JP3360557B2 - Fusion DNA sequence, fusion protein and method for expressing the protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to expression of a fusion protein. More specifically, a fusion DNA sequence containing a DNA sequence encoding a thermostable protein, the fusion DN
The present invention relates to a fusion protein expressed by the A sequence and a method for expressing the fusion protein.

[0002]

2. Description of the Related Art Advances in genetic engineering have made it possible to analyze proteins that have been purified from natural products at the gene level and artificially amplify the target protein (Itakura).
et.al, Science, 198 1056 (1977)). Thioredoxin invented thereafter (hereinafter referred to as TRX in the present specification) (Table 5) and glutathione-S
-Transferase (hereinafter referred to as GST in the present specification) (Japanese Patent Application Laid-Open No. 1-503441);
By applying the NA sequence, it is possible to express a protein that is originally difficult to express, and a technique for expressing a fusion protein has been widely used.

[0003] However, although TRX and GST can be applied to the fusion expression of various difficult-to-express proteins, GS originally intended to express a soluble fusion protein was used.
In T, depending on the protein to be fused, the fusion protein may become insoluble and the productivity may decrease, and the fusion protein fused with TRX may cause problems such as a non-specific reaction. It has been desired to provide a fusion protein having excellent properties.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel fusion DNA sequence excellent in operability and productivity for expressing a target protein or peptide, and expressed from the fusion DNA sequence. Fusion protein and the fusion DN
An object of the present invention is to provide a method for expressing a fusion protein using the A sequence.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the conventional problems, and as a result, have found that a DNA sequence encoding a selected protein or peptide and a DNA sequence encoding a heat-resistant protein are directly linked to each other. Alternatively, it is found that when the fusion protein is expressed from the fusion DNA sequence by indirect fusion, the productivity of the target protein or peptide is increased, and the fusion protein obtains heat resistance and the purification operation is simplified.
The present invention has been completed.

That is, the present invention provides a fusion DNA sequence containing a DNA sequence encoding a heat-resistant protein or peptide, a fusion protein expressed by the fusion DNA sequence, and the DN.
The present invention relates to a method for expressing a fusion protein using the A sequence.

[0007] The fusion protein of the present invention has high solubility and can maintain the heat resistance derived from the heat-resistant protein gene. Due to such properties of the fusion protein, when purifying the fusion protein, unnecessary substances can be easily removed by heat treatment, and the fusion protein can be obtained in good yield.

[0008] In the case of TRX derived from Escherichia coli and GST derived from Schistosoma japonicum widely used as a fusion protein, Escherichia coli and Schistosoma japonicum can live in mammals and other organisms. When the fusion protein is used as an antigen for an immune reaction, a non-specific reaction due to Escherichia coli or Schistosoma japonicum may occur. In contrast, since the fusion protein of the present invention uses a thermostable protein derived from a thermophilic bacterium that cannot be inhabited in living organisms of mammals and other organisms, the fusion protein of the present invention is used as an antigen for an immune reaction. Another major feature is that non-specific reactions derived from the fusion protein hardly occur.

Hereinafter, the present invention will be described in detail.

[0010] DNA encoding the heat-resistant protein of the present invention
Sequence refers to a DNA sequence that encodes a protein that does not thermally denature above 55 ° C., preferably above 75 ° C. Specific phenomena of thermal denaturation include protein inactivation and insolubilization. Examples of a DNA sequence encoding a protein that does not thermally denature at 55 ° C. or higher include, for example, a DNA of a thermophilic bacterium that can live at 55 ° C. or higher.
Sequences can be mentioned. It is preferable to use a DNA sequence of a so-called highly thermophilic bacterium that can live at 75 ° C. or higher because of the properties of the expressed protein and the ease of post-treatment. Examples of highly thermophilic bacteria include, for example, Thermophilus, Sulfolobos, Filococcus, Thermotoga, Filobraclam, Filodictium, Thermococcus,
Thermodiscus, Methanothermas, Methanococcus (FEMS. MICRO. BIOL. REV., Vol.75, p117-124, (199
MICROBIOL., Vol. 47, p627-658, (199)
3)) and the like. Examples of the heat-resistant protein include, for example, adenylate key derived from
Kinase (Sulfolobus acidocaldalius from Adenylate Ki
nase: Arch. Biochem. Biophys., Vol. 307, p405-410,
(1993)) (hereinafter referred to as AK), DNA polymerase derived from Thermophilus, and ferredoxin derived from Pyrococcus furiosis (Pyrococcus furio).
Ferredoxin from sus: Biochemistry, Vol.31, p1192-11
96, (1992)) (hereinafter referred to as FDX), glucosidase derived from Pyrococcus furiosus (Glucosidase derived from Pyrococcus furiosus), rubredoxin derived from Pyrococcus furiosis (Pyrococcus f)
Rubredoxin from uriosus: Biochemistry, Vol. 30, p10
885-10895, (1991)), Glutamate dehydrogenase from Pyrococcus furiosus (from Pyrococcus furiosus)
Glutamate dehydrogenase: Gene, Vol.132, p189-197,
(1988)), glyceraldehyde phosphate dehydrogenase from Methanothermus ferrividus (Metanothermus fervid
Glyceraldehyde 3-phosphate dehydrogenase: G from us
ene, Vol. 64, p189-197, (1988)), a glutamic acid synthase derived from Methanococcus borate (Metanococcus v
Glutamate synthetase from olate: Res.Microbiol.,
Vol.140, P355-371, (1989)), L-lactate dehydrogenase from Thermotoga maritina (L-lactate dehydrogenase from Thermotoga maritina)
lactate dehydrogenase: Eur. J. Biochem., Vol.216,
p709-715, (1993)), an elongation factor derived from Thermococcus cereal (from Thermococcus celer)
Elongation Factor 1-alpha: Nucleic acid res., Vo
l.18, p3989, (1990)).
The heat-resistant protein encoded by the DNA sequence of the present invention is not limited to these. The DNA encoding the thermostable protein of the present invention can be purified from these highly thermophilic bacteria, but can also be synthesized based on a known DNA sequence. The DNA synthesis of thermostable proteins is performed by the β-cyanoethylphosphoramidite method (Sinha et.al, Nucleic
Acids Bos., 12, 4539, (1984)), Letsinger. R. Le
Known techniques such as the method described in t. al, J. Am. Chem. Soc., 88 5319 (1966) can be used as appropriate. In an example which is one embodiment of the present invention, the DNAs of FDX and AK derived from S. phylococcus having the amino acid sequences shown in Sequence Listings 1 and 3 were synthesized by the β-cyanoethyl phosphoramidite method. The synthesized DNA sequences are shown in Sequence Listings 2 and 4.

The DNA sequence encoding the selected target protein or peptide of the present invention is not limited to a specific DNA sequence. Any DNA sequence can be used as long as it encodes a protein or peptide desired to be expressed as a fusion protein. The present invention is particularly useful when expressing a protein or peptide in which it is difficult to obtain a required expression level with DNA itself encoding the selected target protein or peptide.

The fusion DNA sequence of the present invention can be fused using a known method such as a ligation method, a linker ligation method and the like. In the fusion, the 5 ′ end of the DNA sequence of the selected target protein or peptide may be directly fused to the 3 ′ end of the DNA sequence of the heat-resistant protein, or may be indirectly fused as necessary. In the case of indirect fusion, a linker sequence is inserted between the 5 'end of the DNA sequence encoding the target protein or peptide and the 3' end of the DNA sequence encoding the heat-resistant protein or peptide. The linker sequence can be selectively cleaved or digested by a known chemical method, enzymatic method, or the like, in addition to a sequence encoding a polypeptide for binding a target protein or peptide to a heat-resistant protein. Sequences encoding polypeptides can be used. When a linker sequence is inserted between the DNA sequence encoding the target protein or peptide and the DNA sequence encoding the heat-resistant protein, after expressing the fusion protein,
The linker sequence can be cleaved or digested by a chemical method such as Bromcian or the like or an enzymatic method such as thrombin or Factor Xa to purify only the selected target protein or peptide portion.

[0013] To express the fusion protein of the present invention, ordinary genetic engineering techniques can be used. For example, the fusion DNA sequence of the present invention is inserted into a vector suitable for expression, and the vector is introduced into a culture host to induce expression of the fusion protein. After increasing the host by culturing or the like, the target fusion protein or peptide is obtained through purification means such as host disruption and column operation. The host cell to be used may be any cell such as a bacterial cell, a eukaryotic cell, or a mammalian cell as long as it can express a heterologous protein or peptide.For example, Escherichia coli, yeast, Bacillus subtilis,
Baculovirus, COS cells and the like can be mentioned.

The fusion protein of the present invention can be used as it is as a fusion protein, or can be used after separating and purifying the target protein or peptide portion.

