JP3533857B2 - Treponema pallidum fusion antigen and method for measuring anti-treponema pallidum antibody using the fusion antigen - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fusion antigen of Treponema pallidum.

[0002]

[Prior Art] Syphilis is Treponema pallidum.
Paris dam Treponema pallidum, hereinafter T
(sometimes referred to as p). Tp is a kind of spirochete, and it has been confirmed that several surface antigens are present on the cell surface as Tp antigens (The Journal of Immunology, Vol.129,
p.833-838, 1982; The Journal of Immunology, Vol.12
9, p.1287-1291, 1982; Journal of Clinical Microbiol
ogy, Vol.21, p.82-87, 1985; Journal of Clinical Mi
crobiology, Vol.30, p.115-122, 1992). When Tp is infected, antibodies against these surface antigens are produced in the blood. Therefore, syphilis is diagnosed by examining the presence or absence of anti-Tp antibody in the blood.

[0003] In order to examine the presence or absence of anti-Tp antibody, it is common to use an immunoassay applying an antigen-antibody reaction between Tp antigen and anti-Tp antibody in the blood of a patient. For, a large amount of Tp antigen is required. However, since Tp cannot be cultured in large quantities in vitro, a method of inoculating Tp into a testis of a rabbit and purifying a Tp antigen from a rabbit testis infected with Tp has been conventionally used (Acta Pathol Microbiol Scand [B], Vol.83, p.157-
160, 1975). In this method, since Tp is cultured in the living body of a rabbit, there are problems such as individual differences of rabbits and contamination of purified impurities, and it is very difficult to obtain a large amount of Tp antigen with good reproducibility.

Due to the progress of genetic engineering technology in recent years, Tp
The technology to clone the gene encoding the surface antigen of Tp and artificially mass-produce Tp antigen has advanced (Scie
nce, Vol.216, p.522-523, 1982; Infection and Immuni
ty, Vol.36, p.1238-1241,1982; Infection and Immuni
ty, 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; Journal of Bacteriolog
y, Vol.162, p1227-1237, 1985; Infection and Immuni
ty, Vol.54, p.500-506, 1986; Infection and Immunit
y, Vol.56, p.71-78, 1988; Infection and Immunity,
Vol.57, p.2612-2623, 1989; Infection and Immunity,
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; Tokuyohei 2-5004
03). Using such genetic engineering technology, Tp antigen can be produced in large quantities without using live animals, but depending on the type of Tp surface antigen, the gene encoding the antigen hardly expresses the antigen. There is something. Therefore, when such a substance that is difficult to be expressed only by the gene encoding the target substance is used, thioredoxin derived from Escherichia coli (hereinafter sometimes referred to as TRX) or glutathione-derived from Schistosoma japonicum- S-transferase (hereinafter referred to as GS
A method of expressing a target substance with a fusion gene obtained by fusing a gene such as T) with a gene of the target substance has been proposed (Tables 5 to 507209 and 1 to 5).
03441), 17kDa which is one of the surface antigens of Tp
GST15k in which GST is fused to antigen or 15kDa antigen
It was also found that Da antigen and GST17kDa antigen show high sensitivity when used for the measurement of anti-Tp antibody (Japanese Patent Application No. 7-63365).

However, since Escherichia coli and Schistosoma japonicum can live in the human body, there are not a few humans who have in the blood a factor that reacts with TRX and GST.
In such a case, a positive reaction occurs even without Tp infection, which obviously affects the diagnosis. Currently, syphilis can be sufficiently treated by developing effective antibiotics, and it is desired to diagnose and treat the infection as quickly and accurately as possible. Therefore, it has been required to provide a method for measuring Tp antigen and Tp antibody that can exhibit higher sensitivity and specificity.

[0006]

Therefore, it is an object of the present invention to provide a Tp antigen which exhibits higher sensitivity and specificity than conventional methods.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the conventional problems, the present inventors have found that several kinds of surface antigens are fused from a gene encoding a fused Tp surface antigen. When the Tp fusion antigen was expressed, the expression amount of the Tp antigen increased more than before, and the fusion antigen was further treated with anti-Tp
The present invention has been completed by discovering the surprising fact that anti-Tp antibodies can be measured with higher sensitivity by using as an antigen in an antibody measuring method than a measuring system using each surface antigen alone. That is, the present invention provides a fusion antigen of Tp.

The present invention will be described in detail below. There are surface antigens of various molecular weights on the cell surface of Tp, and the major ones are 15 kDa, 17 kDa, 42 kDa, and 4 kDa, respectively.
Antigens such as 7 kDa are known (The Journal of Imm
unology, Vol.129, p.833-838, 1982; The Journal of
Immunology, Vol.129, p.1287-1291, 1982; Journal of
Clinical Microbiology, Vol.21, p.82-87, 1985; Jou
rnal of Clinical Microbiology, Vol.30, p.115-122,1
992). These antigens are TpN by Norris et al.
15, TpN17, TpN44.5 (a), TpN47
(Microbiological. Reviews, Vol.57,
p.750-779) (hereinafter, referred to as TpN47 4
7K, TpN17 may be described as 17K, and TpN15 may be described as 15K). The genes encoding these surface antigens have already been cloned, and the antigens have been genetically produced. The amino acid sequences of these have also been determined (Molecular Microbiology, Vol.4, p.1371-
1379, 1990; Infection and Immunity, Vol.61, p.1202
-1210, 1993; Journal of Bacteriology, Vol.162, p.1
227-1237, 1992; Infection and Immunity, Vol.57, p.3
708-3714, 1989).

The surface antigen of Tp used in the fusion antigen of the present invention means all surface antigens existing on the cell surface of Tp, and examples thereof include antigens such as 47K, 17K and 15K. Further, the surface antigen used in the present invention can be appropriately modified and used within a range not impairing its antigenicity. For example, there is a signal peptide of about 20 amino acids at the N-terminus of the DNA sequence encoding the surface antigen of Tp, but the signal peptide is deleted in the native antigen. It is considered that this is because the signal peptide is cleaved by signal peptidase II after completion of DNA translation of the surface antigen (Microbial Pathogenes
is, Vol.7, p.175-188, 1989; Infectionand Immunity,
Vol.61, p.1202-1210, 1993; Molecular Microbiology,
Vol.4, p.1371-1379, 1990; Infection and Immunity,
Vol.60, p.1568-1576, 1993). In the fusion antigen of the present invention, an antigen containing such an N-terminal signal peptide or an antigen containing no such signal peptide can be used as a fusion antigen. In addition, for example, in 47K, it has been reported that an additional 9 amino acids may be added after the C-terminal of 434 amino acids of the main body (Infection and Im
munity, Vol. 60, p1568-1576, 1992). In such a case, a DNA fragment encoding 47K is prepared by using a sense primer designed not to contain a signal peptide and an antisense primer containing a DNA sequence corresponding to 9 amino acids after the C-terminus. ,
It can also be used as 47K.

The Tp fusion antigen of the present invention is an antigen expressed by fusing these surface antigen genes with each other. Tp
In fusing the surface antigen genes, the combination may be any antigen as long as it is a Tp surface antigen,
There is no limitation on the combination of different antigens, the same antigens, and the like to be fused.

