JPH11192089A - Syphilis treponema-fused dna sequence and expression of syphilis treponema antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new sequence, wherein a DNA sequence encoding a signal peptide-added syphilis treponema surface antigen is fused with a DNA sequence encoding a secretion-related enzyme, affording an antigen useful as e.g. an immunoassay reagent enabling highly sensitive assay. SOLUTION: This sequence is a new syphilis treponema-fused DNA sequence, wherein, a DNA sequence encoding a signal peptide-added syphilis treponema surface antigen is fused with a DNA sequence encoding a secretion-related enzyme, and useful for the production of a large amount of a lipid-bound and highly reactive syphilis treponema antigen. By using this antigen as an immunoassay reagent, the syphilis treponema can be assayed in a more specific and highly sensitive way. This DNA sequence is obtained by specifically amplifying the DNA sequence for the signal-syphilis treponema antigen in the native DNA sequence for syphilis treponema, through a PCR technique using a primer and fusing the resultant DNA sequence for the signal-syphilis treponema antigen with the DNA sequence for a secretory enzyme through e.g. ligation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA sequence fused with Treponema pallidum and a method for expressing a Treponema pallidum antigen using the DNA sequence.

The Treponema pallidum fusion DNA sequence of the present invention is obtained by fusing a DNA sequence encoding a surface antigen of Treponema pallidum to which a signal peptide is added and a DNA sequence encoding a secretion-related enzyme. In addition, the method of expressing the syphilitic treponemal antigen of the present invention is to perform expression using the syphilis treponemal fusion DNA sequence of the present invention described above. An antigen having high reactivity in measurement can be easily obtained in a large amount.

[0003]

2. Description of the Related Art Syphilis is known as Treponema pallidum (Treponema
Paridam Treponema pallidum, hereinafter referred to as Tp. )
Is an infection caused by Tp is a kind of spirochetes, and it has been confirmed that several types of surface antigens exist on the cell surface as Tp antigens, and the main ones are molecular weights of 15 kDa, 17 kDa, 42 kDa, and 47 kDa.
Antigens such as Da are known (The Journal of Immunol
ogy, Vol.129, p.833-838, 1982; The Journal of Immu
nology, Vol.129, p.1287-1291, 1982; Journalof Clin
ical Microbiology, Vol. 21, p. 82-87, 1985; Journal
of Clinical Microbiology, Vol. 30, p. 115-122, 1992)
.

[0004] These antigens have been identified by Norris et al.
N15, TpN17, TpN44.5 (a), TpN4
7 (Microbiological. Reviews, Vol.
57, p.750-779) (hereinafter TpN47 is 47K, TpN
17 is called 17K and TpN15 is called 15K. ). Genes encoding these surface antigens have already been cloned, and antigens have been produced by genetic engineering. In addition, their amino acid sequences have been determined (Molecula
r Microbiology, Vol. 4, p. 1371-1379, 1990; Infectio
n and Immunity, Vol.61, p.1202-1210, 1993; Journal
of Bacteriology, Vol.162, p.1227-1237, 1992; Infe
ction and Immunity, Vol.57, p.3708-3714, 1989).

[0005] When infected with Tp, antibodies against these antigens are produced in the blood. Therefore, whether or not the cells are infected with syphilis is determined by examining the blood for the presence of anti-Tp antibodies.

In order to examine the presence or absence of an anti-Tp antibody, an immunoassay method utilizing an antigen-antibody reaction between a Tp antigen and an anti-Tp antibody in a patient's blood is generally used. Although a large amount of anti-Tp antigen is required to perform an immunoassay for an anti-Tp antibody, recent advances in genetic engineering techniques have made it possible to obtain a Tp antigen artificially by cloning the Tp antigen.
Mass production of p antigen has become possible (Scie
nce, Vol.216, p.522-523, 1982; Infection and Immun
ity, Vol. 36, p. 1238-1241, 1982; Infectionand Immun
ity, Vol. 41, p. 709-721, 1983; Infection and Immunit
y, Vol. 42, p. 435-445, 1983; Infection and Immunit
y, Vol.42, p.187-196, 1983; Journalof Bacteriolog
y, Vol.162, p.1227-1237, 1985; Infection and Immun
ity, Vol. 54, p. 500-506, 1986; Infection and Immuni
ty, Vol. 56, p. 71-78, 1988; Infection and Immunity,
Vol.57, p.2612-2623, 1989; Infection and Immunit
y, Vol. 57, p. 3708-3714, 1989; Molecular Microbiolog
y, Vol.4, p.1371-1379, 1990; Infection and Immunit
y, Vol.58, p.1697-1704, 1990; Infection and Immuni
ty, Vol.61, p.1202-1210, 1993;
03).

[0007] The method for producing the Tp antigen can be performed by a general method in genetic engineering. That is, first, a target DNA sequence is cloned, the obtained DNA sequence encoding the Tp antigen is inserted into a vector, and then the vector is introduced into a host cell. And
A large amount of Tp antigen has been obtained by culturing and expressing the host cell into which the vector has been introduced. Also, a Dp encoding a Tp antigen having a unique signal peptide at the N-terminus
When the NA sequence was used, due to the action of a secretion-related enzyme possessed by the host cell, a considerable amount of a lipid-bound Tp antigen was produced, unlike the above-mentioned Tp antigen.

[0008]

However, the above-mentioned Tp antigen obtained in large quantities has lower reactivity than the natural antigen obtained from the cells, and the Tp antigen to which lipids are bound has lower reactivity. High but only small quantities were obtained, which was not sufficient for industrial use. Currently, syphilis can be sufficiently treated with antibiotics, and it is desired that the infection be diagnosed accurately and treated early. for that reason,
There has been a need for a method that can measure with higher sensitivity and specificity. Accordingly, an object of the present invention is to provide a fusion DNA sequence which can be used to obtain a lipid-bound Tp antigen exhibiting higher sensitivity and specificity than before, and a lipid-bound Tp antigen by using the DNA sequence. The purpose is to provide a method for expressing a large amount.

[0009]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that, using genetic engineering techniques, a DNA sequence encoding a Treponema pallidum antigen to which a signal peptide is added and a DNA sequence encoding a secretion-related enzyme of a protein. A method for efficiently obtaining a lipid-bound immunoreactive Tp antigen by expressing using a fusion DNA sequence obtained by combining the above and simultaneously expressing the respective proteins, and completing the present invention. Reached.

Hereinafter, the present invention will be described in detail. That is, the present invention provides a DNA sequence encoding a Tp antigen to which a signal peptide is added as described above (hereinafter referred to as a signal sequence).
It is called Tp antigen DNA sequence. ) And a DNA sequence encoding a secretion-related enzyme of a protein (hereinafter referred to as a secretory enzyme DNA sequence) and a syphilis treponema fusion DNA sequence (hereinafter referred to as a Tp fusion DNA sequence).