[0015]

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

Example 1 FDX expression vector pWF6A
Preparation of FDX known DNA of FDX derived from Phyrococcus furiosis
Using eight 53-mer primers prepared using a DNA synthesizer (Model 392, manufactured by Perkin Elmer) based on the sequence, a FDX gene derived from P. phyllococcus fuliosis was synthesized by an assembling PCR method. Taq polymerase (manufactured by Toyobo Co., Ltd.) was used for the assembling PCR method, and was performed at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute.
A total of 248 bp of bases were amplified under the conditions of 30 cycles for 1 minute. 5 'restriction enzyme NdeI site at the end of the 3' restriction enzyme EcoRI site at the end, the C-terminal by adding a thrombin cleavage site. The Nd of a 4.6 Kb pW6A vector prepared from pGEMEX-1 (promega) and pGEX-2T (Pharmacia Biotech) was prepared from this fragment.
pWF6A was prepared as a vector expressing FDX by integrating into eI and EcoRI sites. FIG. 1 shows a detailed view of pW6A, and FIG. 2 shows a detailed view of pWF6A. pWF6A
Is at the NdeI and EcoRI sites, 67 amino acids from FDX, 10 amino acids from the thrombin cleavage site and p
It contains the gene for a fusion protein consisting of 96 amino acids including 19 amino acids derived from the W6A multiple cloning site. The DNA sequence of the inserted fragment was confirmed using a DNA sequence kit (Sequenase Kit Ver. 2.0, manufactured by Amersham United States Biochemical). The DNA sequence of FDX inserted into pW6A and the amino acid sequence encoded by the sequence are shown in SEQ ID NOS: 1 and 2 in pW6A.
The DNA sequence of 6A is shown in Sequence Listing 5. The sequence listing, as No. 1 to ATG restriction enzyme sites NdeI, shows a sequence up to but not including the stop codon of the multicloning site
Was . After introducing pWF6A into host Escherichia coli, the cells were cultured in 1% bactotryptone, 0.5% yeast extract, 1% sodium chloride, 50 μg / ml ampicillin, pH 7.5 medium (hereinafter referred to as LB medium in the present specification). After culturing for 1 hour, 1 mM isopropylthiogalactopyranoside (hereinafter referred to as IPTG in this specification) was added, and the cells were cultured for 2 hours to induce expression. 10 mM Tris-
Hydrochloric acid pH 7.5, 1 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA in the present specification) (hereinafter, this buffer is referred to as the TE buffer in the present specification) were added, and the mixture was sonicated, and the mixture was subjected to ultrasonic crushing. % Sodium dodecyl sulfate polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE in the present specification). A band was observed at about 22 Kda by Coomassie brilliant blue staining (hereinafter, referred to as CBB staining in the present specification), and it was recognized as FDX of Filococcus furiosis which formed a dimer.

Example 2 Purification of FDX After introducing pWF6A prepared in Example 1 into host E. coli,
The LB medium was cultured at 37 ° C. The concentration of Escherichia coli in the culture solution was adjusted to turbidity with an absorbance of about 1.0 at a wavelength of 600 nm by preliminary culture, and then 1 mM IPTG was added to induce expression. After culturing for 3 hours, centrifugation was performed to collect Escherichia coli. 50 mM Tris-HCl buffer pH 8.0 was added to the recovered E. coli.
(Hereinafter referred to as Tris buffer) in 20
0 ml was added, and sonication was performed under ice cooling. The expressed fusion protein was recovered in the supernatant as a soluble component after centrifugation. When the supernatant was subjected to a heat treatment at 85 ° C. for 15 minutes, about 80% of the E. coli protein was thermally denatured and precipitated, and 90% or more of FDX was recovered in the centrifuged supernatant after the heat treatment.

[0018] The supernatant was mixed with Qs equilibrated with Tris buffer.
The product was subjected to ion exchange purification using an FF anion exchange column (Pharmacia Biotech). When eluted with a column equilibration buffer containing sodium chloride, FDX was recovered in a fraction eluted at a concentration of about 0.3 M sodium chloride. Then this FD
The X fraction was purified with a resource RPC reverse phase column (Pharmacia Biotech) equilibrated with 20 mM sodium hydroxide. After elution with acetonitrile, purified FDX was recovered in a fraction eluted with about 10% acetonitrile.

Reference Example 1 Purification of TRX pWT8A prepared as a TRX-expressing vector in the same manner as pWF6A prepared in Example 1 was introduced into host Escherichia coli, and then cultured in LB medium at 37 ° C. After the same expression induction as in Example 1, E. coli was recovered by centrifugation. Osmotech shock was applied to the recovered E. coli, and TRX in the periplasmic fraction was extracted. Extraction TRX is 20m
The first purification was performed using a resource RPC reverse phase column (Pharmacia Biotech) equilibrated with M sodium hydroxide. About 10% when eluted with acetonitrile
TRX was collected in the fraction eluted with ア セ ト 20% acetonitrile.
The collected TRX was dialyzed against 4M guanidine hydrochloride, and then subjected to a second purification using a reversed-phase column under the same conditions. As in the first run, the fraction eluted with about 10% to 20% acetonitrile was purified T
RX was recovered.

Example 3 F by Western blotting
Specificity test of DX and TRX Example 2
The reactivity of the FDX purified in Example 1 with the TRX purified in Reference Example 1 was examined.

Using a solubilized product of SDS of Escherichia coli DH5α, a supernatant of an ultrasonically crushed product and an SDS solubilized product of Escherichia coli transfected with pW50 vector (made in house) as immunogens, a total of 9 kinds of anti-Escherichia coli rabbit sera were prepared for three rabbits each. did. Example 2
The FDX purified in Example 1 and the TRX purified in Reference Example 1 were subjected to SDS-PAGE by the Lemley method, and then transferred to a nitrocellulose membrane. After blocking the remaining protein-adsorbed portion on the nitrocellulose membrane with PBS containing 1% skim milk, the above nine anti-Escherichia coli rabbit sera, each diluted 500-fold as a primary antibody, were subjected to peroxidase (peroxidase) as a secondary antibody. Western blotting was performed using a labeled anti-rabbit antibody (hereinafter referred to as POD in the present specification). For color development, 4-chloro-1-naphthol and hydrogen peroxide were used. No band reacting with the anti-Escherichia coli rabbit antibody was found in the portion corresponding to the FDX molecular weight.
In the part corresponding to the RX molecular weight, 9 kinds of anti-Escherichia coli rabbit sera
Six species were used as immunogens, using the supernatant of the ultrasonically crushed product of Escherichia coli DH5α and the solubilized SDS of Escherichia coli transfected with the pW50 vector.

As described above, human specimen H was used as the primary antibody.
25 cases of TLV-I / II mixed panel 204 serum (Boston Biomedica) were each diluted 50-fold, and Western blotting was performed using POD-labeled anti-human IgG as a secondary antibody. No reactivity was confirmed at the site where FDX was transcribed, but two cases out of 25 cases were confirmed at the site where TRX was transcribed. Table 1 shows the results
Shown in

[0023]

[Table 1]

Example 4 Specificity test of FDX and TRX by ELISA using human specimens FDX purified in Example 2 and TRX purified in Reference Example 1 on ELISA plates (manufactured by Becton Dickinson)
Was sensitized at 25 μg / ml in 50 μl portions.

After blocking the remaining protein-adsorbed portion on the wells of the ELISA plate with PBS containing 1% skim milk, a 500-fold diluted human sample manufactured by Boston Biomedica Inc. used in Example 3 as the primary antibody was used. Using POD-labeled anti-human IgG as the secondary antibody,
A specificity test was performed by the ELISA method. AB for coloring
TS and hydrogen peroxide were used. Measurement result is wavelength 405n
m and the difference in absorbance at a wavelength of 492 nm (the difference in absorbance was described as A405 / 492 nm). In the reaction with the sample, FDX did not have a sample exceeding twice the blank, whereas TRX confirmed that the sample exceeded twice the blank in 6 out of 25 cases. Unlike FRX derived from Escherichia coli, nonspecific reaction and cross-reaction derived from Escherichia coli were not observed in FDX derived from Pyrococcus furiosis. The results are shown in FIG.

Example 5 FDX fusion HTLV-Ip19
Expression of fusion protein and FDX-fused HTLV-II p19 fusion protein Genomic DNA was extracted from infected cell lines expressing HTLV-I and HTLV-II by a method such as Molecular Cloning J. Sambrook. Next, EcoRI, Bam
PCR was performed in the same manner as in Example 1 using primers to which an HI site was added, and p1 of each gag region was
About 9 bp of 9 DNA fragments were obtained, these were incorporated into pWF6A, and pWFIP19 was produced as a vector expressing HTLV-I p19, and pWFFIIP19 was produced as a vector expressing HTLV-II p19. FDX-fused HTLV-Ip19 and FD inserted into vector
The DNA sequences of X-fused HTLV-IIp19 are shown in Sequence Listings 6 and 8, and the amino acid sequences encoded by the DNA sequences are shown in Sequence Listings 7 and 9. These vectors were introduced into Escherichia coli in the same manner as in Example 1 to induce expression of each fusion protein. A sample for electrophoresis was prepared under the same conditions as in Example 1, and after 12.5% SDS-PAGE by the Lemley method, one gel
BB staining and the other one were transferred to a nitrocellulose membrane by the method described in Example 3. As a primary antibody, an anti-native HTLV-Ip19 monoclonal antibody (GIN-7 antibody, Tanaka, Y. et. Al Gann., 74, 327-330, (1983)) or an anti-native HTLV-IIp19 monoclonal antibody, Was reacted with the fusion protein in the same manner as in Example 3 and color-developed with 4-chloro-1-naphthol and hydrogen peroxide. A fusion protein with which a monoclonal antibody corresponding to each fusion protein reacted was used. Was observed. These fusion proteins produced a band at about 34 Kda at the same position as the CBB stained gel. FDX fusion HTLV-Ip19 and FDX
The expression level of the fused HTLV-II p19 was increased by about several hundred times as compared with the case where p19 of HTLV-I and p19 of HTLV-II were directly expressed.