To express the fusion antigen of the present invention, conventional genetic engineering techniques can be used. That is, genomic DNA is extracted from Tp increased with a testicle of a rabbit or the like, and this is used as a template. Using the primers prepared based on the known DNA sequence, DNA fragments encoding the respective surface antigens were obtained by the PCR method, and these DNA fragments were fused by the recombinant PCR method or the DNA ligase to give a fusion antigen. Obtain the corresponding DNA fragment. The vector into which this DNA fragment has been inserted is inserted into a host such as Escherichia coli and the number of this bacterium is increased by culturing or the like, and then the expression of the fusion antigen is induced. The fusion antigen of interest is obtained through purification means such as host disruption and electrophoresis (Molecular Clon
ing A LABORATORY MANUAL SECOND EDITION 1989).

To insert the DNA fragment into the vector, for example, N
deI, SacI, EcoRI, SalI, XhoI,
Restriction enzyme recognition sites such as BamHI, KpnI, and HindIII can be used to insert the DNA fragment of the Tp antigen to be fused.

The DNA fragment to be inserted into the vector is
The DNA fragments to be fused can be inserted separately, or those in which the DNA fragments have been directly linked by the recombinant PCR method or the like in advance can be inserted. When a DNA fragment is inserted using these restriction enzyme recognition sites, a short amino acid may be sandwiched between the inserted DNA fragments as a linker. This linker is a recombinant PC
The DNA fragment can be fused by the R method and then inserted into the multicloning site of the expression vector to remove it, but the fusion antigen may be expressed with the linker still contained.

The fusion antigen of the present invention includes, for example, fusion antigens of 47K, 17K and 15K corresponding to N-terminal to C-terminal antigens (hereinafter referred to as 47).
-17-15), 47K, 15K and 17K.
Fusion antigen (hereinafter referred to as 47-15-
17), a fusion antigen fused with 15K, 17K and 47K (hereinafter referred to as 15-17-47), a fusion antigen fused with 15K, 47K and 17K (hereinafter referred to as herein). 15-47-17), 17K, a fusion antigen fused with 15K and 47K (hereinafter referred to as 17-15-47 in the present specification),
A fusion antigen fused with 17K, 47K and 15K (hereinafter referred to as 17-47-15 in the present specification), a fusion antigen fused with 47K and 17K (hereinafter referred to as 47 in the present specification)
-17), a fusion antigen in which 47K and 15K are fused (hereinafter referred to as 47-15), 1
A fusion antigen fused with 7K and 47K (hereinafter referred to as 17-47 in the present specification), a fusion antigen fused with 17K and 15K (hereinafter referred to as 17-15 in the present specification), 15K and 47K. Fused fusion antigen (hereinafter referred to as 15-47 herein), 15K and 17K
Fusion antigen (hereinafter referred to as 15-17 in the present specification), two 15K fusion antigens (hereinafter referred to as 15-15), and three 15K fusions Fusion antigen (hereinafter referred to as 15-15 in the present specification)
-15), a fusion antigen in which four 15Ks are fused (hereinafter referred to as 15-15-15-15 in the present specification), and the like.

Generally, in the anti-Tp antibody measuring system, 47K,
Since Tp major antigens such as 17K and 15K are used as a mixture, the required antigens must be purified separately. By using the fusion antigen of the present invention, a fusion antigen containing a plurality of antigens can be purified as a single antigen and easily obtained.

Since the 17K and 15K antigens are hardly expressed by the gene encoding the antigens themselves, they are expressed as fusion proteins with proteins such as TRX and GST. However, a fusion antigen expressed by a fusion gene containing a gene of a heterologous antigen other than the Tp antigen is
It can cause unexpected non-specific reactions not derived from the Tp antigen. The fusion antigen of the present invention is an antigen in which only the Tp antigen is fused, and among the expressed fusion antigens, T
Since there is no heterologous antigen other than p-antigen, it is very excellent as an antigen for specifically measuring anti-Tp antibody.

In the case of an antibody measuring method for diagnosing virus infection, an example is known in which one fusion antigen in which a plurality of allogeneic antigens are already fused is used as an antigen for antibody measurement (He.
patology, Vol.14, p.381-387, 1991). However, the fusion antigen of the present invention has much higher reactivity with the anti-Tp antibody than when each surface antigen is used alone,
By using the fusion antigen in an anti-Tp antibody measuring system, an anti-Tp antibody measuring method having an unprecedentedly high measuring sensitivity can be provided. Further, in the present invention, the surface antigens of 17K and 15K among the major surface antigens are hardly expressed only by the gene, and when the fusion antigen is combined with other Tp antigen, the expression level is surprisingly increased. There is a fact to be said.

The fusion antigen of the present invention can be used in a method for measuring anti-Tp antibody. What is the method for measuring anti-Tp antibody?
What is the reaction method as long as it is a measurement method using the fusion antigen as an antigen of the anti-Tp antibody measurement method and is based on an immune reaction between the anti-Tp antibody in the sample and the fusion antigen? Good. Various immunoassay methods are well known to those skilled in the art, and for example, if they are classified according to reaction format, agglutination method, turbidimetric method, sandwich method, competitive method, etc. An immunoassay method, a luminescence immunoassay method, a radioimmunoassay method, etc. can be mentioned.
Among them, ELISA by agglutination method that does not require dedicated equipment, enzyme immunoassay method suitable for operation of many samples
Methods, luminescence immunoassays, etc., which are becoming more and more highly sensitive and automated, are particularly preferred.

To use the fusion antigen of the present invention for the measurement of anti-Tp antibody, for example, in the case of the agglutination method, the fusion antigen of the present invention is sensitized to a carrier such as latex, gelatin particles, magnetic particles and the like, and non-specific adsorption is carried out. Block the site. The amount of anti-Tp antibody in the sample can be measured by reacting the sample with the fusion antigen-sensitized carrier for a certain period of time, and then detecting the agglutination caused by the immune reaction using the turbidity, the agglutination image and the like as indicators.

In the case of the ELISA method, the fusion antigen of the present invention is sensitized to the wells of a microtiter plate to block nonspecific adsorption sites. A sample is added to the wells of the fusion antigen-sensitized plate and reacted for a certain period of time. After washing the inside of the wells, an anti-human immunoglobulin antibody labeled with an enzyme such as peroxidase is reacted. After washing the inside of the well,
The amount of anti-Tp antibody in the sample can be measured by adding a substrate for the labeling enzyme, performing an enzymatic reaction, and measuring the enzymatic activity.

Incidentally, such agglutination method and ELISA method are well known in the technical field, and the measuring method of the present invention is not limited thereto. Examples of the sample to be used in the measuring method of the present invention include body fluids such as human and animal sera to be diagnosed for Treponema pallidum, and diluted products thereof.

[0022]

The present invention will be described in more detail with reference to Reference Examples and Examples. Reference Example 1 Preparation of Anti-47K Monoclonal Antibody, Anti-17K Monoclonal Antibody, and Anti-15K Monoclonal Antibody 47K and 17K or 15K N as surface antigens of Tp
GST17K or GST15K with GST fused to the end
And were each expressed in E. coli and purified. Hybridomas were prepared by cell fusion of spleen cells collected from mice immunized with each recombinant antigen and mouse myeloma cells. Each hybridoma was cultured, and a hybridoma that reacts with the recombinant antigen and also with the native antigen was screened by ELISA using the above recombinant antigen and Western blot using the native antigen. The screened hybridomas were cloned to establish cell lines. The established clone is injected into the peritoneal cavity of a mouse, ascites is collected, and the ascites is purified to purify the anti-47K monoclonal antibody (hereinafter referred to as 47K Mab in the present specification) and anti-17K monoclonal antibody (hereinafter referred to in the present specification). Then 17K
Mab) and anti-15K monoclonal antibody (hereinafter referred to as 15K Mab).