In the Tp fusion DNA sequence obtained by fusing the signal-Tp antigen DNA sequence and secretory enzyme DNA sequence of the present invention, the DNA sequence encoding the signal peptide is T
Since it is essential to bind to the DNA sequence encoding the p antigen, it is preferable to obtain the signal-Tp antigen DNA sequence first to obtain the Tp fusion DNA sequence. The signal-Tp antigen DNA sequence is composed of a signal peptide, T-terminal from the N-terminal side to the C-terminal side of the expressed protein.
D encoding each of them so that they are expressed in the order of p antigens
The NA sequence is arranged.

As a method for obtaining the fusion DNA sequence, P
CR method, recombinant PCR method, ligation method,
A linker ligation method and the like can be mentioned.

For example, when the PCR method is used, the signal-Tp antigen DNA sequence in the natural Tp DNA sequence is amplified by using a primer to amplify only the signal-Tp antigen DNA sequence, thereby obtaining a signal-Tp antigen DN.
A sequence can be obtained.

When the recombinant PCR method is used, a signal DNA sequence and a Tp antigen DNA sequence are separately obtained, and one of the DNA sequences is complementary to the other DNA sequence on the side to which the other is to be bound. By synthesizing the sequence and mixing both to perform PCR, the signal-
A Tp antigen DNA sequence can be obtained.

Furthermore, a signal-Tp antigen DNA sequence can be obtained by chemically synthesizing a DNA sequence.

Next, the obtained signal-Tp antigen DNA
Fusion DNA obtained by fusing a DNA sequence with a secretory enzyme DNA sequence
Sequence (in this case, the DNA sequence encoding the secretory enzyme may be either the 5 'end or the 3' end of the signal-Tp antigen DNA sequence, or may be linked to both sides when there are a plurality of secretory enzymes. Can be performed by the same operation using the above-mentioned recombinant PCR method, ligation method, linker ligation method and the like. The Tp fusion DNA sequence can also be obtained by chemically synthesizing a DNA sequence as described above.

Further, as another method for obtaining a Tp fusion DNA sequence in which a signal-Tp antigen DNA sequence and a secretory enzyme DNA sequence are fused, DNA sequences encoding the respective sequences are sequentially introduced into a vector using restriction enzymes. Tp fusion DNA sequences arranged in any order can also be obtained.

The method of expressing the Tp antigen of the present invention can be carried out by a method generally used for the Tp fusion DNA sequence obtained as described above. For example, a protein can be synthesized by incorporating the fusion DNA sequence into a vector by a ligation method or the like, introducing the obtained vector into a host cell, then culturing the host cell and inducing expression.

By performing this expression, a Tp antigen to which a signal peptide is added (hereinafter, referred to as a signal-Tp antigen) and a secretion-related enzyme (hereinafter, referred to as a secretory enzyme) are synthesized on the ribosome. The synthesized signal-Tp antigen is efficiently transported onto the membrane by the action of the simultaneously synthesized secretory enzyme and passes through the membrane. Thereafter, the lipid is added to the Tp antigen and the signal peptide is cleaved on the membrane of the host cell to obtain a lipid-bound Tp antigen.

The signal peptide is a peptide essential for permeation of a protein into a cell membrane in the above expression method.
Consists of ~ 30 amino acid residues. Near the N-terminus, there is a positive charge due to a basic amino acid residue, and as the number of positive charges increases, the rate of membrane permeation increases (J. Biol. Che.
m., Vol. 265, 2873-2880, 1990; J. Biol. Chem., Vol.
267, 4882-4888, 1992; J. Biol. Chem., Vol. 267, 123.
75-12379, 1992). Also, there is a wide hydrophobic region rich in hydrophobic amino acid residues in the center of the signal peptide,
Increasing its hydrophobicity increases the efficiency of membrane permeation (J. Bioenerg. Biomember., Vol. 22, 233-169, 1991;
J. Biol. Chem., Vol. 265, 4358-4363, 1990). Therefore, the number of positive charges at the N-terminus and the degree of hydrophobicity are important factors in order to increase the membrane permeation initiation efficiency, and it is preferable that the number of positive charges is large and the degree of hydrophobicity is high.

For binding lipids, the four amino acids on the C-terminal side of the signal peptide are Leu-XY-Cys.
Are preferable in terms of their efficiency (Microbia
l Pathogenesis, Vol. 7, 175-188, 1989). In the amino acid sequence, X and Y may be the same amino acid,
They may be different, and their types are not particularly limited. T
When the signal peptide is cleaved from the p antigen, the bond between Y and Cys in the amino acid sequence is cleaved.

The enzyme relating to secretion in the present invention has a function of transferring a Tp antigen to which a signal peptide which is a precursor of a lipid-bound Tp antigen is added, onto the membrane of a host cell,
These are individual enzymes that have the function of passing through a membrane.

For example, when the host cell is Escherichia coli, secretory enzymes are SecA, SecB, SecD, and Se.
Sec enzymes such as cE, SecF, SecG, and SecY. When secreting the Tp antigen in this system, S
It is preferable that ecA, SecE, and SecY enzymes are present. In order to increase the membrane permeation rate and further promote secretion, it is more preferable that SecG is further present in these enzymes.

The DNA sequence encoding the signal peptide, Tp antigen and secretion-related enzyme used in the Tp fusion DNA sequence of the present invention is a DNA sequence described in the Sequence Listing of this specification.
Even if one or several DNAs of the NA sequence or the known DNA sequence are deleted, substituted or added, the DNA used in the present invention as long as the functions of the expressed proteins are substantially the same. Included in the array.

[0025]

EXAMPLES The present invention will be described in more detail with reference to Examples and Examples.