Example 6 FDX fusion HTLV-Ip20
E (gp21) fusion protein and HTLV-IIp20E
(Gp21) Expression of fusion protein In the same manner as in Example 5, HTLV-I and HTLV-
Using the DNA of the II infected cell, the env
About 500 bp of the p20E (gp21) DNA fragment of the region was obtained. These DNA fragments were prepared using the pWF prepared in Example 1.
6A was inserted into the EcoRI and BamHI sites, and HTLV
PWFIE as a vector expressing p20E of -I
1. pWFIIE10 was prepared as a vector expressing p20E of HTLV-II. F inserted into the vector
DX-fused HTLV-Ip20E and FDX-fused HTLV
-The DNA sequence of IIp20E is shown in Sequence Listings 10 and 12,
The amino acid sequences encoded by the DNA sequences are shown in Sequence Listings 11 and 13. These vectors were introduced into Escherichia coli, and FDX-fused HTLV-Ip20E under the same conditions as in Example 1.
Expression of the fusion protein (hereinafter referred to as FDX-20 (I)) and the FDX-fused HTLV-IIp20E fusion protein (hereinafter referred to as FDX-20 (II)) was induced. Escherichia coli was sonicated in the same manner as in Example 1, and 12.5% SDS-P
After AGE, one gel was stained with CBB, and the other gel was transferred to a nitrocellulose membrane at 120 mA for 3 hours. After blocking the remaining protein-adsorbed portion on the nitrocellulose membrane with a phosphate buffer containing 1% BSA, native HTLV-I and HTLV-II p20E
(Gp21) anti-p20E (gp21) monoclonal antibody (F-10 Sugamura, K. et. Al J. Immuno
l, 132, 3180-3184 (1984)), 1 μg / ml was reacted with the fusion protein for 1 hour at room temperature, and then the POD-labeled anti-mouse I
gG was reacted at room temperature for 1 hour. Subsequently, when color was developed with 4-chloro-1-naphthol and hydrogen peroxide,
Expression of the fusion protein reacting with the anti-p20E (gp21) monoclonal antibody corresponding to each fusion protein was observed. These fusion proteins produced a band around 32 Kda at the same position as the CBB stained gel.

FDX-20 (I) and FDX-20 (I
I) Both p20E of HTLV-I and HTLV-II
Compared to the case where p20E was directly expressed, the expression level increased about several hundred times.

Example 7 FDX-20 (I) and FDX
Purification of -20 (II) fusion protein Each of pWFIE1 and pWFIIE10 prepared in Example 6 was introduced into host Escherichia coli, and then cultured under LB medium conditions at 37 ° C. E. coli concentration of the culture solution
After adjusting the turbidity to an absorbance of about 1.0 at 0 nm, 1 mM IP
TG was added to induce expression. Three hours after the addition of IPTG, the cells were centrifuged to collect the bacteria. 1 for the recovered E. coli
% Triton X100, 200 ml of 50 mM Tris-HCl pH 8.0 buffer containing 2 M urea were added, and the mixture was subjected to ultrasonic crushing under ice cooling. Centrifuge to remove insolubles (inclusio
n body) was collected. 4M guanidine hydrochloride 10mM
The insoluble was solubilized with a dithiothreitol (hereinafter referred to as DTT) solution. Resource R equilibrated with 20% acetonitrile, 20 mM sodium hydroxide
The solubilized product was purified using a PC reverse phase column (Pharmacia Biotech). When acetonitrile was eluted, purified FDX-20 (I) and FDX-20 (II) fusion proteins were recovered in fractions eluted with about 30-40% acetonitrile, respectively.

Reference Example 2 TRX-fused HTLV-Ip20
Purification of E fusion protein and TRX fusion HTLV-II p20E fusion protein The TRX expression vector pWT8A prepared in Reference Example 1
As in Example 6, e of HTLV-I or HTLV-II
pWTIE1 was introduced by introducing the p20E (gp21) of the nv region.
And pWTIIE10 were prepared and expressed. The TRX-fused HTLV was purified by a purification method using a resource RPC reverse phase column (manufactured by Pharmacia Biotech) in the same manner as in Example 7.
-Ip20E fusion protein (hereinafter referred to as TRX-
20 (I)) and TRX-fused HTLV-II
p20E fusion protein (hereinafter referred to as TRX-20
(Described as (II)).

Example 8 Reactivity test of fusion protein (1) Test by Western blot method FDX-20 (I) and FDX-2 purified in Example 7
0 (II) and TRX-20 (I) purified in Reference Example 2.
And TRX-20 (II) were used to compare the reactivity with a human HTLV specimen in Western blotting.

As a primary antibody, a human specimen HTLV-I / II mixed panel 50 manufactured by Boston Biomedica was used.
The double dilution was subjected to Western blotting in the same manner as in Example 3 using POD-labeled anti- human IgG as a secondary antibody. FDX-20 (I) and FDX-20 (II) and T
RX-20 (I) and TRX-20 (II) reacted with similar samples. Table 2 shows the results.

[0033]

[Table 2]

(2) Comparison by ELISA method FDX-20 (I) and FDX-2 purified in Example 7
0 (II) and TRX-20 (I) purified in Reference Example 2.
And TRX-20 (II) were sensitized at a concentration of 3 μg / ml to an ELISA plate (manufactured by Becton Dickinson) in an amount of 50 μl.

Example 4 using this ELISA plate
Similarly, a human sample manufactured by Boston Biomedica, which was diluted 500 times as the primary antibody, was subjected to ELISA using POD-labeled anti-human IgG as the secondary antibody. FDX
-20 (I) and FDX-20 (II), TRX-2
0 (I) and TRX-20 (II) reacted with similar samples. The results are shown in FIGS.

(3) Concentration Dependence Test by ELISA In order to examine the reactivity test for anti-p20E (gp21) monoclonal antibody and negative serum, FDX-20 (I) and FDX-20 (II) purified in Example 7 were examined. )When,
TRX-20 (I) and TRX-2 purified in Reference Example 2
0 (II) was reduced from 10 μg / ml to そ れ ぞ れ, respectively.
Prepare a dilution series and prepare an ELISA plate (Becton
(Deckinson) (50 μl each).

Using this ELISA plate, a 500-fold diluted anti-p20E (gp21) monoclonal antibody was used as a primary antibody, and POD-labeled anti-mouse I was used as a secondary antibody.
ELISA was performed using gG. ELISA was performed on the negative serum in the same manner as in Example 4. There was no difference in the reactivity with the monoclonal antibody, and the reactivity with the negative serum was lower in the fusion protein with FDX in both HTLV-I and HTLV-II. The results are shown in FIGS.

Reference Example 3 GST and Treponema pallidum 15K
Preparation of fusion protein with da antigen Genomic DNA was extracted from syphilis bacteria (Treponema pallidum nichols strain) purified from rabbit testes subcultured with syphilis bacteria. Using this as a template, based on the known DNA sequence,
DNA synthesizer (Model 392, manufactured by PerkinElmer)
Was used to prepare a primer. Using this primer, a 15 Kda surface antigen (hereinafter, referred to as Tp15) of Treponema pallidum (hereinafter, referred to as Tp) in a thermal cycler (Model PJ1000, manufactured by PerkinElmer) using this primer. DNA encoding
A fragment of about 370 bp was amplified. This DNA fragment is called pW
6A was inserted into the EcoRI site of a GST expression vector pWG6A in which the GST DNA sequence was inserted, and GST and T
fusion protein with p15 (hereinafter referred to as GST-1
5 described below) was obtained. The DNA sequence of GST-15 inserted into the vector is shown in Sequence Listing 14, and the amino acid sequence encoded by the DNA sequence is shown in Sequence Listing 15. This was introduced into Escherichia coli in the same manner as in Example 1 to induce the expression of GST-15. A sample for electrophoresis was prepared under the same conditions as in Example 1;
After 2.5% SDS-PAGE, one gel was stained with CBB and the other was transferred to a nitrocellulose membrane by the method described in Example 3. Anti-Tp15 monoclonal antibody as primary antibody, POD-labeled anti-mouse IgG as secondary antibody
Was reacted in the same manner as in Example 3 to develop color with 4-chloro-1-naphthol and hydrogen peroxide.
A band was generated at about the same position as the CBB stained gel at about 42 Kda.

Reference Example 4 Preparation of fusion protein of TRX and Tp15 The DNA fragment of Tp15 amplified in Reference Example 3 was converted into pW6A
Was inserted into the EcoRI site of a TRX expression vector pWT8A in which the TRX DNA sequence was inserted, and TRX and Tp1
Thus, a vector pWTTp15 expressing a fusion protein with No. 5 (hereinafter referred to as TRX-15 in the present specification) was obtained.
The DNA sequence of TRX-15 inserted into the vector is shown in Sequence Listing 16, and the amino acid sequence encoded by the DNA sequence is shown in Sequence Listing 17. This was introduced into Escherichia coli in the same manner as in Example 1 to induce the expression of TRX-15. A sample for electrophoresis was prepared under the same conditions as in Example 1 and 12.5 by the Remley method.
After% SDS-PAGE, one gel was stained with CBB and the other was transferred to a nitrocellulose membrane by the method described in Example 3. An anti-Tp15 monoclonal antibody was used as a primary antibody, and a POD-labeled anti- mouse IgG was used as a secondary antibody. The reaction was carried out in the same manner as in Example 3, and color development was performed using 4-chloro-1-naphthol and hydrogen peroxide. A band was formed around the same position at about 27 Kda.

Example 9 Preparation of Fusion Protein of FDX and Tp15 The DNA fragment of Tp15 amplified in Reference Example 3 was used in Example 1
EcoR of the FDX expression vector pWF6A prepared in
I, a vector pWFTp15 which was integrated into the BamHI site and expressed a fusion protein of FDX and Tp15 (hereinafter referred to as FDX-15 in the present specification) was obtained. The DNA sequence of FDX-15 inserted into the vector is shown in Sequence Listing 18
The amino acid sequence encoded by the DNA sequence is shown in Sequence Listing 1.
It is shown in FIG. This was introduced into E. coli in the same manner as in Example 1 and FD
X-15 expression was induced. A sample for electrophoresis was prepared under the same conditions as in Example 1, and 12.5% S
After DS-PAGE, one gel was stained with CBB and the other was transferred to a nitrocellulose membrane by the method described in Example 3. An anti-Tp15 monoclonal antibody as a primary antibody,
When POD-labeled anti-mouse IgG was used as a secondary antibody and reacted in the same manner as in Example 3 to perform color development with 4-chloro-1-naphthol and hydrogen peroxide, a band was generated at about the same position as the CBB-stained gel at about 30 Kda. .

