JPH0789992A - Immunogenic artificial polypeptide - Google Patents

Abstract

PURPOSE:To provide an antigen-site polypeptide usable as a vaccine for human influenza A virus and provide a gene coding the polypeptide. CONSTITUTION:This immunogenic artificial polypeptide has essentially the same antigenicity as the stem region of the hemagglutinin molecule of a human influenza A virus and is free from the spherical region of the hemagglutinin molecule. A gene coding the immunogenic artificial polypeptide. The virus has, e.g., HIN1, H2N2 and H3N2 subtypes to provide polypeptides having essentially the same antigenicity as that of each stem region. These immunogenic artificial polypeptides are also separable from a protease-treatment product of the above hemagglutinin molecule. The polypeptide is insusceptible to the variation of antigenicity caused by the variation of the spherical region.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide containing an antigenic site recognized by an antibody to a stem region in a hemagglutinin molecule of human influenza A virus, and a gene encoding the polypeptide.

[0002]

BACKGROUND OF THE INVENTION There are three types of influenza virus (A, B and C), which cause a worldwide pandemic of influenza, and many deaths are caused by human influenza A virus. Influenza A virus is further classified into many subtypes according to the antigenicity of viral surface proteins haemagglutinin (hereinafter abbreviated as HA) and neuraminidase (hereinafter abbreviated as NA), and H1N1 is a human influenza A virus. Three types are currently known: subclass, H2N2 subclass, and H3N2 subclass. HA of this influenza A virus is a globular region (
It is composed of two different regions, a head region) and a stem region, and the globular region contains a receptor binding site for the virus to bind to target cells.
A is involved in the hemagglutination activity of A, while the stem region contains a fusion peptide required for membrane fusion between the viral envelope and the endosomal membrane of cells and is involved in the fusion activity [Wiley et al., Annual. Review of Biochemistry (Ann. Rev. Biochem.), Volume 56,
365-394 (1987)]. The anti-HA antibodies conventionally obtained as anti-HA antibodies that recognize the H1N1 subtype and H2N2 subtype all recognize the globular region of HA. However, this region is the site where antigenic variation is most likely to occur, and therefore these antibodies do not commonly react with subtypes of human influenza A virus, and with the antigenic change of HA of the virus, It loses recognizability. On the other hand, Green et al. Synthesized a polypeptide from the amino acid sequence of the stem region of one HA of the H3N2 subtype, and obtained an antibody against this polypeptide. Japanese activity is weak (Table 59-50
1714), and the polypeptide itself used as an antigen did not react with rabbit antiviral sera obtained by immunization with H3N2 subtype, and there was also a problem in terms of antigenicity [Cell, No. 28, pp. 477-487 (1982)]. HA of influenza A virus is cleaved at one site by protease,
Although the infectivity of the virus is activated, the larger polypeptide after cleavage of the protease is HA1, and the smaller one is H.
It is called A2. Of these, HA2 is said to have few antigenic mutations due to influenza A virus subtypes. Grate H. (Glathe H.) et al. In East German Patent Application Publication No. 228737 describe that HA2 was taken out by subjecting virus particles to trypsin treatment after acid treatment or only reducing agent treatment. However, these operations destroy the three-dimensional structure of the HA molecule and make it irreversibly denatured, and the resulting HA2 has no original three-dimensional structure. Moreover, the above publication does not disclose that the obtained product was specifically confirmed to be effective as a vaccine.

[0003]

[Problems to be Solved by the Invention] Human influenza A
Type virus periodically changes the type of HA and NA, causing a pandemic, and even if vaccinated before the winter season, when the influenza is outbreak, another type of virus causes an influenza pandemic, so the effect of the vaccine Can often not be expected. However, if it is possible to obtain an antigenic site in the HA molecule or NA molecule that is common to virus subtypes and is unlikely to cause antigenic mutation, this antigenic site will be useful as a vaccine. It is an object of the present invention to provide an antigenic site polypeptide which can be used as a vaccine, which is recognized by an antibody recognizing a subtype of influenza A virus, and a gene encoding the polypeptide.

[0004]

[MEANS FOR SOLVING THE PROBLEMS] To outline the present invention, the first invention of the present invention has substantially the same antigenicity as the stem region in the HA molecule of human influenza A virus, and HA
The present invention relates to an immunogenic artificial polypeptide lacking a globular region in a molecule. The second invention of the present invention relates to a gene encoding the immunogenic artificial polypeptide of the first invention of the present invention.

As a result of earnest studies, the present inventors have found that the H1N1 subtype and H2N of human influenza A virus are
Polypeptides containing a commonly conserved antigenic site in each stem region of two subtype HA molecules, and H
It was found that a polypeptide containing a commonly conserved antigenic site in the stem region of the 3N2 subtype HA molecule is extremely useful as a vaccine, and the polypeptide is useful for genetic engineering production of the polypeptide. The present invention has been completed by creating a gene encoding

An immunogenic polypeptide having substantially the same antigenicity as the stem region in the HA molecule of the human influenza A virus of the present invention and lacking the globular region in the HA molecule is a protein. A typical example is a polypeptide in which the globular region of the HA molecule has been engineered to be deleted. The gene of the present invention obtained by specifically deleting the globular region from the gene encoding the HA molecule is It may be a polypeptide expressed by using it. Furthermore, these polypeptides only have to have a three-dimensional structure recognized by an antibody that specifically recognizes the stem region in the HA molecule, and even if a part of them is deleted, another polypeptide has a different amino acid sequence. May be added. Further, it may be partially digested with protease during protein engineering production or genetic engineering production. That is, in the present invention, having substantially the same antigenicity as the stem region in the HA molecule means having the antigenicity of both the HA1 portion and the HA2 portion that can be effectively used as a vaccine in the stem region of the HA molecule. It means that you have to. Therefore, the above-mentioned Grate H. et al. HA obtained by denaturation and destruction of the original three-dimensional structure, like those obtained
Polypeptides consisting only of 2 are not included. In the present invention, examples of the most effective immunogenic artificial polypeptide for vaccine include the following. (1) TGLRN polypeptide sequence represented by at least SEQ ID NO: 1 in the sequence listing and SEQ ID NO: 2 in the sequence listing in the molecule
Immunogenicity characterized in that it has a GITNKVNSVIEK polypeptide sequence represented by Artificial polypeptide. (2) TGMRN polypeptide sequence represented by at least SEQ ID NO: 3 in the sequence listing and SEQ ID NO: 4 in the sequence listing in the molecule
An immunity characterized by having a QINGKLNR (L / V) IEK polypeptide sequence represented by Primitive artificial polypeptide. (3) The immunogenic artificial polypeptide of the first invention of the present invention, characterized in that it is separated from a protease-treated product of a hemagglutinin molecule of human influenza A virus.

Human influenza A virus H1N
An antibody that recognizes a common site in the HA molecular stem region of the 1 subtype and the H2N2 subtype and neutralizes the virus is
As a monoclonal antibody, it can be prepared as follows. Mammals such as mice, guinea pigs and rabbits are immunized with the following antigens. The following substances can be used as the antigen.

Viral particles selected from the H1N1 subtype or the H2N2 subtype can be used. As the H1N1 subtype, for example, A / Bangkok /
10/83, A / Yamagata / 120/86, A / Osaka
/ 930/88, A / Suita / 1/89 (above, Osaka University Research Institute for Microbial Diseases), A / PR / 8/34 [Influenza (H1N1), ATCC VR-95], A
1 / FM / 1/47 [Influenza A (H1N1),
ATCC VR-97], A / New Jersey / 8/76
[Influenza A (H1N1), ATCC VR-89
7], A / NWS / 33 [Influenza A (H1N
1), ATCCVR-219], A / Weiss / 43 [influenza A (H1N1), ATCCVR-96],
A / WS / 33 [Influenza A (H1N1), AT
CC VR-825]. Examples of H2N2 subtypes are A / Okuda / 57, A / Adachi / 2/57,
A / Kumamoto / 1/65, A / Kaizuka / 2/65, A
/ Izumi / 5/65 (above, preserved strain of the Institute for Microbial Diseases, Osaka University), A2 / Japan / 305/57 [Influenza A (H2N2), ATCC VR-100]. Alternatively, an HA molecule obtained from these viruses, an HA polypeptide prepared by gene recombination technology, or a recognition site of the antibody of the present invention, that is, an antigen site of a stem region in the HA molecule is contained in the molecule. It can be immunized with a recombinant polypeptide or a synthetic polypeptide containing in its molecule the antigenic site of the stem region in the HA molecule.

Next, spleen cells obtained from the immunized animal are fused with, for example, mouse myeloma cells, and from the obtained hybridomas, cells producing antibodies having the following properties (A) to (C) are selected, It can be prepared by culturing the cells. (A) It has binding and neutralizing activities to the viruses of the above H1N1 subtype and H2N2 subtype. (B) H3N2 subtype virus, such as A / Fuku
oka / C29 / 85, A / Sichuan / 2/87, A / Ib
araki / 1/90, A / Suita / 1/90, A / Kitaky
ushu / 159/93 (above, preserved strain of the Institute for Microbial Diseases, Osaka University), A / PortChalmers / 1/73 [Influenza A (H3N2), ATCC VR-810], A2 /
Aichi / 2/68 [Influenza A, ATCC VR
-547], and B-type viruses such as B / Nagasaki
/ 1/87 (Osaka University Research Institute for Microbial Diseases), B / Al
len / 45 [Influenza B, ATCC VR-10
2] has no binding property or neutralizing activity. (C) H of H1N1 subtype and H2N2 subtype
Although it recognizes the A molecule, it does not inhibit the hemagglutination activity involving the globular region in the HA molecule, but does inhibit the membrane fusion activity involving the stem region in the HA molecule.

The preparation of this hybridoma is carried out based on Nature, Vol. 256, pp. 495-497 (1975). Balb for immunization
/ C mouse, Balb / c mouse and other mouse F1
A mouse or the like is used. Immunization against one mouse
For example, virus particles (100 to 1000 HA units) are used as an antigen, and the treatment is performed, for example, three times in 2 to 5 months. The breeding of mice and the collection of splenocytes follow conventional methods.

As the myeloma cell, SP2 / 0-Ag
14 (ATCC CRL1581), p3 x 63Ag8
U. 1 (ATCC CRL1597), p3 x 63Ag
8 (ATCC TIB9), p3 × 63-Ag8.65.
3 (ATCC CRL1580) and the like are preferably used. Spleen cells and myeloma cells were mixed at a ratio of 1: 1 to 10: 1, and fusion was performed with NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (v / v)] and molecular weight 1
It is carried out by incubating the mixture of both cells at 35 to 37 ° C for 1 to 5 minutes in a phosphate buffer (pH 7.2 to 7.4) containing 000 to 6000 polyethylene glycol. The fused cells are selected using HAT medium as the growing cells. Cloning of the fused cells is repeated at least 3 times by the limiting dilution method.

When the hybridoma is cultured in the same manner as ordinary animal cells, the antibody of the present invention can be obtained in the medium as a result. In addition, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a pristane-treated nude mouse or Balb / c mouse and proliferating it. That is, 0.5 to 1 mg of pristane was inoculated into the abdominal cavity of these mice, and then 5 to 10 ⁶ to 1 × 10 ⁷ hybridomas were transplanted to the abdominal cavity in the second to third weeks. Ascites usually accumulates after 7 to 10 days and is collected. Monoclonal antibodies in culture and ascites fluid are purified by conventional means.

The obtained monoclonal antibody neutralizes the virus by recognizing the stem region of HA molecule of H1N1 subtype and H2N2 subtype and inhibiting the membrane fusion activity of the virus. It has properties.

(A) By staining test, MDCK infected with H1N1 subtype and H2N2 subtype respectively
It recognizes cells (ATCC CCL34) and not MDCK cells infected with the H3N2 subtype. In the staining test, four kinds of antibodies (monoclonal antibody of the present invention, rabbit anti-mouse immunoglobulin G serum, goat anti-rabbit immunoglobulin G serum, peroxidase-rabbit antiperoxidase complex) were used, and Journal of Clinical Microbiology (J. Clin. Microbiol),
Vol. 28, pp. 1308 to 1313 (1990).

(B) By immunoprecipitation method, HA molecules of H1N1 subtype and H2N2 subtype are recognized and H3
It does not recognize HA molecules of the N2 subtype.

(C) In the hemagglutination test, the hemagglutination activity of each of the H1N1 subtype, H2N2 subtype and H3N2 subtype is not inhibited.

(D) Recognizing a common conserved region unique to the stem region in H1N1 subtype and H2N2 subtype HA molecules, which is identified by genetic analysis encoding the HA molecule, It does not recognize the common storage area specific to the trunk area.

The gene encoding the HA molecule is analyzed as follows. MDCK cells are infected with virus particles, the infected cells are collected the next day, and viral RNA is extracted from the cells using guanidine isothiocyanate. Next, H1
N-, H2N2, H3N2 subtype (-) chain RN
An oligonucleotide primer containing a sequence complementary to the 3'end of A, for example, primer 5 represented by SEQ ID NO: 5 in the sequence listing was prepared, and cDNA was prepared using the primer.
To synthesize. To amplify the cDNA, H1N1,
An oligonucleotide primer containing a sequence complementary to the 3'end of the (+) strand RNA of each of H2N2 and H3N2 subtypes, for example, primer 6 represented by SEQ ID NO: 6 in the sequence listing was prepared, and the primer and primer 5 PCR using cDNA (Polymerase chain reaction)
The method enables efficient amplification. Amplified DNA
After separating the HA gene of about 1.7 kbp from it by agarose gel electrophoresis, use a second PC using, for example, primers 5 and 6.
Perform R and centrifuge the amplified DNA using 20% (w / v) polyethylene glycol 6000 / 2.5M NaCl to obtain a purified precipitate fraction. Next, a sequence primer selected from HA gene sequences of each subclass of virus was prepared, the primer was labeled with [γ- ³² P] ATP, and the labeled primer and the above-mentioned purified fraction were annealed. The dideoxy method using a thermal cycler [BioTechniques, Vol. 9,
66-72 (1990)].

For example, primers 7 to 14 represented by SEQ ID NOS: 7 to 14 in the sequence listing are sequence primers for H1N1 subtype, and SEQ ID NO: 1 in the sequence listing.
Primers 15-23 represented by 5-23 are H
A sequence primer for 2N2 subtype,
Primers 24 to 26 represented by SEQ ID NOs: 24 to 26 in the sequence listing are sequence primers for H3N2 subtype. In addition, by using the primers 9 and 13 as PCR primers and the primers 11 and 12 as sequence primers, H1
A part of the gene encoding the stem region of the HA molecule of N1 subtype is efficiently amplified and analyzed. Also, PCR
By using the primers 17 and 21 as the primers for use and the primers 19 and 20 as the sequence primers, a part of the gene encoding the stem region of the HA molecule of the H2N2 subtype is efficiently amplified,
Parsed. Further, by using the primers 24 and 26 as PCR primers and the primers 25 and 26 as sequence primers, H3N
A part of the gene encoding the stem region of the HA molecule of two subtypes is efficiently amplified and analyzed.