Reference Example 1 Treponema pallidu
m, Nicols strain) were subcultured from rabbit testes and purified by Percoll density centrifugation (Sex. Transm. Dis.,
Vol. 11, p. 275-286, 1984) and centrifuged again to separate the precipitate from 50 mM Tris-Cl (pH 8.5), 5 mM
M EDTA, 3% OTG (n-octyl-β-D-
thioglucoside) and eluted syphilis antigen overnight. After centrifugation, the supernatant is taken and subjected to Superdex 200 at 10 mM phosphoric acid (pH 7.0).
A buffer of 0.15 M NaCl, 0.5% OTG was flowed to fractionate. The fraction containing 47K was applied to hydroxyapatite, and 10 mM phosphoric acid (pH 7.0), 0.3 mM
A buffer of% OTG was flowed to fractionate. The pass fraction was further adjusted to pH 9.7 with 20 mM Tris-Cl and applied to hydroxyapatite. The pass fraction was collected, concentrated and used for the following experiments. In addition, the syphilis antigen 47K (hereinafter referred to as 47C2) which does not contain genes encoding the Shine-Dalgarno sequence and the signal sequence at the 5 'end of the DNA fragment.
Called. ) Was prepared. This sequence is different from the DNA fragment encoding 47K prepared in Reference Example 5 in that it does not contain the above two sequences, and therefore no addition of lipid occurs and the native antigen n
-47K does not occur. This gene was inserted into the expression vector pW6A prepared in Reference Example 3. This vector is called pW
Named 6A-47C2. pW6A-47C2 was introduced into Escherichia coli strain BL21 (DE3), and the cells were cultured.
Fusion antigen 1 mM IPTG (Isopropyl-1-
thio-β-D-galactopyranosid
After e) was added, expression was induced, cells were disrupted, and centrifugation was performed. Purification was carried out according to the method of Norgard et al.
INFECTION AND IMMUNITY, Ap
r. 1992, pp 1568-1576) and purified with hydroxyapatite. Native 47K and recombinant antigen 47C2 having no lipid at the N-terminal were applied in an amount of 0.2 μg each, and subjected to reducing SDS electrophoresis on a 12.5% polyacrylamide gel. Next, Western blot was performed using a 500-fold diluted human TPPA-positive serum as a primary antibody. The results are shown in FIG. In all 11 sera, the native antigen 47K extracted from the syphilis subcultured in rabbit testis subculture was the recombinant antigen 47C2
The reactivity was better than that.

Reference Example 2 Preparation of Anti-47K Monoclonal Antibody and Anti-17K Monoclonal Antibody GS at the N-terminal of 47K and 17K as Tp surface antigen
Each of GST47K and GST17K fused with T was expressed in E. coli and purified. Spleen cells collected from mice immunized with the obtained recombinant antigen were fused with mouse myeloma cells to prepare hybridomas. The hybridomas were cultured, and hybridomas that reacted with the recombinant antigens and also reacted with the native antigens were screened by ELISA using the above-mentioned recombinant antigens and Western blot using the native antigens. The screened hybridomas were cloned to establish cell lines. After injecting the established clone into the mouse intraperitoneal cavity to prepare ascites, the ascites is collected and purified to obtain an anti-47K monoclonal antibody (hereinafter, referred to as 47KMab) and an anti-17K monoclonal antibody (hereinafter, referred to as 17KMab). Obtained.

(Reference Example 3) Preparation of Expression Vector pW6A The nucleotide sequence encoding the tac promoter and GST was removed from the vector pGEX2T manufactured by Pharmacia, and ge was removed from the T7 promoter of pGEMEX-1 manufactured by Promega.
Ne10, the multiple cloning site, and the nucleotide sequence up to the T7 transcriptional terminator were inserted. The nucleotide sequence of gene10 was removed from the obtained vector, and a lac operator synthesized using a DNA synthesizer (Model 381A manufactured by Applied Biosystems) was inserted immediately after the T7 promoter. The expression vector thus obtained was named pW6A. A detailed diagram of pW6A is shown in FIG.

(Reference Example 4) Preparation of Primers for Producing Various Fusion Antigen Expression Vectors In order to prepare the vectors for expressing each fusion antigen prepared in Examples 1 and 3 below, the following primers were used. It was synthesized using a DNA synthesizer (Model 381A manufactured by Applied Biosystems).

5 '------------------------------------ 3' Nde I 47K-5 'end 24 bases Primer 1 GTGTA CATATG AAAGTGAAATACGCACTACTTTCT (35 bases)

BamHI 47K-3 ′ terminal 24 bases Primer 2 GACACC GGATCC CTACTGGGCCACTACCTTCGCACG (36 bases) (antisense)

NdeI 17K-5 ′ terminal side 22 bases Primer 3 TCT CATATG AAAGGATCTGTCCGCGCGCTGT (31 bases)

BamHI 17K-3 ′ terminal 24 bases Primer 4 CAG GGATCC CTATTTCTTTGTTTTTTTGAGCAC (33 bases) (antisense)

BamHI SecA-5 ′ terminal side 18 bases Primer 5 TTT GGATCC CTCCGCAACGCGGGGCGT (27 bases)

XbaI SecA-3 ′ terminal 22 bases Primer 6 GT TCTAGA TTATTGCAGGCGGCCATGGCAC (30 bases) (antisense)

XbaI SecY-5 ′ terminal 21 bases Primer 7 CG TCTAGA CTGGCGGTAAAATCGAGGAAT (29 bases)

NotI SecY-3 ′ terminal 22 bases Primer 8 GG GCGGCCGC TTATCGGCCGTAGCCTTTCAGG (32 bases) (antisense)

NotI SecE-5 ′ terminal 20 bases Primer 9 GG GCGGCCGC TTTGTAGAAAACTTCTGACA (30 bases)

EcoT22I SecE-3 ′ terminal 20 bases Primer 10 CA ATGCAT TCAGAACCTCAGGCCAGTGA (28 bases) (antisense)

BamHI SecG-5 ′ terminal 20 bases Primer 11 ATTGG GGATCC GCAACTCCGCAAGGAACAGG (31 bases)

SecG-3 ′ 21 bases Primer 12 TTAGTTCGGGATATCGCTGGT (21 bases) (antisense)

SecG-3 ′ terminal 15 bases SecA-5 ′ terminal 18 bases Primer 13 GATATCCCGAACTAA CTCCGCAACGCGGGGCGT (33 bases)

Base primer 14 for prlA4 (SecY mutation) TGCTTATCATTGTTGTCGTGAT (22 bases)

Base primer 15 for prlA4 (SecY mutation) CAATGATAAGCAGTGAGGTCCCACC (25 bases) (antisense)

Reference Example 5 Tp47K and 17K genes and Escherichia coli secretion-related enzymes SecG, SecA, prlA
4 (SecY mutant), SecE gene and S
Preparation of ecG-SecA Cassette Gene a) Purification of rabbits from subcultured syphilis (Treponema pallidum, Nicols strain) by Percoll density centrifugation (Sex. Transm. Dis., Vol. 11, p. 275-286) , 19
84) Then, genomic DNA was extracted by the SDS-proteinase K / phenol / chloroform method.

B) Reference Example 4 as a sense primer by polymerase chain reaction (PCR)
And the primer 4 prepared in Reference Example 4 was selected as an antisense primer, and syphilis antigen 17K (I
nfection and Immunity, Vol.61, p.1202-1210, 1993)
Was prepared. The 5 'end of this DNA fragment does not contain a Shine-Dalgarno sequence. The nucleotide sequence is shown in Sequence Listing 1.