Example 10 FDX-15, GST-15
Test of FDX-1 and TRX-15 FDX-1 produced in Example 9, Reference Example 3 and Reference Example 4
5, after introducing vectors expressing GST-15 and TRX-15 into host E. coli, 1 liter of LB medium
The cells were cultured at 7 ° C. The concentration of Escherichia coli in the culture was adjusted to a turbidity of about 1.0 absorbance at a wavelength of 600 nm as a preliminary culture, and then 1 mM IPTG was added to induce expression. After the cells were collected by centrifugation, the cells were added to Tris buffer 20
0 ml was added, and the mixture was subjected to sonication under ice cooling, and then the fusion proteins were collected in the centrifugation supernatants. 800 µl each
Take each, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃
For 13 minutes. After centrifugation, each sample was separated into a supernatant and a precipitate, and analyzed by SDS-PAGE and Western blotting. 1% skim milk was used as a blocking agent for Western blotting, and an anti-TP rabbit antibody was used as a primary antibody. A POD-labeled anti-rabbit antibody was used as a secondary antibody, and 4-chloro-1-naphthol and hydrogen peroxide were used as color formers. The color development result of the Western blot was confirmed with a densitometer. The results are shown in FIGS. TRX-15 and GST-15 partially precipitated due to heat denaturation at 40 ° C., and about 80% at 60 ° C. and about 100% at 70 ° C. FDX-15 hardly causes precipitation due to heat denaturation at 40 ° C to 80 ° C,
At 100C almost 100% was present in the supernatant.

Example 11 Purification of FDX-15 by Heat Treatment The pWFTp15 prepared in Example 9 was introduced into host Escherichia coli, and then cultured under the conditions of 1 liter of LB medium at 37 ° C. The concentration of Escherichia coli in the culture was adjusted to a turbidity of about 1.0 absorbance at a wavelength of 600 nm as a preliminary culture, and then 1 mM IPTG was added to induce expression. The cells were collected by centrifugation.
200 ml of Tris buffer was added to the cells, and the cells were disrupted by ultrasonic disruption treatment, and FDX-15 was recovered in the supernatant of the centrifugation. Then, using a hot plate and a thermostat,
Heat treatment was performed for 10 minutes, and FDX-15 was collected in the centrifuged supernatant. The heat-treated supernatant was purified using a QFF anion exchange column (Pharmacia Biotech) equilibrated with a Tris buffer. When elution was performed with a column equilibration buffer solution containing sodium chloride, FDX-15 was recovered in a fraction eluted with a sodium chloride concentration of about 0.3 M to 0.4 M. Next, 10 mM DTT was added to the QFF collected fraction, and 20 mM DTT was added.
Purification was performed using a resource RPC reverse phase column (Pharmacia Biotech) equilibrated with a sodium hydroxide solution.
When eluted with acetonitrile, about 20% ~
FDX-15 was recovered in the fraction eluted with 25% acetonitrile. The reverse-phase recovered fraction was concentrated using Centriprep (manufactured by Amicon) and subjected to gel filtration using a Superdex 200 column (manufactured by Pharmacia Biotech). 6M urea, 0.5M sodium chloride, 20mM Tris-HCl p
When eluted with H8.0 buffer, purified FDX-15 was recovered at a molecular weight of about 50,000. By heat treatment at 60 ° C., about 80% of the E. coli protein was precipitated by heat denaturation, and FDX-1
As for No. 5, almost 100% was recovered in the supernatant even at 70 ° C., and the degree of purification was increased about 5-fold by the heat treatment alone.

Further, pWGTp15 prepared in Reference Example 3
Was introduced into host Escherichia coli, and then GST-purified by ordinary column operation without induction and heat treatment without induction and expression operations.
Western blotting of FDX-15 and heat-purified FDX-15 was performed in the same manner as in Example 10 using an anti-Tp rabbit antibody. FDX-15 despite heat treatment purification
Was shown to retain reactivity. FIG. 10 shows the results.
Shown in

Example 12 AK expression vector pW6AK
Preparation of Sulfarobos acidocardarius AK by assembling PCR using 16 53-mer primers prepared using a DNA synthesizer (manufactured by PerkinElmer) based on the known DNA sequence of Sulfarobos-derived AK Was synthesized. Ta for assembly PCR
q polymerase (manufactured by Toyobo) at 94 ° C-1
A total of 630 bp was amplified under the conditions of 30 minutes, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and 30 cycles. A restriction enzyme NdeI site at the 5 ′ end, a restriction enzyme EcoRI site at the 3 ′ end,
A thrombin cleavage site was added to the C terminal. This fragment is p
4.6 Kb prepared from GEMEX-1 (manufactured by Promega) and pGEX-2T (manufactured by Pharmacia Biotech)
Was inserted into the NdeI and EcoRI sites of the pW6A vector, and pW6AK was produced as a vector expressing AK. FIG. 11 shows a detailed diagram of pW6AK. pW6AK contains 194 amino acids from AK, 10 amino acids from thrombin cleavage site and pW at NdeI and EcoRI sites.
It contains a gene for a fusion protein consisting of 223 amino acids including 19 amino acids derived from the 6A multiple cloning site. The DNA sequence of the inserted fragment was confirmed using a DNA sequence kit (Sequenase Kit Ver. 2.0, manufactured by Amersham United States Biochemical). The DNA sequence of AK inserted into pW6A is shown in Sequence Listing 3.
In Table 4, the amino acid sequence encoded by the DNA sequence is shown.
Shown in After introduction into host Escherichia coli, the cells were cultured in an LB medium for 2 hours, and then added with 1 mM IPTG and cultured for 2 hours to induce expression. A TE buffer solution was added to the E. coli precipitate, and the mixture was sonicated and subjected to 15% SDS-PAGE by the Lemley method. A band was confirmed at about 40 Kda by CBB staining.

Example 13 Purification of AK After introducing pW6AK prepared in Example 12 into host Escherichia coli, it was cultured in LB medium at 37 ° C. The concentration of Escherichia coli in the culture solution was adjusted to about 1.0 at a wavelength of 600 nm in the preliminary culture.
After the turbidity of 1 mM, 1 mM IPTG was added to induce expression. After culturing for 3 hours, centrifugation was performed to collect Escherichia coli.
200 ml of Tris buffer was added to the recovered Escherichia coli and subjected to ultrasonic crushing under ice cooling. The expressed fusion protein is
After centrifugation, it was recovered in the supernatant as a soluble component. When the supernatant was heat-treated at 65 ° C. for 10 minutes, about 70%
Of the E. coli protein was heat denatured and precipitated in the centrifuged supernatant after heat treatment.
0% or more of AK was recovered.

The supernatant was purified on a hydroxyapatite column (Bio-Rad) equilibrated with Tris buffer. When eluted with a sodium phosphate buffer, AK was collected in a fraction eluted at a concentration of about 0.2 M sodium phosphate. Next, the AK fraction was purified by gel filtration using a Superdex 200 26/60 column equilibrated with 6 M urea, 0.5 M sodium chloride, 20 mM Tris-HCl buffer, pH 9.4. Purified AK was recovered in a fraction having a molecular weight of about 20,000.

Example 14 Preparation of Fusion Protein of AK and Tp15 The DNA fragment of Tp15 amplified in Reference Example 3 was used in Example 1
EcoR of AK expression type vector pW6AK prepared in Step 2.
I, a vector pW6AKTp15 that was integrated into the BamHI site and expressed a fusion protein of AK and Tp15 (hereinafter referred to as AK-15 in the present specification) was obtained. The DNA sequence of AK-15 inserted into the vector is shown in Sequence Listing 20, and the amino acid sequence encoded by the DNA sequence is shown in Sequence Listing 21. This was introduced into Escherichia coli in the same manner as in Example 1 and AK-15 was introduced.
Was induced. A sample for electrophoresis was prepared under the same conditions as in Example 1 and 12.5% SDS-P by the Laemmli method was used.
After AGE, one gel was stained with CBB and the other was transferred to a nitrocellulose membrane by the method described in Example 3. An anti-Tp15 monoclonal antibody was used as a primary antibody, and a POD-labeled anti-mouse IgG was used as a secondary antibody. The reaction was carried out in the same manner as in Example 3, and color development was performed using 4-chloro-1-naphthol and hydrogen peroxide. A band was formed around the same position at about 40 Kda.

Example 15 Purification of AK-15 by Heat Treatment After introducing pWAKTp15 prepared in Example 14 into host Escherichia coli, the cells were cultured under the conditions of 1 liter of LB medium and 37 ° C. As a preculture, the concentration of E. coli in
m, the turbidity was adjusted to about 1.0, and then 1 mM IPTG
Was added to induce expression. The cells were collected by centrifugation. 50 mM glycine-sodium hydroxide pH
200 ml of 10.0 buffer was added, the cells were disrupted by ultrasonic disruption treatment, and AK-15 was recovered in the centrifuged supernatant. Next, heat treatment was performed at 60 ° C. for 10 minutes using a hot plate, and AK-15 was collected in the centrifuged supernatant. 4M urea 5
After dialysis against 0 mM sodium acetate buffer pH 6.0, the heat-treated supernatant was purified using an SFF cation exchange column (Pharmacia Biotech) equilibrated with the same buffer. When elution was carried out with an equilibration buffer containing sodium chloride, AK-15 was recovered in a fraction eluted with a sodium chloride concentration of about 0.2 M to 0.4 M. The collected AK-15 fraction was
Superdex 200 equilibrated with 6M urea 0.5M sodium chloride 20mM Tris-HCl buffer pH 9.4
Gel filtration and purification were performed on a 26/60 column. Purified AK-15 was recovered in a fraction having a molecular weight of about 40,000.

