAAGATT 60 GAAAGCACGT GTAAAATGTT TCCCGCGCAAGTCTAGTTAG ATGTTTTAAA ACACCTTTGC GGCCCGAGTT GTTTGCGTAC 180 GTGACTAGCG AAGAAGATGT GTGGACCGCA GAACAGATAG TAAAACAAAA CCCTAGTATT 240 GGAGCAATAA TCGATTTAAC CAACACGTCT AAATATTATG ATGGTGTGCA TTTTTTGCGG 300 GCGGGCCTGT TATACAAAAA AATTCAAGTA CCTGGCCAGA CTTTGCCGCC TGAAAGCATA 360 GTTCAAGAAT TTATTGACAC GGTAAAAGAA TTTACAGAAA AGTGTCCCGG CATGTTGGTG 420 GGCGTGCACT GCACACACGG TATTAATCGC ACCGGTTACA TGGTGTGCAG ATATTTAATG 480 CACACCCTGG GTATTGCGCC GCAGGAAGCC ATAGATAGAT TCGAAAAAGC CAGAGGTCAC 540 AAAATTGAAA GACAAAATTA CGTTCAAGAT TTATTAATTT AATTAATATT ATTTGCATTC 600 TTTAACAAAT ACTTTATCCT ATTTTCAAAT TGTTGCGCTT CTTCCAGCGA ACCAAAACTA 660 TGCTTCGCTT GCTCCGTTTA GCTTGTAGCC GATCAGTGGC GTTGTTCCAA TCGACGGTAG 720 GAT TAGGCCG GATATTCTCC ACCACAATGT TGGCAACGTT GATGTTACGT TTATGCTTTT 780 GGTTTTCCAC GTACGTCTTT TGGCCGGTAA TAGCCGTAAA CGTAGTGCCG TCGCGCGTCA 840 CGCACAACAC CGGATGTTTG CGCTTGTCCG CGGGGTATTG AACCGCGCGA TCCGACAAAT 900 CCACCACTTT GGCAACTAAA TCGGTGACCT GCGCGTCTTT TTTCTGCATT ATTTCGTCTT 960 TCTTTTGCAT GGTTTCCTGG AAGCCGGTGT ACATGCGGTT TAGATCAGTC ATGACGCGCG 1020 TGACCTGCAA ATCTTTGGCC TCGATCTGCT TGTCCTTGAT GGCAACGATG CGTTCAATAA 1080 ACTCTTGTTT TTTAACAAGT TCCTCGGTTT TTTGCGCCAC CACCGCTTGC AGCGCGTTTG 1140 TGTGCTCGGT GAATGTCGCA ATCAGCTTAG TCACCAACTG TTTGCTCTCC TCCTCCCGTT 1200 GTTTGATCGC GGGATCGTAC TTGCCGGTGC AGAGCACTTG AGGAATTACT TCTTCTAAAA 1260 GCCATTCTTG TAATTCTATG GCGTAAGGCA ATTTGGACTT CATAATCAGC TGAATCACGC 1320 CGGATTTAGT AATGAGCACT GTATGCGGCT GCAAATACAG CGGGTCGCCC CTTTTCACGA 1380 CGCTGTTAGA GGTAGGGCCC CCATTTTGGA TGGTCTGCTC AAATAACGAT TTGTATTTAT 1440 TGTCTACATG AACACGTATA GCTTTATCAC AAACTGTATA TTTTAAACTG TTAGCGACGT 1500 CCTTGGCCAC GAACCGGACC TGTTGGTCGC GCTCTAGCAC GTACCGCAGG TTGAACGTAT 1560 CTTCTCCAAA TT TAAATTCT CCAATTTTAA CGCGAGCCAT TTTGATACAC GTGTGTCGAT 1620 TTTGCAACAA CTATTGTTTT TTAACGCAAA CTAAACTTAT TGTGGTAAGC AATAATTAAA 1680 TATGGGGGAA CATGCGCCGC TACAACACTC GTCGTTATGA ACGCAGACGG CGCCGGTCTC 1740 GGCGCAAGCG GCTAAAACGT GTTGCGCGTT CAACGCGGCA AACATCGCAA AAGCCAATAG 1800 TACAGTTTTG ATTTGCATAT TAACGGCGAT TTTTTAAATT ATCTTATTTA ATAAATAGTT 1860 ATGACGCCTA CAACTCCCCG CCCGCGTTGA CTCGCTGCAC CTCGAGCAGT TCGTTGACGC 1920 CTTCCTCCGT GTGGCCGAAC ACGTCGAGCG GGTGGTCGAT GACCAGCGGC GTGCCGCACG 1980 CGACGCACAA GTATCTGTAC ACCGAATGAT CGTCGGGCGA AGGCACGTCG GCCTCCAAGT 2040 GGCAATATTG GCAAATTCGA AAATATATAC AGTTGGGTTG TTTGCGCATA TCTATCGTGG 2100 CGTTGGGCAT GTACGTCCGA ACGTTGATTT GCATGCAAGC CGAAATTAAA TCATTGCGAT 2160 TAGTGCGATT AAAACGTTGT ACATCCTCGC TTTTAATCAT GCCGTCGATT AAATCGCGCA 2220 ATCGAGTCAA GTGATCAAAG TGTGGAATAA TGTTTTCTTT GTATTCCCGA GTCAAGCGCA 2280 GCGCGTATTT TAACAAACTA GCCATCTTGT AAGTTAGTTT CATTTAATGC AACTTTATCC 2340 AATAATATAT TATGTATCGC ACGTCAAGAA TTAACAATGC GCCCGTTGTC GCATCTCAAC 2400 ACGACTATGA TAGAGATC AA ATAAAGCGCG AATTAAATAG CTTGCGACGC AACGTGCACG 2460 ATCTGTGCAC GCGTTCCGGC ACGAGCTTTG ATTGTAATAA GTTTTTACGA AGCGATGACA 2520 TGACCCCCGT AGTGACAACG ATCACGCCCA AAAGAACTGC CGACTACAAA ATTACCGAGT 2580 ATGTCGGTGA CGTTAAAACT ATTAAGCCAT CCAATCGACC GTTAGTCGAA TCAGGACCGC 2640 TGGTGCGAGA AGCCGCGAAG TATGGCGAAT GCATCGTATA ACGTGTGGAG TCCGCTCATT 2700 AGAGCGTCAT GTTTAGACAA GAAAGCTACA TATTTAATTG ATCCCGATGA TTTTATTGAT 2760 AAATTGACCC TAACTCCATA CACGGTATTC TACAATGGCG GGGTTTTGGT CAAAATTTCC 2820 GGACTGCGAT TGTACATGCT GTTAACGGCT CCGCCCACTA TTAATGAAAT TAAAAATTCC 2880 AATTTTAAAA AACGCAGCAA GAGAAACATT TGTATGAAAG AATGCGTAGA AGGAAAGAAA 2940 AATGTCGTCG ACATGCTGAA CAACAAGATT AATATGCCTC CGTGTATAAA AAAAATATTG 3000 AACGATTTGA AAGAAAACAA TGTACCGCGC GGCGGTATGT ACAGGAAGAG GTTTATACTA 3060 AACTGTTACA