C) Next, Reference Example 4 was used as a sense primer.
Primer 1 prepared in Reference Example 4 was selected as an antisense primer, and syphilis antigen 47K (Infec) was selected using genomic DNA of syphilis as a template.
tion and Immunity, Vol. 60, p. 1568-1576, 1992). 5 'of this DNA fragment
The end does not include the Shine-Dalgarno sequence. The nucleotide sequence is shown in Sequence Listing 2.

D) In addition, a miniprep method (current protocols) was used from Escherichia coli BL21 (DE3).
in molecular biology unit
2.4 published by John Wil
ey & Sons) to extract genomic DNA.

E) Reference Example 4 as a sense primer by polymerase chain reaction (PCR)
The primer 11 prepared in Reference Example 4 was selected as an antisense primer, and the secretion-related enzyme Se was selected using E. coli genomic DNA as a template.
A DNA fragment encoding cG (EMVO J., Vol. 13, pp. 3272-3277, 1994) was prepared. The 5 'end of this DNA fragment contains the Shine-Dalgarno sequence. The nucleotide sequence is shown in Sequence Listing 3.

F) Next, Reference Example 4 as a sense primer
The primer 5 prepared in Reference Example 4 was selected as an antisense primer, and the secretion-related enzyme SecA was selected using E. coli genomic DNA as a template.
(JOURNAL OF BACTERIOLOGY, Vol. 170, pp. 3404-3414, 19
88) was prepared. This DNA
The 5 'end of the fragment contains the Shine-Dalgarno sequence.
The nucleotide sequence is shown in Sequence Listing 4.

G) Next, as a sense primer, Reference Example 4
The primer 7 prepared in Example 4 was selected as an antisense primer, the primer 8 prepared in Reference Example 4 was selected, and Escherichia coli genomic DNA was used as a template.
(Biomembr., Vol. 22, pp. 353-367, 1990). The 5 'end of this DNA fragment contains the Shine-Dalgarno sequence. The nucleotide sequence is shown in Sequence Listing 5.
Shown in

Next, Reference Example 4 was used as a sense primer by the polymerase chain reaction (PCR) method.
Primer 7 prepared in Reference Example 4 as an antisense primer,
Using the gene encoding SecY prepared in Reference Example 5 as a template, prlA4 (SecY mutant) (BIO / T
ECHNOLOGY, VOL. 12, pp. 178-140, 1994). This is named SecY-2 gene.

Similarly, the primer 14 prepared in Reference Example 4 as a sense primer and the primer 8 prepared in Reference Example 4 as an antisense primer were selected by PCR, and the gene encoding SecY prepared in Reference Example 5 was selected. Using prlA4 (SecY mut as a template)
ant) The gene used as the material for production was amplified. This is named SecY-3 gene.

By the recombinant PCR method, the primer 7 prepared in Reference Example 4 as a sense primer and the primer 8 prepared in Reference Example 4 as an antisense primer were selected.
2. Both of the genes encoding SecY-3 were added to the reaction solution as templates, and recombinant PCR was performed to bind these genes. This is called prlA4 (SecY
mutant). The 5 'end of this DNA fragment contains the Shine-Dalgarno sequence. In this nucleotide sequence, the G at position 1251 has been replaced with A.

H) Reference Example 4 as a sense primer
The primer 9 prepared in Example 4 was selected as the antisense primer, the primer 10 prepared in Reference Example 4 was selected, and the secretion-related enzyme Sec was selected using E. coli genomic DNA as a template.
A DNA fragment encoding E (Genes Dev., Vol. 3, p. 1035-1044, 1989) was prepared. The 5 'end of this DNA fragment contains the Shine-Dalgarno sequence. The nucleotide sequence is shown in Sequence Listing 6.

I) Reference Example 4 as a sense primer by polymerase chain reaction (PCR)
Primer 11 prepared in Reference Example 4, and primer 12 prepared in Reference Example 4 as an antisense primer,
Using the gene encoding SecG prepared in Reference Example 5 as a template, a gene serving as a material for producing a SecG-SecA gene cassette was amplified. This is designated as SecG-2 gene. Similarly, by PCR, primer 13 prepared in Reference Example 4 as a sense primer and primer 6 prepared in Reference Example 4 as an antisense primer were selected, and the gene encoding SecA prepared in Reference Example 5 was used as a template. , A gene used as a material for preparing a SecG-SecA gene cassette was amplified. This is Sec
Named A-2 gene. By a recombinant PCR method, the primer 11 prepared in Reference Example 4 as a sense primer and the primer Example 4 as an antisense primer were used.
The primer 6 prepared in Step 5 was selected, and both of the genes encoding SecG-2 and SecA-2 prepared in Reference Example 5 were added to the reaction solution as templates, and a gene serving as a material for preparing a SecG-SecA gene cassette was prepared. Was amplified. Next, the recombinant PCR in Reference Example 4 was performed to bind these genes. This is named SecG-SecA gene cassette.

(Example 1) 17K produced in Reference Example 5,
SecA, SecG, prlA4 (SecY muta
nt) and the gene encoding SecE were inserted into the vector in this order. A system that simultaneously expresses these five antigens and enzymes is described below as 17K: SecG: SecA: p.
rlA4 (SecY mutant): Named as SecE expression system. 17K: SecG: SecA: prlA4
(SecY mutant): Expression vectors pW6A and 17 were used to create vectors for the SecE expression system.
The gene encoding K is replaced with restriction enzymes NdeI and BamHI.
After ligation, the gene encoding 17K was inserted into the expression vector pW6A. This vector is named pW6A-17K. Then, pW6A-17K
And SecA-encoding genes are BamHI and XbaI.
After ligation, the gene encoding SecA was ligated and inserted into the vector pW6A-17K. This vector is named pW6A-17K-SecA. Subsequently, pW6A-17K-SecA and prlA4 (SecY
Mutant) is replaced with the restriction enzyme XbaI.
Digested with NotI and ligated to prlA4
The gene encoding (SecY mutant) was inserted into the vector pW6A-17K-SecA. This vector was constructed using pW6A-17K-SecA-prlA4 (Se
cY mutant). Subsequently, pW6A-1
7K-SecA-prlA4 (SecY mutan
t) and the gene encoding SecE by the restriction enzyme NotI
After digestion with EcoT22 and ligating,
Was transformed into the vector pW6A-17K-Se
It was inserted into cA-prlA4 (SecY mutant). This vector was constructed using pW6A-17K-SecA-pr.
Named lA4 (SecY mutant) -SecE.