The common conserved region in HA molecules among virus subtypes is represented by SEQ ID NO: 1 in the sequence listing of the stem region in HA molecules of H1N1 subtype and H2N2 subtype found by the present inventors. And the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing. FIG. 1 is a schematic diagram of the tertiary structure of the HA molecule [Wiley et al., Nature, No. 289].
Vol. Pp. 373-378 (1981)], and H1N1.
The position of the common conserved region in the HA molecule between subtypes and H2N2 subtypes is shown. Both polypeptide sequences shown in the regions A and B in the figure are located close to each other in the center of the stem region of the HA molecule as shown in FIG.
The monoclonal antibody C179 produced by oma C179 [FERM BP-4517] is a TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region of the HA molecule and GITNKVNSVIEK represented by SEQ ID NO: 2 in the sequence listing. Recognizes polypeptide sequences.

(E) In the neutralizing activity test, it inhibits the plaque forming ability or focus forming ability of the H1N1 subtype and H2N2 subtype, but does not inhibit the plaque forming ability or focus forming ability of the H3N2 subtype. The neutralizing activity test is performed by the pull reduction neutralization test or the influenza virus rapid focus reduction neutralization test described in the above-mentioned Journal of Clinical Microbiology. That is, mixing the antibody and virus,
After incubating for a certain period of time, MDCK cells were infected, and the neutralizing ability was judged by the decrease of appearance of plaques or foci.

(F) In the fusion activity test, the membrane fusion activity of H1N1 subtype and H2N2 subtype is inhibited, but the membrane fusion activity of H3N2 subtype is not inhibited. Fusion activity test is Nature, Volume 300, 658-65
Follow the method described on page 9 (1982). That is, CV
-1 cells (ATCC CCL70) were infected with the virus, then treated with an antibody, and the ability to inhibit fusion activity was determined by the presence or absence of polykaryon formation.

The monoclonal antibody C179 binds to the stem region of the HA molecule, inhibits the membrane fusion activity of the H1N1 and H2N2 subtypes, and strongly neutralizes the infectivity of the virus strain. Therefore, it binds to the stem region in HA molecules of H1N1 and H2N2 subtypes,
A polypeptide that induces an antibody (hereinafter abbreviated as C179 type antibody) that inhibits the membrane fusion activity of H1N1 subtype and H2N2 subtype and strongly neutralizes the infectivity of the virus should be used as a vaccine for influenza. You can That is, if a polypeptide that has substantially the same antigenicity as the stem region in the HA molecule of H1N1 subtype and H2N2 subtype and induces C179 type antibody is used as an immunogen, H1N1 subtype and H2N2 subtype It is possible to prevent and treat the influenza epidemic. As the immunogenic polypeptide, an HA molecule prepared from H1N1 subtype, H2N2 subtype, or an HA polypeptide prepared by gene recombination technology can be used. A polypeptide having a stem region in a HA molecule as a constituent component (hereinafter abbreviated as a stem region polypeptide) in which a globular region in the HA molecule is deleted is an immunogen that specifically induces a C179 type antibody. Is particularly effective as a sex polypeptide.

This stem region polypeptide is a HA molecule or H
The globular region of the A polypeptide can be obtained by enzymatically limiting decomposition and removal. For example, HA purified from viral particles of H1N1 subtype or H2N2 subtype
The stem region polypeptide can be prepared by subjecting the molecule to limited degradation with a protease. Alternatively, the stem region polypeptide may be prepared and used by treating virus particles, a split vaccine obtained by inactivating virus particles, and an extract obtained by treating virus particles with a surfactant, respectively.

The protease used is preferably a proteinase capable of decomposing the globular region without losing the antigenicity of the stem region in the HA molecule. The proteinase that can be used in the present invention is proteinase K (EC3.4.21.EC), which is an alkaline proteinase produced by Tritirachium album.
14) and (manufactured by Boehringer) are mentioned as an example. The stem region polypeptide of the present invention can be prepared using a proteinase showing a result equivalent to this proteinase K. In addition, the proteinase and the peptidase may be combined, and the proteinase treatment may be followed by the peptidase treatment. In addition, since HA molecules take a strong trimer in solution, they are less susceptible to protease digestion, and therefore HA
HA in the presence of denaturing agents such as guanidine hydrochloride and urea
By treating the molecule with protease, H
Protease treatment of the A molecule can be performed. The concentration of the denaturant may be any concentration as long as it does not cause irreversible denaturation of the target stem region polypeptide and that protease also acts. When using urea as a denaturant, 0.1-8
Protease digestion may be performed in the presence of urea in the range of M, preferably 1-3M. The protease treatment can also be performed using a resin such as sepharose on which protease is immobilized, and the protease-immobilized resin can be easily removed by centrifugation after the reaction is completed. The denaturant and low molecular weight substances in the reaction solution can be removed by dialysis treatment, and a HA-molecule protease-treated substance can be prepared. The molecular weight of the protease-treated HA molecule can be measured by performing gel electrophoresis.

The target stem region polypeptide can be confirmed by measuring the binding between the protease-treated product and the C179 type antibody and the hemagglutination activity of the protease-treated product. The stem region polypeptide obtained by protease treatment has substantially the same antigenicity (binding property to C179 type antibody) as the stem region in the HA molecule, and lacks the biological activity (hemagglutination activity) of the globular region. It is a lost polypeptide and is composed of a polypeptide portion derived from the stem region of HA1 and a polypeptide portion derived from HA2 in the HA molecule. In this regard, the Grate H. These vaccines are essentially different.

Further, a polypeptide having substantially the same antigenicity as the stem region in a natural HA molecule and lacking the globular region in the HA molecule is prepared by gene recombination technology or chemical synthesis method. , May be used. For example, from RNA of a virus of H1N1 subtype or H2N2 subtype, HA
A gene is prepared, the site encoding the globular region in the gene is cleaved and removed with a restriction enzyme, and then the gene is integrated into a vector and expressed in animal cells such as CV-1 cells to obtain the desired polypeptide. be able to. In addition, after the HA gene is incorporated into a vector, the site encoding the globular region in the HA gene can be selectively deleted by the PCR method, and a vector containing the gene can be used to For example, the desired polypeptide can be obtained by expressing the stem region polypeptide in CV-1 cells. The antigenicity of these stem region polypeptides can be confirmed by the binding of the C179 type antibody. Intramolecular H1N as a stem region polypeptide
It may be any one as long as it has a common conserved region in the stem region in HA molecules of 1 subtype and H2N2 subtype and can induce C179 type antibody. The stem region polypeptide having a common conserved region has, for example, a TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing and a GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing in the molecule, Polypeptides may be prepared and used in which the configurations of the sequences are those in the native HA molecule that have substantially the same antigenicity as these sequences. The stem region polypeptide may be one in which amino acids have been deleted, substituted, inserted, transposed, added or rearranged within a range that does not affect its immunogenicity. For example, the C-terminal portion of the stem region polypeptide and Alternatively, a part of the N-terminal region polypeptide may be deleted, or a signal peptide of the HA molecule or a part of the globular region may be added.

Human influenza A virus H3N
An antibody that specifically recognizes a common site in the HA subregions of two subtypes can be prepared as a monoclonal antibody as follows. Mammals such as mice, guinea pigs and rabbits are immunized with the following antigens. The following substances can be used as the antigen. H3N2
Viral particles selected from subtypes can be used. Or HA obtained from these viruses
H prepared by using a molecule or gene recombination technology
A polypeptide, or a recombinant polypeptide containing the recognition site of the antibody of the present invention, that is, the antigenic region of the stem region in the HA molecule in the molecule, or containing the antigenic region of the stem region in the HA molecule in the molecule It can be immunized with a synthetic polypeptide.

Next, the spleen cells obtained from the immunized animal are
For example, it can be prepared by fusing with mouse myeloma cells, selecting cells producing an antibody having the following properties (D) to (F) from the resulting hybridoma, and culturing the cells. (D) It has a binding property to a subtype virus of H3N2. (E) It does not bind to H1N1 and H2N2 subtype viruses. (F) H3N2 subtype HA molecule is recognized, but H
It does not inhibit the hemagglutination activity involving the globular region in the A molecule.

The obtained monoclonal antibody recognizes the stem region of the HA molecule of the H3N2 subtype, and has the following properties in detail.

(G) The staining test recognizes MDCK cells infected with H3N2 subtype (ATCCCCL34), but does not recognize MDCK cells infected with H1N1 subtype and H2N2 subtype.

(H) The H3N2 subtype HA molecule was recognized by the immunoprecipitation method, and H1N1 subtype and H2 were recognized.
It does not recognize HA molecules of the N2 subtype.

(I) In the hemagglutination test, the hemagglutination activity of each of the H1N1 subtype, H2N2 subtype and H3N2 subtype is not inhibited.

(J) Recognizing a common conserved region unique to the stem region in the HA molecule of the H3N2 subtype, which is identified by genetic analysis encoding the HA molecule, and It does not recognize the common storage area specific to the trunk area.

The common conserved region in the H3N2 subtype HA molecule is the TGMRN polypeptide sequence represented by SEQ ID NO: 3 in the sequence listing of the stem region in the H3N2 subtype HA molecule found by the present inventors. And the QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the sequence listing. FIG. 2 shows a schematic diagram of the tertiary structure in the HA molecule [Willie et al., Supra] and the position of the common conserved region in the HA molecule of the H3N2 subtype. Both polypeptide sequences shown in the A'region and B'region in the figure are located close to each other in the center of the stem region in the HA molecule as shown in FIG. -4516)
The monoclonal antibody AI3C produced by Escherichia coli is TGMRN represented by SEQ ID NO: 3 in the sequence listing of the stem region in the HA molecule.
QING represented by the polypeptide sequence and SEQ ID NO: 4 in the sequence listing
It recognizes the KLNR (L / V) IEK polypeptide sequence.

A polypeptide that induces an antibody (hereinafter abbreviated as AI3C type antibody) that specifically recognizes a common conserved region peculiar to the stem region in the HA molecule of the H3N2 subtype should be used as a vaccine for influenza. You can That is, when a polypeptide that has substantially the same antigenicity as the stem region in the HA molecule of H3N2 subtype and induces AI3C type antibody is used as an immunogen, the outbreak of H3N2 subtype influenza can be prevented or treated. be able to. The immunogenic polypeptide may be H3N2
HA molecules prepared from subtypes and HA polypeptides prepared by gene recombination techniques can be used, but HA is likely to become an antigenic determinant and easily cause antigenic mutation.
A stem region polypeptide having a stem region of an HA molecule in which the globular region of the molecule is deleted as a constituent is particularly effective as an immunogenic polypeptide that specifically induces an AI3C type antibody.

This stem region polypeptide is an HA molecule or H
The globular region of the A polypeptide can be obtained by enzymatically limiting decomposition and removal. For example, the HA region purified from H3N2 subtype virus particles is subjected to limited degradation with a protease to prepare the stem region polypeptide. Alternatively, the stem region polypeptide may be prepared and used by treating virus particles, a split vaccine obtained by inactivating virus particles, and an extract obtained by treating virus particles with a surfactant, respectively. The protease used is preferably a proteinase capable of degrading the globular region without losing the antigenicity of the stem region in the HA molecule. Examples of proteinases that can be used in the present invention include the above-mentioned proteinase K. The stem region polypeptide of the present invention can be prepared using a proteinase showing a result equivalent to this proteinase K. In addition, the proteinase and the peptidase may be combined, and the proteinase treatment may be followed by the peptidase treatment. In addition, since the HA molecule takes a strong trimer in the solution, it is less susceptible to protease digestion,
Therefore, by treating the HA molecule with a protease in the presence of a denaturing agent such as guanidine hydrochloride or urea, the HA molecule can be efficiently treated with a protease. The concentration of the denaturant may be any concentration as long as it does not cause irreversible denaturation of the target stem region polypeptide and that protease also acts. When urea is used as a denaturant, protease digestion may be performed in the presence of urea in the range of 0.1 to 8M, preferably 1 to 3M. The protease treatment can also be performed using a resin such as sepharose on which protease is immobilized, and the protease-immobilized resin can be easily removed by centrifugation after the reaction is completed. The denaturant and low molecular weight substances in the reaction solution can be removed by dialysis treatment, and a HA-molecule protease-treated substance can be prepared. The molecular weight of the protease-treated HA molecule can be measured by performing gel electrophoresis.

The target stem region polypeptide can be confirmed by measuring the binding between the protease-treated product and the C179 type antibody and the hemagglutination activity of the protease-treated product. The stem region polypeptide obtained by protease treatment has substantially the same antigenicity (binding to AI3C type antibody) as the stem region in the HA molecule, and lacks the biological activity (hemagglutination activity) of the globular region. The lost polypeptide is composed of a polypeptide portion derived from the stem region of HA1 in the HA molecule and a polypeptide portion derived from HA2.

Further, a polypeptide having substantially the same antigenicity as the stem region in the natural HA molecule and lacking the globular region in the HA molecule was prepared by gene recombination technology or chemical synthesis method. , May be used. For example, HA gene was prepared from RNA of H3N2 subtype virus, and the site encoding the globular region in the gene was cleaved and removed with a restriction enzyme, and then the vector was integrated into a vector and used in animal cells such as CV-1 cells. The desired polypeptide can be obtained by expressing. In addition, after the HA gene is incorporated into a vector, the site encoding the globular region in the HA gene can be selectively deleted by the PCR method, and a vector containing the gene can be used to For example, the desired polypeptide can be obtained by expressing the stem region polypeptide in CV-1 cells.
The antigenicity of these stem region polypeptides can be confirmed by the binding property of AI3C type antibody.

As the stem region polypeptide, H in the molecule
It may be any one as long as it has a common conserved region in the stem region of HA molecule of 3N2 subtype and can induce AI3C type antibody. Examples of the stem region polypeptide having a common conserved region include TG represented by SEQ ID NO: 3 in the sequence listing in the molecule.
MRN polypeptide sequence and a QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the sequence listing, the configuration of which sequence is substantially identical to these sequences in the native HA molecule. A polypeptide having a configuration with antigenicity may be prepared and used. The stem region polypeptide may be one in which amino acids have been deleted, substituted, inserted, transposed, added or rearranged within a range that does not affect its immunogenicity. For example, the C-terminal portion of the stem region polypeptide and And / or a part of the N-terminal region polypeptide may be deleted, a signal peptide of HA molecule,
A part of the spherical portion region may be added.

When these polypeptides are used as vaccines, their antigenicity can be increased by selecting an appropriate carrier. As the carrier, for example, albumin, polyamino acid, etc. may be used. The vaccine of the present invention may be administered by a conventional active immunization method, that is, a prophylactically or therapeutically effective and immunogenic amount is administered once or multiple times in a manner compatible with the dosage formulation. In addition, the vaccine may be formulated as usual, and may contain an adjuvant for improving the immune response. The dose of the administered polypeptide depends on the nature of the vaccine used, the concentration in the preparation, the route of administration, etc., but is usually 1 μg to 100 mg, preferably 10 μg to 10 mg for an adult.
It suffices to administer mg.

The anti-influenza virus antibody used in the present invention will be described below with reference to Reference Examples. Reference example A 1. Preparation of virus A / PR / 8/34, A / Ba as H1N1 subtype
ngkok / 10/83, A / Yamagata / 120/86, A
/ Osaka / 930/88, A / Suita / 1/89, Al
/ FM / 1/47 was used. A / Okuda / 57, A / Adachi / 2/57, A / Kumamo as H2N2 subtype
to / 1/65, A / Kaizuka / 2/65, A / Izumi /
5/65 was used. A2 / Ai as H3N2 subtype
chi / 2/68, A / Fukuoka / C29 / 85, A / Si
chuan / 2/87, A / Ibaraki / 1/90, A / Suita
/ 1/90, A / Kitakyushu / 159/93 was used. B / Nagasaki / 1/87 was used as the B virus. Each virus was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs and cultured at 34 ° C for 4 days, and then each virus was collected.