TTGCAAACGT GGTTTCGTGT GCCAAGTGTG AAAACCGATG TTTAATCAAG 3120 GCTCTGACGC ATTTCTACAA CCACGACTCC AAGTGTGTGG GTGAAGTCAT GCATCTTTTA 3180 ATCAAATCCC AAGATGTGTA TAAACCACCA AACTGCCAAA AAATGAAAAC TGTCGACAAG 3240 CTCTGTCCGT TTGCTGGCAA CTG CAAGGGT CTCAATCCTA TTTGTAATTA TTGAATAATA 3300 AAACAATTAT AAATGCTAAA TTTGTTTTTT ATTAACGATA CAAACCAAAC GCAACAAGAA 3360 CATTTGTAGT ATTATCTATA ATTGAAAACG CGTAGTTATA ATCGCTGAGG TAATATTTAA 3420 AATCATTTTC AAATGATTCA CAGTTAATTT GCGACAATAT AATTTTATTT TCACATAAAC 3480 TAGACGCCTT GTCGTCTTCT TCTTCGTATT CCTTCTCTTT TTCATTTTTC TCCTCATAAA 3540 AATTAACATA GTTATTATCG TATCCATATA TGTATCTATC GTATAGAGTA AATTTTTTGT 3600 TGTCATAAAT ATATATGTCT TTTTTAATGG GGTGTATAGT ACCGCTGCGC ATAGTTTTTC 3660 TGTAATTTAC AACAGTGCTA TTTTCTGGTA GTTCTTCGGA GTGTGTTGCT TTAATTATTA 3720 AATTTATATA ATCAATGAAT TTGGGATCGT CGGTTTTGTA CAATATGTTG CCGGCATAGT 3780 ACGCAGCTTC TTCTAGTTCA ATTACACCAT TTTTTAGCAG CACCGGATTA ACATAACTTT 3840 CCAAAATGTT GTACGAACCG TTAAACAAAA ACAGTTCACC TCCCTTTTCT ATACTATTGT 3900 CTGCGAGCAG TTGTTTGTTG TTAAAAATAA CAGCCATTGT AATGAGACGC ACAAACTAAT 3960 ATCACAAACT GGAAATGTCT ATCAATATAT AGTTGCTGAT ATCATGGAGA TAATTAAAAT 4020 GATAACCATC TCGCAAATAA ATAAGTATTT TACTGTTTTC GTAACAGTTT TGTAATAAAA 4080 AAACCTATAA ATATTCCGGA TTATTCATA C CGTCCCACCA TCGGGCGCGG ATCCATGTCT 4140 ATTTCATCTT CTTCAGGACC TGACAATCAA AAAAATATCA TGTCTCAAGT TCTGACATCG 4200 ACACCCCAGG GCGTGCCCCA ACAAGATAAG CTGTCTGGCA ACGAAACGAA GCAAATACAG 4260 CAAACACGTC AGGGTAAAAA CACTGAGATG GAAAGCGATG CCACTATTGC TGGTGCTTCT 4320 GGAAAAGACA AAACTTCCTC GACTACAAAA ACAGAAACAG CTCCACAACA GGGAGTTGCT 4380 GCTGGGAAAG AATCCTCAGA AAGTCAAAAG GCAGGTGCTG ATACTGGAGT ATCAGGAGCG 4440 GCTGCTACTA CAGCATCAAA TACTGCAACA AAAATTGCTA TGCAGACCTC TATTGAAGAG 4500 GCGAGCAAAA GTATGGAGTC TACCTTAGAG TCACTTCAAA GCCTCAGTGC CGCGCAAATG 4560 AAAGAAGTCG AAGCGGTTGT TGTTGCTGCC CTCTCAGGGA AAAGTTCGGG CTCCGCAAAA 4620 TTGGAAACAC CTGAGCTCCC CAAGCCCGGG GTGACACCAA GATCAGAGGT TATCGAAATC 4680 GGACTCGCGC TTGCTAAAGC AATTCAGACA TTGGGAGAAG CCACAAAATC TGCCTTATCT 4740 AACTATGCAA GTACACAAGC ACAAGCAGAC CAAACAAATA AACTAGGTCT AGAAAAGCAA 4800 GCGATAAAAA TCGATAAAGA ACGAGAAGAA TACCAAGAGA TGAAGGCTGC CGAACAGAAG 4860 TCTAAAGATC TCGAAGGAAC AATGGATACT GTCAATACTG TGATGATCGC GGTTTCTGTT 4920 GCCATTACAG TTATTTCTAT TGTTGCTGCT ATTT TTACAT GCGGAGCTGG ACTCGCTGGA 4980 CTCGCTGCGG GAGCTGCTGT AGGTGCAGCG GCAGCTGGAG GTGCAGCAGG AGCTGCTGCC 5040 GCAACCACGG TAGCAACACA AATTACAGTT CAAGCTGTTG TCCAAGCGGT GAAACAAGCT 5100 GTTATCACAG CTGTCAGACA AGCGATCACC GCGGCTATAA AAGCGGCTGT CAAATCTGGA 5160 ATAAAAGCAT TTATCAAAAC TTTAGTCAAA GCGATTGCCA AAGCCATTTC TAAAGGAATC 5220 TCTAAGGTTT TCGCTAAGGG AACTCAAATG ATTGCGAAGA ACTTCCCCAA GCTCTCGAAA 5280 GTCATCTCGT CTCTTACCAG TAAATGGGTC ACGGTTGGGG TTGGGGTTGT AGTTGCGGCG 5340 CCTGCTCTCG GTAAAGGGAT TATGCAAATG CAGCTCTCGG AGATGCAACA AAACGTCGCT 5400 CAATTTCAGA AAGAAGTCGG AAAACTGCAG GCTGCGGCTG ATATGATTTC TATGTTCACT 5460 CAATTTTGGC AACAGGCAAG TAAAATTGCC TCAAAACAAA CAGGCGAGTC TAATGAAATG 5520 ACTCAAAAAG CTACCAAGCT GGGCGCTCAA ATCCTTAAAG CGTATGCCGC AATCAGCGGA 5580 GCCATCGCTG GCGCAGCATA AAAGCTAGAA TTCCGGAGCG GCCGCTGCAG ATCTGATCCT 5640 TTCCTGGGAC CCGGCAAGAA CCAAAAACTC ACTCTCTTCA AGGAAATCCG TAATGTTAAA 5700 CCCGACACGA TGAAGCTTGT CGTTGGATGG AAAGGAAAAG AGTTCTACAG GGAAACTTGG 5760 ACCCGCTTCA TGGAAGACAG CTTCCCCATT GTTAACGACC AAGAAGTGAT GGATGTTTTC 5820 CTTGTTGTCA ACATGCGTCC CACTAGACCC AACCGTTGTT ACAAATTCCT GGCCCAACAC 5880 GCTCTGCGTT GCGACCCCGA CTATGTACCT CATGACGTGA TTAGGATCGT CGAGCCTTCA 5940 TGGGTGGGCA GCAACAACGA GTACCGCATC AGCCTGGCTA AGAAGGGCGG CGGCTGCCCA 6000 ATAATGAACC TTCACTCTGA GTACACCAAC TCGTTCGAAC