Reference Example 2 Preparation of Expression Vector pW6A The tac promoter and the nucleotide sequence encoding GST were removed from the vector pGEX2T manufactured by Pharmacia, and the T7 promoter of pGEMEX-1 manufactured by Promega was removed from the promoter.
The nucleotide sequences up to ne10, the multi-cloning site, and the T7 transcriptional terminator were inserted. The nucleotide sequence of gene10 was removed from the obtained vector, and a lac operator synthesized by a DNA synthesizer (Model 381A manufactured by Applied Biosystems) was inserted immediately after the T7 promoter to partially modify the multicloning site. The expression vector thus created is
It was named W6A. A detailed view of pW6A is shown in FIG.

Reference Example 3 Preparation of Primers for Preparing Various Fusion Antigen Expression Vectors In order to prepare the vectors for expressing each fusion antigen prepared in Examples 1 to 16 and Reference Example 4 below, The primer was synthesized using a DNA synthesizer (Model 381A manufactured by Applied Biosystems).

[0025]                5 '------------------------------------ 3'                       Nde I 47K-5 'terminal 18 bases Primer 1 TAGCC CATATG GGCTCGTCTCATCATGAG (29 bases) (sense)

[0026]                 15K-5 'end 17 bases 47K-3' end side 20 bases Primer 2 ATAGAACTAAATGAACA AGACACACGGGATAGGACAC (37 bases) (Antisense)

[0027]                 15K-5 'terminal side 17 bases Primer 3 TGTTCATTTAGTTCTATCCC (20 bases) (sense)

[0028]                 17K-5 'end 15 bases 15K-3' end 20 bases Primer 4 TGTGCACGAGACACA CCTGCTAATAATGGCTTCCT (35 bases) (Antisense)

[0029]                 17K-5 'terminal 20 bases Primer 5 TGTGTCTCGTGCACAACCGT (20 bases) (sense)

[0030]                       BamHI 17K-3 'terminal 21 bases Primer 6 GATCC GGATCC CTA TTTCTTTGTTTTTTTGAGCAC (35 bases) (Antisense) stop codon

[0031]                       Nde I 15K-5 'terminal 18 bases Primer 7 TAGCC CATATG TGTTCATTTAGTTCTATC (29 bases) (sense)

[0032]                       Nde I 17K-5 'terminal 18 bases Primer 8 TAGCC CATATG TGTGTCTCGTGCACAACC (29 bases) (sense)

[0033]                       BamHI 15K-3 'end 17 bases Primer 9 GATCC GGATCC CTA CCTGCTAATAATGGCTT (31 bases) (Antisense) stop codon

[0034]                       BamH I 47K-3 'end 17 bases Primer 10 GATCC GGATCC CTA AGACACACGGGATAGGA (31 bases) (Antisense) stop codon

[0035]                        Sal I 15K-5 'end 18 bases Primer 11 CGAGGC GTCGAC TGTTCATTTAGTTCTATC (30 bases) (sense)

[0036]                      Sal I 47K-5 'terminal 18 bases Primer 12 GAAC GTCGAC TGTGGCTCGTCTCATCAT (28 bases) (sense)

[0037]                      Xho I 47K-3 'end 18 bases Primer 13 CTTG CTCGAG AGACACACGGGATAGGAC (28 bases) (Antisense)

[0038]                      Sal I 17K-5 'terminal 18 bases Primer 14 AGTA GTCGAC TGTGTCTCGTGCACAACC (28 bases) (sense)

[0039]                      Xho I 17K-3 'terminal 21 bases Primer 15 CAGA CTCGAG TTTCTTTGTTTTTTTGAGCAC (31 bases) (Antisense)

[0040]                      Sal I 17K-3 'terminal 21 bases Primer 16 CAGA GTCGAC TTTCTTTGTTTTTTTGAGCAC (31 bases) (Antisense)

[0041]                      Sal I 15K-3 'end 18 bases Primer 17 GCTA GTCGAC CCTGCTAATAATGGCTTC (28 bases) (Antisense)

[0042]                      Sac I 47K-3 'terminal 20 bases Primer 18 CGTA GAGCTC AGACACACGGGATAGGACAC (30 bases) (Antisense)

[0043]                      Sac I 17K-5 'terminal 20 bases Primer 19 TAGC GAGCTC TGTGTCTCGTGCACAACCGT (30 bases) (sense)

[0044] Primer 20        BamHI I Base that encodes 9 amino acids 47K-3 'end 18 bases  GATCC GGATCC CTA AGACACACGGGATAGGACACCCCTCTT CTGGGCCACTACCTTCGC               Stop codon (59 bases) (Antisense)

[0045]                       Sal I 47K-3 'end 18 bases Primer 21 CTCTT GTCGAC AGACACACGGGATAGGAC (29 bases) (Antisense)

[0046]                      BamHI 15K-3 'end 18 bases Primer 22 CCGG GGATCC CCTGCTAATAATGGCTTC (28 bases) (Antisense)

[0047]                      BamHI 15K-5 'end 18 bases Primer 23 CCGG GGATCC TGTTCATTTAGTTCTATC (28 bases) (sense)

[0048]                      Kpn I 15K-3 'end 18 bases Primer 24 CCGG GGTACC CTA CCTGCTAATAATGGCTTC (31 bases) (Antisense) stop codon

[0049]                      Kpn I 15K-3 'end 18 bases Primer 25 CCGG GGTACC CCTGCTAATAATGGCTTC (28 bases) (Antisense)

[0050]                      Kpn I 15K-5 'terminal 18 bases Primer 26 CCGG GGTACC TGTTCATTTAGTTCTATC (28 bases) (sense)

[0051]                      Hind III 15K-3 'terminal 15 bases Primer 27 CCGG AAGCTT CTA CCTGCTAATAATGGC (28 bases) (Antisense) stop codon

[0052]                      EcoR I 17K-3 'terminal 21 bases Primer 28 GACT GAATTC TTTCTTTGTTTTTTTGAGCAC (31 bases) (Antisense)

[0053]                       EcoR I 15K-5 'terminal 21 bases Primer 29 GGTG GAATTC TGTTCATTTAGTTCTATCCCG (31 bases) (sense)

Reference Example 4 Preparation of Tp47K, 17K and 15K Genes Bacteria were purified from rabbit testes by subculture of syphilis (Treponema pallidum, Nicols strain) by Percoll density centrifugation (Sex. Transm. Dis., Vol. 11, p.275-286, 1984)
Then, the genomic DNA was extracted by the SDS-proteinase K / phenol / chloroform method. By the polymerase chain reaction (PCR) method, the primer 7 prepared in Reference Example 3 was selected as a sense primer, and the primer 9 prepared in Reference Example 3 was selected as an antisense primer, and the extracted genomic DNA was used as a template for 15K (Molecular Microbiology, Vol.4, p.1371-
1379, 1990) was prepared. Next, the primer 8 prepared in Reference Example 3 was selected as the sense primer, and the primer 6 prepared in Reference Example 3 was selected as the antisense primer.
DN coding unity, Vol.61, p.1202-1210, 1993)
The A fragment was created. Further, the primer 1 prepared in Reference Example 3 was selected as the sense primer, and the primer 20 prepared in Reference Example 3 was selected as the antisense primer.
7K (Infection and Immunity, Vol.60, p.1568-1576,
1992) was prepared. In 47K, it is reported that an additional 9 amino acids may be added after the C-terminal of 434 amino acids in the body (Infectio
n and Immunity, Vol.60, p1568-1576, 1992), as shown in FIG. 2, when amplifying a 47K DNA fragment, the nucleotide sequence corresponding to this 9 amino acid was added to the antisense primer used. Was prepared and used. In addition, DNA fragments encoding 15K and 17K are shown in FIG.