The vector pW6A-17K-SecA-p
rlA4 (SecY mutant) -SecE was digested with restriction enzymes BamHI and XbaI, followed by agarose gel electrophoresis using a gel containing ethidium bromide, and the resulting pW6A-17K-prlA4 (SecY mutan) was obtained.
t) Among the bands of -SecE (SecA has been removed) and SecA, pW6A-17K-prlA
Only the band of 4 (SecY mutant) -SecE (SecA was removed) was cut out and purified with GeneClean II (Funakoshi). Next, SecG-Se
After digesting the cA gene cassette with the restriction enzymes BamHI and XbaI, it is ligated and the SecG-SecA gene cassette is inserted into the vector pW6A-17K-prlA4 (S
ecYmutant)-Inserted into SecE (SecA has been removed). This vector is called pW6A-17K
-SecG-SecA-prlA4 (SecY mut
ant) -SecE. A schematic diagram of the resulting vector is shown in FIG. Each of the vectors into which the gene was inserted was introduced into Escherichia coli DH5α by the calcium chloride method. After culturing the cells in an LB medium containing ampicillin, the desired vector was obtained by the alkaline SDS method.

(Example 2) pW6A obtained in Example 1
-17K-SecG-SecA-prlA4 (SecY
Mutant) -SecE and pW6A-17K were separately introduced into different E. coli strains BL21 (DE3), and the cells were cultured using an LB medium containing ampicillin. The fusion antigen was induced to induce expression by adding 1 mM IPTG, and the cells were disrupted, centrifuged, and recovered as a precipitate. 1
Syphilis bacteria subcultured in 7K and rabbit testes and purified by Percoll density centrifugation were subjected to reducing SDS electrophoresis on a 15% polyacrylamide gel. Thereafter, Western blotting was performed using the serum of a rabbit cultured with syphilis in a testis as a primary antibody in Reference Example 5, and expression of an antigen reactive with the serum was observed. Here, two bands are observed in the 17K expression system. These bands are, from the top, a 17K antigen (hereinafter referred to as p-17K), which has a signal sequence called a premature type and has no lipid added thereto, and a native type. This is an antigen (hereinafter, referred to as n-17K) to which a signal sequence is removed and a lipid is added. The migration position of the n-17K antigen coincided with that of native 17K syphilis extracted from rabbit testis. Next, the 17K expression system and 17K
K: SecG: SecA: prlA4 (SecY mu
tant): The expression product of the SecE expression system was subjected to reducing SDS electrophoresis on a 15% polyacrylamide gel. afterwards,
Western blotting was performed using the serum of a rabbit cultured with testicular syphilis in Example 1 as a primary antibody. FIG. 4 shows the results. 17K: SecG: SecA: prlA4
(SecY mutant): 17 in the SecE expression system
Compared with the K expression system, the ratio of the expression level of native type n-17K was significantly increased. Further, it was confirmed by the same Western blotting that p-17K and n-17K reacted with the anti-17K monoclonal antibody prepared in Reference Example 2.

(Example 3) 47K produced in Reference Example 5,
SecA, SecG, prlA4 (SecY muta
nt) and the gene encoding SecE were inserted into the vector in this order. A system that simultaneously expresses these five antigens and enzymes is as follows: 47K: SecG: SecA: prlA4
(SecY mutant): Named as SecE expression system. 47K: SecG: SecA: prlA4 (Sec
Y mutant): To create vectors for the SecE expression system, the vector pW6A-17K-SecG
-SecA-prlA4 (SecY mutant)-
SecE was digested with restriction enzymes NdeI and BamHI, and then subjected to agarose gel electrophoresis using a gel containing ethidium bromide, and the resulting pW6A-SecG-SecA-prl appeared.
A4 (SecY mutant) -SecE (the gene encoding 17K has been removed) band and 17K
PW6A-SecG among the bands of the gene encoding
-SecA-prlA4 (SecY mutant)-
Only the band of SecE (from which the gene encoding 17K had been removed) was cut out and purified using GeneClean II (Funakoshi). The gene encoding 47K was digested with restriction enzymes NdeI and BamHI, ligated, and the 47K gene was transformed into the vector pW6A-SecG-Se.
cA-prlA4 (SecY mutant) -Sec
E (17K has been removed). This vector was constructed using pW6A-47K-SecG-SecA-prlA.
4 (SecY mutant) -Name as SecE.
A schematic diagram of the resulting vector is shown in FIG. For comparison, an expression system of only 47K was prepared. Expression vector pW
After the genes encoding 6A and 47K were digested with restriction enzymes NdeI and BamHI, they were ligated and the gene encoding 47K was inserted into the expression vector pW6A. This vector is named pW6A-47K. A schematic diagram of the resulting vector is shown in FIG. Each of the vectors into which the gene was inserted was transformed into Escherichia coli DH by the calcium chloride method.
After culturing the cells in an LB medium containing ampicillin introduced into 5α, the desired vector was obtained by the alkaline SDS method.

(Example 4) pW6A obtained in Example 3
-47K-SecG-SecA-prlA4 (SecY
Mutant) -SecE and pW6A-47K were separately introduced into different E. coli strains BL21 (DE3), and the cells were cultured using an LB medium containing ampicillin. The fusion antigen was recovered as a precipitate after induction of expression, disruption of cells, and centrifugation. Syphilis bacteria subcultured at 47K and rabbit testes and purified by Percoll density centrifugation were subjected to reducing SDS electrophoresis on a 7.5% polyacrylamide gel. Then, Western blot was performed using the serum of a rabbit cultured with syphilis in a testis as a primary antibody in Reference Example 5,
Expression of antigen reactive with serum was observed. Where 47K
In the expression system, two bands are observed. These bands each have a signal sequence called a pre-mature type from the top and have no lipid added to the 47K antigen (hereinafter, p-type).
47K) and an antigen to which a signal sequence called a native type is removed and a lipid is added (hereinafter referred to as n-4).
7K). The migration position of the n-47K antigen coincided with that of rabbit testis-extracted syphilis. Next, a 47K expression system and 47K: SecG: SecA: prlA4 (Sec
6. Y mutant): Expression product of SecE expression system
Reduction SDS electrophoresis was performed on a 5% polyacrylamide gel. Thereafter, Western blotting was performed using the serum of a rabbit cultured with syphilis in the testis in Example 1 as a primary antibody. The results are shown in FIG. 47K: SecG: Sec
A: prlA4 (SecY mutant): SecE
In the expression system, the native type n-4 was compared with the 47K expression system.
The ratio of 7K expression increased. In addition, p-47K and n-47K were found in Reference Example 2 by the same Western blot.
It was confirmed that it reacted with the anti-47K monoclonal antibody prepared in the above.