AGTTCATCGA TCGTGTCATC 6060 TGGGAGAACT TCTACAAGCC CATCGTTTAC ATCGGTACCG ACTCTGCTGA AGAGGAGGAA 6120 ATTCTCCTTG AAGTTTCCCT GGTGTTCAAA GTAAAGGAGT TTGCACCAGA CGCACCTCTG 6180 TTCACTGGTC CGGCGTATTA AAACACGATA CATTGTTATT AGTACATTTA TTAAGCGCTA 6240 GATTCTGTGC GTTGTTGATT TACAGACAAT TGTTGTACGT ATTTTAATAA TTCATTAAAT 6300 TTATAATCTT TAGGGTGGTA TGTTAGAGCG AAAATCAAAT GATTTTCAGC GTCTTTATAT 6360 CTGAATTTAA ATATTAAATC CTCAATAGAT TTGTAAAATA GGTTTCGATT AGTTTCAAAC 6420 AAGGGTTGTT TTTCCGAACC GATGGCTGGA CTATCTAATG GATTTTCGCT CAACGCCACA 6480 AAACTTGCCA AATCTTGTAG CAGCAATCTA GCTTTGTCGA TATTCGTTTG TGTTTTGTTT 6540 TGTAATAAAG GTTCGACGTC GTTCAAAATA TTATGCGCTT TTGTATTTCT TTCATCACTG 6600 TCGTTAGTGT ACAATTGACT CGACGTAAAC ACGTTAAATA AAGCT TGGAC ATATTTAACA 6660 TCGGGCGTGT TAGCTTTATT AGGCCGATTA TCGTCGTCGT CCCAACCCTC GTCGTTAGAA 6720 GTTGCTTCCG AAGACGATTT TGCCATAGCC ACACGACGCC TATTAATTGT GTCGGCTAAC 6780 ACGTCCGCGA TCAAATTTGT AGTTGAGCTT TTTGGAATTA TTTCTGATTG CGGGCGTTTT 6840 TGGGCGGGTT TCAATCTAAC TGTGCCCGAT TTTAATTCAG ACAACACGTT AGAAAGCGAT 6900 GGTGCAGGCG GTGGTAACAT TTCAGACGGC AAATCTACTA ATGGCGGCGG TGGTGGAGCT 6960 GATGATAAAT CTACCATCGG TGGAGGCGCA GGCGGGGCTG GCGGCGGAGG CGGAGGCGGA 7020 GGTGGTGGCG GTGATGCAGA CGGCGGTTTA GGCTCAAATG TCTCTTTAGG CAACACAGTC 7080 GGCACCTCAA CTATTGTACT GGTTTCGGGC GCCGTTTTTG GTTTGACCGG TCTGAGACGA 7140 GTGCGATTTT TTTCGTTTCT AATAGCTTCC AACAATTGTT GTCTGTCGTC TAAAGGTGCA 7200 GCGGGTTGAG GTTCCGTCGG CATTGGTGGA GCGGGCGGCA ATTCAGACAT CGATGGTGGT 7260 GGTGGTGGTG GAGGCGCTGG AATGTTAGGC ACGGGAGAAG GTGGTGGCGG CGGTGCCGCC 7320 GGTATAATTT GTTCTGGTTT AGTTTGTTCG CGCACGATTG TGGGCACCGG CGCAGGCGCC 7380 GCTGGCTGCA CAACGGAAGG TCGTCTGCTT CGAGGCAGCG CTTGGGGTGG TGGCAATTCA 7440 ATATTATAAT TGGAATACAA ATCGTAAAAA TCTGCTATAA GCATTGTAAT TTCGCTATCG 7500 TTTACCGTGC CGATATTTAA CAACCGCTCA ATGTAAGCAA TTGTATTGTA AAGAGATTGT 7560 CTCAAGCTCC GCACGCCGAT AACAAGCCTT TTCATTTTTA CTACAGCATT GTAGTGGCGA 7620 GACACTTCGC TGTCGTCGAC GTACATGTAT GCTTTGTTGT CAAAAACGTC GTTGGCAAGC 7680 TTTAAAATAT TTAAAAGAAC ATCTCTGTTC AGCACCACTG TGTTGTCGTA AATGTTGTTT 7740 TTGATAATTT GCGCTTCCGC AGTATCGACA CGTTCAAAAA ATTGATGCGC ATCAATTTTG 7800 TTGTTCCTAT TATTGAATAA ATAAGATTGT ACAGATTCAT ATCTACGATT CGTCATGGCC 7860 ACCACAAATG CTACGCTGCA AACGCTGGTA CAATTTTACG AAAACTGCAA AAACGTCAAA 7920 ACTCGGTATA AAATAATCAA CGGGCGCTTT GGCAAAATAT CTATTTTATC GCACAAGCCC 7980 ACTAGCAAAT TGTATTTGCA GAAAACAATT TCGGCGCACA ATTTTAACGC TGACGAAATA 8040 AAAGTTCACC AGTTAATGAG CGACCACCCA AATTTTATAA AAATCTATTT TAATCACGGT 8100 TCCATCAACA ACCAAGTGAT CGTGATGGAC TACATTGACT GTCCCGATTT ATTTGAAACA 8160 CTACAAATTA AAGGCGAGCT TTCGTACCAA CTTGTTAGCA ATATTATTAG ACAGCTGTGT 8220 GAAGCGCTCA ACGATTTGCA CAAGCACAAT TTCATACACA ACGACATAAA ACTCGAAAAT 8280 GTCTTATATT TCGAAGCACT TGATCGCGTG TATGTTTGCG ATTACGGATT GTGCAA ACAC 8340 GAAAACTCAC TTAGCGTGCA CGACGGCACG TTGGAGTATT TTAGTCCGGA AAAAATTCGA 8400 CACACAACTA TGCACGTTTC GTTTGACTGG TACGCGGCGT GTTAACATAC AAGTTGCTAA 8460 CCGGCGGTTC GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA 8520 TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA 8580 GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA AACCTGTCGT 8640 GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT 8700 CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT 8760 CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA 8820 ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG TTGCTGGCGT 8880 TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT 8940 GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC 9000 GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA 9060 GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG GTCGTTCGCT 9120 CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA 9 180 ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG 9240 GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC 9300 CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG CGCTCTGCTG AAGCCAGTTA 9360 CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT GGTAGCGGTG 9420 GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA GAAGATCCTT 9480 TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA GGGATTTTGG 9540 TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA TGAAGTTTTA 9600 AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC TTAATCAGTG 9660 AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA CTCCCCGTCG 9720 TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA ATGATACCGC 9780 GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG 9840 AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG 9900 AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG 9960 GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT 10020 C AAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC 10080 CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC 10140 ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA 10200 CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG GCGTCAATAC 10260 GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA AAACGTTCTT 10320 CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG TAACCCACTC 10380 GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA 10440 CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA 10500 TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT 10560 ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA 10620 AAGTGCCACC TGACGTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT AAAAATAGGC 10680 GTATCACGAG GCCCTTTCGT CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA 10740 TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC AGACAAGCCC 10800 GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCTG GCTTAACTAT GCGGCATCAG 10860 AGCAGATTGT ACTGAGAGTG CACCATATGC GGTGTGAAAT ACCGCACAGA TGCGTAAGGA 10920 GAAAATACCG CATCAGGCGC CATTCGCCAT TCAGGCTGCG CAACTGTTGG GAAGGGCGAT 10980 CGGTGCGGGC CTCTTCGCTA TTACGCGCCAGC TGGCGATGACTC GGGATGTCGATC GGGATGTCGATC GGGATGTCAG

SEQ ID NO: 11 Sequence length: 9595 Sequence type: nucleic acid Number of strands: double-stranded Sequence type: other nucleic acid Plasmid sequence TCGAGCAGTT CGTTGACGCC TTCCTCCGTG TGG
CCGAACA CGTCGAGCGG GTGGTCGATG 60 ACCAGCGGG TGCCGCACGC GACGCACAAG TAT
CTGTACA CCGAATGATC GTCGGGCGAA 120 GGCACGTCGGG CCTCCAAGTG GCAATATTGG CAA
ATTCGAA AATATATACA GTTTGGGTTTGT 180 TTGCGCATAT CTATCGTGGC GTTGGGCATG TAC
GTCCGAA CGTTGATTTG CATGCAAGCC 240 GAAATTAAATCATTGCGATT AGTGCGATTA AAA
CGTTGTA CATCCCTCGCT TTTAATCATG 300 CCGTCGATTA AATCGCGCAA TCGAGTCAAG TGA
TCAAGT GTTGGAATAAT GTTTTCTTTG 360 TATTCCCGAG TCAAGCGCAG CGCGTTATTTT AAC
AAAACTAG CCATCTTGTA AGTTAGTTTC 420 ATTTAATGCA ACTTTATCCA ATAATATATT ATG
TATCGCA CGTCAAGAAT TAACAATGCG 480 CCCGTTGTCG CATCTCAACA CGACTATGAT AGA
GATCAAA TAAAGCGCGA ATTAAATAGC 540 TTGCGACGCA ACGTGCACGA TCTGTGCACG CGT
TCCGGCA CGAGCTTTGA TTGTAATAAG 600 TTTTTACGAA GCGATGATCAT GACCCCCGTA GTG
ACAACGA TCACGCCCAAAAGAACTGCC 660 GACTACAAAA TTACCGAGTA TGTCGGGTGAC GTT
AAAACTA TTAAGCCATC CAATCGACCG 720 TTAGTCGAAT CAGGACCCGCT GGTGCGAGAA GCC
GCGAAGT ATGGCGAATG CATCGTATAA 780 CGTGTGGAGGT CCGCTCCATTA GAGCGTCATG TTT
AGACAAG AAAGCTACCAT ATTTAATTGA 840 TCCCGATGAT TTTATTGATA AATTGACCCT AAC