Example 1 15-17 Preparation of fusion antigen expression vector a) Primer 7 prepared in Reference Example 3 was selected as a sense primer, and primer 4 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1μ
PCR was performed using M and 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase (TaKaRa) at 94 ° C. for 1 minute, −55 ° C. for 2 minutes, −72 ° C. for 3 minutes, and 30 cycles. Then, the DNA fragment 1 was prepared. b) Using the primers 5 and 6 prepared in Reference Example 3 and the DNA fragment encoding 17K prepared in Reference Example 4,
PCR was performed in the same manner as in Example 1-a) to prepare DNA fragment 2. c) Using 1 ng each of the DNA fragments 1 and 2 prepared in Examples 1-a) and b), 1 μM each of the primers 7 and 6 prepared in Reference Example 3, and 2.5 units of the Taq polymerase at 94 ° C. 1 minute-55 ° C 2 minutes-72 ° C 3
After 40 minutes and 40 cycles, PCR was further performed under the conditions of 72 ° C. for 15 minutes and 1 cycle to prepare a DNA fragment encoding the 15-17 fusion antigen. d) The pW6A prepared in Reference Example 2 and the 15-17 fusion antigen gene prepared in Example 1-c) were combined with NdeI (NEW EN
5 units made by GLAND BIOLAB and BamHI (NEW EN
5 units of GLAND BIOLAB) were cut at 37 ° C. for 1 hour. e) pW6A and 15-17 prepared in Example 1-d)
The fusion antigen gene was reacted with Ligation Kit ver.1 manufactured by TaKaRa under the condition of 16 ° C. for 2 hours. After completion of the reaction, this reaction solution was used to transform Escherichia coli DH5α, which is a competent cell prepared by the Hanahan method, to give 50 μg.
The strain was spread on LB agar medium containing / ml ampicillin, and cultured overnight at 37 ° C. From the colonies of the ampicillin-resistant bacterium that appeared, a colony having a 15-17 fusion antigen expression vector incorporating the 15-17 fusion antigen gene was selected, and this colony was incubated overnight at 37 ° C in an LB liquid medium containing ampicillin. Shake culture, collect cells, and use alkaline SD
The target vector was purified by the S method. The results are shown in Fig. 4.

Example 2 Preparation of 15-47 fusion antigen expression vector a) Primer 7 prepared in Reference Example 3 as a sense primer and primer 1 as an antisense primer
7 was selected. Using 1 μM of each primer, 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of the Taq polymerase, 94 ° C. for 1 minute and −55 ° C. 2
A PCR method was carried out under the conditions of 30 minutes at −72 ° C. for 3 minutes to prepare a DNA fragment 3. b) DNA fragment 3 produced in Example 2-a) and Reference Example 2
The pW6A prepared in 1. was used with NdeI5 unit and Sal
It cut | disconnected at 37 degreeC for 1 hour by 5 units of I (made by Toyobo).
After completion of the reaction, DNA fragment 3 was inserted into pW6A prepared in Reference Example 2 under the same conditions as in Example 1-e). c) The primer 12 prepared in Reference Example 3 was selected as the sense primer, and the primer 10 was selected as the antisense primer. 1 μM of each primer and Reference Example 4
PCR was performed using 0.1 ng of 47K gene obtained in 1. and 2.5 units of Taq polymerase under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes, 30 cycles.
NA fragment 4 was obtained. d) DNA fragment 3 insertion pW6 prepared in Example 2-b)
A and the DNA fragment 4 prepared in Example 2-c) were cleaved with the SalI5 unit and the BamHI5 unit, respectively, at 37 ° C. for 1 hour. After completion of the reaction, the DNA fragment 4 was inserted into the DNA fragment 3 inserted pW6A under the same conditions as in Example 1-e) to obtain a 15-47 fusion antigen expression vector. The results are shown in Fig. 4.

Example 3 Preparation of 17-47 Fusion Antigen Expression Vector a) Primer 8 prepared in Reference Example 3 was selected as a sense primer, and primer 15 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of 17K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase were used to
PCR was performed under the conditions of 4 cycles of 1 minute-55 degrees C of 2 minutes-72 degrees C of 3 minutes for 30 cycles to prepare DNA fragment 5. b) DNA fragment 5 obtained in Example 3-a) and Reference Example 2
The pW6A prepared in step 1 was used as the NdeI5 unit and X
Five units of hoI (manufactured by Toyobo Co., Ltd.) were cut at 37 ° C for 1 hour. After completion of the reaction, pW6 was prepared under the same conditions as in Example 1-e).
DNA fragment 5 was inserted into A. c) The DNA fragment 4 obtained in Example 2-c) is the above Sa
11-unit and BamHI5-unit, and Example 3-
The DNA fragment 5 inserted pW6A obtained in b) is the above Xho.
I5 unit and BamHI5 unit at 37 ℃
It was cut for 1 hour. Then, under the same conditions as in Example 1-e), the DNA fragment 4 was inserted into the DNA fragment 5 insertion pW6A, and 1
A 7-47 fusion antigen expression vector was prepared. The results are shown in Fig. 4.

Example 4 Preparation of 17-15 Fusion Antigen Expression Vector a) Primer 11 prepared in Reference Example 3 was selected as a sense primer, and primer 9 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of the Taq polymerase were used to obtain 9
The PCR method was carried out under the conditions of 4 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes, 30 cycles to obtain DNA fragment 6. b) The DNA fragment 6 prepared in Example 4-a) is the same as Sa described above.
11-unit and BamHI5-unit, and Example 3-
The DNA fragment 5 inserted pW6A prepared in b) is the above Xho.
37 ° C 1 with I5 unit and BamHI5 unit
I disconnected for an hour. After completion of the reaction, the DNA fragment 6 was inserted into the DNA fragment 5 insertion pW6A under the same conditions as in Example 1-e),
A 17-15 fusion antigen expression vector was prepared. The results are shown in Fig. 4.

Example 5 Preparation of 47-15 Fusion Antigen Expression Vector a) Primer 1 prepared in Reference Example 3 was selected as a sense primer, and Primer 21 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of 47K gene obtained in Reference Example 4 and 2.5 units of Taq polymerase at 94 ° C.
The PCR method was carried out under the conditions of 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes and 30 cycles to obtain a DNA fragment 7. b) DNA fragment 7 prepared in Example 5-a) and pW6A
Were cut with the NdeI5 unit and the SalI5 unit, respectively, at 37 ° C. for 1 hour. After completion of the reaction, Example 1
DNA fragment 7 was inserted into pW6A under the same conditions as in (e). c) DNA fragment 6 prepared in Example 4-a) and Example 5
The DNA fragment 7-inserted pW6A prepared in -b) was cut with the SalI5 unit and the BamHI5 unit at 37 ° C for 1 hour. After completion of the reaction, DNA fragment 6 was inserted into pW6A having DNA fragment 7 inserted under the same conditions as in Example 1-e) to prepare a 47-15 fusion antigen expression vector. The results are shown in Fig. 4.