2. Preparation of Monoclonal Antibody (1) A / Okuda / 57 virus (320 HA units) prepared in Reference Example A-1 was suspended in Freund's complete adjuvant before use, and intraperitoneally injected into Balb / c mice at 1-month intervals. Immunization was performed twice by the above method, and one month later, the cells were boosted by intraperitoneal injection using a PBS suspension of the same antigen (320 HA units). Three days later, the spleen was removed from the mouse to prepare splenocytes. P3 for mouse myeloma
X63Ag8 was cultured in a DME medium supplemented with 10% fetal bovine serum for 2 days after passage, and washed with physiological saline before cell fusion to prepare. Next, splenocytes and myeloma cells were mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass was thoroughly loosened, and then stirred to give 1 ml.
Mixed solution of [polyethylene glycol-4000 (2
g), MEM (2 ml), dimethylsulfoxide] and kept at 37 ° C. for 5 minutes, and then MEM was slowly added so that the total amount of the solution was 10 ml. Then, after centrifugation,
The supernatant was removed and the cells were loosely loosened. To this, 30 ml of a normal medium [PRMI-1640 plus 10% fetal bovine serum] was added, and the cells were gently suspended using a measuring pipette.

Dispense the suspension into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO ₂ . Next, HAT medium was added and the cells were cultured for 10 to 14 days. Subsequently, a part of the culture supernatant was collected and a hybridoma was screened.

(2) In order to obtain a monoclonal antibody that cross-reacts between influenza A virus subtypes, the above-mentioned culture supernatant, which has not been diluted, is used as the primary antibody, and three subtypes (H1N1, H2N2, H3N
A staining test of MDCK cells infected with each of 2) was performed. The dyeing test was performed according to the method described in the above-mentioned Journal of Clinical Microbiology. That is, 96-well microtiter plate (Falcon 30
72: Becton Dickinson), each subtype strain of human influenza A virus (H1N1: A)
/ Yamagata / 120/86, H2N2: A / Okuda / 5
MDCK cells infected with 7, H3N2: A / Fukuoka / C29 / 85) were washed with PBS (pH 7.4) and fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were treated with four kinds of antibodies [the above-mentioned culture supernatant containing a monoclonal antibody, a 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), goat anti-rabbit immunoglobulin G serum (organotechnica). (Manufactured by ORGANOTECHNICA), a 500-fold diluted solution, manufactured by Organotechnica Co., Ltd.
00-fold diluted solution], the reaction was continuously performed for 40 minutes, and the treated cells were washed with PBS. Finally, the peroxidase reaction was performed by the method of Graham, Karnovsky, using 0.01% H ₂ O ₂ and 0.3 mg / ml 3,3'-diaminobenzidine tetrahydrochloride in PBS. Of His Chemistry and Sight Chemistry (J. Histochem. Cytochem.), 14th
Vol. 291-302 (1966)]. The stained cells were observed by an ordinary light microscope, and antibodies that recognize MDCK cells infected with H1N1 subtype and MDCK cells infected with H2N2 subtype were selected. Next, the cells in the hole in which the cells confirmed to have the antibody production are taken out and subjected to the limiting dilution method three times, the desired cells are cloned, and the cloned hybridoma strain is Hy
Bridoma C179 and the monoclonal antibody produced by the hybridoma were designated as monoclonal antibody C179.

The Hybridoma C179 is a Hybridoma.
It has been designated as C179 and has been deposited as FERM BP-4517 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(3) Balb / c mice treated with pristane were intraperitoneally administered with the above hybridoma strain at 5 × 10 ⁶ mice / mouse. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare an ascites fluid. About 1 mg of the monoclonal antibody C179 (hereinafter simply abbreviated as C179) was contained in 1 ml of ascites fluid. C179
Was an IgG2a type antibody purified with Protein A-Sepharose 4B (Pharmacia).

3. Properties of Monoclonal Antibody (1) A 100-fold dilution of ascites fluid described in Reference Example A-2- (3) was serially diluted, and the staining test described in Reference Example A-2- (2) was performed to recognize C179 antigen. The sex was examined. H1N1
Subtypes are A / PR / 8/34, A / Bangkok
/ 10/83, A / Yamagata / 120/86, A / Osak
a / 930/88, A / Suita / 1/89, A1 / FM
/ 1/47, A / Okuda / as the H2N2 subtype
57, A / Adachi / 2/57, A / Kumamoto / 1/6
5, A / Kaizuka / 2/65, A / Izumi / 5/65,
A / Aichi / 2/68, A / as H3N2 subtype
Fukuoka / C29 / 85, A / Sichuan / 2/87, A
/ Ibaraki / 1/90, A / Suita / 1/90, A / Ki
takyushu / 159/93 and B / Nagasaki / 1/87 were used as influenza B viruses. C179
Recognized all H1N1 and H2N2 subtypes, but not H3N2 subtype, B virus.

(2) The neutralizing activity of the antibody was measured by the above-mentioned influenza virus rapid focus reduction neutralization test using the archives of virology (Arch. Virol.
), 86, 129-135 (1985), Microbiology and Immunology (Microbiol. I).
mmunol.), 29, 327-335 (198).
It carried out according to 5). As an antibody, Reference Example A-2-
Using the ascites fluid of (3), the antibody was added with a 3-fold volume of a solution of a receptor-degrading enzyme (RDE: Takeda Pharmaceutical Co., Ltd.) before use, reacted at 37 ° C for 18 hours, and then heated at 56 ° C for 45 minutes. The RDE was inactivated by the treatment and finally prepared as a 16-fold diluted solution of the ascites fluid, which was used as a test solution, and the measurement was carried out as follows.

That is, MDC is applied to a 96-well microplate.
10 ⁴ K cells / well were dispensed, and the next day, the antibody (16
4-fold dilution of (double dilution) and the respective virus solutions described in Reference Example A-3- (1) prepared in 30 focus forming units / hole were mixed in equal amounts and incubated at 37 ° C. for 1 hour. Next, 25 μl of this mixed solution was dispensed into each well of the microtiter plate containing the MDCK cells, and the mixture was kept for 30 minutes for 3 minutes.
It was kept warm at 7 ° C. Then, the solution in each well was removed, each well was washed with PBS, and then MEM containing 0.5% tragacanth gum (Wako Pure Chemical Industries, Ltd.) and 5 μg / ml trypsin was added. After incubating at 37 ° C. for 20 to 24 hours, the additive solution was removed, each well was washed with PBS, and the cells were treated with absolute ethanol at room temperature for 10 minutes to fix the cells. Next, it was dried and cells were stained according to the staining test described in Reference Example A-2- (2). After staining, the cells were washed with tap water, dried, and the number of stained foci was counted under an optical microscope.

C179 inhibited the focus formation of all H1N1 and H2N2 subtypes and showed strong virus neutralizing activity, while it did not affect the focus formation of H3N2 subtype and B virus. In addition, similar results were shown in the pull reduction neutralization test.

(3) The hemagglutination inhibitory activity (HI) of the antibody was determined as follows. RDE as in Reference Example A-3- (2)
A serial dilution was prepared using the treated antibody (32-fold dilution), and then each virus (1) described in Reference Example A-3- (1) was prepared.
6 HA units) and allowed to react for 30 minutes at room temperature. Then, chicken red blood cells were added and mixed well, and the effect of the antibody on the hemagglutination activity of each virus was examined. C179
Did not affect the hemagglutination activity of all subtype viruses.

(4) Antibody fusion inhibitory activity was measured by the above-mentioned Nature, Vol. 300, pp. 658-659 (198).
The method of 2) was modified and performed. That is, a monolayer culture of CV-1 cells was infected with each virus strain described in Reference Example A-3- (1). After 24 hours, cells were washed twice with DMEM and then trypsin added (10 μm).
g / ml) in DMEM for 15 minutes at 37 ° C.
Next, the cells were washed twice with DMEM, and then Reference Example A-2-
Add the DMEM diluted solution of the ascites fluid of (3),
It was kept warm for 0 minutes. Thereafter, the cells were adjusted to pH 5.0 with fusion medium (PRMI without Na ₂ CO ₃ , 0.2% bovine serum albumin, 10 mM MES, 10 mM HEPE).
S) at 37 ° C. for 2 minutes, followed by washing twice with DMEM to remove the fusion medium, followed by incubation with DMEM containing 2% fetal bovine serum at 37 ° C. for 3 hours. Next, the cells were fixed with anhydrous methanol and stained with Giemsa, and then the presence or absence of polykaryon formation was determined by an optical microscope. C179
Blocked the polykaryon formation of all H1N1 and H2N2 subtypes, but not the H3N2 and B virus polykaryon formation.

As described above, C179 is H1N1 subtype, H
The antibody specifically recognized the 2N2 subtype, inhibited viral membrane fusion, and exhibited neutralizing activity. The results are shown in Table 1.

[0055]

[Table 1]

The numbers in the table are the dilution factors of the ascites fluid of Reference Example A-2- (3), and the staining titer was the maximum dilution factor of the ascites fluid capable of staining cells in the staining test, and the neutralizing activity. Shows the maximum dilution factor of the ascites fluid capable of suppressing the appearance of foci up to half of the number of foci in the control antibody-free group.
In addition, + means that polykaryon formation is completely inhibited by the antibody solution of the ascites fluid diluted 1000 times, and − means 10
This means that no inhibition was observed even when the ascites fluid diluted twice was used. In addition, no HI activity was observed using the 32-fold diluted ascites fluid.

4. Determination of Epitope (1) It was determined by immunoprecipitation that the recognition protein of C179 was an HA molecule. That is, H2N2
After subtype A / Okuda / 57 was adsorbed and infected with MDCK cells for 30 minutes, 10 μCi of methionine in the medium was added.
The infected cells were labeled by culturing for 24 hours in MEM substituted with " ³⁵ S] methionine.
PA buffer [50 mM Tris (pH 7.4), 150 mM N
aCl, 1 mM EDTA, 1% nonidet P-40, 1
% Deoxycholic acid, 0.1% SDS]. Subsequently, the insoluble matter was removed by centrifugation to obtain a supernatant. Next, the supernatant was mixed with C179 and incubated at 4 ° C. for 1 hour, Protein A-Sepharose CL4B beads were added, and the mixture was kept at room temperature for 2 hours to adsorb the immunoprecipitate to the beads. The beads were then collected and washed 5 times with RIPA buffer,
Boiled to liberate the C179 binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate, dried, and autoradiographed. The labeled protein to which C179 binds was identified as the A / Okuda / 57 HA molecule from its electrophoretic pattern. Similar tests for H1N1 subtype, other H2
N2 subtype and H3N2 subtype were used respectively. C179 is all H1N1 subtypes and H2
Was specifically immunoprecipitated with HA molecules of N2 subtype,
It did not bind to the H3N2 subtype HA molecule.

(2) MDCK cells infected with the H1N1 subtype or the H2N2 subtype were cultured in the presence of C179 to obtain an antigen mutant strain showing no sensitivity to C179. That is, A / Su of H1N1 subtype as parent strain
ita / 1/89, H2N2 subtype A / Izumi / 5
MDC infected with the virus by using E./65
K cells were cultivated in the presence of C179, and mutant strains capable of growing even in the presence of C179 were purely separated from plaques of MDCK cells, and A / Suita / 1/89 mutant strains were A / Su.
The mutant strains of ita / 1/89 (R) and A / Izumi / 5/65 were designated as A / Izumi / 5/65 (R), respectively. The 2 mutants showed no reactivity to C179 in the staining test and the neutralization test. In addition, both mutant strains are MD
The plaque-forming ability in CK cells was also weak, it did not show pathogenicity in experimental animal mice, and it was a weakly infected strain that could grow only in cultured cells.

(3) In order to identify the antigen recognition site of the antibody, the gene encoding the HA molecule (hereinafter abbreviated as HA gene) was analyzed. (A) Synthesis of primer: primer 5 to primer 2
6 was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-2SW column)
And purified by Sep-pak C18 to obtain about 50 μg of each DNA.

(B) MDCK cells infected with A / Suita / 1/89 were collected, guanidine isothiocyanate was added, and the cells were lysed by repeatedly injecting 5 times with a syringe. Layer the cell extract after lysis on cesium chloride solution,
Ultracentrifugation was performed. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer solution, treated with phenol and chloroform, and then precipitated with ethanol, and the recovered RNA was used as a sample of viral genomic RNA. Then using primer 5 c
DNA synthesis, followed by primer 5, primer 6
Was used to amplify the synthetic cDNA by the PCR method. Next, the amplified cDNA was separated by agarose gel electrophoresis and the 1.7 kbp cDNA band corresponding to the HA gene was eluted. Using this cDNA with primers 5 and 6,
Further amplified by PCR method, 20% amplified fragment
(W / v) Polyethylene glycol 6000, 2.5M
An NaCl solution was added at 60% (v / v) and the mixture was centrifuged to obtain a purified precipitate fraction.

Next, H labeled with [γ- ³² P] ATP
The nucleotide sequence of the purified gene was analyzed by the dideoxy method using the thermal cycler described in the above Biotechnics using the 1N1 subtype sequencing primers, primers 7 to 14, as follows. Ie 2
95 pmol of primer and 1 pmol of purified fragment
Annealing was carried out by heating at ℃ for 3 minutes and quenching. Tack polymerase is then added to the buffer in a buffer containing deoxynucleotides and dideoxynucleotides.
The temperature was kept at 10 ° C for 10 minutes to carry out a polymerase extension reaction.
Then, in order to completely carry out the extension reaction, the reaction was transferred to a thermal cycler reaction. Thermal cycler conditions are 90
A cycle of 1 ° C., 1 minute, 55 ° C., 2 minutes, 72 ° C., 3 minutes was repeated 10 times. Next, the cycle-finished product was heated in the presence of formamide at 95 ° C. for 3 minutes and then rapidly cooled in ice, and electrophoresed on an 8% denaturing polyacrylamide gel. After the electrophoresis, the gel is dried, exposed with an X-ray film, and then the base sequence is read.
The base sequence of the entire A gene was determined.

(C) HA of A / Suita / 1/89 (R)
Analysis of the nucleotide sequence of the gene was carried out by referring to Reference Example A-4- (3)-
According to the method of (b), the base sequence of the whole HA gene was determined and compared with the HA gene of the parent strain. The HA gene of the mutant strain had three nucleotide substitutions. That is, H of the parent strain shown in SEQ ID NO: 27 in the sequence listing
G of the base number 627 of the A gene is A and base number 73
The G at position 6 was mutated to A, and the C at base number 1018 was mutated to A. The HA molecule is cleaved at one site by a protease to activate the infectivity of the virus. The larger polypeptide after cleavage of the protease is called HA1 and the smaller one is called HA2. And both of these polypeptides are linked by an S-S bond, but this time, the mutations are 189, 225 and 31 of HA1.
It was accompanied by amino acid substitutions at position 8. Amino acid residues 189 and 225 were in a highly variable region, and the substitution at position 318, Thr → Lys (ACA → AAA at the nucleotide level), was responsible for the non-responsiveness of the mutant strain to C179. The amino acid number of the HA molecule in the present specification is based on the H3 numbering method described in Virus, Volume 11, 257-266 (1961).