[0062]

According to the present invention, a large amount of highly reactive Tp antigen to which lipids are bound can be obtained. When this antigen is used as an immunoassay reagent, it is more specific and more sensitive than conventional ones. High measurements can be made. In addition, a stable supply of antigen is ensured by using genetic engineering technology.

[0063]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 486 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid sequence TCTCATATGA AAGGATCTGT CCGCGCGCGCTG TGCGCGTTCC TTGGTGTTGG AGCGCTCGGT 30 60 AGCGCTTTGT GTGTCTCGTG CACAACCGTG TGTCGCCACGCC 120 AAGGTAGAGT GCGCGTTGAA GGGAGGTATC TTTCGGGGTA CGCTACCTGC GGCCGATTGC 150 180 CCGGGAATCG ATACGACTGT GACGTTCAAC GCGGATGGCA CTGCGCAAAA GGTAGAGCTT 210 240 GCCCTTGAGA AGAAGTCGGC ACCTTCTCCT CTTACGTATC GCGGTACGTG GATGGTACGT 270 300 GAAGACGGAA TTGTCGAACT CTCGCTTGTG TCCTCGGAGC AATCGAAGGC ACCGCACGAG 330 360 AAAGAGCTGT ACGAGCTGAT AGACAGTAAC TCCGTTCGCT ACATGGGCGC TCCCGGCGCA 390 420 GGAAAGCCTT CAAAGGAGAT GGCGCCGTTT TACGTGCTCA AAAAAACAAA GAAATAGGGA 450 480 TCCCTG

[0064]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 1325 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid sequence GTGTACATAT GAAAGTGAAA TACGCACTAT TTTCTGCCGG AGCGCTGCAG TTGTTGGTTG 30 60 TAGGCTGTGG CTCGTCTCAT CATGAGTAGC ACTATGGCAG TG 120 ACTACTGGGC CGGGGAGTTG GGGCAGAGTA GGGACGTGCT TTTGGCGGGT AATGCCGAGG 150 180 CGGACCGCGC GGGGGATCTC GACGCAGGCA TGTTCGATGC AGTTTCTCGC GCAACCCACG 210 240 GGCATGGCGC GTTCCGTCAG CAATTTCAGT ACGCGGTTGA GGTATTGGGC GAAAAGGTTC 270 300 TCTCGAAGCA GGAGACCGAA GACAGCAGGG GAAGAAAAAA GTGGGAGTAC GAGACTGACC 330 360 CAAGCGTTAC TAAGATGGTG CGTGCCTCTG CGTCATTTCA GGATTTGGGA GAGGACGGGG 390 420 AGATTAAGTT TGAAGCAGTC GAGGGTGCAG TAGCGTTGGC GGATCGCGCG AGTTCCTTCA 450 480 TGGTTGACAG CGAGGAATAC AAGATTACGA ACGTAAAGGT TCACGGTATG AAGTTTGTCC 510 540 CAGTTGCGGT TCCTCATGAA TTAAAAGGGA TTGCAAAGGA GAAGTTTCAC TTCGTGGAAG 570 600 ACTCCCGCGCGT TACGGAGAAT ACCAACGGCC TTAAGACAAT GCTCGATCGATG GATAGTGATCGATG GATAGTGATCGATG GATAGTGATCGAGTAATC GCC CGCACGACCT TGTGGTAGAC ACGGTGGGTA 690 720 CCGGTTACCA CAGCCGTTTT GGTTCGGACG CAGAGGCTTC TGTGATGCTG AAAAGGGCTG 750 780 ATGGCTCTGA GCTGTCGCAC CGTGAGTTCA TCGACTATGT GATGAACTTC AACACGGTCC 810 840 GCTACGACTA CTACGGTGAT GACGCGAGCT ACACCAATCT GATGGCGAGT TATGGCACCA 870 900 AGCACTCTGC TGACTCCTGG TGGAAGACAG GAAGAGTGCC CCGCATTTCG TGTGGTATCA 930 960 ACTATGGGTT CGATCGGTTT AAAGGTTCAG GGCCGGGATA CTACAGGCTG ACTTTGATTG 990 1020 CGAACGGGTA TAGGGACGTA GTTGCTGATG TGCGCTTCCT TCCCAAGTAC GAGGGGAACA 1050 1080 TCGATATTGG GTTGAAGGGG AAGGTGCTGA CCATAGGGGG CGCGGACGCG GAGACTCTGA 1110 1140 TGGATGCTGC AGTTGACGTG TTTGCCGATG GACAGCCTAA GCTTGTCAGC GATCAAGCGG 1170 1200 TGAGCTTGGG GCAGAATGTC CTCTCTGCGG ATTTCACTCC CGGCACTGAG TACACGGTTG 1230 1260 AGGTTAGGTT CAAGGAATTC GGTTCTGTGC GTGCGAAGGT AGTGGCCCAG TAGGGATCCG 1290 1320 GTGTC

[0065]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 369 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid sequence ATTGGGGATC CGCAACTCCG CAAGGAACAG GTTGATTATG TATGAAGCTC TTTTAGTAGT 30 60 TTTCCTTATT GTGGCAATTG GCCTTGAATC 120 TGATATGGGA GCCTCCTTCG GAGCAGGCGC TTCCGCTACG CTGTTTGGTT CAAGTGGTTC 150 180 TGGTAACTTC ATGACCCGCA TGACGGCGCT GCTGGCAACG TTATTCTTCA TCATCAGTCT 210 240 GGTGCTGGGT AACATCAATA GCAAAACAACACATAGCACGATCAGGAGATCAGGAGATCGATCAGGAGATCAGGAGATCAGGCGATCAGGCGATCAGGAGATCAAGG

[0066]

[Sequence list]