Example 6 Preparation of 47-17 fusion antigen expression vector a) Primer 14 prepared in Reference Example 3 was selected as a sense primer, and primer 6 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of 17K gene obtained in Reference Example 4 and 2.5 units of Taq polymerase at 94 ° C.
PCR was carried out under the conditions of 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes for 30 cycles to prepare a DNA fragment 8. b) DNA fragment 8 prepared in Example 6-a) and Example 5
The DNA fragment 7-inserted pW6A prepared in -b) was cut with the SalI5 unit and the BamHI5 unit at 37 ° C for 1 hour. After completion of the reaction, DNA fragment 8 was inserted into pW6A having DNA fragment 7 inserted under the same conditions as in Example 1-e) to prepare a 47-17 fusion antigen expression vector. The results are shown in Fig. 4.

Example 7 Preparation of 47-15-17 Fusion Antigen Expression Vector a) Primer 11 prepared in Reference Example 3 was selected as a sense primer, and primer 6 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of the 15-17 fusion antigen expression vector prepared in Example 1, and Taq polymerase 2.5 described above.
Using the unit, 94 ℃ 1 minute -55 ℃ 2 minutes -72 ℃ 3
PCR was performed under the condition of 30 minutes and 30 minutes for DNA fragment 9
Got b) DNA fragment 9 prepared in Example 7-a) and Example 5
The DNA fragment 7-inserted pW6A prepared in -b) was cut with the SalI5 unit and the BamHI5 unit at 37 ° C for 1 hour. After the completion of the reaction, the DNA fragment 9 was inserted into the DNA fragment 7-inserted pW6A under the same conditions as in Example 1-e) to prepare a 47-15-17 fusion antigen expression vector. Results are shown in FIG.

Example 8 Preparation of 17-47-15 Fusion Antigen Expression Vector a) Primer 12 prepared in Reference Example 3 was selected as a sense primer, and Primer 2 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of 47K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase were used to obtain 9
PCR was performed under the conditions of 4 cycles of 1 minute-55 degrees C of 2 minutes-72 degrees C of 3 minutes and 30 cycles to prepare a DNA fragment 10. b) The primer 3 prepared in Reference Example 3 was selected as the sense primer, and the primer 9 prepared in Reference Example 3 was selected as the antisense primer. Each primer 1μ
Using M, 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of the Taq polymerase, 94
The PCR method was carried out under the conditions of 30 ° C. for 1 minute at −55 ° C. for 2 minutes and −72 ° C. for 3 minutes to prepare a DNA fragment 11. c) DNA fragment 10 prepared in Examples 8-a) and b)
And 11 each of 1 ng, each of the primers 12 and 9 prepared in Reference Example 3 at 1 μM and 2.5 units of Taq polymerase described above at 94 ° C for 1 minute-55 ° C for 2 minutes-7.
After 40 cycles at 2 ° C for 3 minutes, PCR was performed under the conditions of 72 ° C for 15 minutes and 1 cycle to prepare a gene encoding the 47-15 fusion antigen. d) The gene encoding the 47-15 fusion antigen obtained in Example 8-c) contains the SalI5 unit and BamHI.
5 units, DNA fragment 5 prepared in Example 3-b)
The inserted pW6A contains the XhoI5 unit and the Bam.
It cut | disconnected at 37 degreeC 1 hour with HI5 unit. After the reaction,
DNA fragment 5 insertion pW6A under the same conditions as in Example 1-e)
The gene encoding the 47-15 fusion antigen was inserted into
A vector for expressing the 7-47-15 fusion antigen was prepared. Results are shown in FIG.

Example 9 Preparation of 17-15-47 Fusion Antigen Expression Vector a) Primer 8 prepared in Reference Example 3 was selected as a sense primer, and primer 17 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of the 17-15 fusion antigen expression vector prepared in Example 4, and Taq polymerase 2.5 described above.
Using the unit, 94 ℃ 1 minute -55 ℃ 2 minutes -72 ℃ 3
PCR is performed under the condition of 30 minutes and 30 cycles for DNA fragment 1
2 was produced. b) The DNA fragment 12 prepared in Example 9-a) and pW6A prepared in Reference Example 2 were digested with the NdeI5 unit and the SalI5 unit, respectively, at 37 ° C. for 1 hour.
After completion of the reaction, pW6A was added with D under the same conditions as in Example 1-e).
NA fragment 12 was inserted. c) DNA fragment 4 prepared in Example 2-c) and Example 9
The DNA fragment 12-inserted pW6A prepared in -b) was cleaved with the SalI5 unit and the BamHI5 unit, respectively, at 37 ° C for 1 hour. After completion of the reaction, Example 1-e)
DNA fragment 4 was inserted into pW6A in which DNA fragment 12 was inserted under the same conditions as above to prepare a 17-15-47 fusion antigen expression vector. Results are shown in FIG.

Example 10 Preparation of 15-47-17 fusion antigen expression vector a) Primer 12 prepared in Reference Example 3 was selected as a sense primer, and primer 13 prepared in Reference Example 3 was selected as an antisense primer. 1 μM of each primer and 0.1 ng of 47K gene prepared in Reference Example 4
And 2.5 units of the Taq polymerase,
PCR was performed under conditions of 94 ° C. for 1 minute, −55 ° C. for 2 minutes, −72 ° C. for 3 minutes, and 30 cycles to prepare DNA fragment 13. b) The DNA fragment 13 prepared in Example 10-a) and pW6A prepared in Reference Example 2 were cleaved with the SalI5 unit and the XhoI5 unit, respectively, at 37 ° C. for 1 hour. After completion of the reaction, pW6A was prepared under the same conditions as in Example 1-e).
DNA fragment 13 was inserted into c) The DNA fragment 8 prepared in Example 6 is the SalI5
Unit and BamHI5 unit, Example 10-b).
The DNA fragment 13-inserted pW6A prepared in 1.
It cut | disconnected by 5 unit and said BamHI5 unit at 37 degreeC for 1 hour. After completion of the reaction, the DNA fragment 8 was inserted into the DNA fragment 13-inserted pW6A under the same conditions as in Example 1-e). (Hereinafter, referred to as DNA fragment 13-8 inserted pW6A.) D) DNA fragment 3 prepared in Example 2-a) and Example 1
0-c) DNA fragment 13-8 insertion pW6A
Were cut with the NdeI5 unit and the SalI5 unit, respectively, at 37 ° C. for 1 hour. After completion of the reaction, Example 1
The DNA fragment 3 was inserted into the DNA fragment 13-8-inserted pW6A under the same conditions as in -e) to obtain a 15-47-17 fusion antigen expression vector. Results are shown in FIG.