(D) A / Izumi / 5/65 and A / Izum
The analysis of the nucleotide sequence of each HA gene of i / 5/65 (R) was carried out according to the method of Reference Example A-4- (3)-(b), provided that a H2N2 subtype sequencing primer and primer were used. 15-23 was performed. A /
The nucleotide sequence of the HA gene of Izumi / 5/65 is shown in SEQ ID NO: 28 in the sequence listing. The HA gene of the mutant strain had one nucleotide substitution. The T at the base number 1197 of the HA gene of the parent strain shown in SEQ ID NO: 28 is mutated to A, and this mutation involves an amino acid substitution at the 52nd position of HA2. This 52nd substitution, Val → Gl
u (GTA → GAA at the nucleotide level) was responsible for the non-responsiveness of the mutant strain C179.

(E) A / PR / 8 of H1N1 subtype
/ 34, A / Bangkok / 10/83, A / Yamagata / 1
20/86, A / Osaka / 930/88, H2N2 subtype A / Okuda / 57, A / Adachi / 2/57, A
/ Kumamoto / 1/65, A / Kaizuka / 2/65, H3
N2 / Subtype A2 / Aichi / 2/68, A / Fukuok
a / C29 / 85, A / Sichuan / 2/87, A / Ibar
H of HA molecule of aki / 1/90, A / Suita / 1/90
In order to identify the amino acid sequence near the 318th position of A1 and the amino acid sequence near the 52nd position of HA2, a part of the base sequence of each HA gene was analyzed.

Reference Example A for the H1N1 subtype
RNA of each virus according to the method of 4- (3)-(b)
A genomic cDNA was synthesized, and PCR amplification was performed on this cDNA using primers 9 and 13. Using the obtained DNA fragment as a template and primers 11 and 12,
The base sequence was determined by the dideoxy method using a thermal cycler.

Regarding the H2N2 strain, Reference Example A-4-
According to the method of (3)-(b), cDNA of RNA genome of each virus was synthesized, and PCR amplification was performed on this cDNA using primers 17 and 21. DN obtained
Using the A fragment as a template and the primers 19 and 20, the nucleotide sequence was similarly determined by the dideoxy method.

Regarding the H3N2 strain, Reference Example A-4-
CDNA for viral genomic RNA was synthesized according to the method of (3)-(b), and PCR amplification was performed for this cDNA using primers 24 and 26. DN obtained
Using the A fragment as a template and the primers 25 and 26, the nucleotide sequence was similarly determined by the dideoxy method.

For the H1N1 subtype and the H2N2 subtype, 31 of the HA1 region represented by SEQ ID NO: 1 in the sequence listing is used.
The TGLRN polypeptide sequence of 8th to 322nd (A region) and the GITNKVNSVIEK polypeptide sequence of 47th to 58th of HA2 region (B region) represented by SEQ ID NO: 2 in the sequence listing are conserved, while H3N2 is conserved. The subtype includes 318 of the HA1 region represented by SEQ ID NO: 3 in the sequence listing.
-322 TGMRN polypeptide sequence (A 'region)
And 47-of the HA2 region represented by SEQ ID NO: 4 in the sequence listing.
The 58 th QINGKLNR (L / V) IEK polypeptide sequence (B 'region) is conserved. Area A and A'area are 1
The amino acids are different, and there is a difference of 5 to 6 amino acid residues in the B region and B'region. The difference between these two regions is the difference in antigen recognition of the antibody, and in the serological and fusion inhibition tests,
This is the reason why the antibody could not react with the H3N2 subtype.

As shown in FIG. 1, the TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the A region and the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing of the B region correspond to the HA molecule. They are located close to each other in the center of the stem region, C179 recognizes both sequences, and the site is an epitope of C179. C179 is this H
It binds to the stem region of the A molecule, inhibits the membrane fusion function of the HA molecule, and neutralizes the virus.

H1N1 subtype: SEQ ID NO: 2 in Sequence Listing
Base numbers 1017 to 1031 of the HA gene of A / Suita / 1/89 shown in 7 encode the A region, and base number 1
191-1226 code the B region. SEQ ID NO: 29 in the sequence listing shows a part of HA gene of A / PR / 8/34, and its nucleotide numbers 76 to 90 encode A region,
The base numbers 250 to 285 encode the B region. SEQ ID NO: 30 in the sequence listing is A / Bangkok / 10/83
Of the HA gene, base numbers 76 to 90 thereof encode the A region, and base numbers 250 to 285 thereof encode the B region. Sequence number 31 in the sequence listing is A / Yama
A part of the HA gene of gata / 120/86 is shown, and its base numbers 76 to 90 encode the A region, and its base number 2
50 to 285 encode the B region. SEQ ID NO: 32 in the sequence listing shows a part of HA gene of A / Osaka / 930/88, its base numbers 76 to 90 encode A region, and its base numbers 250 to 285 encode B region. .

H2N2 subtype: SEQ ID NO: 2 in Sequence Listing
Base numbers 1007 to 1021 of the HA gene of A / Izumi / 5/65 shown in 8 encode the A region, and base number 1
181-1216 code the B region. SEQ ID NO: 33 of the sequence listing shows a part of HA gene of A / Okuda / 57, and its nucleotide numbers 94 to 108 encode A region,
The base numbers 268 to 303 encode the B region. Sequence number 34 in the sequence listing is H of A / Adachi / 2/57
A part of the A gene is shown, and its base numbers 103 to 117 encode the A region and its base numbers 277 to 312 encode the B region. Sequence number 35 in the sequence listing is A / Kuma
A part of the HA gene of moto / 1/65 is shown, and its base numbers 104 to 118 encode the A region, and its base number 2
78 to 313 encode the B region. SEQ ID NO: 36 in the sequence listing shows a part of HA gene of A / Kaizuka / 2/65, the base numbers 88 to 102 encode the A region, and the base numbers 262 to 297 encode the B region. .

H3N2 subtype: SEQ ID NO: 3 in Sequence Listing
7 is A2 / Aichi / 2/68, SEQ ID NO: 38 is A / Fuku
oka / C29 / 85, SEQ ID NO: 39 is A / Sichuan / 2
/ 87, SEQ ID NO: 40 is A / Ibaraki / 1/90, SEQ ID NO: 41 is a part of the HA gene of A / Suita / 1/90, and the respective base numbers 84 to 98 are A '.
It encodes a region, and each base number 258 to 293 encodes a B'region.

5. Preventive effect against influenza virus A mouse infection experiment of influenza virus was conducted, and C1
The preventive effect of 79 was investigated. C on 10 Balb / c mice
179 PBS solution (1 mg / ml) was intraperitoneally administered at 1 ml per mouse. A1 / FM / 1/47 (4000 HA units) of H1N1 subtype diluted 1000 times after 1 day
Was inoculated intranasally with 25 μl each. As a control, 12 mice were inoculated with the virus only.

As shown in FIG. 3, 8 mice in the control group died (2 mice after 5 days, 5 mice after 6 days, 1 mouse after 8 days). Also, other surviving mice were markedly debilitated. On the other hand, the C179-administered mice were normal, and all were well after 14 days. FIG. 3 is a diagram showing the survival rate of the C179-administered group and the non-administered group, in which the vertical axis represents the survival rate and the horizontal axis represents the number of days after virus infection.

Reference Example B 1. Preparation of virus A / whistling swan / shim as H5N3 subtype
ane / 476/83, A / for H6N6 subtype
whistling swan / shimane / 37/80, H7N7 subtype is A / turfted duck / shimane / 124R
/ 80, A / turky / Onta for H8N4 subtype
rio / 6118/68, A / chicken / Germany "N" / 49 was used as the H10N7 subtype, and each virus was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs,
After culturing at 34 ° C. for 4 days, each virus was collected. These virus strains are all conserved strains of the Institute for Microbial Diseases of Osaka University, and the HA molecule of A / chicken / Germany "N" / 49 has the amino acid sequences represented by SEQ ID NOs: 3 and 4 in the sequence listing. Has been done.

2. Preparation of Monoclonal Antibody (1) A2 / Aichi / 2/68 prepared in Reference Example A-1
Virus (320 HA units) was suspended in Freund's complete adjuvant prior to use, and immunized twice by intraperitoneal injection of Balb / c mice at 1-month intervals, and 1 month later, the antigen (320 HA units) A PBS suspension was used for boosting by intraperitoneal injection. Three days later, the spleen was removed from the mouse to prepare splenocytes. P for mouse myeloma
3 × 63Ag8 was cultured in DME medium supplemented with 10% fetal bovine serum for 2 days after subculture, and washed with physiological saline before cell fusion to prepare. Next, spleen cells and myeloma cells were mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass was thoroughly loosened, and then stirred while stirring 1
ml mixed solution [polyethylene glycol-4000 (2
g), MEM (2 ml), dimethylsulfoxide] and kept at 37 ° C. for 5 minutes, and then MEM was slowly added so that the total amount of the solution was 10 ml. Then, after centrifugation,
The supernatant was removed and the cells were loosely loosened. To this, 30 ml of a normal medium [PRMI-1640 plus 10% fetal bovine serum] was added, and the cells were gently suspended using a measuring pipette.

Dispense the suspension into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO ₂ . Next, HAT medium was added and the cells were cultured for 10 to 14 days. Subsequently, a part of the culture supernatant was collected and a hybridoma was screened.

(2) H3 of influenza A virus
In order to obtain a monoclonal antibody that reacts with the A'region and B'region conserved in N2 type, the above-mentioned undiluted culture supernatant was used as a primary antibody, and three subtypes (H3N2, H10N7, A staining test of MDCK cells infected with each of H1N1) was performed. The dyeing test was performed according to the method described in Reference Example A-2- (2). That is, 96-well microtiter plate (Falcon 3
072: Becton Dickinson), each subtype strain of human influenza A virus (H3N2:
A2 / Aichi / 2/68, H10N7: A / chicken /
Germany "N" / 49, H1N1: A / PR / 8/3
4) MDCK cells infected with PBS (pH 7.4)
After washing with water, it was fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were treated with four kinds of antibodies [the above-mentioned culture supernatant containing a monoclonal antibody, a 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), goat anti-rabbit immunoglobulin G serum (organotechnica). (Manufactured by K.K.) and a 1000-fold diluted solution of peroxidase-rabbit anti-peroxidase complex (manufactured by Organotechnica)] for 40 minutes each, and the treated cells were washed with PBS. Finally, the peroxidase reaction was carried out using 0.01% H ₂ O ₂ and 0.3 mg / ml 3,3′-diaminobenzidine tetrahydrochloride in PBS, using Graham, Karnovsky.
I went the same way. The stained cells were observed under a normal light microscope to find out that H3N2 subtype-infected MDCK cells and H
Antibodies that respectively recognize 10N7 subtype-infected MDCK cells were selected. Next, the cells in the hole in which the cells confirmed to produce the antibody are taken out, the limiting dilution method is performed 3 times, the target cells are cloned, and the cloned hybridoma strain is Hybridoma AI3C, which the hybridoma produces. The monoclonal antibody was designated as monoclonal antibody AI3C. The product name of the monoclonal antibody AI3C is monoclonal antibody F49 (Takara Shuzo).

This Hybridoma AI3C is a Hybridoma A
It has been deposited as FERM BP-4516 at the Institute of Biotechnology, Institute of Biotechnology, designated as I3C.

(3) Balb / c mice treated with pristane were intraperitoneally administered with the above hybridoma strain at 5 × 10 ⁶ mice / mouse. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare an ascites fluid. 1 ml of ascites fluid contained about 5 mg of monoclonal antibody AI3C (hereinafter, simply abbreviated as AI3C). AI3C
Was purified with Protein A-Sepharose 4B (Pharmacia).

3. Properties of Monoclonal Antibody (1) A 100-fold dilution of ascites fluid described in Reference Example B-2- (3) was serially diluted, and the staining test described in Reference Example A-2- (2) was performed to recognize AI3C antigen. The sex was examined. H1N1
Subtypes are A / PR / 8/34, A / Bangkok
/ 10/83, A / Yamagata / 120/86, A / Osak
a / 930/88, A / Suita / 1/89, A1 / FM
/ 1/47, A / Okuda / as the H2N2 subtype
57, A / Adachi / 2/57, A / Kumamoto / 1/6
5, A / Kaizuka / 2/65, A / Izumi / 5/65,
A / Aichi / 2/68, A / as H3N2 subtype
Fukuoka / C29 / 85, A / Sichuan / 2/87, A
/ Ibaraki / 1/90, A / Suita / 1/90, A / Ki
Using takyushu / 159/93 and B / Nagasaki / 1/87 as influenza B virus, reference example B-
The virus described in 1 was also used. AI3C is all H3N
It recognized two subtypes and A / chicken / German "N" / 49, and did not recognize the other subtype, type B virus.

(2) The hemagglutination inhibitory activity (HI) of the antibody was determined as follows. RDE as in Reference Example A-3- (2)
A serial dilution was prepared using the treated antibody (32-fold dilution), and then mixed with each virus (16 HA units) described in Reference Example B-3- (1) and Reference Example B-1 at room temperature. 30
Let react for minutes. Then, chicken red blood cells were added and mixed well, and the effect of the antibody on the hemagglutination activity of each virus was examined. AI3C did not affect the hemagglutinating activity of all subtype viruses.

Determination of Epitope (1) It was determined by immunoprecipitation that the AI3C recognition protein was an HA molecule. That is, H3N2
Subtype A / Aichi / 2/68 for 30 minutes MDCK
After adsorbing and infecting cells, methionine in the medium was
Infected cells were labeled by incubating for 24 hours in MEM substituted with μCi of " ³⁵ S] methionine.Then, the cells were collected, and then RIPA buffer [50 mM Tris (pH 7.4), 150 mM was added.
NaCl, 1 mM EDTA, 1% nonidet P-4
0, 1% deoxycholic acid, 0.1% SDS]. Subsequently, the insoluble matter was removed by centrifugation to obtain a supernatant. Next, the supernatant was mixed with AI3C, incubated at 4 ° C. for 1 hour, Protein A-Sepharose CL4B beads were added, and the mixture was kept at room temperature for 2 hours to adsorb the immunoprecipitate to the beads. The beads were then collected and washed 5 times with RIPA buffer, then boiled to release the AI3C binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate, dried, and autoradiographed. The labeled protein to which AI3C binds is A2 / Aichi / 2/6 based on its electrophoretic pattern.
8 HA molecules were identified. Similar tests were carried out using the H1N1 subtype, H2N2 subtype, other H3N2 subtypes, and the virus described in Reference Example B-1, respectively. AI3C is all H3N2 subtypes and A / ch
It was specifically immunoprecipitated with HA molecules of icken / Germany "N" / 49, but showed no binding with HA molecules of H1N1 and H2N2 subtypes.

[0084]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Antigen Polypeptide (1) Synthesis of Primer: H2N2 Subtype (-)
An oligonucleotide primer containing a sequence complementary to the 3'end of the strand RNA (primer 27, primer 2
8), and an oligonucleotide primer containing a sequence complementary to the 3'end of the (+) strand RNA (primer 2
9, primer 30) was synthesized using a DNA synthesizer,
After deprotection, ion exchange HPLC (TSK gel, DEA
Purified by E-2SW column, and separated by Sep-Pak (Sep-p)
ak) Desalted with C18 to obtain about 50 μg of each DNA. The nucleotide sequences of the primers 27 to 30 are shown in SEQ ID NOs: 42 to 45 of the sequence listing.