When Western blotting was performed using an anti-Tp rabbit antibody in the same manner as in Example 11, it was shown that AK-15 retained reactivity despite heat treatment. The result is shown in FIG.

[0050]

As described above, according to the present invention, a fusion D which is more operable and more productive than a conventional DNA sequence encoding a fusion protein.
It is possible to provide an NA sequence, a fusion protein expressed from the fusion DNA sequence, and a method for expressing a fusion protein using the DNA sequence.

[0051]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 291 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAAGGCCACC GAAGGAAAGGCCACC 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCGTC 210 240 GACCTCGAGG GATCCGGGCC CTCTAGATGC GGCCGCATGC ATGGTACC A

[0052] SEQ ID NO: 2 Length of sequence: 9 6 sequence type: amino acid Topology: linear sequence type: Gene expression protein sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Val Asp Leu Glu Gly Ser Gly Pro Ser Arg Cys 80 85 90 Gly Arg Met His Gly Thr 95

SEQ ID NO: 3 Sequence length: 672 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA ATGAAGATTG GTATTGTAAC TGGTATCCCT GGTGTAGGGA AAAGTACTGT CTTGGCTAAA 30 60 GTTAAAGAGA TATTGGATAA TCAAGGTATA AATAACAAGA TCATAAATTA TGGAGATTTT 90 120 ATGTTAGCAA CAGCATTAAA ATTAGGCTAT GCTAAAGATA GAGACGAAAT GAGAAAATTA 150 180 TCTGTAGAAA AGCAGAAGAA ATTGCAGATT GATGCGGCTA AAGGTATAGC TGAAGAGGCA 210 240 AGAGCAGGTG GAGAAGGATA TCTGTTCATA GATACGCACG CTGTGATACG TACACCCTCT 270 300 GGATATTTAC CTGGTTTACC GTCAGATATA ATTACAGAAA TAAATCCGTC TGTTATCTTT 330 360 TTACTGGAAG CTGATCCTAA GATAATATTA TCAAGGCAAA AGAGAGATAC AACAAGGAAT 390 420 AGAAATGATT ATAGTGACGA ATCAGTTATA TTAGAAACCA TAAACTTCGC TAGATATGCA 450 480 GCTACTGCTT CTGCAGTATT AGCCGGTTCT ACTGTTAAGG TAATTGTAAA CGTGGAAGGA 510 540 GATCCTAGTA TAGCAGCTAA TGAGATAATA AGGTCTATGA AGGGTGGTTC TTCTCTGGTT 570 600 CCGCGTGGAC TGGAATTCGT CGACCTCGACGTC CGACCTCGAGCG TAG ACC GTACCT AA

[0054] SEQ ID NO: 4 Length of sequence: 22 3 SEQ type: amino acid Topology: linear sequence type: Gene expression protein sequence Met Lys Ile Gly Ile Val Thr Gly Ile Pro Gly Val Gly Lys Ser 1 5 10 15 Thr Val Leu Ala Lys Val Lys Glu Ile Leu Asp Asn Gln Gly Ile 20 25 30 Asn Asn Lys Ile Ile Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala 35 40 45 Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Glu Met Arg Lys Leu 50 55 60 Ser Val Glu Lys Gln Lys Lys Leu Gln Ile Asp Ala Ala Lys Gly 65 70 75 Ile Ala Glu Glu Ala Arg Ala Gly Gly Glu Gly Tyr Leu Phe Ile 80 85 90 Asp Thr His Ala Val Ile Arg Thr Pro Ser Gly Tyr Leu Pro Gly 95 100 105 Leu Pro Ser Asp Ile Ile Thr Glu Ile Asn Pro Ser Val Ile Phe 110 115 120 Leu Leu Glu Ala Asp Pro Lys Ile Ile Leu Ser Arg Gln Lys Arg 125 130 135 Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val Ile 140 145 150 Leu Glu Thr Ile Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala 155 160 165 Val Leu Ala Gly Ser Thr Val Lys Val Ile Val Asn Val Gl u Gly 170 175 180 Asp Pro Ser Ile Ala Ala Asn Glu Ile Ile Arg Ser Met Lys Gly 185 190 195 Gly Ser Ser Leu Val Pro Arg Gly Leu Glu Phe Val Asp Leu Glu 200 205 210 Gly Ser Gly Pro Ser Arg Cys Gly Arg Met His Gly Thr 215 220

SEQ ID NO: 5 Sequence length: 4557 Sequence type: Number of nucleic acid strands: Double strand Topology: Circular Sequence type: Other nucleic acid Synthetic DNA sequence ATGGCTAGCG AATTCGTCGA CCTCGAGGGA TCCGGGCCCT CTAGATGCGG CCGCATGCAT 30 60 GGTACCTAAC TAACTAAGCT TGAGTATTCT ATAGTGAC CTAAATCCCA GCTTGATCCG 90 120 GCTGCTAACA AAGCCCGAAA GGAAGCTGAG TTGGCTGCTG CCACCGCTGA GCAATAACTA 150 180 GCATAACCCC TTGGGGCCTC TAAACGGGTC TTGAGGGGTT TTTTGCTGAA AGGAGGAACT 210 240 ATATCCGGAT AACCTGGCGT AATAGCGAAG AGGCCCGCAC CGAATTAATT CATCGTGACT 270 300 GACTGACGAT CTGCCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC 330 360 TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG 390 420 GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCCAGTCA CGTAGCGATA 450 480 GCGGAGTGTA TAATTCTTGA AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 510 540 ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCGCGCTTTTCGGGGA AATGTGCGCG 570 600 GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAGATCTCTC 0 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 690 720 GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA 750 780 CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 810 840 TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA 870 900 TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTGTTGAC GCCGGGCAAG 930 960 AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 990 1020 CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA 1050 1080 TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 1110 1140 CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 1170 1200 TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGCAGCA ATGGCAACAA 1230 1260 CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 1290 1320 ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 1350 1380 GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 1410 1440 TGGGGCCAGA TGGTAAGCC C TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA 1470 1500 CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT 1530 1560 AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 1590 1620 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 1650 1680 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 1710 1740 CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 1770 1800 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 1830 1860 CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 1890 1920 CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 1950 1980 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 2010 2040 GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 2070 2100 AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 2130 2160 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 2190 2220 GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC 2250 2280 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 2310 2340 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 2370 2400 CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC 2430 2460 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCTG ATGCGGTATT 2490 2520 TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATAAA TTCCGACACC ATCGAATGGT 2550 2580 GCAAAACCTT TCGCGGTATG GCATGATAGC GCCCGGAAGA GAGTCAATTC AGGGTGGTGA 2610 2640 ATGTGAAACC AGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG 2670 2700 TTTCCCGCGT GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG 2730 2760 CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG GCGGGCAAAC 2790 2820 AGTCGTTGCT GATTGGCGTT GCCACCTCCA GTCTGGCCCT GCACGCGCCG TCGCAAATTG 2850 2880 TCGCGGCGAT TAAATCTCGC GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG 2910 2940 AACGAAGCGG CGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA 2970 3000 GTGGGCTGAT CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT 3030 3060 GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC AACAGTATTA 3090 3120 TTTTCTCCCA TGAAGACGGT ACGCGACTGG GCGTGGAGCA TCTGGTCGCA TTGGGTCACC 3150 3180 AGCAAATCGC GCTGTTAGCG GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG 3210 3240 GCTGGCATAA ATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT 3270 3300 GGAGTGCCAT GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA 3330 3360 CTGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC ATTACCGAGT 3390 3420 CCGGGCTGCG CGTTGGTGCG GATATCTCGG TAGTGGGATA CGACGATACC GAAGACAGCT 3450 3480 CATGTTATAT CCCGCCGTTA ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA 3510 3540 GCGTGGACCG CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC 3570 3600 CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC 3630 3660 GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC 3690 3720 AGTGAGCGCA ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC 3750 3780 TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCGCG GATAACAATT TCACACAGGA 3810 3840 AACAGCTATG ACCATGATTA CGGATTCACT GGCCGTCGTT TTACAACGTC GTGACTGGGA 3870 3900 AAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTTTCG CCAGCTGGCG 3930 3960 TAATAGCGAA GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA 3990 4020 ATGGCGCTTT GCCTGGTTTC CGGCACCAGA AGCGGTGCCG GAAAGCTGGC TGGAGTGCGA 4050 4080 TCTTCCTGAG GCCGATACTG TCGTCGTCCC CTCAAACTGG CAGATGCACG GTTACGATGC 4110 4140 GCCCATCTAC ACCAACGTAA CCTATCCCAT TACGGTCAAT CCGCCGTTTG TTCCCACGGA 4170 4200 GAATCCGACG GGTTGTTACT CGCTCACATT TAATGTTGAT GAAAGCTGGC TACAGGAAGG 4230 4260 CCAGACGCGA ATTATTTTTG ATGGCGTTGG AATTACGTTA TCGACTGCAC GGTGCACCAA 4290 4320 TGCTTCTGGC GTCAGGCAGC CATCGGAAGC TGTGGTATGG CTGTGCAGGT CGTAAATCAC 4350 4380 TGCATAATTC GTGTCGCTCA AGGCGCACTC CCGTTCTGGA TAATGTTTTT TGCGCCGACA 4410 4440 TCATAACGGT TCTGGCAAAT GGGAATTGGG AAATTAATAC GACTCACTAT ATGGAATTGT 4470 4500 GAGCGGATAA CAATTCCTAG AAATAATTTT GTTTAACTTT AAGAAGGAGA TATACAT 4530