Example 11 Preparation of 15-17-47 fusion antigen expression vector a) Primer 7 prepared in Reference Example 3 was selected as a sense primer, and primer 16 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of the 15-17 fusion antigen expression vector prepared in Example 1, and Taq polymerase 2.5 described above.
Using the unit, 94 ℃ 1 minute -55 ℃ 2 minutes -72 ℃ 3
PCR is performed under the condition of 30 minutes and 30 cycles for DNA fragment 1
4 was produced. b) The DNA fragment 14 prepared in Example 11-a) and pW6A prepared in Reference Example 2 were cleaved with the NdeI5 unit and the SalI5 unit, respectively, at 37 ° C. for 1 hour. After completion of the reaction, pW6A was prepared under the same conditions as in Example 1-e).
DNA fragment 14 was inserted into c) DNA fragment 4 prepared in Example 2-c) and Example 1
The DNA fragment 14-inserted pW6A prepared in 1-b) was cut with the SalI5 unit and the BamHI5 unit at 37 ° C. for 1 hour. After completion of the reaction, Example 1-
Under the same conditions as in e), DNA was inserted into pW6A of DNA fragment 14.
Fragment 4 was inserted to prepare a 15-17-47 fusion antigen expression vector. Results are shown in FIG.

Example 12 Preparation of 47-17-15 fusion antigen expression vector a) Primer 1 prepared in Reference Example 3 was selected as a sense primer, and primer 18 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of 47K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase described above
The PCR method was carried out under the conditions of 30 ° C. for 1 minute at −55 ° C. for 2 minutes and −72 ° C. for 3 minutes to prepare a DNA fragment 15. b) The DNA fragment 15 prepared in Example 12-a) and pW6A prepared in Reference Example 2 were cleaved with 5 units of the SacI (Toyobo Co., Ltd.) at 37 ° C. for 1 hour, and then the Nde was further separated.
Cut with I5 unit for 1 hour. After completion of the reaction, Example 1
DNA fragment 15 was inserted into pW6A under the same conditions as in (e). c) The primer 19 prepared in Reference Example 3 was selected as the sense primer, and the primer 28 prepared in Reference Example 3 was selected as the antisense primer. 1 μM of each primer and 0.1 ng of 17K gene prepared in Reference Example 4
And 2.5 units of the Taq polymerase,
PCR was performed under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes, 30 cycles to prepare DNA fragment 16. d) The primer 29 prepared in Reference Example 3 was selected as the sense primer, and the primer 9 prepared in Reference Example 3 was selected as the antisense primer. Each primer 1
μM, 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of the Taq polymerase were used to obtain 9
PCR was performed under the conditions of 4 cycles of 1 minute-55 degrees C of 2 minutes-72 degrees C of 3 minutes for 30 cycles to prepare a DNA fragment 17. e) The DNA fragment 16 obtained in Example 12-c) was digested with the SacI5 unit at 37 ° C. for 1 hour, and then Eco.
It cut | disconnected at 37 degreeC 1 hour with 5 units of RI (made by Toyobo). The DNA fragment 17 obtained in Example 12-d) was digested with the EcoRI5 unit and the BamHI5 unit at 37 ° C. for 1 hour. The DNA fragment 15-inserted pW6A prepared in Example 12-b) was digested with the SacI5 unit at 37 ° C. for 1 hour, and further digested with the BamHI5 unit at 37 ° C. for 1 hour. After completion of the reaction, Example 1-
Under the same conditions as in e), DNA was inserted into pW6A of DNA fragment 15.
Fragment 16 and DNA fragment 17 were inserted simultaneously, 47-17
A -15 fusion antigen expression vector was obtained. Results are shown in FIG.

Example 13 15 Preparation of vector for expressing antigen a) Primer 7 prepared in Reference Example 3 was selected as a sense primer, and primer 9 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1μ
PCR was performed using M and 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase (TaKaRa) at 94 ° C. for 1 minute, −55 ° C. for 2 minutes, −72 ° C. for 3 minutes, and 30 cycles. Then, the DNA fragment 18 was prepared. b) DNA fragment 18 and pW6A prepared in Reference Example 2
Each was cut with the NdeI5 unit and the BamHI5 unit at 37 ° C. for 1 hour. After completion of the reaction, Example 1-
Under the same conditions as in e), insert the DNA fragment 18 into pW6A,
A vector for expressing 15 antigens was obtained. Results are shown in FIG.

Example 14 Preparation of 15-15 Antigen Expression Vector a) Primer 11 prepared in Reference Example 3 was selected as a sense primer, and primer 9 prepared in Reference Example 3 was selected as an antisense primer. Each primer 1
μM, 0.1 ng of the 15K gene prepared in Reference Example 4 and 2.5 units of Taq polymerase (TaKaRa) were used to perform PCR under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes, 30 cycles. Then, the DNA fragment 19 was prepared. b) DNA fragment 19 and 15-47 obtained in Example 10
-17 fusion antigen expression vector,
A unit and the BamHI5 unit were cut at 37 ° C. for 1 hour. After completion of the reaction, D under the same conditions as in Example 1-e)
The DNA fragment 19 was inserted into the 15-47-17 fusion antigen expression vector from which the NA fragment 47-17 had been removed.
A vector for expressing 5 fusion antigens was obtained. Results are shown in FIG.

Example 15 Preparation of 15-15-15 Antigen Expression Vector a) Primer 11 prepared in Reference Example 3 was selected as a sense primer, and primer 22 prepared in Reference Example 3 was selected as an antisense primer. 1 μM of each primer and 0.1 ng of 15K gene prepared in Reference Example 4
And 2.5 units of Taq polymerase (manufactured by TaKaRa) were used to perform the PCR method under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes for 30 cycles to prepare a DNA fragment 20. b) DNA fragment 20 and 15-47 obtained in Example 10
-17 fusion antigen expression vector,
A unit and the BamHI5 unit were cut at 37 ° C. for 1 hour. After completion of the reaction, D under the same conditions as in Example 1-e)
The DNA fragment 20 was inserted into the 15-47-17 fusion antigen expression vector from which the NA fragment 47-17 was removed (hereinafter, referred to as
DNA fragment 15-15 insert designated pW6A). c) The primer 23 prepared in Reference Example 3 was selected as the sense primer, and the primer 24 prepared in Reference Example 3 was selected as the antisense primer. 1 μM of each primer and 0.1 ng of 15K gene prepared in Reference Example 4
And 2.5 units of Taq polymerase (TaKaRa) were used to perform a PCR method under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes for 30 cycles to prepare a DNA fragment 21. d) DNA fragment 21 obtained in Example 15-c) and DNA fragment 15-15 insertion pW prepared in Example 15-b)
6A with 5 units of KpnI (manufactured by Toyobo Co., Ltd.) at 37 ° C.
After cutting for a period of time, the BamHI5 unit was further used at 37 ° C 1
I disconnected for an hour. After the reaction was completed, the DNA fragment 21 was inserted into pW6A inserted into the DNA fragment 15-15 under the same conditions as in Example 1-e).
Was inserted to obtain a vector for expressing 15-15-15 fusion antigen. Results are shown in FIG.