 SEQ ID NO: 4 Sequence length: 2754 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid sequence TTTGGATCCC TCCGCAACGC GGGGCGTTTG AGATTTTATT ATGCTAATCA AATTGTTAAC 30 60 TAAAGTTTTC GGTAGTCGTA ACGATCGCAC CCTGGCAGTCGCATCCTGGCAGTCGCAT 120 CATCAATGCC ATGGAACCGG AGATGGAAAA ACTCTCCGAC GAAGAACTGA AAGGGAAAAC 150 180 CGCAGAGTTT CGTGCACGTC TGGAAAAAGG CGAAGTGCTG GAAAATCTGA TCCCGGAAGC 210 240 TTTCGCCGTG GTACGTGAGG CAAGTAAGCG CGTCTTTGGT ATGCGTCACT TCGACGTTCA 270 300 GTTACTCGGC GGTATGGTTC TTAACGAACG CTGCATCGCC GAAATGCGTA CCGGTGAAGG 330 360 AAAAACCCTG ACCGCAACGC TGCCTGCTTA CCTGAACGCA CTAACCGGTA AAGGCGTGCA 390 420 CGTAGTTACC GTCAACGACT ACCTGGCGCA ACGTGACGCC GAAAACAACC GTCCGCTGTT 450 480 TGAATTCCTT GGCCTGACTG TCGGTATCAA CCTGCCGGGC ATGCCAGCAC CGGCAAAGCG 510 540 CGAAGCTTAC GCAGCTGACA TCACTTACGG TACGAACAAC GAATACGGCT TTGACTACCT 570 600 GCGCGACAAC ATGGCGTTCA GCCCTGAAGA ACGTGTACAG CGTAAACTGC ACTATGCGCTGA GATCTA GTCGCTGA 630 TCGA TGAAGCGCGT ACACCGCTGA TCATTTCCGG 690 720 CCCGGCAGAA GACAGCTCGG AAATGTATAA ACGCGTGAAT AAAATTATTC CGCACCTGAT 750 780 CCGTCAGGAA AAAGAAGACT CCGAAACCTT CCAGGGCGAA GGCCACTTCT CGGTGGACGA 810 840 AAAATCTCGC CAGGTGAACC TGACCGAACG TGGTCTGGTG CTGATTGAAG AACTGCTGGT 870 900 GAAAGAGGGC ATCATGGATG AAGGGGAGTC TCTGTACTCT CCGGCCAACA TCATGCTGAT 930 960 GCACCACGTA ACGGCGGCGC TGCGCGCTCA TGCGCTGTTT ACCCGTGACG TCGACTACAT 990 1020 CGTTAAAGAT GGTGAAGTTA TCATCGTTGA CGAACACACC GGTCGTACCA TGCAGGGCCG 1050 1080 TCGCTGGTCC GATGGTCTGC ACCAGGCTGT GGAAGCGAAA GAAGGTGTGC AGATCCAGAA 1110 1140 CGAAAACCAA ACGCTGGCTT CGATCACCTT CCAGAACTAC TTCCGTCTGT ATGAAAAACT 1170 1200 GGCGGGGATG ACCGGTACTG CTGATACCGA AGCTTTCGAA TTTAGCTCAA TCTACAAGCT 1230 1260 GGATACCGTC GTTGTTCCGA CCAACCGTCC AATGATTCGT AAAGATCTGC CGGACCTGGT 1290 1320 CTACATGACT GAAGCGGAAA AAATTCAGGC GATCATTGAA GATATCAAAG AACGTACTGC 1350 1380 GAAAGGCCAG CCGGTGCTGG TGGGTACTAT CTCCATCGAA AAATCGGAGC TGGTGTCAAA 1410 1440 CGAACTGACC AAAGCCGGTA TTAAGCACAA CGTCCTGAAC GCCAAAT TCC ACGCCAACGA 1470 1500 AGCGGCGATT GTTGCTCAGG CAGGTTATCC GGCTGCGGTG ACTATCGCGA CCAATATGGC 1530 1560 GGGTCGTGGT ACAGATATTG TGCTCGGTGG TAGCTGGCAG GCAGAAGTTG CCGCGCTGGA 1590 1620 AAATCCGACC GCAGAGCAAA TTGAAAAAAT TAAAGCCGAC TGGCAGGTAC GTCACGATGC 1650 1680 GGTACTGGAA GCAGGTGGCC TGCATATCAT CGGTACCGAG CGTCACGAAT CCCGTCGTAT 1710 1740 CGATAACCAG TTGCGCGGTC GTTCTGGTCG TCAGGGGGAT GCTGGTTCTT CCCGTTTCTA 1770 1800 CCTGTCGATG GAAGATGCGC TGATGCGTAT TTTTGCTTCC GACCGAGTAT CCGGCATGAT 1830 1860 GCGTAAACTG GGTATGAAGC CAGGCGAAGC CATTGAACAC CCGTGGGTGA CTAAAGCGAT 1890 1920 TGCCAACGCC CAGCGTAAAG TTGAAAGCCG TAACTTCGAC ATTCGTAAGC AACTGCTGGA 1950 1980 ATATGATGAC GTGGCTAACG ATCAGCGTCG CGCCATTTAC TCCCAGCGTA ACGAACTGTT 2010 2040 GGATGTCAGC GATGTGAGCG AAACCATTAA CAGCATTCGT GAAGATGTGT TCAAAGCGAC 2070 2100 CATTGATGCC TACATTCCAC CACAGTCGCT GGAAGAAATG TGGGATATTC CGGGGCTGCA 2130 2160 GGAACGTCTG AAGAACGATT TCGACCTCGA TTTGCCAATT GCCGAGTGGC TGGATAAAGA 2190 2220 ACCAGAACTG CATGAAGAGA CGCTGCGTGA GCGCATTCTG GCGCAGTCCA TCGAAGT GTA 2250 2280 TCAGCGTAAA GAAGAAGTGG TTGGTGCTGA GATGATGCGT CACTTCGAGA AAGGCGTCAT 2310 2340 GCTGCAAACG CTTGACTCCC TGTGGAAAGA GCACCTGGCA GCGATGGACT ATCTGCGTCA 2370 2400 GGGTATCCAC CTGCGTGGCT ACGCACAGAA AGATCCGAAG CAGGAATACA AACGTGAATC 2430 2460 GTTCTCCATG TTTGCAGCGA TGCTGGAGTC GTTGAAATAT GAAGTTATCA GTACGCTGAG 2490 2520 CAAAGTTCAG GTACGTATGC CTGAAGAGGT TGAGGAGCTG GAACAACAGC GTCGTATGGA 2550 2580 AGCCGAGCGT TTAGCGCAAA TGCAGCAGCT TAGCCATCAG GATGACGACT CTGCAGCCGC 2610 2640 AGCTGCACTG GCGGCGCAAA CCGGAGAGCG CAAAGTAGGA CGTAACGATC CTTGCCCGTG 2670 2700 CGGTTCTGGT AAAAAATACA AGCAGTGCCA TGGCCGCCTG CAATAATCTA GAAC 2730

[0067]

[Sequence list]