SEQ ID NO: 6 Sequence length: 672 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCATG 210 240 GGCCAAATCT TTTCCCGTAG CGCTAGCCCT ATTCCGCGGC CGCCCCGGGG GCTGGCCGCT 270 300 CATCACTGGC TTAACTTCCT CCAGGCGGCA TATCGCCTAG AACCCGGTCC CTCCAGTTAC 330 360 GATTTCCACC AGTTAAAAAA ATTTCTTAAA ATAGCTTTAG AAACACCGGT CTGGATCTGC 390 420 CCCATTAACT ACTCCCTCCT AGCCAGCCTA CTCCCAAAAG GATACCCCGG CCGGGTGAAT 450 480 GAAATTTTAC ACATACTCAT CCAAACCCAA GCCCAGATCC CGTCCCGCCC CGCGCCGCCG 510 540 CCGCCGTCAT CCTCCACCCA CGACCCCCCG GATTCTGACC CACAAATCCC CCCTCCCTAT 570 600 GTTGAGCCTA CAGCCCCC TAGTCGATCCGCC CGCATCTCGCGATCGATCTCGACGTC 0 CATGGTACCT AA

SEQ ID NO: 7 Sequence length: 209 Sequence type: amino acid Topology: linear Sequence type: gene expression protein sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Met Gly Gln Ile Phe Ser Arg Ser Ala Ser Pro 80 85 90 Ile Pro Arg Pro Pro Arg Gly Leu Ala Ala His His Trp Leu Asn 95 100 105 Phe Leu Gln Ala Ala Tyr Arg Leu Glu Pro Gly Pro Ser Ser Tyr 110 115 120 Asp Phe His Gln Leu Lys Lys Phe Leu Lys Ile Ala Leu Glu Thr 125 130 135 Pro Val Trp Ile Cys Pro Ile Asn Tyr Ser Leu Leu Ala Ser Leu 140 145 150 Leu Pro Lys Gly Tyr Pro Gly Arg Val Asn Glu Ile Leu His Ile 155 160 165 Leu Ile Gle Thr Gln Ala Gln Ile Pro Ser Arg Pro Ala Pr o Pro 170 175 180 Pro Pro Ser Ser Ser Thr His Hisp Pro Pro Asp Ser Asp Pro Gln 185 190 195 Ile Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gln Val Leu 200 205

Sequence number: 8 Sequence length: 690 Sequence type: Number of nucleic acid chains: Double-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCATG 210 240 GGACAAATCC ACGGGCTTTC CCCAACTCCA ATACCCAAAG CCCCCAGGGG GCTATCAACC 270 300 CACCACTGGC TTAACTTTCT CCAGGCTGCT TACCGCTTGC AGCCTAGGCC CTCCGATTTC 330 360 GACTTCCAGC AGCTACGACG CTTTCTAAAA CTAGCCCTTA AAACGCCCAT TTGGCTAAAT 390 420 CCTATTGACT ACTCGCTTTT AGCTAGCCTT ATCCCCAAGG GATATCCAGG AAGGGTGGTA 450 480 GAGATTATAA ATATCCTTGT CAAAAATCAA GTCTCCCCTA GCGCCCCCGC CGCCCCAGTT 510 540 CCGACACCTA TCTGCCCTAC TACTACTCCT CCGCCACCTC CCCCCCCTTC CCCGGAGGCC 570 600 CATGTTCCCC CCCCTGCTCTACGT GGAACCCCACGCTCACCGTGTGAGACCCC 0 TCTAGATGCG GCCGCATGCA TGGTACCTAA 690

[0059] SEQ ID NO: 9 Length of sequence: 215 Type of sequence: amino acid Topology: linear sequence type: Gene expression protein sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Met Gly Gln Ile His Gly Leu Ser Pro Thr Pro 80 85 90 Ile Pro Lys Ala Pro Arg Gly Leu Ser Thr His His Trp Leu Asn 95 100 105 Phe Leu Gln Ala Ala Tyr Arg Leu Gln Pro Arg Pro Ser Asp Phe 110 115 120 Asp Phe Gln Gln Leu Arg Arg Phe Leu Lys Leu Ala Leu Lys Thr 125 130 135 Pro Ile Trp Leu Asn Pro Ile Asp Tyr Ser Leu Leu Ala Ser Leu 140 145 150 Ile Pro Lys Gly Tyr Pro Gly Arg Val Val Glu Ile Ile Asn Ile 155 160 165 Leu Val Lys Asn Gln Val Ser Pro Ser Ala Pro Ala Ala Pr o Val 170 175 180 Pro Thr Pro Ile Cys Pro Thr Thr Thr Pro Pro Pro Pro Pro Pro 185 190 195 Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr Val Glu Pro Thr 200 205 210 Thr Thr Gln Cys Phe 215

SEQ ID NO: 10 Sequence length: 810 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCGCA 210 240 GTACCGGTGG CGGTCTGGCT TGTCTCCGCC CTGGCCATGG GAGCCGGAGT GGCTGGCAGG 270 300 ATTACCGGCT CCATGTCCCT CGCCTCAGGA AAGAGCCTCC TACATGAGGT GGACAAAGAT 330 360 ATTTCCCAAT TAACTCAAGC AATAGTCAAA AACCACAAAA ATCTGCTCAA AATTGCACAG 390 420 TATGCTGCCC AGAACAGACG AGGCCTTGAT CTCCTGTTCT GGGAGCAAGG AGGATTATGC 450 480 AAAGCATTAC AAGAACAGTG CTGTTTTCTA AATATTACTA ATTCCCATGT CTCAATACTA 510 540 CAAGAGAGAC CCCCCCTTGA AAATCGAGTC CTGACTGGCT GGGGCCTTAA CTGGGACCTT 570 600 GGCCTCTCAC AGTGGCTCAGGCTCTCAGGCTCTCAGGCTCTCAGGCTCTCAGGCAGTCAGGCTCTCAGGCGCTCAGGCAGTCAGGCTCGCAGGCAGTCAGGGTCGCGCGCGCTCGCAGGCGCTCGCGCGCTCGCAGGCGCTCGCAGGCGCTCGCGGTCGCAGGCTCTCGGCTGCGGTCGCAGGGCT 0 660 CTTCTTGTTA TCCTTGCAGG ACCATGCATC CTCCGTCAGC TACGACACCT CCCCTCGCGC 690 720 GTCAGATACC CCCATTACTC TCTTATAAAC CCTGAGTCAT CCCTGTAAGG ATCCGGGCCC 750 780 TCTAGATGCG GCCGCATGCA TGGTACCTAA 810

SEQ ID NO: 11 Sequence Length: 255 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Ala Val Pro Val Ala Val Trp Leu Val Ser Ala 80 85 90 Leu Ala Met Gly Ala Gly Val Ala Gly Arg Ile Thr Gly Ser Met 95 100 105 Ser Leu Ala Ser Gly Lys Ser Leu Leu His Glu Val Asp Lys Asp 110 115 120 Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys Asn Leu 125 130 135 Leu Lys Ile Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp 140 145 150 Leu Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys Ala Leu Gln Glu 155 160 165 Gln Cys Cys Phe Leu Asn Ile Thr Asn Ser His Val Ser Ile Leu 170 175 180 Gln Glu Arg Pro Pro Leu Glu Asn Arg Val Leu Thr Gly Trp Gly 185 190 195 Leu Asn Trp Asp Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala Leu 200 205 210 Gln Thr Gly Ile Thr Leu Val Ala Leu Leu Leu Leu Val Ile Leu 215 220 225 Ala Gly Pro Cys Ile Leu Arg Gln Leu Arg His Leu Pro Ser Arg 230 235 240 Val Arg Tyr Pro His Tyr Ser Leu Ile Asn Pro Glu Ser Ser Leu 245 250 255

SEQ ID NO: 12 Sequence length: 816 Sequence type: nucleic acid Number of strands: double strand Topology: linear strand Sequence type: other nucleic acid Synthetic DNA sequence ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCGCC 210 240 GTTCCAATAG CAGTGTGGCT TGTCTCCGCC CTAGCGGCCG GAACAGGTAT CGCTGGTGGA 270 300 GTAACAGGCT CCCTATCTCT GGCTTCCAGT AAAAGCCTTC TCCTCGAGGT TGACAAAGAC 330 360 ATCTCCCACC TTACCCAGGC CATAGTCAAA AATCATCAAA ACATCCTCCG GGTTGCACAG 390 420 TATGCAGCCC AAAATAGACG AGGATTAGAC CTCCTATTCT GGGAACAAGG GGGTTTGTGC 450 480 AAGGCCATAC AGGAGCAATG TTGCTTCCTC AACATCAGTA ACACTCATGT ATCCGTCCTC 510 540 CAGGAACGGC CCCCTCTTGA AAAACGTGTC ATCACCGGCT GGGGACTAAA CTGGGATCTT 570 600 GGACTGTCTCGC AATGGGTCTCAG AGATGCCTCCAGGAAGG 660 CTCCTCGTCA TATTGTTTGG CCCCTGTATC CTCCGCGCCAAA TCCAGGCCCT TCCACAGCGG 690 720 TTACAAAACC GACATAACCA GTATTCCCTT ATCAACCCAG AAACCATGCT ATAAGGATCC 750 780 GGGCCCTCTA GATGCGGCCG CATGCATGGT ACCTAA 810

SEQ ID NO: 13 Sequence Length: 257 Sequence Type: Amino Acid Topology: Linear Sequence Type: Gene Expression Protein Sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Ala Val Pro Ile Ala Val Trp Leu Val Ser Ala 80 85 90 Leu Ala Ala Gly Thr Gly Ile Ala Gly Gly Val Thr Gly Ser Leu 95 100 105 Ser Leu Ala Ser Ser Lys Ser Leu Leu Leu Glu Val Asp Lys Asp 110 115 120 Ile Ser His Leu Thr Gln Ala Ile Val Lys Asn His Gln Asn Ile 125 130 135 Leu Arg Val Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp 140 145 150 Leu Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys Ala Ile Gln Glu 155 160 165 Gln Cys Cys Phe Leu Asn Ile Ser Asn Thr His Val Ser Val Leu 170 175 180 Gln Glu Arg Pro Pro Leu Glu Lys Arg Val Ile Thr Gly Trp Gly 185 190 195 Leu Asn Trp Asp Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala Leu 200 205 210 Gln Thr Gly Ile Thr Ile Leu Ala Leu Leu Leu Leu Val Ile Leu 215 220 225 Phe Gly Pro Cys Ile Leu Arg Gln Ile Gln Ala Leu Pro Gln Arg 230 235 240 Leu Gln Asn Arg His Asn Gln Tyr Ser Leu Ile Asn Pro Glu Thr 245 250 255 Met Leu