(2) MD infected with A / Okuda / 57
CK cells were collected, guanidine isothiocyanate was added, and the cells were lysed by repeating injection and injection 5 times with a syringe. The cell extract after lysis was overlaid with a cesium chloride solution and subjected to ultracentrifugation. The precipitate at the bottom of the centrifuge tube is dissolved in a buffer solution, treated with phenol and chloroform, and then precipitated with ethanol.
Sample. Next, using primer 5, cDNA
Was synthesized, and subsequently, the synthetic cDNA was amplified by PCR using the primers 27 and 29. Next, the amplified cDNA was separated by agarose gel electrophoresis, and the 1.8 kbp cDNA band corresponding to the HA gene was eluted. This cDNA was further amplified by PCR using primers 28 and 30, and a secondary amplification D of 1.8 kbp was obtained.
NA fragment is 20% (w / v) polyethylene glycol 6000, 2.5M NaCl solution is 60% (v / v)
v) Addition and centrifugation were performed to obtain a purified precipitate fraction.

(3) The base sequence of the HA gene of A / Okuda / 57 was analyzed according to the method of Reference Example A-4- (3)-(b, d). The nucleotide sequence and amino acid sequence of the HA gene of A / Okuda / 57 are shown in SEQ ID NO: 46 of the sequence listing. In SEQ ID NO: 46, base numbers 1 to 5 are sequences derived from primer 30, base numbers 6 to 48 are non-coding regions, base numbers 49 to 93 are signal peptide coding regions, and base numbers 94 to 231 are stem regions. A region encoding the N-terminal side, base numbers 232 to 873 are regions encoding the globular region, base numbers 874 to 1734 are regions encoding the C-terminal side of the stem region, base numbers 1735 to
1775 is a non-coding region, base number 1776-
1783 is a sequence derived from primer 30.

(4) Preparation of plasmid (a) The ends of the 1.8 kbp secondary amplified DNA fragment prepared in Example 1- (3) were blunted using T4 DNA polymerase. Next, the blunted fragment and the SmaI digestion product (2.7 kbp) of the plasmid pHSG299 (Takara Shuzo) were ligated with T4 DNA ligase to prepare a plasmid in which the HA gene was incorporated. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was separated from the obtained kanamycin-resistant transformant. The plasmid was named plasmid pH2-299.

Escherichia coli JM109 strain transformed with the plasmid pH2-299 is Escherichia coli JM109 / pH2-299.
It is named and displayed and has been deposited as FERM P-13431 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

(B) Plasmid pEF-BOS [Nucleic Acids Rese
arch), vol. 18, p. 5322 (1990)], Aa
A 2.6 kbp B fragment obtained by digesting the tII digested product and the plasmid pMAM _neo-s (manufactured by Clontech) with BamHI.
The amHI DNA fragment was ligated with T4 DNA ligase to prepare a shuttle vector plasmid for animal cells and E. coli in which the 2.6 kbp BamHI DNA fragment was incorporated. Next, Escherichia coli JM109 was transformed with the plasmid, and a plasmid was isolated from the obtained transformant resistant to ampicillin.
It was named F-BOS / NeoA.

The plasmid pH2-299 was digested with NheI to prepare a 1.8 kbp DNA fragment having the sequence of nucleotides 4 to 1780 of the cDNA represented by SEQ ID NO: 46 in the sequence listing. Next, the DNA fragment and the XbaI digestion product (7.9 kbp) of the plasmid pEF-BOS / NeoA were ligated with T4 DNA ligase to prepare a shuttle vector having the HA gene incorporated therein. Next, with this vector E. coli JM109
The strain was transformed, and the plasmid was isolated from the obtained transformant resistant to ampicillin. This plasmid was designated as plasmid pEBNaH2. In addition, Escherichia coli JM109 strain transformed with the plasmid was used as Escherichia coli JM109.
It was named / pEBNaH2.

(C) Oligonucleotide primers (primer 31,
The primer 32) was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-).
2SW column) and purified, Sep-pak (Sep-pa)
k) Desalting with C18 gave about 50 μg of each DNA. The primer 31 corresponds to the DNA sequence of nucleotide numbers 207 to 231 of the HA gene represented by SEQ ID NO: 46 in the sequence listing.
The primer is an antisense strand primer for RNA, and the primer 32 is a sense strand primer for mRNA corresponding to the DNA sequence of base numbers 874 to 899.

By using the primer 31 and the primer 32, only the region encoding the globular region of the HA gene was removed from the plasmid pH2-299 at 3.8 kbp.
Attempts were made to amplify the above DNA fragment by the PCR method. Es
cherichia coli JM109 / pH2-299 (FERM P-134
31), a plasmid pH2-299 was prepared, and PCR was carried out using 0.05 pmol of the plasmid, 50 pmol of each of primer 31 and primer 32. PCR conditions are 90
A cycle of 1 minute, 55 ° C., 2 minutes, 72 ° C., 3 minutes was repeated 25 times to prepare a 3.8 kbp DNA amplification product.
Next, this 3.8 kbp DNA fragment was phosphorylated with T4 kinase, blunt-ended with T4 DNA polymerase, and ligated with T4 DNA ligase to prepare a circular plasmid. Next, with the plasmid, E. coli J
M109 was transformed, and a plasmid was isolated from the obtained kanamycin-resistant transformant. This plasmid was designated as plasmid p299H2S _nc .

A plasmid of p299H2S _nc was digested with NheI to prepare a 1.1 kbp DNA fragment from which the region encoding the globular region (corresponding to base numbers 232 to 873 of SEQ ID NO: 46) was removed from the HA gene. did. The base sequence of the fragment is SEQ ID NO: 4 in the sequence listing.
The amino acid sequence encoded by the fragment is shown in SEQ ID NO: 50 in the sequence listing. Next, this 1.1 kbp DNA fragment and the shuttle vector plasmid pEF-BOS /
The XbaI digestion product of NeoA (7.9 kbp) was ligated with T4 DNA ligase, and a plasmid in which the region coding the N-terminal side of the stem region of the HA gene and the DNA coding the C-terminal side were directly bound was incorporated. It was made. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was separated from the obtained ampicillin-resistant transformant. The plasmid was named pENH2dHO1.

The plasmid was transformed with pENH2dHO1 Escherichia coli JM109 strain was Escherichia coli JM109 / pENH2dHO1.
It is named and displayed and has been deposited as FERM BP-4190 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

(5) Expression of polypeptide: Escherichia
From E. coli JM109 / pENH2dHO1 (FERM BP-4190), a plasmid pENH2dHO1 incorporating a gene encoding a polypeptide lacking the globular region of the HA molecule was prepared, and from Escherichia coli JM109 / pEBNaH2, an HA molecule was encoded. A plasmid pEBNaH2 containing the gene was prepared. About 5 × 10 ⁶ CV-1 cells were treated with trypsin, then washed once with 20 ml of 10% FCS-MEM (manufactured by Dulbecco) and twice with PBS, and then washed with 1 ml of PBS.
Suspended in. 0.8 ml of this was placed in a cuvette for Gene Pulser, a gene transfer device manufactured by Bio-Rad, 30 μg of plasmid pENH2dHO1 was added, stirred with a Pasteur pipette, and allowed to stand at 0 ° C. for 10 minutes. Set the cuvette in Gene Pulser, a gene transfer device, and set it to 250
After treating with V and 960 μFD, the cells were allowed to stand at 0 ° C. for 10 minutes to transform CV-1 cells. The transformed cells were then placed in 30 ml of 10% FCS-MEM and 5
The cells were cultured in a petri dish having a diameter of 6 cm each for 2 days. The cultured CV-1 cells were washed with PBS (pH 7.4) and then fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were mixed with 4 kinds of antibodies [C179, a rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica) 1000-fold diluted solution, and a goat anti-rabbit immunoglobulin G serum (manufactured by Organotechnica) 500-fold diluted solution. , 1000-fold diluted solution of peroxidase-rabbit peroxidase complex (manufactured by Organotechnica)] for 40 minutes each, and the treated cells were washed with PBS. Finally, the peroxidase reaction was carried out by the method of Graham, Karnovsky using 0.01% H ₂ O ₂ and 0.3 mg / ml of 3,3'-diaminobenzidine tetrahydrochloride in PBS. It was C transformed with plasmid pENH2dHO1
V-1 cells were stained brown, and the cells lacked the globular region of the HA molecule, but expressed a polypeptide in which the higher order structure of the stem region was normally retained.

Since this expressed polypeptide lacks the globular region that is likely to become an antigenic determinant and easily causes antigenic mutation, it can specifically recognize the stem regions of H1N1 subtype and H2N2 subtype, It can be used as a vaccine for inducing an antibody that neutralizes.

Using the plasmid pEBNaH2, CV-
One cell was transformed, the transformant was cultured, and a staining test using C179 of the cultured cell was performed. Plasmid pE
CV-1 cells transformed with BNaH2 were stained brown and
This cell also expressed an HA polypeptide having a normal conformation in the stem region.

Example 2 Preparation of Antigen Polypeptide (1) Synthesis of Primer: H3N2 Subtype (-)
Oligonucleotide primer (primer 33) containing a sequence complementary to the 3'end of the strand RNA, and (+)
An oligonucleotide primer containing a sequence complementary to the 3'end of the strand RNA (primer 34, primer 3
5) was synthesized using a DNA synthesizer, and after deprotection, purified by ion exchange HPLC (TSK gel, DEAE-2SW column), desalted with Sep-pak C18, and each DNA was separated. 50 μg was obtained. Primer 33-
The respective nucleotide sequences of 35 are SEQ ID NOs: 51 to 5 in the sequence listing.
3 shows.

(2) MDCK cells infected with A2 / Aichi / 2/68 were collected, guanidine isothiocyanate was added, and the cells were lysed by repeatedly injecting 5 times with a syringe. Layer the cell extract after lysis on cesium chloride solution,
Ultracentrifugation was performed. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer solution, treated with phenol and chloroform, and then precipitated with ethanol, and the recovered RNA was used as a sample of viral genomic RNA. Then using primer 5 c
DNA was synthesized, and then the synthetic cDNA was amplified by PCR using the primers 33 and 34. Next, the amplified cDNA was separated by agarose gel electrophoresis, and the 1.8 kbp cDNA band corresponding to the HA gene was eluted. This cDNA is used as primers 33, 35
Was further amplified by the PCR method, and the 1.8 kbp secondary amplified DNA fragment was 20% (w / v) polyethylene glycol 6000 and 60% of 2.5 M NaCl solution.
(V / v) was added and the mixture was centrifuged to obtain a purified precipitate fraction.

(3) Analysis of the base sequence of the HA gene of A2 / Aichi / 2/68 was carried out according to Reference Example A-4- (3)-(b,
It carried out according to the method of d). A2 / Aichi / 2/68 H
The nucleotide sequence and amino acid sequence of the A gene are shown in SEQ ID NO: 54 of the sequence listing. In SEQ ID NO: 54, base numbers 1 to 8
Is a sequence derived from primer 33, base numbers 9 to 36 are non-coding regions, base numbers 37 to 84 are signal peptide coding regions, base numbers 85 to 246 are regions coding the N-terminal side of the stem region, and base numbers 247-90
3 is a region encoding the spherical region, base numbers 904 to 1
734 is a region encoding the C-terminal side of the stem region, base numbers 1735 to 1769 are non-coding regions, and base numbers 1770 to 1777 are sequences derived from primer 35.

(4) Preparation of plasmid (a) The ends of the 1.8 kbp secondary amplified DNA fragment prepared in Example 2- (3) were blunted using T4 DNA polymerase. Next, a blunted fragment and a HincII digestion product of the plasmid pUC118 (Takara Shuzo) (1.8
Kbp) was ligated with T4 DNA ligase and H
A plasmid in which the A gene was integrated was prepared. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was separated from the obtained ampicillin-resistant transformant. The plasmid was designated plasmid pU118H3 _xxn.

The E. coli JM109 strain transformed with the plasmid pU118H3 _xxn is Escherichia coli JM109 / pU118H3.
_It is named and displayed as _xxn, and has been deposited as FERM P-13567 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(C) Oligonucleotide primers represented by SEQ ID NOs: 55 and 56 in the sequence listing (primer 36,
The primer 37) was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-
2SW column) and purified, Sep-pak (Sep-pa)
k) Desalting with C18 gave about 50 μg of each DNA. The primer 36 corresponds to the DNA sequence of base numbers 227 to 246 of the HA gene represented by SEQ ID NO: 54 in the sequence listing.
The primer is an antisense strand primer for RNA, and the primer 37 is a primer for the sense strand for mRNA corresponding to the DNA sequence of base numbers 904 to 923.

By using the primers 36 and 37, an attempt was made to amplify a 4.3 kbp DNA fragment from the plasmid pU118H3 _xxn by excluding the region encoding the globular region of the HA gene by the PCR method. Esch
erichia coli JM109 / pU118H3 _xxn (FERM P-13
567) Plasmid PU118H3 _xxn were prepared from, the plasmid 0.05Pmol, primer 36, a PCR using primers 37 each 50pmol was performed. PCR conditions are 90
A cycle of 1 ° C., 1 minute, 55 ° C., 2 minutes, 72 ° C., 3 minutes was repeated 25 times to prepare a DNA amplification product of 4.3 kbp.
Next, this 4.3 kbp amplified DNA fragment was phosphorylated with T4 kinase, blunt-ended with T4 DNA polymerase, and ligated with T4 DNA ligase to prepare a circular plasmid. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was separated from the obtained ampicillin-resistant transformant. This plasmid was designated as plasmid p118H3dHO1 _xxn .

Plasmid p118H3dHO1 _xxn was _added to XbaI, N
A region encoding the globular region from the HA gene by cleavage with heI (base number 247 of SEQ ID NO: 54)
A DNA fragment of 1.1 kbp was prepared in which (corresponding to ˜903) was removed. The nucleotide sequence of the fragment is shown in SEQ ID NO: 57 of the sequence listing, and the amino acid sequence encoded by the fragment is shown in SEQ ID NO: 58 of the sequence listing. Next, this 1.1kb
The DNA fragment of p and the XbaI digestion product (7.9 kbp) of the shuttle vector plasmid pEF-BOS / NeoA were treated with T4.
Ligation with DNA ligase was performed to prepare a plasmid in which a DNA in which the N-terminal side coding region of the HA gene stem region and the C-terminal side coding region were directly bound was incorporated. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was separated from the obtained ampicillin-resistant transformant. The plasmid was named pENH3dHO1.

The Escherichia coli JM109 strain transformed with the plasmid pENH3dHO1 is designated and designated as Escherichia coli JM109 / pENH3dHO1 and has been deposited as FERM BP-4518 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

(5) Expression of polypeptide: Escherichia
From p. coli JM109 / pENH3dHO1 (FERM BP-4518), a plasmid pENH3dHO1 incorporating a gene encoding a polypeptide lacking the globular region of the HA molecule was prepared. About 5 × 10 ⁶ CV-1 cells were treated with trypsin, washed once with 20 ml of 10% FCS-MEM (manufactured by Dalbeco) and twice with PBS, and suspended in 1 ml of PBS.
0.8 ml of this was placed in a cuvette for Gene Pulser, a gene transfer device of Bio-Rad, and plasmid pENH3d was added.
30 μg of HO1 was added, and the mixture was stirred with a Pasteur pipette and left standing at 0 ° C. for 10 minutes. Set the cuvette in Gene Pulser gene transfer device, 250V, 960μF
After the treatment under the conditions of D, the mixture was allowed to stand at 0 ° C for 10 minutes, and then CV-
One cell was transformed. Next, transform the transformed cells into 3
Place in 0 ml of 10% FCS-MEM, 5 ml each, diameter 6 cm
The cells were cultured for 2 days in a petri dish.