 SEQ ID NO: 5 Sequence length: 1368 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid sequence CGTCTAGACT GGCGGTAAAA TCGAGGAATA AGTAGCAGAT GGCTAAACAA CCGGGATTAG 30 60 ATTTTCAAAG TGCCAAAGGT GGCTTAGTGCGCTCGAA 120 GTGCGCTGAT TGTGTTCCGT ATTGGCTCTT TTATTCCGAT CCCTGGTATT GATGCCGCTG 150 180 TACTTGCCAA ACTGCTTGAG CAACAGCGAG GCACCATCAT TGAGATGTTT AACATGTTCT 210 240 CTGGTGGTGC TCTCAGCCGT GCTTCTATCT TTGCTCTGGG GATCATGCCG TCGGCGTCGA 270 300 TCATTATCCA GCTGCTGACG GTGGTTCACC CAACGTTGGC AGAAATTAAG AAAGAAGGGG 330 360 AGTCTGGTCG TCGTAAGATC AGCCAGTACA CCCGCTACGG TACCCTGGTG CTGGCAATAT 390 420 TCCAGTCGAT CGGTATTGCT ACCGGTCTGC CGAATATGCC TGGTATGCAA GGCCTGGTGA 450 480 TTAACCCGGG CTTTGCATTC TACTTCACCG CTGTTGTAAG TCTGGTCACA GGAACCATGT 510 540 TCCTGATGTG GTTGGGCGAA CAGATTACTG AACGAGGTAT CGGCAACGGT ATTTCAATCA 570 600 TTATCTTCGC CGGTATTGTC GCGGGACTCC CGCCAGCCAT TGCCTCAC TGCGTCACTC ATCGAGCAGC GTGT TGCTGTTGGT TGCAGTATTA GTATTTGCAG 690 720 TGACGTTCTT TGTTGTATTT GTTGAGCGTG GTCAACGCCG CATTGTGGTA AACTACGCGA 750 780 AACGTCAGCA AGGTCGTCGT GTCTATGCTG CACAGAGCAC ACATTTACCG CTGAAAGTGA 810 840 ATATGGCGGG GGTAATCCCG GCAATCTTCG CTTCCAGTAT TATTCTGTTC CCGGCGACCA 870 900 TCGCGTCATG GTTCGGGGGC GGTACTGGTT GGAACTGGCT GACAACAATT TCGCTGTATT 930 960 TGCAGCCTGG GCAACCGCTT TATGTGTATG CGTCTGCAAT CATCTTCTTC TGTTTCTTCT 990 1020 ACACGGCGTT GGTTTTCAAC CCGCGTGAAA CAGCAGATAA CCTGAAGAAG TCCGGTGCAT 1050 1080 TTGTACCAGG AATTCGTCCG GGAGAGCAAA CGGCGAAGTA TATCGATAAA GTAATGACCC 1110 1140 GCCTGACCCT GGTTGGTGCG CTGTACATTA CCTTTATCTG CCTGATCCCG GAGTTCATGC 1170 1200 GTGATGCAAT GAAAGTACCG TTCTACTTCG GTGGGACCTC ACTGCTTATC GTTGTTGTCG 1230 1260 TGATTATGGA CTTTATGGCT CAAGTGCAAA CTCTGATGAT GTCCAGTCAG TATGAGTCTG 1290 1320 CATTGAAGAA GGCGAACCTG AAAGGCTACG GCCGATAAGC GGCCGCCC 1350

[0068]

[Sequence list]

 SEQ ID NO: 6 Sequence length: 431 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Other nucleic acid Sequence GGGCGGCCGC TTTGTAGAAA ACTTCTGACA GGTTGGTTTA TGAGTGCGAA TACCGAAGCT 30 60 CAAGGAAGCG GGCGCGGCTGGGTCTGAGG 120 GTGGCGATTG TCGGCAACTA CCTTTATCGC GACATTATGC TGCCGCTGCG TGCGCTGGCC 150 180 GTAGTAATTC TGATTGCTGC AGCGGGTGGT GTCGCGCTGT TAACGACAAA AGGTAAAGCT 210 240 ACCGTTGCTT TTGCCCGTGA AGCGCGTACC GAAGTCCGTA AGGTCATTTG GCCGACTCGC 270 300 CAGGAAACAT TGCACACCAC GCTGATTGTG GCTGCGGTTA CCGCAGTAAT GTCACTGATC 330 360 CTGTGGGGAC TGGATGGTAT TCTGGTTCGC CTGGTATCCT TTATCACTGG CCTGAGGTTC 390 420 TGAATGCATT G

[Brief description of the drawings]

FIG. 1 shows the reactivity of 47K and 47C2.

FIG. 2 is a detailed diagram of the expression vector pW6A.

FIG. 3 (a) is a detailed view of the expression vector pW6A-17K. (B) is a detailed view of the expression vector pW6A-47K. (C) Expression vector pW6A-17K-SecG-Se
It is a detailed diagram of cA-prlA4-SecE. (D) Expression vector pW6A-47K-SecG-Se
It is a detailed diagram of cA-prlA4-SecE.

FIG. 4 is a graph showing the effect of simultaneous expression of a recombinant antigen and a secretion-related enzyme at 17K.

FIG. 5 is a graph showing the effect of simultaneous expression of a recombinant antigen and a secretion-related enzyme at 47K.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/53 G01N 33/53 D 33/577 33/577 B (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 (C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (9)

[Claims] 1. A syphilis-Treponemal fusion DNA sequence in which a DNA sequence encoding a surface antigen of T. syphilis to which a signal peptide is added and a DNA sequence encoding a secretion-related enzyme are fused. 2. The treponema syphilis fusion DNA sequence according to claim 1, wherein the secretion-related enzyme is a Sec enzyme. 3. The method according to claim 1, wherein the Sec enzyme is SecA, SecG and S.
3. The syphilis treponemal fusion DNA sequence according to claim 1 or 2, which is ecY. 4. The syphilis treponemal fusion DNA sequence according to claim 3, further comprising SecE as a Sec enzyme. 5. A surface antigen of treponema pallidum to which a DNA sequence is added with a signal peptide, SecG, SecA, S
5. The Treponemal syphilis fusion DNA sequence according to claim 4, which is a fused sequence of DNA sequences encoding ecY and SecE, wherein the DNA sequences are fused in the above order. 6. The surface antigen of Treponema pallidum having a molecular weight of 15
The syphilis-Treponemal fusion DNA according to any one of claims 1 to 5, which is an antigen selected from kDa, 17 kDa, and 47 kDa.
Array. 7. The surface antigen of Treponema pallidum has a molecular weight of 17
7. The Treponema pallidum fusion DNA sequence according to claim 6, which is a kDa antigen. 8. The surface antigen of Treponema pallidum has a molecular weight of 47.
7. The Treponema pallidum fusion DNA sequence according to claim 6, which is a kDa antigen. 9. A method for expressing a Treponema pallidum antigen using the Treponema pallidum DNA sequence according to any one of claims 1 to 8.