SEQ ID NO: 14 Sequence length: 1119 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGTCCCCTA TACTAGGTTA TTGGAAAATT AAGGGCCTTG TGCAACCCAC TCGACTTCTT 30 60 TTGGAATATC TTGAAGAAAA ATATGAAGAG CATTTGTATG AGCGCGATGA AGGTGATAAA 90 120 TGGCGAAACA AAAAGTTTGA ATTGGGTTTG GAGTTTCCCA ATCTTCCTTA TTATATTGAT 150 180 GGTGATGTTA AATTAACACA GTCTATGGCC ATCATACGTT ATATAGCTGA CAAGCACAAC 210 240 ATGTTGGGTG GTTGTCCAAA AGAGCGTGCA GAGATTTCAA TGCTTGAAGG AGCGGTTTTG 270 300 GATATTAGAT ACGGTGTTTC GAGAATTGCA TATAGTAAAG ACTTTGAAAC TCTCAAAGTT 330 360 GATTTTCTTA GCAAGCTACC TGAAATGCTG AAAATGTTCG AAGATCGTTT ATGTCATAAA 390 420 ACATATTTAA ATGGTGATCA TGTAACCCAT CCTGACTTCA TGTTGTATGA CGCTCTTGAT 450 480 GTTGTTTTAT ACATGGACCC AATGTGCCTG GATGCGTTCC CAAAATTAGT TTGTTTTAAA 510 540 AAACGTATTG AAGCTATCCC ACAAATTGAT AAGTACTTGA AATCCAGCAA GTATATAGCA 570 600 TGGCCTGGTCAGTCAGTCAGGCGGTCGGTCAG 630 660 CTGGTTCCGC GTGGATCGGA ATTCTGTTCA TTTAGTTCTA TCCCGAATGG CACGTACCGG 690 720 GCGACGTATC AGGATTTTGA TGAGAATGGT TGGAAGGACT TTCTCGAGGT TACTTTTGAT 750 780 GGTGGCAAGA TGGTGCAGGT GGTTTACGAT TATCAGCATA AAGAAGGGCG GTTTAAGTCC 810 840 CAGGACGCTG ACTACCATCG GGTCATGTAT GCATCCTCGG GCATAGGTCC TGAAAAGGCC 870 900 TTCAGAGAGC TCGCCGATGC TTTGCTTGAA AAGGGTAATC CCGAGATGGT GGATGTGGTC 930 960 ACCGGTGCAA CTGTTTCTTC CCAGAGTTTC AGGAGGTTGG GTCGTGCGCT TCTGCAGAGT 990 1020 GCGCGGCGCG GCGAGAAGGA AGCCATTATT AGCAGGTAGG AATTCGTCGA CCTCGAGGGA 1050 1080 TCCGGGCCCT CTAGATGCGG CCGCATGCAT GGTACCTAA 1110

SEQ ID NO: 15 Sequence length: 352 Sequence type: amino acid Topology: linear Sequence type: gene expression protein sequence Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln 1 5 10 15 Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu 20 25 30 His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys 35 40 45 Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp 50 55 60 Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile 65 70 75 Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala 80 85 90 Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly 95 100 105 Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val 110 115 120 Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp 125 130 135 Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His 140 145 150 Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met 155 160 165 Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys 170 175 180 Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser 185 190 195 Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe 200 205 210 Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly 215 220 225 Ser Glu Phe Cys Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr Arg 230 235 240 Ala Thr Tyr Gln Asp Phe Asp Glu Asn Gly Trp Lys Asp Phe Leu 245 250 255 Glu Val Thr Phe Asp Gly Gly Lys Met Val Gln Val Val Tyr Asp 260 265 270 Tyr Gln His Lys Glu Gly Arg Phe Lys Ser Gln Asp Ala Asp Tyr 285 280 280 His Arg Val Met Tyr Ala Ser Ser Gly Ile Gly Pro Glu Lys Ala 290 295 300 Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly Asn Pro Glu 305 310 315 Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser Gln Ser Phe 320 325 330 Arg Arg Leu Gly Arg Ala Leu Leu Gln Ser Ala Arg Arg Gly Glu 335 340 345 Lys Glu Ala Ile Ile Ser Arg 350

SEQ ID NO: 16 Sequence length: 858 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGTTACACC AACAACGAAA CCAACACGCC AGGCTTATTC CTGTGGAGTT ATATATGAGC 30 60 GATAAAATTA TTCACCTGAC TGACGACAGT TTTGACACGG ATGTACTCAA AGCGGACGGG 90 120 GCGATCCTCG TCGATTTCTG GGCAGAGTGG TGCGGTCCGT GCAAAATGAT CGCCCCGATT 150 180 CTGGATGAAA TCGCTGACGA ATATCAGGGC AAACTGACCG TTGCAAAACT GAACATCGAT 210 240 CAAAACCCTG GCACTGCGCC GAAATATGGC ATCCGTGGTA TCCCGACTCT GCTGCTGTTC 270 300 AAAAACGGTG AAGTGGCGGC AACCAAAGTG GGTGCACTGT CTAAAGGTCA GTTGAAAGAG 330 360 TTCCTCGACG CTAACCTGGC GGAGCTCGGT GGTTCTTCTC TGGTTCCGCG TGGATCGGAA 390 420 TTCTGTTCAT TTAGTTCTAT CCCGAATGGC ACGTACCGGG CGACGTATCA GGATTTTGAT 450 480 GAGAATGGTT GGAAGGACTT TCTCGAGGTT ACTTTTGATG GTGGCAAGAT GGTGCAGGTG 510 540 GTTTACGATT ATCAGCATAA AGAAGGGCGG TTTAAGTCCC AGGACGCTGA CTACCATCGG 570 600 GTCATGTACG CATCCTTCGGAGCTAGCTAGTCAGTCAGTCGATCGATCAGTCGATCGATCAGTCGATCGATCAGTCGATCAGTCGATCGATCGTGATCGATCGTTCGAGGCTGACGTATCA 660 TTGCTTGAAA AGGGTAATCC CGAGATGGTG GATGTGGTCA CCGGTGCAAC TGTTTCTTCC 690 720 CAGAGTTTCA GGAGGTTGGG TCGTGCGCTT CTGCAGAGTG CGCGGCGCGG CGAGAAGGAA 750 780 GCCATTATTA GCAGGTAGGA ATTCGTCGAC CGCGTCGATCCGCGCTCGCCGG

SEQ ID NO: 17 Sequence Length: 265 Sequence Type: Amino Acid Topology: Linear Sequence Type: Gene Expression Protein Sequence Met Leu His Gln Gln Arg Asn Gln His Ala Arg Leu Ile Pro Val 1 5 10 15 Glu Leu Tyr Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 20 25 30 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp 35 40 45 Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile 50 55 60 Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 65 70 75 Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly 80 85 90 Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 95 100 105 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu 110 115 120 Phe Leu Asp Ala Asn Leu Ala Glu Leu Gly Gly Ser Ser Leu Val 125 130 135 Pro Arg Gly Ser Glu Phe Cys Ser Phe Ser Ser Ile Pro Asn Gly 140 145 150 Thr Tyr Arg Ala Thr Tyr Gln Asp Phe Asp Glu Asn Gly Trp Lys 155 160 165 Asp Phe Leu Glu Val Thr Phe Asp Gly Gly Lys Met Val Gln Val 170 175 180 Val Tyr Asp Tyr Gln His Lys Glu Gly Arg Phe Lys Ser Gln Asp 185 190 195 Ala Asp Tyr His Arg Val Met Tyr Ala Ser Ser Gly Ile Gly Pro 200 205 210 Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly 215 220 225 Asn Pro Glu Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser 230 235 240 Gln Ser Phe Arg Arg Leu Gly Arg Ala Leu Leu Gnu Ser Ala Arg 245 250 255 Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg 260 265

SEQ ID NO: 18 Sequence length: 672 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 30 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 90 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 150 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCTGT 210 240 TCATTTAGTT CTATCCCGAA TGGCACGTAC CGGGCGACGT ATCAGGATTT TGATGAGAAT 270 300 GGTTGGAAGG ACTTTCTCGA GGTTACTTTT GATGGTGGCA AGATGGTGCA GGTGGTTTAC 330 360 GATTATCAGC ATAAAGAAGG GCGGTTTAAG TCCCAGGACG CTGACTACCA TCGGGTCATG 390 420 TATGCATCCT CGGGCATAGG TCCTGAAAAG GCCTTCAGAG AGCTCGCCGA TGCTTTGCTT 450 480 GAAAAGGGTA ATCCCGAGAT GGTGGATGTG GTCACCGGTG CAACTGTTTC TTCCCAGAGT 510 540 TTCAGGAGGT TGGGTCGTGC GCTTCTGCAG AGTGCGCGGC GCGGCGAGAAAAGAGATCGCTG TAG 600GA 0 660 CATGGTACCT AA