Cultured CV-1 cells were treated with PBS (pH 7.
After washing with 4), it was fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were diluted with four kinds of antibodies (AI3C, rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica) at 1000 times dilution and goat anti-rabbit immunoglobulin G serum (manufactured by Organotechnica) at 500 times dilution. , A peroxidase-rabbit peroxidase complex (manufactured by Organotechnica Co., Ltd., 1000-fold dilution), and the reaction was continued for 40 minutes, and the treated cells were washed with PBS. ₂
O ₂ and 0.3 mg / ml of 3,3′-diaminobenzidine tetrahydrochloride in PBS were used, and Graham, Karnovsky (Gr.
aham, Karnovsky). Plasmid pENH3d
CV-1 cells transformed with HO1 were stained brown, and the cells lacked the globular region of the HA molecule, but expressed a polypeptide in which the higher order structure of the stem region was normally retained. Since this expressed polypeptide is likely to become an antigenic determinant and lacks the globular region where antigenic mutation easily occurs,
It can be used as a vaccine that induces an antibody that specifically recognizes the stem region of the H3N2 subtype.

Example 3 Preparation of Antigen Polypeptide (1) Preparation of HA Molecule A / Yamagata / 32/89 virus particles (40 mg) prepared in Reference Example A-1 was used in 27 ml of 5 mM Tris-HCl.
After suspending in (pH 8.0) and adding 3 ml of 20% NP-40, the mixture was kept at 37 ° C for 30 minutes. Then, after centrifugation, the supernatants were collected, and a 0.8 μm filter unit (Millex-
PF: manufactured by Millipore) was used for filtration. Then
The ion exchange membrane (memSep DEAE: manufactured by Millipore) equilibrated with 5 mM Tris-HCl (pH 8.0) was loaded with the filtrate and washed with the same buffer. Further, HA molecules were eluted using the same buffer containing 1 M NaCl.

(2) Proteinase Treatment of HA Molecule The HA molecule (2.6 μg) prepared in Example 3- (1) was treated with N.
-Ethylmorpholine buffer (pH 7.5), 4 pm each
ol lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.),
Digestion was performed with V8 protease (manufactured by Sigma) and chymotrypsin (manufactured by Boehringer) at 37 ° C. for 1 hour. HA molecule prepared in Example 3- (1) (2.6 μg)
Was kept at 42 ° C. for 1 hour in the presence of 2 M urea to denature it. Next, using 4 pmol of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin (manufactured by Boehringer), proteinase K (manufactured by Boehringer), pronase (manufactured by Boehringer), thermolysin (manufactured by Boehringer), 50 mM tris-hydrochloric acid buffer solution. After digesting at 37 ° C for 12 hours in (pH 7.6),
Dialysis was performed against PBS. A part of these digested liquids was taken, and the digested fragments were analyzed by the dot blot method using C179 and SDS-polyacrylamide gel electrophoresis. The dot blot method was performed as follows. 1μ of digestion solution on a nitrocellulose filter (manufactured by MSI)
1 was loaded and dried. The same operation was repeated 5 times, a total of 5 μl of digestive fluid was loaded, and then blocking was performed using Block Ace (Yukirushi Co., Ltd.). Then, these were reacted with a 500-fold diluted solution of the C179 solution at room temperature for 1 hour each. It was washed with a Tris-hydrochloric acid buffer solution (pH 7.6) containing 0.02% Tween 20, and further washed with a Tris-hydrochloric acid buffer solution (pH 7.6) for 10 minutes three times. Further, these were treated with 50 parts of alkaline phosphatase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Orgenix).
The mixture was reacted with a 0-fold diluted solution at room temperature for 1 hour and washed in the same manner. Finally, the alkaline phosphatase reaction was carried out using 5-bromo-4-chloro-3-indolyl phosphate and carbonic acid-sodium carbonate solution of nitroblue tetrazolium (pH 9.0) in the presence of 1 mM MgCl ₂ . . As a result, in the absence of urea, most of the HA molecules were undigested regardless of which protease was used. When the HA molecule denatured with urea is used as a substrate,
It was not digested with V8 protease, thermolysin or pronase. Excessive digestion with lysyl endopeptidase, chymotrypsin, and subtilisin leads to C179
The antigenicity against was completely lost. On the other hand, when proteinase K was used, the HA molecule was digested, and production of a fragment polypeptide capable of binding to C179 was observed.

(3) Preparation of Stem Region Polypeptide The HA molecule (250 μg / 1) prepared in Example 3- (1)
400 μl) to 100 μl of 1M Tris-HCl (pH
7.6), add 500 μl of 8M urea each, 1 hour, 4
It was kept warm at 2 ° C. To this solution, 2000 μl of immobilized proteinase K gel was added, and the mixture was shaken for 7 hours for 37 hours.
It was kept warm at ℃. Next, the reaction solution obtained by centrifugation was dialyzed against PBS for 12 hours to obtain a stem region polypeptide. The immobilized proteinase K gel was prepared as follows. 4 mg proteinase K in 1 ml H ₂
It was dissolved in O, adjusted to pH 5.0 with 0.1N HCl, added with 1 ml of ECH-Sepharose (Pharmacia), and 1 ml of 0.2M EDC (pH 5.0).
The temperature was kept at 4 ° C for 24 hours. The gel was washed 3 times with 10 ml of PBS to obtain immobilized proteinase K gel.

(4) Properties of Stem Region Polypeptide Using the stem region polypeptide described in Example 3- (3) as a test sample, the antigenicity to C179 was examined by the ELISA method. That is, a dilution solution of the stem region polypeptide was added to a microtiter plate (Maxisorp: Nunc), fixed at 37 ° C. for 1 hour and 30 minutes, and then blocked using Block Ace (Yukirushi). It was These were then treated with two types of antibodies [C179 solution (10 mg / ml)
And a 500-fold dilution of peroxidase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Kappel)] for 1 hour and 30 minutes, and then PBS.
Washed with. Finally, the peroxidase reaction was carried out with 0.03% H ₂ O ₂ and 1 mg / ml o-phenylenediamine dihydrochloride in citric acid-phosphate solution (pH 5.2).
The amount of antigen was calculated from the absorbance of the reaction solution at 492 nm. The HA molecule described in Example 3- (1) was used as a standard substance. From the results of the ELISA method, the stem region polypeptide is H
It was revealed that it shows the same antigenicity as the A molecule. The hemagglutination activity (HA value) of the stem region polypeptide was measured as follows. U-shaped 96-well bottom plate (Falcon 39
11: Becton Dickinson Co., Ltd.), the sample solution was serially diluted 2-fold with PBS, and an equivalent amount of 0.5% was added to this.
The chicken red blood cell suspension was added and stirred well. After reacting at room temperature for 1 hour, aggregation of red blood cells was observed. The maximum dilution ratio in which aggregation was observed was defined as HA value. The HA value of the stem region polypeptide is 1/1 of the HA value of the HA molecule.
It was 000 or less.

As described above, the stem region polypeptide prepared by the protease treatment has the same antigenicity as the HA molecule,
Moreover, it was revealed that the blood coagulation activity derived from the globular region has substantially disappeared. This polypeptide is easy to serve as an antigenic determinant and has a digested globular region that easily causes antigenic mutation.
It can be used as a vaccine that specifically recognizes the stem region of the H2N2 subtype and induces antibodies that neutralize the virus.

Example 4 Preparation of Antigen Polypeptide (1) Preparation of HA molecule A / Kitakyushu / 159/93 prepared in Reference Example A-1
Virus particles (40 mg) of 27 ml of 5 mM Tris-HC
It was suspended in 1 (pH 8.0), 3 ml of 20% NP-40 was added, and the mixture was kept at 37 ° C. for 30 minutes. Then after centrifugation,
The supernatant was collected and filtered using a 0.8 μm filter unit (Millex-PF: manufactured by Millipore). Next, an ion exchange membrane (memSep DEAE: manufactured by Millipore) equilibrated with 5 mM Tris-HCl (pH 8.0) was loaded with the filtrate and washed with the same buffer. Further, HA molecules were eluted using the same buffer containing 1 M NaCl.

(2) Proteinase treatment of HA molecule The HA molecule (2.6 μg) prepared in Example 4- (1) was treated with
4 each in N-ethylmorpholine buffer (pH 7.5)
pmol lysyl endopeptidase, V8 protease,
Digestion was performed with chymotrypsin for 1 hour at 37 ° C. HA molecule prepared in Example 4- (1) (2.6 μg)
Was denatured by incubating at 42 ° C for 1 hour in the presence of 2M urea. Next, using 4 pmol of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin, proteinase K, pronase, and thermolysin, 5
In 0 mM Tris-HCl buffer (pH 7.6) at 37 ° C, 1
After digesting for 2 hours, it was dialyzed against PBS.
A part of these digested liquids was taken and the digested fragments were analyzed by the dot blot method using AI3C and the SDS-polyacrylamide gel electrophoresis method. As a result, in the absence of urea, most of the HA molecules were undigested regardless of which protease was used. H modified with urea
When the A molecule was used as a substrate, it was not digested with V8 protease, thermolysin or pronase. Lysyl endopeptidase, chymotrypsin, and subtilisin were excessively digested, and the antigenicity to AI3C was completely lost. On the other hand, when proteinase K was used, the HA molecule was digested, and production of a fragment polypeptide having the ability to bind to AI3C was observed.

(3) Preparation of stem region polypeptide HA molecule prepared in Example 4- (1) (250 μg / 1
400 μl) to 100 μl of 1M Tris-HCl (pH
7.6), add 500 μl of 8M urea each, 1 hour, 4
It was kept warm at 2 ° C. To this solution, 2000 μl of immobilized proteinase K gel was added, and the mixture was shaken for 7 hours for 37 hours.
It was kept warm at ℃. Next, the reaction solution obtained by centrifugation was dialyzed against PBS for 12 hours to obtain a stem region polypeptide.

(4) Properties of Stem Region Polypeptide Using the stem region polypeptide described in Example 4- (3) as a test sample, the antigenicity against AI3C was examined by the ELISA method. That is, a dilution solution of the stem region polypeptide was added to a microtiter plate (Maxisorp: Nunc), fixed at 37 ° C. for 1 hour and 30 minutes, and then blocked using Block Ace (Yukirushi). It was These are then treated with two types of antibodies [AI3C solution (10 mg / ml)
And a 500-fold dilution of peroxidase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Kappel)] for 1 hour and 30 minutes, and then PBS.
Washed with. Finally, the peroxidase reaction was carried out with 0.03% H ₂ O ₂ and 1 mg / ml o-phenylenediamine dihydrochloride in citric acid-phosphate solution (pH 5.2).
The amount of antigen was calculated from the absorbance of the reaction solution at 492 nm. The HA molecule described in Example 4- (1) was used as a standard substance. From the results of the ELISA method, the stem region polypeptide is H
It was revealed that it shows the same antigenicity as the A molecule. The hemagglutination activity (HA value) of the stem region polypeptide was measured as follows. U-shaped 96-well bottom plate (Falcon 39
11: Becton Dickinson Co., Ltd.), the sample solution was serially diluted 2-fold with PBS, and an equivalent amount of 0.5% was added to this.
The chicken red blood cell suspension was added and stirred well. After reacting at room temperature for 1 hour, aggregation of red blood cells was observed. The maximum dilution ratio in which aggregation was observed was defined as HA value. The HA value of the stem region polypeptide is 1/1 of the HA value of the HA molecule.
It was 000 or less.

As mentioned above, the stem region polypeptide prepared by the protease treatment has the same antigenicity as the HA molecule,
Moreover, it was revealed that the blood coagulation activity derived from the globular region has substantially disappeared. Since the globular region of the polypeptide is likely to serve as an antigenic determinant and the antigenic mutation is likely to occur, it can be used as a vaccine for inducing an antibody that specifically recognizes the stem region of the H3N2 subtype. .

Example 5 Preventive Effect Against Influenza Virus Escherishia coli JM109 / pENH2dH01 (FERM BP
-4190), H of A / Okuda / 57 (H2N2)
A plasmid pENH2dH01 incorporating a gene encoding a polypeptide lacking the globular region of the A molecule was prepared.
After treating about 5 × 10 ⁶ CV-1 cells with trypsin,
1% with 20 ml of 10% FCS-MEM (manufactured by Dulbecco)
Once, washed twice with PBS, and suspended in 1 ml of PBS. 0.8 ml of this was placed in a cuvette for Gene Pulser, a gene transfer device of Bio-Rad, and plasmid pENH2dH0
30 μg of 1 was added, and the mixture was stirred with a Pasteur pipette and left standing at 0 ° C. for 10 minutes. Set the cuvette in Gene Pulser gene transfer device, 250V, 960μF
After the treatment under the conditions of D, the mixture was allowed to stand at 0 ° C for 10 minutes, and then CV-
One cell was transformed. Next, 6
Place in 0 ml of 10% FCS-MEM, 5 ml each, diameter 6 cm
It was cultured in a petri dish. On day 3 of culture, the expression of the polypeptide was confirmed by a staining test using C179, and the cells expressing the polypeptide were treated with PBS containing trypsin, and then the cells were collected by centrifugation. Next, the collected cells were suspended in PBS and intraperitoneally administered to each of 10 4-week-old female BALB / c mice as a vaccine at 1 × 10 ⁵ cells. Two weeks later, a second immunization was performed in the same manner. Incidentally using CV-1 cells in pENH2dH01 as a control non-transformed, likewise 10 mice to 1 × 10 ⁵ cells, respectively 2
It was administered once intraperitoneally. Al / FM one week after the final immunization
/ 1/47 (H1N1) was added to each mouse at 25 μl (8
X 10 ⁴ FFU) was intranasally inoculated with each mouse, and thereafter, the viability of the mice was examined daily. The results are shown in Fig. 4. As shown in FIG. 4, immunization was carried out with CV-1 cells expressing the antigen polypeptide,
In the vaccinated mouse group (indicated by a black circle in FIG. 4), 7 out of 10 mice survived on the 15th day after inoculation with the highly virulent strain Al / FM / 1/47. On the other hand, in the control mouse group (indicated by a black triangle in FIG. 4), 9 out of 10 mice died. FIG. 4 is a diagram showing the survival rate of the antigen polypeptide-administered group and the non-administered group, wherein the vertical axis represents the survival rate and the horizontal axis represents the number of days after virus infection.

As described above, it was revealed that the antigenic polypeptide lacking the globular region of the HA molecule can be a vaccine against the H1N1 subtype virus even if it is derived from the H2N2 subtype. Since this antigenic polypeptide lacks the globular region that is likely to become an antigenic determinant and is prone to antigenic mutation, the H1N1 subtype,
It can be used as a vaccine that specifically recognizes the stem region of the H2N2 subtype and induces antibodies that neutralize the virus.

Example 6 Preventive Effect Against Influenza Virus Using the stem region polypeptide described in Example 3 as a test sample, the protective effect against influenza virus infection was examined. The stem region polypeptide was suspended in PBS and intraperitoneally administered to 4-week-old female BALB / c mice at an amount of 10 μg / 0.5 ml. A total of 3 when the same dose was administered intraperitoneally at weekly intervals
Immunized twice. Only PBS was administered to the control group.
10 days after the final immunization, A1 / FM / 1/47 (H1N
1) 25 μl of virus per mouse (2.0 × 10 ³
FFU) was intranasally inoculated with each mouse, and changes in body weight of living and dead mice and surviving mice were observed. As shown in FIG. 5, the reduction rate of the average body weight of the mice immunized with the stem region polypeptide was significantly lower than that of the control group. As shown in FIG. 6, 5 out of the 11 mice in the control group died within 7 days after the virus inoculation, but 8 out of 10 mice immunized with the stem region polypeptide were 14 after the virus inoculation. The cells survived even day after day, and the survival rate was 14% 14 days after the virus inoculation. On the other hand, the survival rate of the control group 14 days after virus inoculation was 55%. FIG. 5 is a diagram showing changes in body weight of the stem region polypeptide-administered group and the control group, the vertical axis represents the average body weight of mice alive in each group, and the horizontal axis represents the number of days after virus inoculation. FIG. 6 is a diagram showing survival rates of the stem region polypeptide administration group and the control group, the vertical axis shows the survival rate, and the horizontal axis shows the number of days after virus inoculation. As described above, it was revealed that the stem region polypeptide can be a vaccine against influenza virus.