TCCATAC ACGGTATTCT ACAATGGCGG 900 GGTTTTGGTC AAAATTTCCG GACTGCCGATT GTA
CATGCTG TTAACGGCTC CGCCCACTAT 960 TAATGAAATT AAAATTCCCA ATTTTAAAAAA ACG
CAGCAAG AGAACAATTTT GTATGAAAGA 1020 ATGCGTAGAA GGAAAGAAAAA ATGTCGTCGA CAT
GCTGAAC AACAAGATTA ATAGCCCTC 1080 GTGTATAAAA AAAATTATTGA ACGATTTGAA AGA
AAACAAT GTACCGCGCG GCGGTATGTA 1140 CAGGAAGAGG TTTACTACTAA ACTGTTACAT TGC
AAACGTG GTTTCGTGTG CCAAGGTGTGA 1200 AAAACCGATGT TTAATCAAGG CTCTGACGCA TTT
CTACAAAC CACGACTCCA AGTGTGTGGG 1260 TGAAGTCATG CATCTTTTAA TCAATCCCCA AGA
TGTGTAT AAAACCACCAA ACTGCCAAAA 1320 AATGAAAACT GTCGACAAGC TCTGTCCGGTTGC
TGGCAAC TGCAAGGGTC TCAATCCTAT 1380 TTGTAATTAT TGAATAATAA AACAAATTATA AAT
GCTAAAT TTGTTTTTTTA TTAACGATAC 1440 AAACCAAACG CAACAAGAAC ATTTGTAGTA TTA
TCTATAAA TTGAAAACGC GTAGTTTAAA 1500 TCGCTGAGGT AATATTTAAA ATCATTTTTCA AAT
GATTCAC AGTTAATTTG CGACAATATA 1560 ATTTTATTTT CACATAAACT AGACGCCTTG TCG
TCTCTCTT CTTCGTATTC CTTCCTCTTTTT 1620 TCATTTTTCT CCTCATAAAAA ATTAACATAG TTA
TTATCGT ATCCATATAT GTATCTATCG 1680 TATAGAGTAA ATTTTTTGTT GTCATAAATA TAT
AGTTCTT TTTTAATGGGG GTGTAGTAGTA 1740 CCGCTGCGCA TAGTTTTTCT GTAATTTACA ACA
GTGCTAT TTTCTGGTAG TTCTTCGGG 1800 TGTGTTGCTT TAATTATTAAA ATTTATAATAA TCA
ATGAATT TGGGATCGTC GGTTTTGTAC 1860 AATATGTTGC CGGCATAGTA CGCAGCTTTCT TCT
AGTTCAA TTACACCATT TTTTAGCAGC 1920 ACCGGATTAA CATAACTTTTC CAAAATGTTG TAC
GAACCGT TAAACAAAAA CAGTTCACCT 1980 CCCTTTTCTA TACTATTGTC TGCGAGCAGGT TGT
TGTTTGT TAAAAAATAAC AGCCATTGTA 2040 ATGAGACGCA CAAACTAATA TCACCAAACTG GAA
AGTTCTA TCAATADATA GTTGCTGATA 2100 TCATGGAGAT AATTAAAATG ATAACCCATCT CGC
AAAATAAA TAAGTATTTT ACGTTTTTCG 2160 TAACAGTTTT GTAATAAAAA AACCTATAAA TAC
GGATCTG TATTCATGTC CCCTATAGAT 2220 CCGATGGGAC ATCATCATCA TCATCACGGA AGG
AGAAGGG CCAGTGTTGC GGCGGGGAATT 2280 TTGGTCCCTC GTGGAAGCCC AGGACTCGAT GGC
ATATGCT CGAGATCCAT GTCTATTTCA 2340 TCTCTTTCAG GACCTGACAA TCAAAAAAAT ATC
ATGTTCTC AAGTTCTGAC ATCGACACCC 2400 CAGGGCGTGC CCCAACAAGA TAAGCTGTCT GGC
AACGAAA CGAAGCAAAT ACAGCAAACA 2460 CGTCAGGGGTA AAAACACTGA GATGGAAAGC GAT
GCCACTA TTGCTGGTGC TTCTGGAAAAA 2520 GACAAAACTTT CCCGACTAC AAAAAACAGAAAACA
GCTCCAC AACAGGGAG TGCTGCTGGGG 2580 AAAGAATCCT CAGAAAGTCA AAAGAGCAGGT GCT
GACTCT GAGTATCAG AGCGGCTGCT 2640 ACTACAGCAT CAAATACTGC AACAAAAATT GCT
ATGCAGA CCCTATTGA AGAGCGGAGC 2700 AAAAGTATGGG AGTCTACCTT AGATCACTTT CAA
AGCCTCA GTGCCGCGCA AATGAAAGAA 2760 GTCGAAGCGG TTGTTTGTTGC TGCCCTCTCA GGG
AAAAGTT CGGGCTCCGC AAAATTGGAA 2820 ACACCTGGAC TCCCCAAGCC CGGGGTGACA CCA
AGATCAG AGGTTATCGA AATCGGACTC 2880 GCGCTTGCTA AAGCAATTCA GACATTGGGGA GAA
GCCACAA AATCGTCCT ATCTACTACTAT 2940 GCAAGTACAC AAGCACAAGC AGACCAAAACA AAT
AAAACTAG GTCTAGAAAA GCAAGCGATA 3000 AAAATCGATA AAGAACGAGA AGAATACCAAA GAG
ATGAAGG CTGCCGAACA GAAGTCTCAAA 3060 GATCTCGAAG GAACAATGGA TACTGTCAAT ACT
GTGATGGA TCGCGGTTTTC TGTTGCCATT 3120 ACAGTTATTT CTATTGTTGC TGCTATTTTTT ACA
TGCGGAG CTGGATCCGC TGGACTCGCT 3180 GCGGGAGCTG CTGTAGGTGC AGCGGCAGCT GGA
GGTGCAG CAGGAGCTGC TGCCGCAACC 3240 ACGGTAGCAA CACAAATTAC AGTTCAAGCT GTT
GTCCAAG CGGTGAAACA AGCTGTTATC 3300 ACAGCTGTCA GACAAGCGAT CACCGCGGCT ATA
AAAGCGG CTGTCAAATC TGGAATAAAA 3360 GCATTTATCA AAACTTAGT CAAAGCGGATT GCC
AAAGCCA TTTCTAAAGG AATCTCTAAG 3420 GTTTTCGCTA AGGGAACTCA AATGATTGGCG AAG
AACTTCC CCAAGCTCTC GAAAGTCATC 3480 TCGTCTCTTA CCAGTAAATG GGTCACGGGTTT GGG
GTTGGGGG TTGTAGTTGC GGGCCCTGCT 3540 CTCGGGTAAAG GGATTATGCCA AATGCAGCTC TCG
GAGATCGC AACAAAAGCGT CGCTCAATTT 3600 CAGAAAGAAG TCGGAAAAACT GCAGGCTGCG GCT
GATATGA TTTCTATGTTT CACTCAATTT 3660 TGGCAACAGG CAAGTAAAAT TGCCTCAAAA CAA
ACAGGCG AGTCTAATGA AATGACTCAA 3720 AAAGCTCCA AGCTGGGCGCTCAAATCCTT AAA
GCGTATAG CCGCAATCAG CGGGACCATC 3780 GCTGGCGCAG CATAAAAGCT AGAATTTCGA GGA