Example 16 Preparation of 15-15-15-15 antigen expression vector a) Primer 23 prepared in Reference Example 3 was selected as a sense primer, and primer 25 prepared in Reference Example 3 was selected as an antisense primer. 1 μM of each primer and 0.1 ng of 15K gene prepared in Reference Example 4
And 2.5 units of Taq polymerase (TaKaRa) were used to perform a PCR method under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes for 30 cycles to prepare a DNA fragment 22. b) DNA fragment 22 obtained in Example 16-a) and DNA fragment 15-15 insertion pW prepared in Example 15-b).
6A was cut with the KpnI5 unit at 37 ° C. for 1 hour, and further cut with the BamHI5 unit at 37 ° C. for 1 hour. After the reaction was completed, DNA was prepared under the same conditions as in Example 1-e).
DNA fragment 22 was inserted into fragment 15-15-inserted pW6A (hereinafter referred to as DNA fragment 15-15-15-inserted pW6A). c) The primer 26 prepared in Reference Example 3 was selected as the sense primer, and the primer 27 prepared in Reference Example 3 was selected as the antisense primer. 1 μM of each primer and 0.1 ng of 15K gene prepared in Reference Example 4
And 2.5 units of Taq polymerase (TaKaRa) were used to perform a PCR method under the conditions of 94 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 3 minutes for 30 cycles to prepare a DNA fragment 23. d) The DNA fragment 23 obtained in Example 16-c) and the DNA fragment 15-15-15-inserted pW6A prepared in Example 16-b) were cleaved with the KpnI5 unit at 37 ° C. for 1 hour, and then HindIII ( It was cut at 37 ° C. for 1 hour with 5 units manufactured by Toyobo Co., Ltd. After completion of the reaction, Example 1-e)
DNA fragment 15-15-15 insertion pW6A under the same conditions as
DNA fragment 23 was inserted into to obtain a 15-15-15-15 fusion antigen expression vector. Results are shown in FIG.

Example 17 Expression of various fusion antigens Escherichia coli BL which is a competent cell prepared by the Hanahan method using each expression vector obtained in Examples 1 to 12
21 (DE3) was transformed and 50 μg / ml
Spread the fungus on LB agar medium containing ampicillin 37
Cultured overnight at ° C. From the emerged ampicillin-resistant bacterial colonies, select a colony having a fusion antigen expression vector incorporating each fusion antigen gene, and select 50 μg / ml.
It was shake-cultured overnight at 37 ° C. in 4 ml of LB liquid medium containing ampicillin. 50 μg / m 2 of this cultured bacterium
1 L LB liquid medium containing 1 ampicillin at 37 ° C for 3
After shaking culture for 1 hour, 1 mM IPTG (isopropyl-β-D (-)-thiogalactopyranoside) was added as a final concentration,
Further, the cells were cultured with shaking at 37 ° C. overnight to induce the expression of each fusion antigen.

Example 18 Purification of Fusion Antigen Among the fusion antigens expressed in Example 17, 47-17-1
5 fusion antigen, 47-15-17 fusion antigen, 15-17-
The 47 and 15-17 fusion antigens were purified.
47-17-15 fusion antigen of 3 antigen fusion type, 47-15
For the -17 fusion antigen and the 15-17-47 fusion antigen, Escherichia coli after antigen expression was ultrasonically disrupted, the precipitate was solubilized with urea after centrifugation, and gel filtration was performed using Superdex-200 (Pharmacia). Purification was carried out by chromatography, ion exchange chromatography using Q-Sepharose (Pharmacia), and adsorption chromatography using ceramic hydroxyapatite (Asahi Optical Co., Ltd.). Regarding the two-antigen fusion type 15-17 fusion antigen, Escherichia coli after antigen expression was ultrasonically disrupted, and after centrifugation, the supernatant was denatured with urea and ion-exchange chromatography using Q-Sepharose (Pharmacia). Graphy, ceramic hydroxyapatite (Asahi Optical Co., Ltd.)
Purification was carried out by adsorption chromatography using and ion-exchange chromatography using SP-Sepharose (Pharmacia). Each antigen has a purity of 95% or more from 1 liter of culture solution, and 47-17-15 fusion antigen is 7 m.
g, 47-15-17 fusion antigen 10 mg, 15-17
-47 fusion antigen is 20 mg, 15-17 fusion antigen is 10 mg
mg was obtained. Each purified fusion antigen was subjected to reducing SDS-polyacrylamide gel electrophoresis and stained with Coomassie. The results are shown in Fig. 7. The three-antigen fusion type antigen has an amino acid sequence of 7
The 15-17 fusion antigen was predicted to have a molecular weight of 5K and the molecular weight of 29K, but the results of electrophoresis confirmed that the purified antigens each had the expected molecular weight. Also, 47 KMa produced in Reference Example 1
When Western blotting was performed using b, 17K Mab and 15K Mab, it was confirmed that each monoclonal antibody reacted with each antigen in the fusion antigen.

Example 19 Preparation of fusion antigen-sensitized plate Each well of a 96-well ELISA plate (manufactured by BECTON DICKINSON) was diluted with 10 mM PBS and diluted to 0.66 pmol / well. It was sensitized by pouring and leaving it at 4 ° C. overnight. After the sensitization, blocking was performed with 10 mM PBS containing 1% skim milk at 37 ° C. for 2 hours.

Reference Example 5 Preparation of single antigen-sensitized plate Recombinant single antigen 47K expressed and purified in E. coli
Antigen, 17K antigen containing a signal peptide at the N-terminus (hereinafter referred to as S17K antigen) and GST
0.66 pm of each or all 15K antigens in separate wells
sensitized with ol / well. After the sensitization, blocking was performed with 10 mM PBS containing 1% skim milk at 37 ° C. for 2 hours.

Example 20 Reactivity test between fusion antigen and monoclonal antibody 47KMab, 17KMab, 15 prepared in Reference Example 1
10mMP containing 1% skimmed milk and 0.05% Tween 20 (trade name) so that the final concentration of each monoclonal antibody in each of the KMabs or the mixture is 10 μM
After diluting with BS (hereinafter referred to as skim milk-containing PBST), 50 μl was added to each well of the antigen-sensitized plate prepared in Example 19 and Reference Example 5 after washing, and incubated at room temperature for 1.5 hours. After cleaning, skim-containing milk PBST
50 μl of peroxidase-labeled anti-mouse Ig (manufactured by DAKO) diluted 1/1000 with 1 was added and incubated at room temperature for 1.5 hours. After washing, 50 μl of a mixed solution of hydrogen peroxide solution and ABTS was added to develop color for 3 minutes. After stopping the reaction, the absorbance at 405 nm was measured with a spectrophotometer. The results are shown in Table 1. As shown in Table 1, the fusion antigen showed an activity equal to or higher than that of the single antigens 47K, S17K and GST15K.

[0076]

[Table 1]

Example 21 Comparative test of reactivity between fusion antigen and single antigen Tp diluted 1/1000 with skim milk containing PBST
50 μl of positive sera (purchased from Boston Biomedica Inc.) and negative sera were added to each well after washing the antigen-sensitized plates prepared in Example 19 and Reference Example 5 at room temperature.
Incubated for 5 hours. After cleaning, skim milk containing P
50 μl of peroxidase-labeled anti-human IgG (manufactured by BIO SOURCE) diluted 1/1000 with BST was added, and the mixture was incubated at room temperature for 1.5 hours. After washing, 50 μl of a mixed solution of hydrogen peroxide solution and ABTS was added to develop color for 3 minutes. After stopping the reaction, the absorbance at 405 nm was measured with a spectrophotometer. The results are shown in Table 2. The fusion antigen had a higher reaction with the positive serum than with the single antigen and those sensitized by mixing the single antigen, and did not react with the negative serum at all.

[0078]

[Table 2]

Example 22 Reactivity test between fusion antigen and positive serum Tp positive serum (24 cases (purchased from Boston Biomedica Inc.)) and negative serum (1 case) were prepared in the same manner as in Example 21 except that the coloring time was 30 minutes. Measured. The results are shown in Table 3. The reaction of the fusion antigen with 24 positive sera was significantly higher than that with the negative sera.