[0123]

INDUSTRIAL APPLICABILITY The present invention provides an immunogenic polypeptide capable of producing an antibody that specifically binds to a stem region of a subtype HA molecule of human influenza A virus, and a gene encoding the polypeptide. . The polypeptide can be genetically and enzymatically supplied in a large amount, and is not particularly affected by antigenic changes due to changes in the HA molecular globular region, and thus is particularly useful as a vaccine for influenza prevention.

[0124]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 5 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment

SEQ ID NO: 2 Sequence Length: 12 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment

SEQ ID NO: 3 Sequence Length: 5 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment

SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Xaa at position 9 Val or Leu.

SEQ ID NO: 5 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence AGCAAAAGCA GGGGATAAT 19

SEQ ID NO: 6 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence AGTAGAAACA AGGGTGTTTT T 21

SEQ ID NO: 7 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence TCTTTTCGAG TACTGTGTCA ACA 23

SEQ ID NO: 8 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GCCCCACTAC AATTGGGGAA ATG 23

SEQ ID NO: 9 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence TTTTACAGAA ATTTGCTATG GCTG 24

SEQ ID NO: 10 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ACTCCCCTAT TGTGACTGGG TGTA 24

SEQ ID NO: 11 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GGTTATCATC ATCAGAATGA AC 22

SEQ ID NO: 12 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGTTCACCTT GTTTGTAATC CCGT 24

SEQ ID NO: 13 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CCATTTTTTA CTCTTTCCAT GCAT 24

SEQ ID NO: 14 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ATCTACTCAA CTGTCGCCAG TTCA 24

SEQ ID NO: 15 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TTGTGTCGAC CTTCTCTGTG GAA 23

SEQ ID NO: 16 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence TGTAGCATTG CCGGATGGCT 20

SEQ ID NO: 17 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ATTATCCGGT TGCCAAAGGA TCG 23

SEQ ID NO: 18 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GAGAGCACTG GTAATCTGTT GCA 23

SEQ ID NO: 19 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CCATCAAATG CCTTTTGAGT GGA 23

SEQ ID NO: 20 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ACTAGAAGCT CAGCATTGTA TGT 23

SEQ ID NO: 21 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CATGCATTCA TCATCACATT TGTG 24

SEQ ID NO: 22 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CATACTTGGG ATAATCATAC GTC 23

SEQ ID NO: 23 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GCCATTTATG CTACAGTAGC AGG 23

SEQ ID NO: 24 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GATCAGATTG AAGTGACTAA TGCT 24

SEQ ID NO: 25 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GAATGCATCA CTCCAAATGG AAGC 24

SEQ ID NO: 26 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence AGGTCCTGAA TTCTCCCTTC TAC 23

SEQ ID NO: 27 Sequence length: 1754 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Suita / 1/89 SEQ GGATAATAAA TACAACCAAA ATGAAAGCAA AACTACTAGT CCTGTTATGT GCATTTACAG 60 CTACAGATGC AGACACAATA TGTATAGGCT ACCATGCGAA CAACTCAACC GACACTGTTG 120 ACACAGTACT TGAGAAGAAC GTGACAGTGA CACACTCTGT CAACCTACTT GAGGACAGTC 180 ACAACGGAAA ACTATGTCGA CTAAAAGGAA TAGCCCCACT ACAATTGGGT AATTGCAGCA 240 TTGCCGGATG GATCTTAGGA AACCCAGAAT GCGAATCACT GTTTTCTAAG GAATCATGGT 300 CCTACATTGC AGAAACACCA AACTCCGAGA ATGGAACATG TTACCCAGGG TATTTCGCCG 360 ACTATGAGGA ACTGAGGGAG CAATTGAGTT CAGTATCATC ATTCGAGAGA TTCGAAATAT 420 TCCCCAAAGA AAGCTCATGG CCCAACCACA CCGTAACCAA AGGAGTAACG GCATCATGCT 480 CCCATAATGG GAAAAGCAGT TTTTACAGAA ATTTGCTATG GCTGACGGGGAGAAGAATGGCT 540 TGTACCCAAA TCTGAGCAAG TCCTATGTCACATCCTGTTGCACACCTGTTGTT ACGACAGAGA GAAAGAAGTCCTTGTACTAT 600GA T TCACATTATA GCAGGAGATT CACCCCAGAA ATAGCAAAAA 720 GACCCAAAGT AAGAGGTCAA GAAGGAAGAA TTAACTACTA CTGGACTCTG CTGGAACCCG 780 GGGACACAAT AATATTTGAG GCAAATGGAA ATCTAATAGC GCCATGGTAT GCTTTCGCAC 840 TGAGTAGAGG CTTTGGGTCA GGAATCATCA CCTCAAACGC ATCAATGGAT GAATGTGACG 900 CGAAGTGTCA AACACCCCAG GGAGCTATAA ACAGTAGTCT TCCTTTCCAG AATGTACACC 960 CAGTCACAAT AGGAGAGTGT CCAAAGTATG TCAGGAGTAC AAAATTAAGG ATGGTTACAG 1020 GACTAAGGAA CATCCCATCC ATTCAATCCA GAGGTTTGTT TGGAGCCATT GCCGGTTTCA 1080 TTGAAGGGGG GTGGACTGGA ATGATAGATG GATGGTATGG TTATCATCAT CAGAATGAAC 1140 AAGGATCTGG CTATGCTGCG GATCAAAAAA GCACACAAAA TGCCATTAAC GGAATTACAA 1200 ACAAGGTGAA TTCTGTAATC GAGAAAATGA ACACTCAATT CACAGCTGTG GGCAAAGAAT 1260 TCAACAAATT AGAAAGAAGG ATGGAATACT TAAATAAAAA AGTTGATGAT GGATTTCTGG 1320 ACATTTGGAC ATATAATGCA GAATTGTTGG TTCTACTGGA AAATGAAAGG ACTTTGGATT 1380 TTCATGACTC AAATGTGAAG AATCTGTATG AGAAAGTAAA AAGCCAATTA AAGAATAATG 1440 CCAAAGAAAT AGGATACGGG TGTTTTGAAT TCTACCACAA GTGTAACAAT GAATGCATGG 1500 AAAGTGTGAA AAATGGAACT TATGACTAT C CAAAATATTC CGAGGAATCA AAGTTAAACA 1560 GGGAAAAAAT TGATGGAGTG AAATTGGAAT CAATGGGAGT CTATCAGATT CTGGCGATCT 1620 ACTCAACTGT CGCCAGTTCA CTGGTGCTTT TGGTAATCCATGTGCATCGACTGAGTCCATAGC CAGGAGCAATC AGCTTCTGGA 1680 TGTTCAATGCATGC

SEQ ID NO: 28 Sequence length: 1728 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Izumi / 5/65 SEQ ATAGACAACC AAAAGCATAA CAATGGCCAT CATCTATCTC ATACTCCTGT TCACAGCAGT 60 GAGGGGGGAC CAGATATGCA TTGGATACCA TGCCAATAAT TCCACAGAAA AGGTCGACAC 120 AATTCTAGAG CGGAATGTCA CTGTGACTCA TGCCAAGGAC ATCCTTGAGA AGACCCACAA 180 CGGAAAGCTA TGCAAACTAA ACGGAATCCC TCCACTTGAA CTAGGGGACT GTAGCATTGC 240 CGGATGGCTC CTTGGAAATC CAGAATGTGA TAGGCTTCTA AGGGTGCCAG AATGGTCCTA 300 TATAATGGAG AAAGAAAACC CGAGATACAG TTTATGTTAC CCAGGCAACT TCAATGACTA 360 TGAAGAATTG AAACATCTCC TCAGCAGCGT AAAACATTTC GAGAAAGTAA AGATTCTGCC 420 CAAAGATAGA TGGACACAGC ATACAACAAC TGGAGGTTCA AAGGCCTGCG CAGTGTCAGG 480 TAAACCATCA TTCTTCAGGA ACATGGTCTG GCTGACACAGAGCATGAGCATGAGCATGAGCATGAGCATGAGCATGAGCATGAGCATGAGCATGAGCATG A ACAAAAGGTC AATCCCTGAA ATAGCAGCAA GGCCTAAAGT 720 GAATGGACTA GGAAGTAGAA TGGAATTCTC TTGGACCCTC TTGGATGTGT GGGACACCAT 780 AAATTTTGAG AGCACTGGTA ATCTAGTTGC ACCAGAGTAT GGATTCAAAA TATCGAAAAG 840 AGGTAGTTCA GGGATCATGA AGACAGAAGG AACACTTGGG AACTGTGAGA CCAAATGCCA 900 AACTCCTTTG GGAGCAATAA ATACAACACT ACCTTTTCAC AATGTCCACC CACTGACAAT 960 AGGTGAATGC CCCAAATATG TAAAATCGGA GAAATTGGTC TTAGCAACAG GACTAAGGAA 1020 TGTTCCCCAG ATTGAATCAA GAGGATTGTT TGGGGCAATA GCTGGCTTTA TAGAAGGAGG 1080 ATGGCAAGGA ATGGTTGATG GTTGGTATGG ATACCATCAC AGCAATGACC AGGGATCAGG 1140 GTATGCAGCA GACAAAGAAT CCACTCAAAA GGCATTTGAT GGAATCACCA ACAAGGTAAA 1200 TTCTGTGATT GAAAAGATGA ACACCCAATT TGAAGCTGTT GGGAAAGAAT TCAATAATTT 1260 AGAGAAAAGA CTGGAGAACT TGAACAAAAA GATGGAAGAC GGGTTTCTAG ATGTGTGGAC 1320 ATACAATGCT GAGCTTCTAG TTCTGATGGA AAATGAGAGG ACACTTGACT TCCATGATTC 1380 TAATGTCAAG AACCTGTATG ATAAAGTCAG AATGCAGCTG AGAGACAACG TCAAAGAACT 1440 AGGAAATGGA TGTTTTGAAT TTTATCACAA ATGTGACGAT GAATGCATGA ATAGTGTGAA 1500 AAACGGGACG TATGATTATC CCAAGTATG A AGAAGAATCT AAACTAAATA GAAATGAAAT 1560 CAAAGGGGTA AAATTGAGCA GCATGGGGGT TTACCAAATT CTTGCCATTT ATGCTACAGT 1620 TGCAGGTTCT CTGTCACTGG CAATCATGAT GGCTGGGATC TCTTTCTGGA TGTGCTCCTA 1680 CGGGTCTCTG CATGTGATTAATATACT

SEQ ID NO: 29 Sequence length: 442 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / PR / 8/34 SEQ CCTTTCCAGA ATATACACCC AGTCACAATA GGAGAGTGCC CAAAATACGT CAGGAGTGCC 60 AAATTGAGGA TGGTTACAGG ACTAAGGAAC ATCCCGTCCA TTCAATCCAG AGGTCTATTT 120 GGAGCCATTG CCGGTTTTAT TGAAGGGGGA TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA GGGATCAGGC TATGCAGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTTATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAATTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTAGT TCTACTGGAA 420 AATGAAAGGA CTCTGGATTT CC 442

SEQ ID NO: 30 Sequence length: 424 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Bangkok / 10/83 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTACGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT TGAAGGGGGA TGGACTGGAA TGATAGATGG ATGGTATCGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT TCTACTGGAA 420 AATG 424

SEQ ID NO: 31 Sequence length: 424 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Yamagata / 120/86 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT TGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT CCTACTGGAA 420 AATG 424

SEQ ID NO: 32 Sequence length: 429 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Osaka / 930/88 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT AGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GAATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT TCTACTGGAA 420 AATGAAAGG 429

SEQ ID NO: 33 Sequence length: 400 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Okuda / 57 Sequence GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG TGAGTGCCCC 60 AAATATGTAA AATCGGAGAA GTTGGTCTTA GCAACAGGAC TAAGGAATGT TCCCCAGATT 120 GAATCAAGAG GATTGTTTGG GGCAATAGCT GGTTTTATAG AAGGAGGATG GCAAGGAATG 180 GTTGACGGTT GGTATGGATA CCATCACAGC AATGACCAGG GATCAGGGTA TGCAGCAGAC 240 AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC TGTGATTGAA 300 AAGATAAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCG GTAACTTAGA GAAAAGACTG 360 GAGAACTTGA ACAAAAAGAT GGAAGACGGG TTTCTAGATG 400

SEQ ID NO: 34 Sequence length: 409 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Adachi / 2/57 SEQ CGCCTTGGAG CAATAAATAC AACATTGCCT TTTCACAATG TCCACCCACT GACAATAGGT 60 GAGTGCCCCA AATATGTAAA ATCGGAGAAG TTGGTCTTAG CAACAGGACT AAGGAATGTT 120 CCCCAGATTG AATCAAGAGG ATTGTTTGGG GCAATAGCTG GTTTTATAGA AGGAGGATGG 180 CAAGGAATGG TTGATGGTTG GTATGGATAC CATCACAGCA ATGACCAGGG ATCAGGGTAT 240 GCAGCAGACA AAGAATCCAC TCAAAAGGCA TTTGATGGAA TCACCAACAA GGTAAATTCT 300 GTGATTGAAA AGATGAACAC CCAATTTGAA GCTGTTGGGA AAGAATTCGG TAACTTAGAG 360 AGAAGACTGG AGAACTTGAA CAAAAAGATG GAAGACGGGT TTCTAGATG 409

SEQ ID NO: 35 Sequence length: 410 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kumamoto / 1/65 SEQ CTCCTTTGGA GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG 60 TGAATGCCCC AAATATGTAA AATCGGAGAA ACTGGTCTTA GCAACAGGAC TAAGGAATGT 120 TCCCCAGATT GAATCAAGAG GATTGTTTGG GGCAATAGCT GGCTTTGTAG AAGGAGGATG 180 GCAAGGAATG ATTGATGGTT GGTATGGATA CCATCACAGC AATGATCAGG GATCAGGGTT 240 TGCAGCAGAC AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC 300 TGTGATTGAA AAGATGAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCA ATAATTTAGA 360 GAAAAGACTG GAGAACTTGA ACAAAAGGAT GGAAGACGGG TTTCTAGATG 410

SEQ ID NO: 36 Sequence length: 394 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kaizuka / 2/65 SEQ AATACAACAC TACCTTTTCA CAATGTCCAC CCACTGACAA TAGGTGAATG CCCCAAATAT 60 GTAAAATCGG AGAAATTGGT CTTAGCAACA GGACTAAGGA ATGTTCCCCA GATTGAATCA 120 AGAGGATTGT TTGGGGCAAT AGCTGGCTTT ATAGAAGGAG GATGGCAAGG AATGGTTGAT 180 GGTTGGTATG GATACCATCA CAGCAATGAC CAGGGATCAG GGTATGCAGC AGACAAAGAA 240 TCCACTCAAA AGGCATTTGA TGGAATCACC AACAAGGTAA ATTCTGTGAT TGAAAAGATG 300 AACACCCAAT TTGAAGCTGT TGGGAAAGAA TTCAATAATT TAGAGAAAAG ACTGGAGAAC 360 TTGAACAAAA AGATGGAAGA CGGGTTTCTA GATG 394