ATTCAGG CCCTCATGGGG AGCTCGCGGC 3840 CGCCTGCAGGGTACCCCCGG GAGATCTGTA CCG
ACTCTGC TGAAGAGGAG GAAATTCTCC 3900 TTGAAGTTTC CCTGGTGTTC AAAGTAAAGGG AGT
TTGCACC AGAGCACCCT CGTTCACTG 3960 GTCCGGCGTA TTAAAACACG ATACATTGTT ATT
AGTACAT TTATTAAGCG CTAGATTCTG 4020 TGCGTTGTTG ATTTACAGAC AATTGTTGTGTA CGT
ATTTTAA TAATTCATTA AATTTATAAT 4080 CTTTAGGGTG GTATGTTAGA GCGAAAATCA AAT
GATTTTC AGCGTCTTTA TATCTGAATT 4140 TAAATATTAA ATCCTCAATA GATTTGTAAA ATA
GGTTTCG ATTAGTTTCA AACAAGGGGTT 4200 GTTTTTCCGA ACCGATGGGCT GGACTATCTTA ATG
GATTTTC GCTCAACGCC ACAAAACTTG 4260 CCAAAATCTTG TAGCAGCAAT CTAGCTTTGT CGA
TATTCGT TTGGTTTTTTG TTTTGTAATA 4320 AAGGTTCGAC GTCGTTCAAA ATATTATGCCG CTT
TGTATTTCTTTTCATCA CTGTCGTTTAG 4380 TGTACAATTG ACTCGACGTA AACACGTTAA ATA
AAGCTTG GACATATTTA ACATCGGGCG 4440 TGTTAGCTTT ATTAGGCGA TTATCGTCGT CGT CGT
CCCAACC CTCGTCGTTA GAAGTTGCTT 4500 CCGAAGACGA TTTTGCCATA GCCACACGAC GCC
TATTAAT TGTGTCGGCT AACACTCCG 4560 CGATCAAATT TGTAGTTGAG CTTTTTTGGAA TTA
TTTCTGA TTGCGGGCGT TTTTGGGCGG 4620 GTTTCAATCT AACTGTGCCC GATTTTTAATT CAG
ACAACAC GTTAGAAAGC GATGGTGCAG 4680 GCGGTGGTAA CATTTCAGAC GGCAAATCTA CTA
ATGGCGG CGGTGGTGGGA GCTGATGATA 4740 AATCTACCAT CGGTGGAGGC GCAGGGGGGG CTG
GCGGCGG AGGCGGAGGC GGAGGTGGTG 4800 GCGGTGATGC AGAGGCGGTTTAGGCTCAATG
TCTCTTT AGGCAACCA GTGCGCACCT 4860 CAACTATTGT ACTGGTTTCG GGCGCCGTTT TTG
GTTTGAC CGGTCTGAGA CGAGTGCGAT 4920 TTTTTTCGTT TCTAATAGCT TCCAACAAATT GTT
GTCTTGTC GTCTAAAGGT GCAGCGGGTT 4980 GAGGTCCCGT CGGCATTGGT GGAGCGGGCGCG GCA
ATTCGA CATCGATGGGT GGTGGTGGGTG 5040 GTGGAGGCGC TGGAATGTTA GGCACGGGAG AAG
GTGGTGG CGGCGGGTGCC GCCGGTATAA 5100 TTTGTTCTGG TTTAGTTTTGT TCGCGCACGA TTG
TGGGCAC CGGCGCAGGC GCCGCTGGCT 5160 GCACACCGGA AGGTCGTCTG CTTCGAGGCA GCG
CTTGGGGG TGGTGGCAAT TCAATATTAT 5220 AATTGGAATA CAAATCGTAA AAATCTGCTA TAA
GCATGT AATTTCGCTA TCGTTTACCG 5280 TGCCGATTATT TAACACCACCGCTCAATGTAAG CAA
TGTATT GTAAAGAGAT TGTCTCAAGC 5340 TCGGATCGAT CCCGCACGCC GATAACAAGC CTT
TTCATTT TTACTACAGC ATTGTAGTGGG 5400 CGAGACACTT CCGCTGTCGTC GACGTACATG TAT
GCTTTTGT TGTCAAAAC GTCGTTGGCA 5460 AGCTTTAAAA TATTTAAAAG AACATCTCTG TTC
AGCACCA CTGTGTTGTC GTAATGTTTG 5520 TTTTTGATAA TTTGCGCTTC CGCAGTATCG ACA
CGTCTAAAAATTGATG CGCATCAATT 5580 TTGTTGTTCC TATTATTGAA TAAAATAAGAT TGT
ACAGATT CATATCTACG ATTCGTTCATG 5640 GCCACCACAAATGCTACCGCT GCAAACGCTG GTA
CAATTTT ACGAAAACTG CAAAAACGTC 5700 AAAACTCGGT ATAAAATAAT CAACGGGCGC TTT
GGCAAATATCTTATTTT ATCGCACAAG 5760 CCCTAGAGCA AATTGTATTT GCAGAAAACA ATT
TCGGCGC ACAATTTTA CGCTGACGAA 5820 ATAAAAGTTTC ACCAGTTTAAT GAGCGAACCAC CCA
AATTTTTA TAAAAATCTA TTTTAATCAC 5880 GGTTCCATCA ACAACCAAGT GATCGTGATG GAC GAC
TACATTG ACTGTCCCGA TTTATTTTAA 5940 ACACTACAAAA TTAAAGGGCGA GCTTTCGTAC CAA
CTTGTTTA GCAATAATTAT TAGACAGCTG 6000 TGTGAAGCGC TCAACGATTTT GCACAAGCAC AAT
TTCATAC ACAACGACAT AAAACTCGAA 6060 AATGCTTAT ATTTCGAAGC ACTTGATCGC GTG
TATGTTTT GCGATCTACGG ATTGTGCAAA 6120 CACGAAACT CACTTAGCGT GCACGACGGC ACG
TTGGAGT ATTTTAGTCC GGAAAAAATT 6180 CGACACAAA CTATGCACGT TTCGTTTGAC TGG
TACGCGG CGTGTTAACA TACAAGTTGC 6240 TAACCGGCGG CCGACACCCA TTTGAAAAAA GCG
AAGACGA AATGTTGGAC TTGAATAGCCA 6300 TGAAGCGTCCG TCAGCAATAC AATGACATTG GCG
TTTTAAA ACACGTTCGT AACGTTAACG 6360 CTCGTGACTT TGGTTACTGC CTAACAAGAT ACA
ACATGA TTGTAGACTC ACAAATACCA 6420 AACAAATTAT AAAACATGAG TTTTTGTTCGT AAA
AATGCCA CTTGTTTTTAC GAGTAGAATT 6480 AGCTTGGCAC TGGCCGTTCGT TTTACAACGT CGT
GACTGGGG AAAACCCTGG CGTTACCACA 6540 CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCC
AGCTGGC GTAATAGCGA AGAGGCCCGC 6600 ACCGATCCGCC CTTCCCCAACA GTTGCGCAGC CTG
AATGGCG AATGGCGCCT GATGCGGTAT 6660 TTTCTCCTTA CGCATCTGTG CGGTATTTCA CAC