[0080]

[Table 3]

Example 23 15K, 15K-15K, 1
5K-15K-15K, 15K-15K-15K-15
Expression of K 15K, 15K-15 obtained in Examples 13 to 16
K, 15K-15K-15K, 15K-15K-15K
E. coli BL21 (DE
3) was transformed by the Hanahan method. Then 50
The transformed bacteria were plated on LB agar medium containing μg / ml ampicillin and cultured overnight at 37 ° C. 2 ml containing ampicillin containing 50 μg / ml ampicillin was selected from among the colonies of the ampicillin-resistant bacteria that appeared and which had a fusion antigen expression vector into which each fusion antigen gene was incorporated.
LB liquid medium was cultured at 37 ° C. with shaking overnight. After 40 μl of the cultivated bacteria was shake-cultured in 2 ml of LB liquid medium containing 50 μg / ml ampicillin at 37 ° C. for 2 hours,
The final concentration was 1 mM IPTG (isopropyl-β-D (-)-
Thiogalactopyranoside) was added, and the mixture was further cultured by shaking at 37 ° C. overnight to induce the expression of each fusion antigen. 1.5 ml of 2 ml of the Escherichia coli culture broth thus obtained was transferred to a microcentrifuge tube, and a microcentrifuge (Himac CT 15D manufactured by Hitachi, Ltd.) was used to centrifuge at 15,000 rpm for 30 seconds to collect the cells. . 15 for each tube
0 μl TE buffer (10 mM Tris-HCl buffer,
pH 8.0, 1 mM EDTA and 150 μl × 2 sample buffer (1 M Tris-HCl buffer, pH 6.
(8, 20% w / v SDS, 10% v / v β-ME) was added, mixed well, boiled for 5 minutes, and further sonicated for 10 minutes. 2 μl for 13 μl of the sample obtained in this way
50% glycerin was added and mixed.
% SDS gel electrophoresis was performed using a polyacrylamide gel (Laemmli, UK 1970: Nature 227, 680).
After electrophoresis, the gel was Coomassie stained. The result is shown in Fig. 8.
Shown in. When induced overnight, 15K-15K, 15K-
Bands of 15K-15K and 15K-15K-15K-15K were confirmed. Similarly, each antigen whose expression was induced overnight was subjected to reduction SDS electrophoresis using a 10 to 15% polyacrylamide gradient gel, electroblotted on a nitrocellulose membrane, and after blocking, 15K Mab prepared in Reference Example 1 as a primary antibody. Was used for Western blotting. As shown in FIG. 9, 15K, 15K-
15K, 15K-15K-15K, 15K-15K-1
All bands of 5K-15K could be confirmed.

Example 24 15K, 15K-15K, 1
5K-15K-15K, 15K-15K-15K-15
Comparison of K Expression Levels An electrophoretic sample prepared by overnight expression-inducing Escherichia coli was prepared as shown in Example 23, and an electrophoretic marker containing BSA of known concentration (Pharmacia LKB Low Molecular Were).
Right Calibration Kit) was subjected to reducing SDS electrophoresis using a 15% polyacrylimide gel, and after electrophoresis, the gel was stained with Coomassie. The result is shown in FIG. Next, the amount of antigen expressed from the stained band was calculated using a densitometer (AE-6900 densitogram manufactured by Atto). B of known density for lane1 to lane6 in FIG.
The density of the SA band was measured using a densitometer to prepare a calibration curve. The result is shown in FIG. Furthermore, 15K, 15K-15K, 15K-15K-15K, 1
The intensity of the corresponding band of 5K-15K-15K-15K was similarly measured using a densitometer, and the expression level was calculated using the calibration curve in FIG. The results are shown in Table 4. No band is observed for 15K, but 15K-15K
At -15K, the expression level was the highest at 1 μg per lane (equivalent to 23 mg per liter of cell culture). By repeatedly fusing 15K in series as described above, the expression level was increased.

[0083]

[Table 4]

[Brief description of drawings]

FIG. 1 is a diagram showing details of the expressed Betacou pW6A.

FIG. 2 is a diagram showing a procedure for preparing a gene encoding a 47K antigen.

FIG. 3 is a view showing a procedure for preparing genes encoding 15K and 17K antigens.

FIG. 4 is a diagram showing details of a vector expressing a biantigen fusion antigen.

FIG. 5 is a diagram showing details of a vector expressing a triantigen fusion antigen.

FIG. 6 is a diagram showing details of a vector expressing an antigen in which 15K is repeatedly fused.

FIG. 7 is an electropherogram of purified fusion antigen.

FIG. 8 is an electropherogram of 15K repeat fusion antigen.

Figure 9: Western blot of 15K repeat fusion antigen.

FIG. 10 is an electrophoretogram for quantifying a 15K repeat fusion antigen.

FIG. 11 is a graph showing a calibration curve for quantification of bands on an electrophoretic gel prepared using BSA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI G01N 33/571 H01J 61/067 L H01J 61/067 C12P 21/02 (C12P 21/02 C12R 1:19 C12R 1:19) C12N 15/00 ZNAA (72) Inventor Masahisa Okada 2-7-1, Nishi-Shinjuku, Shinjuku-ku, Tokyo Inside Fuji Revio Co., Ltd. (56) Reference JP-A-7-287017 (JP, A) JP-A-62- 48699 (JP, A) JP 9-229938 (JP, A) JP 9-288110 (JP, A) International publication 95/012676 (WO, A1) International publication 95/004145 (WO, A1) (58) ) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/09 C07K 14/20 C07K 19/00 BIOSIS / MEDLINE / WPIDS (STN) EUROPAT (QUESTEL)

Claims (3)

(57) [Claims] 1. A fusion antigen of Treponema pallidum in which 2 to 4 surface antigens of Treponema pallidum are fused. 2. The molecular weight of the surface antigen of Treponema pallidum is
The Treponema pallidum fusion antigen according to claim 1, which is 47 kDa, 17 kDa, or 15 kDa. 3. A fusion antigen, 47 kDa, 15 kDa, 17 in which Treponema pallidum fusion antigen is fused from N-terminal side to C-terminal side in the order of 47 kDa, 17 kDa, 15 kDa.
Fusion antigen, 15kDa, 17k fused in order of kDa
Fusion antigen, 15kD fused in the order of Da and 47kDa
a, a fusion antigen fused in the order of 47kDa, 17kDa, a fusion antigen fused in the order of 17kDa, 15kDa, 47kDa, a fusion antigen fused in the order of 17kDa, 47kDa, 15kDa, a fusion antigen in the order of 47kDa, 17kDa Fusion antigen, fusion antigen in the order of 47kDa, 15kDa, fusion antigen in the order of 17kDa, 47kDa, fusion antigen in the order of 17kDa, 15kDa, fusion antigen in the order of 15kDa, 47kDa Antigen, fusion antigen fused in the order of 15 kDa, 17 kDa, fusion antigen fused in the order of 15 kDa, 15 kDa, fusion antigen fused in the order of 15 kDa, 15 kDa, 15 kDa, or 15 kDa, 15 kDa
The fusion antigen according to claim 2, which is a fusion antigen fused in the order of a, 15 kDa, and 15 kDa.