SEQ ID NO: 19 Sequence Length: 203 Sequence Type: Amino Acid Topology: Linear Sequence Type: Gene Expression Protein Sequence Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp 1 5 10 15 Ala Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp 20 25 30 Glu Gly Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu 35 40 45 Leu Tyr Asn Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser 50 55 60 Ala Ile Thr Ile Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg 65 70 75 Gly Ser Glu Phe Cys Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr 80 85 90 Arg Ala Thr Tyr Gln Asp Phe Asp Glu Asn Gly Trp Lys Asp Phe 95 100 105 Leu Glu Val Thr Phe Asp Gly Gly Lys Met Val Gln Val Val Tyr 110 115 120 Asp Tyr Gln His Lys Glu Gly Arg Phe Lys Ser Gln Asp Ala Asp 125 130 135 Tyr His Arg Val Met Tyr Ala Ser Ser Gly Ile Gly Pro Glu Lys 140 145 150 Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly Asn Pro 155 160 165 Glu Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser Gln Ser 170 175 180 Phe Arg Arg Leu Gly Arg Ala Leu Leu Gln Ser Ala Arg Arg Gly 185 190 195 Glu Lys Glu Ala Ile Ile Ser Arg 200

SEQ ID NO: 20 Sequence length: 1035 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATGAAGATTG GTATTGTAAC TGGTATCCCT GGTGTAGGGA AAAGTACTGT CTTGGCTAAA 30 60 GTTAAAGAGA TATTGGATAA TCAAGGTATA AATAACAAGA TCATAAATTA TGGAGATTTT 90 120 ATGTTAGCAA CAGCATTAAA ATTAGGCTAT GCTAAAGATA GAGACGAAAT GAGAAAATTA 150 180 TCTGTAGAAA AGCAGAAGAA ATTGCAGATT GATGCGGCTA AAGGTATAGC TGAAGAGGCA 210 240 AGAGCAGGTG GAGAAGGATA TCTGTTCATA GATACGCACG CTGTGATACG TACACCCTCT 270 300 GGATATTTAC CTGGTTTACC GTCAGATATA ATTACAGAAA TAAATCCGTC TGTTATCTTT 330 360 TTACTGGAAG CTGATCCTAA GATAATATTA TCAAGGCAAA AGAGAGATAC AACAAGGAAT 390 420 AGAAATGATT ATAGTGACGA ATCAGTTATA TTAGAAACCA TAAACTTCGC TAGATATGCA 450 480 GCTACTGCTT CTGCAGTATT AGCCGGTTCT ACTGTTAAGG TAATTGTAAA CGTGGAAGGA 510 540 GATCCTAGTA TAGCAGCTAA TGAGATAATA AGGTCTATGA AGGGTGGTTC TTCTCTGGTT 570 600 CCGCGTGGAT CGGAATTCTGTCGATCGATG 630 660 TATCAGGATT TTGATGAGAA TGGTTGGAAG GACTTTCTCG AGGTTACTTT TGATGGTGGC 690 720 AAGATGGTGC AGGTGGTTTA CGATTATCAG CATAAAGAAG GGCGGTTTAA GTCCCAGGAC 750 780 GCTGACTACC ATCGGGTCAT GTATGCATCC TCGGGCATAG GTCCTGAAAA GGCCTTCAGA 810 840 GAGCTCGCCG ATGCTTTGCT TGAAAAGGGT AATCCCGAGA TGGTGGATGT GGTCACCGGT 870 900 GCAACTGTTT CTTCCCAGAG TTTCAGGAGG TTGGGTCGTG CGCTTCTGCA GAGTGCGCGG 930 960 CGCGGCGAGA AGGAAGCCAT TATTAGCAGG TAGGGATCCG GGCCCTCTAG ATGCGGCCGC 990 1020 ATGCATGGTA CCTAA

[0071] SEQ ID NO: 21 SEQ Length: 3 30 sequence types: amino acid Topology: linear sequence type: Gene expression protein sequence Met Lys Ile Gly Ile Val Thr Gly Ile Pro Gly Val Gly Lys Ser 1 5 10 15 Thr Val Leu Ala Lys Val Lys Glu Ile Leu Asp Asn Gln Gly Ile 20 25 30 Asn Asn Lys Ile Ile Asn Tyr Gly Asp Phe Met Leu Ala Thr Ala 35 40 45 Leu Lys Leu Gly Tyr Ala Lys Asp Arg Asp Glu Met Arg Lys Leu 50 55 60 Ser Val Glu Lys Gln Lys Lys Leu Gln Ile Asp Ala Ala Lys Gly 65 70 75 Ile Ala Glu Glu Ala Arg Ala Gly Gly Glu Gly Tyr Leu Phe Ile 80 85 90 Asp Thr His Ala Val Ile Arg Thr Pro Ser Gly Tyr Leu Pro Gly 95 100 105 Leu Pro Ser Asp Ile Ile Thr Glu Ile Asn Pro Ser Val Ile Phe 110 115 120 Leu Leu Glu Ala Asp Pro Lys Ile Ile Leu Ser Arg Gln Lys Arg 125 130 135 Asp Thr Thr Arg Asn Arg Asn Asp Tyr Ser Asp Glu Ser Val Ile 140 145 150 Leu Glu Thr Ile Asn Phe Ala Arg Tyr Ala Ala Thr Ala Ser Ala 155 160 165 Val Leu Ala Gly Ser Thr Val Lys Val Ile Val Asn Val Glu Gly 170 175 180 Asp Pro Ser Ile Ala Ala Asn Glu Ile Ile Arg Ser Met Lys Gly 185 190 195 Gly Ser Ser Leu Val Pro Arg Gly Ser Glu Phe Cys Ser Phe Ser 200 205 210 Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp Phe Asp 215 220 225 Glu Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly Gly 230 235 240 Lys Met Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly Arg 245 250 255 Phe Lys Ser Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser 260 265 270 Ser Gly Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala 275 280 285 Leu Leu Glu Lys Gly Asn Pro Glu Met Val Asp Val Val Thr Gly 290 295 300 Ala Thr Val Ser Ser Gln Ser Phe Arg Arg Leu Gly Arg Ala Leu 305 310 315 Leu Gln Ser Ala Arg Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg 320 325 330

[Brief description of the drawings]

FIG. 1 is a detailed diagram of an expression vector pW6A.

FIG. 2 is a detailed diagram of the expression vector pWF6A.

FIG. 3 is a graph showing the reactivity between a fusion protein and a negative sample.

FIG. 4 is a graph showing the reactivity of the HTLV-I fusion protein with a positive specimen.

FIG. 5 is a graph showing the reactivity of the HTLV-II fusion protein with a positive specimen.

FIG. 6 is a graph showing the concentration-dependent reactivity of the HTLV-I fusion protein.

FIG. 7 is a graph showing the concentration-dependent reactivity of the HTLV-II fusion protein.

FIG. 8 is a graph showing the fusion protein activity of the heat-treated supernatant.

FIG. 9 is a graph showing the fusion protein activity of the heat-treated precipitate.

FIG. 10 shows the fusion protein activity after heat treatment purification.

FIG. 11 is a detailed diagram of the expression vector pW6AK.

FIG. 12 shows the fusion protein activity after heat treatment purification.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/21 C12R 1:01 C12R 1:19) C12N 15/00 A (C12N 15/09 C12R 1:01) (C12P 21 / 02 C12R 1:19) (56) References Prog. Biotechnol. (1995), 10, p. 77-84 J.C. Bacteriol. (1994), 176 (15), p. 4790-4703 Archives of Biochemistry and Biophysics (1993), 307 (2), p. 405-410 (58) Fields investigated (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) EUROPAT (QUESTEL) WPI (DIALOG) GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq

Claims (11)

(57) [Claims] 1. A DNA of a heat-resistant protein which is not directly denatured at 55 ° C. or higher and fused directly or indirectly to the 5 ′ end of a DNA sequence encoding a selected protein or peptide.
A fusion DNA sequence containing the A sequence. 2. The DNA sequence of the thermostable protein is 75 ° C.
The above is a DNA sequence of a heat-resistant protein that is not heat-denatured.
The DNA sequence according to claim 1. 3. The DNA sequence according to claim 1 , wherein the DNA sequence of the thermostable protein is a DNA sequence derived from a thermophilic bacterium. 4. The DN according to claim 1 , wherein the DNA sequence of the thermostable protein is a DNA sequence derived from an extremely thermophilic bacterium.
A sequence. 5. The DNA sequence of the thermostable protein is derived from a thermophilus, a sulforobos, a phylococcus, a thermotoga, a filobaclam, a phyllodictium, a thermococcus, a thermodiscus, a methanothermas or a methanococcus. The DNA sequence of
3. The DNA sequence according to 1 or 2 . 6. The DNA sequence of the heat-resistant protein is a DNA sequence derived from a bacterium Filococcus or Sulfarobos.
A DNA sequence according to claim 1 or 2 . 7. The DNA sequence of the thermostable protein is a thermostable ferredoxin or a thermostable adenylate kinase DNA.
The D according to any one of claims 1 to 6, which is a sequence.
NA sequence. 8. The DNA sequence of the thermostable protein is sulfaro.
Boss-derived adenylate kinase, thermophilus
DNA polymerase, Phyrococcus furiosis
Ferredoxin, a substance derived from Phyrococcus furiosis
Lucosidase, rubre from phylococcus furiosis
Doxin, Glutamine from Phyrococcus furiosis
Acid dehydrogenase, glycan derived from Methanothermas ferrividus
Cellaldehyde phosphate dehydrogenase, Methanococcus bore
Glutamate synthase derived from Lactobacillus, thermotogamari
L-lactate dehydrogenase and Thermococcus sele
DNA sequence of Elongation Factor from E. coli
The method according to claim 1, wherein the material is selected from the group consisting of
Is the DNA sequence of 2. 9. DNA sequence of thermostable proteins claims 1 is a DNA sequence of the Staphylococcus bacterium derived ferredoxin or sulfur Lobos subtilis-derived adenylate kinase
9. The DNA sequence according to any one of 8 above. 10. A fusion protein expressed from the DNA sequence according to any one of claims 1 to 9 . 11. A method for expressing a fusion protein using the DNA sequence according to any one of claims 1 to 9 .