CGCATAC GTCAAAGCAA CCATAGTACG 6720 CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTG
TGGTGGT TACGCGCAGC GTGTACCGCTA 6780 CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCG
CTTTCTT CCCTTCCTTT CTCGCCACGT 6840 TCCGCGGCTT TCCCCGTCAA GCTCTAAATC GGG
GGCTCCC TTTAGGGTTC CGATTTAGTG 6900 CTTTACGGCA CCTCACCCCAAAAAAACTTG ATT
TGGGTGA TGGTTCACGT AGTGGGCCAT 6960 CGCCCTGATA GACGGTTTTT CGCCCTTTTGA CGT
TGGAGTC CACGTTCTTT AATAGTGGAC 7020 TCTTGTTCCA AACTGGAACA ACACTCAACC CTA
TCTCGGGG CTATTCTTTTT GATTTATAAG 7080 GGATTTTGCCC GATTTCGGCC TATTGGTTAA AAA
ATGAGCT GATTTAACAA AAAATTTAACG 7140 CGAATTTTAA CAAAATATTA ACGTTTACAA TTT
TATGGGTG CACTCTCAGT ACAATCTGCT 7200 CTGATGCCGC ATAGTTAAGC CAGCCCCGAC ACC
CGCCAAC ACCCGCTGAC GCGCCCTGAC 7260 GGGCTTGTCT GCTCCCGGCA TCCGCTTACA GAC
AAGCTGT GACCGTCTCC GGGAGCTGCA 7320 TGTGTCAGAG GTTTTCACCG TCATCACCGA AAC
GCGCGAG ACGAAAGGGC CTCGGTGATAC 7380 GCCTATTTTA TAGGTTTAAT GTCATGATAAA TAA
TGGTTTC TTAGACGTCA GGTGGCACTT 7440 TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTT
TATTTTTT CTAATACAT TCAAAATAGTGT 7500 ATCC GCTCAT GAGACAATAA CCCTGATAAA TGC
TTCAATA ATATTGAAAA AGGAAGAGTA 7560 TGAGATTACA AATTTCCCGT GTCGCCCTTA TTC
CCTTTTT TGCGGGCATTT TGCCTTCCTG 7620 TTTTTGCTCA CCCAGAAACG CTGGTGGAAAG TAA
AAGATGC TGAAGATCAG TTGGGTGCAC 7680 GAGTGGGTTA CATCGGAACTG GATCTCCAACA GCG
GTAAGAT CCTTGAGAGT TTTCGCCCCG 7740 AAAACGTTT TCCAATGATG AGCACTTTTA AAG
TTCTGCT ATGTGGCGCG GTATTATCCC 7800 GTATTGACGC CGGGCAAGAG CAACTCGGGTC GCC
GCATACA CTATTCTCAG AAT GACT TGG 7860 TTGAGTACTC ACCAGTCACA GAAAAGCATC TTA
CGGATGG CATGACAGTA AGAGAATTAT 7920 GCAGTGCTGC CATAACCCATG AGTGATAACA CTG
CGGCCAA CCTACTTCTG ACAACGATCCG 7980 GAGGACCGAA GGGCTACACC GCTTTTTTGC ACA
ACATGGGG GGATCATTGTA ACTCGCCTTG 8040 ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TAC
CAAACGA CGAGCGTGAC ACCACGATCGC 8100 CTGTAGCAAT GGCAACAACG TTGCGCAAAAC TAT
TAACTGG CGAACTACTT ACTCTAGCTT 8160 CCCGGGCAACA ATTAATAGAC TGGATGGAGG CGG
ATAAAGT TGCAGGCACCA CTTCTGCGCT 8220 CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATA
AATCTGG AGCCGGTGAG CGTGGGTTCTC 8280 GCGGTATCAT TGCAGCACTG GGGCCCAGATG GTA
AGCCCTC CCGTATCGTA GTTATCTACA 8340 CGACGGGGAG TCAGGCACT ATGGATGAAC GAA
ATAGACA GATCGCTGAG ATAGGTGCCT 8400 CACTGATTAA GCATTGGTAA CTGTCAGAACC AAG
TTTACTC ATATAACTTT TAGATTGATT 8460 TAAAACTTCA TTTTTAATTT AAAAGGATCT AGG
TGAAGAT CCTTTTTGAT AATCTCATGA 8520 CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACT
GAGCCGTCAGACCCCGTA GAAAAGATCA 8580 AAGGATCTCTC TTGAGATCCT TTTTTTCTGC GCG
TAATCTG CTGCTTGCAA ACAAAAAAAC 8640 CACCGCTACC AGCGGTGGTT TGTTTGCCGGG ATC
AAGAGCT ACCAACTCTT TTTCCGAAGG 8700 TAACTGGCTT CAGCAGAGCG CAGATACCAAA ATA
CGTTCCT TCTAGTGTAG CCGTAGTTAG 8760 GCCACCACT CAAGAACTCT GTAGCACCGC CTA
CATACCCT CGCTCTGCTA ATCCTGGTTAC 8820 CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTC
TTACCGG GTTGGACTCA AGACGATAGT 8880 TACCGGATAA GGCGCAGCCG TCCGGGCTGAA CGG
GGGGTTC GTGCACACAG CCCAGCTTTGG 8940 AGCGAACGAC CTACACCGAA CTGAGATACC TAC
AGCGTGA GCTATGAGAA AGCGCCACGC 9000 TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGG
TAAGCGG CAGGGTCGGA ACAGGAGAGC 9060 GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGT
ACTTTTA TAGTCCTGTC GGGTTTCGCC 9120 ACCTCTACT TGAGCGTCGA TTTTTGTGAT GCT
CGTCAGG GGGGCGGAGC CTATGGAAAA 9180 ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGG
CCTTTTG CTGGCCTTTTT GCTCACATGT 9240 TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATA
ACCGTAT TACGCCTTT GAGTGAGCTG 9300 ATACCGCTCCG CCGCAGCCGA ACGACCGAGC GCA
GCGAGTTC AGTGAGCGAG GAAGCGGAAG 9360 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGC
GTTGGGCC GATTCATTAA TGCAGCTGGC 9420 ACGACAGGTT TCCCGACTGG AAAGCGGGGCA GTG
AGCGCAA CGCAATTTAAT GTGAGTTAGC 9480 TCACTCATTA GGCACCCCAG GCTTTACACT TTA
TGCTTCC GGCTCGTATG TTGTGTGGAA 9540 TTGTGAGCCG ATAACAATTT CACACAGGAA ACA
GCTATGA CCATGATTAC GAATT 9595

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a process for producing a recombinant baculovirus of the present invention.

FIG. 2 is a diagram of a Western blot of the fusion protein produced in Example 4.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/02 G01N 33/531 A 21/08 33/571 G01N 33/531 A61K 39/395 D 33/571 C07K 14/295 / / A61K 39/395 C12N 5/00 B C07K 14/295 15/00 ZNAA (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (54) [Title of the invention] Histidine tag-Chlamydia Pneumonie antigen polypeptide fusion protein, method for producing the same, DNA encoding the fusion protein, recombinant vector containing the DNA, transformant containing the recombinant vector, recombinant baculovirus, method for producing the same, and anti-Chlamydia pneumoniae D Antibody production method

Claims (14)

[Claims] 1. A polypeptide comprising a sequence comprising at least 5 consecutive amino acids in the peptide of SEQ ID NO: 1 directly or via an amino acid sequence to a peptide comprising at least 6 consecutive histidines A histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein comprising: 2. The fusion protein according to claim 1, wherein the amino acid sequence is a peptide represented by SEQ ID NO: 2. 3. A DNA encoding the fusion protein according to claim 1 or 2, or a DNA complementary thereto. 4. The DNA according to claim 3, which has the nucleotide sequence of SEQ ID NO: 3. A recombinant vector comprising the DNA according to claim 3 or 4. 6. A method comprising: (a) a DNA encoding a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein; and (b) a promoter sequence; and (c) a DNA located upstream of the DNA of (a).
6. The recombinant vector according to claim 5, comprising a DNA capable of causing homologous recombination with the baculovirus DNA, downstream of the DNA of (a) and upstream of the DNA of (b). 7. The pHLTCPN53 plasmid. 8. A transformant containing the recombinant vector according to claim 5 or 6. 9. A transformant deposited under accession number FERM P-16036. 10. A baculovirus DNA comprising (a) a DNA encoding a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein and (b) a promoter sequence, and incorporating a DNA located upstream of the DNA of (a). A recombinant baculovirus consisting of: 11. A method of introducing the recombinant vector and the baculovirus DNA according to claim 6 into insect cells, thereby causing homologous recombination between the recombinant vector and the baculovirus DNA in insect cells. A method for producing a recombinant baculovirus. 12. The pHLTCPN53 plasmid and baculovirus DNA are introduced into insect cells, whereby:
A method for producing a recombinant baculovirus, comprising causing homologous recombination between the plasmid and baculovirus DNA in insect cells. 13. Infecting insect cells with the recombinant baculovirus according to claim 10, culturing the insect cells,
A method for producing a histidine tag-Chlamydia pneumoniae antigen polypeptide fusion protein, comprising obtaining a cell lysate or a cell lysate of cultured insect cells. 14. An anti-Chlamydia cerevisiae, wherein the fusion protein according to claim 1 or 2 is used as an antigen.
A method for producing a pneumoniae antibody.