SEQ ID NO: 37 Sequence length: 329 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A2 / Aichi / 2/68 SEQ ATGACAAGCC CTTTCAAAAC GTAAACAAGA TCACATATGG AGCATGCCCC AAGTATGTTA 60 AGCAAAACAC CCTGAAGTTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCCTATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG ATAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC CATCGACCAA ATCAATGGGA AATTGAACAG GGTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTC 329

SEQ ID NO: 38 Sequence length: 334 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Fukuoka / C29 / 85 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGGTATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334

SEQ ID NO: 39 Sequence length: 329 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Sichuan / 2/87 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGCT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329

SEQ ID NO: 40 Sequence length: 334 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Ibaraki / 1/90 SEQ ATGACAAACC CTTTCAAAAT ATAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334

SEQ ID NO: 41 Sequence length: 329 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Suita / 1/90 SEQ ATGACAAACC CTTTCAAAAT GTAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAAAAA CAAACTAGGG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AACTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329

SEQ ID NO: 42 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GATCTAGAAG CAAAAGCAGG GGTTATACCA 30

SEQ ID NO: 43 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CGGCTAGCAA AAGCAGGGGT TATACCATAG 30

SEQ ID NO: 44 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ACAGATCTAG TAGAAACAAG GGTGTTTTT 29

SEQ ID NO: 45 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CGGCTAGCAG AAACAAGGGT GTTTTTAATT 30

SEQ ID NO: 46 Sequence length: 1783 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Okuda / 57 Sequence CGGCTAGCAA AAGCAGGGGT TATACCATAG AAAACCAAAA 40 GCAAAACA ATG GCC ATC ATT TAT CTC ATT CTC CTG TTC ACA GCA GTG AGA GGG 93 Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly -15 -10 -5 GAC CAG ATA TGC ATT GGA GCC AAT AAT TCC ACA GAG AAG 138 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys 1 5 10 15 GTC GAC ACA ATT CTA GAG CGG AAC GTC ACT GTG ACT CAT GCC AAG 183 Val Asp Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys 20 25 30 GAC ATC CTT GAG AAG ACC CAT AAC GGA AAG TTA TGC AAA CTA AAC 228 Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn 35 40 45 GGA ATC CCT CCA CTT GAA CTA GGG GAC TGT AGC ATT GCC GGA TGG 273 Gly Ile Pro Pro Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp 50 55 60 CTC CTT GGA AAT CCA AAA TGT GAT AGG CTT CTA AGT GTG CCA GAA 318 Leu Leu Gly Asn Pro Lys Cys Asp Arg Leu Leu Ser Val Pro Glu 65 70 75 CGG TCC TAT ATA TTG GAG AAA GAA AAC CCG AGA GAC GGT TTG TGT 363 Arg Ser Tyr Ile Leu Glu Lys Glu Asn Pro Arg Asp Gly Leu Cys 80 85 90 TAT CCA GGC AGC TTC AAT GAT TAT GAA GAA TTG AAA CAT CTC CTC 408 Tyr Pro Gly Ser Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu 95 100 105 AGC AGC GTG AAA CAT TTC GAG AAA GTA AAG ATT CTG CCC AAA GAT 453 Ser Ser Val Lys His Phe Glu Lys Val Lys Ile Leu Pro Lys Asp 110 115 120 AGA TGG ACA CAG CAT ACA ACA ACT GGA GGT TCA CGG GCC TGC GCG 498 Arg Trp Thr Gln His Thr Thr Thr Gly Gly Ser Arg Ala Cys Ala 125 130 135 GTG TCT GGT AAT CCA TCA TTT TTC AGG AAC ATG GTC TGG CTG ACA 543 Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Met Val Trp Leu Thr 140 145 150 AAG GAA GGA TCA GAT TAT CCG GTT GCC AAA GGA TCG TAC AAC AAT 588 Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly Ser Tyr Asn Asn 155 160 165 ACA AGC GGA GAA CAA ATG CTA ATA ATT TGG GGG GTG CAC CAT CCC 633 Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val His His Pr o 170 175 180 ATT GAT GAG ACA GAA CAA AGA ACA TTG TAC CAG AAT GTG GGA ACC 678 Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val Gly Thr 185 190 195 TAT GTT TCC GTA GGC ACA TCA ACA TTG AAC AAA AGG TCA ACC CCA 723 Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr Pro 200 205 210 GAA ATA GCA ACA AGG CCT AAA GTG AAT GGA CAA GGA GGT AGA ATG 768 Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met 215 220 225 GAA TTC TCT TGG ACC CTC TTG GAT ATG TGG GAC ACC ATA AAT TTT 813 Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe 230 235 240 GAG AGT ACT GGT AAT CTA ATT GCA CCA GAG TAT GGA TTC AAA ATA 858 Glu Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile 245 250 255 TCG AAA AGA GGT AGT TCA GGG ATC ATG AAA ACA GAA GGA ACA CTT 903 Ser Lys Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu 260 265 270 GAG AAC TGT GAG ACC AAA TGC CAA ACT CCT TTG GGA GCA ATA AAT 948 Glu Asn Cys Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn 275 280 285 ACA ACA TTA CCT TTT CAC AAT GTC CA C CCA CTG ACA ATA GGT GAG 993 Thr Thr Leu Pro Phe His Asn Val His Pro Leu Thr Ile Gly Glu 290 295 300 TGC CCC AAA TAT GTA AAA TCG GAG AAG TTG GTC TTA GCA ACA GGA 1038 Cys Pro Lys Tyr Val Lys Ser Glu Lys Leu Val Leu Ala Thr Gly 305 310 315 CTA AGG AAT GTT CCC CAG ATT GAA TCA AGA GGA TTG TTT GGG GCA 1083 Leu Arg Asn Val Pro Gln Ile Glu Ser Arg Gly Leu Phe Gly Ala 320 325 330 ATA GCT GGT TTT ATA GAA GGA GGA TGG CAA GGA ATG GTT GAC GGT 1128 Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly 335 330 345 TGG TAT GGA TAC CAT CAC AGC AAT GAC CAG GGA TCA GGG TAT GCA 1173 Trp Tyr Gly Tyr His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala 350 355 360 GCA GAC AAA GAA TCC ACT CAA AAG GCA TTT GAT GGA ATC ACC AAC 1218 Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn 365 370 375 AAG GTA AAT TCT GTG ATT GAA AAG ATA AAC ACC CAA TTT GAA GCT 1263 Lys Val Asn Ser Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala 380 385 390 GTT GGG AAA GAA TTC GGT AAC TTA GAG AAA AGA CTG GAG AAC TTG 1308 Val Gly Lys Glu Phe Gly Asn Leu Glu Lys Arg Leu Glu Asn Leu 395 400 405 AAC AAA AAG ATG GAA GAC GGG TTT CTA GAT GTG TGG ACA TAC AAT 1353 Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn 410 415 420 GCT GAG CTT TTA GTT CTG ATG GAA AAT GAG AGG ACA CTT GAC TTT 1398 Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe 425 430 435 CAT GAT TCT AAT GTC AAG AAT CTG TAT AGT AAA GTC AGA ATG CAG 1443 His Asp Ser Asn Val Lys Asn Leu Tyr Ser Lys Val Arg Met Gln 440 445 450 CTG AGA GAC AAC GTC AAA GAA CTA GGA AAT GGA TGT TTT GAA TTT 1488 Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 455 460 465 TAT CAC AAA TGT GAT GAT GAA TGC ATG AAT AGT GTG AAA AAC GGG 1533 Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly 470 475 480 ACA TAT GAT TAT CCC AAG TAT GAA GAA GAG TCT AAA CTA AAT AGA 1578 Thr Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg 495 500 505 AAT GAA ATC AAA GGG GTA AAA TTG AGC AGC ATG GGG GTT TAT CAA 1623 Asn Glu Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln 510 515 520 ATC CTT GCC ATT TAT GCT ACA GTA GCA GGT TCT ATG TCA CTG GCA 1668 Ile Leu Ala Ile Tyr Ala Thr Val Ala Gly Ser Met Ser Leu Ala 525 530 535 ATC ATG ATG GCT GGG ATC TCT TTC TGG GTG TGC TCC AAC GGG TCT 1713 Ile Met Met Ala Gly Ile Ser Phe Trp Val Cys Ser Asn Gly Ser 540 545 550 CTG CAG TGC AGG ATC TGC ATA TGATTATAAG TCATTTTATA ATTAAAAACA 1764 Leu Gln Cys Arg Ile Cys Ile 555 CCCTTGTTTC TGCTAGCC

SEQ ID NO: 47 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TCCGTTTAGT TTGCATAACT TTCCG 25

SEQ ID NO: 48 Sequence Length: 26 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence TCCGGGATCA TGAAAACAGA AGGAAC 26

SEQ ID NO: 49 Sequence length: 1135 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Okuda / 57 Sequence CTAGCAAAAG CAGGGGTTAT ACCATAGAAA ACCAAAAGCA AAACAATGGC CATCATTTAT 60 CTCATTCTCC TGTTCACAGC AGTGAGAGGG GACCAGATAT GCATTGGATA CCATGCCAAT 120 AATTCCACAG AGAAGGTCGA CACAATTCTA GAGCGGAACG TCACTGTGAC TCATGCCAAG 180 GACATCCTTG AGAAGACCCA TAACGGAAAG TTATGCAAAC TAAACGGATC CGGGATCATG 240 AAAACAGAAG GAACACTTGA GAACTGTGAG ACCAAATGCC AAACTCCTTT GGGAGCAATA 300 AATACAACAT TACCTTTTCA CAATGTCCAC CCACTGACAA TAGGTGAGTG CCCCAAATAT 360 GTAAAATCGG AGAAGTTGGT CTTAGCAACA GGACTAAGGA ATGTTCCCCA GATTGAATCA 420 AGAGGATTGT TTGGGGCAAT AGCTGGTTTT ATAGAAGGAG GATGGCAAGG AATGGTTGAC 480 GGTTGGTATG GATACCATCA CAGCAATGAC CAGGGATCAG GGTATGCAGC AGACAAAGAA 540 TCCACTCAAA AGGCATTTGA TGGAATCACCGAACAGAG ACTAAGACTAGAACTATAGAACTATAACTATAGAGAATATCTCGAGA CGGGTTTCTA GATGTGTGGA CATACAATGC TGAGCTTTTA 720 GTTCTGATGG AAAATGAGAG GACACTTGAC TTTCATGATT CTAATGTCAA GAATCTGTAT 780 AGTAAAGTCA GAATGCAGCT GAGAGACAAC GTCAAAGAAC TAGGAAATGG ATGTTTTGAA 840 TTTTATCACA AATGTGATGA TGAATGCATG AATAGTGTGA AAAACGGGAC ATATGATTAT 900 CCCAAGTATG AAGAAGAGTC TAAACTAAAT AGAAATGAAA TCAAAGGGGT AAAATTGAGC 960 AGCATGGGGG TTTATCAAAT CCTTGCCATT TATGCTACAG TAGCAGGTTC TATGTCACTG 1020 GCAATCATGA TGGCTGGGAT CTCTTTCTGG GTGTGCTCCA ACGGGTCTCT GCAGTGCAGG 1080 ATCTGCATAT GATTATAAGT CATTTTATAA TTAAAAACAC CCTTGTTTCT GCTAG 1135

SEQ ID NO: 50 Sequence Length: 348 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Sequence Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly -15 -10 -5 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys 1 5 10 15 Val Asp Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys 20 25 30 Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn 35 40 45 Gly Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Glu 50 55 60 Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro 65 70 75 Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr 80 85 90 Val Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val 95 100 105 Pro Gln Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 110 115 120 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr 125 130 135 His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu 140 145 150 Ser Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn Lys Val Asn Ser 155 160 165 Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala Val Gly Lys Glu 170 175 180 Phe Gly Asn Leu Glu Lys Arg Leu Glu Asn Leu Asn Lys Lys Met 185 190 195 Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu 200 205 210 Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 215 220 225 Val Lys Asn Leu Tyr Ser Lys Val Arg Met Gln Leu Arg Asp Asn 230 235 240 Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys 245 250 255 Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr Tyr Asp Tyr 260 265 270 Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Ile Lys 275 280 285 Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile 290 295 300 Tyr Ala Thr Val Ala Gly Ser Met Ser Leu Ala Ile Met Met Ala 305 310 315 Gly Ile Ser Phe Trp Val Cys Ser Asn Gly Ser Leu Gln Cys Arg 320 325 330 Ile Cys Ile

SEQ ID NO: 51 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence GATCTAGAAG CAAAGCAGGG GATAATTCTA 30

SEQ ID NO: 52 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ACAGATCTAG TAGAAACAAG GGTGTTTTT 29

SEQ ID NO: 53 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CGGCTAGCAG AAACAAGGGT GTTTTTAATT 30

SEQ ID NO: 54 Sequence length: 1778 Array type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA to genomic RNA Origin:  Organism name: A2 / Aichi / 2/68 Sequence GATCTAGAAG CAAAGCAGGG GATAATTCTA 30 TTAATC ATG AAG ACC ATC ATT GCT TTG AGC TAC ATT TTC TGT CTG GCT CTC 81 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu -15 -10 -5 GGC CAA GAC CTT CCA GGA AAT GAC AAC AGC ACA GCA ACG CTG TGC 127 Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys 1 5 10 CTG GGA CAT CAT GCG GTG CCA AAC GGA ACA CTA GTG AAA ACA ATC 172 Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile 15 20 25 ACA GAT GAT CAG ATT GAA GTG ACT AAT GCT ACT GAG CTA GTT CAG 217 Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln 30 35 40 AGC TCC TCA ACG GGG AAA ATA TGC AAC AAT CCT CAT CGA ATC CTT 262 Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu 45 50 55 GAT GGA ATA GAC TGC ACA CTG ATA GAT GCT CTA TTG GGG GAC CCT 307 Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro 60 65 70 CAT TGT GAT GTT TTT CAA AAT GAG ACA TGG GAC CTT TTC GTT GAA 352 His Cys Asp Val Phe Gln Asn Glu Thr Trp Asp Leu Phe Val Glu 75 80 85 CGC AG C AAA GCT TTC AGC AAC TGT TAC CCT TAT GAT GTG CCA GAT 397 Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp 90 95 100 TAT GCC TCC CTT AGG TCA CTA GTT GCC TCG TCA GGC ACT CTG GAG 442 Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu 105 110 115 TTT ATC ACT GAG GGT TTC ACT TGG ACT GGG GTC ACT CAG AAT GGG 487 Phe Ile Thr Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly 120 125 130 GGA AGC AAT GCT TGC AAA AGG GGA CCT GGT AGC GGT TTT TTC AGT 532 Gly Ser Asn Ala Cys Lys Arg Gly Pro Gly Ser Gly Phe Phe Ser 135 140 145 AGA CTG AAC TGG TTG ACC AAA TCA GGA AGC ACA TAT CCA GTG CTG 577 Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Thr Tyr Pro Val Leu 150 155 160 AAC GTG ACT ATG CCA AAC AAT GAC AAT TTT GAC AAA CTA TAC ATT 622 Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile 165 170 175 TGG GGG ATT CAC CAC CCG AGC ACG AAC CAA GAA CAA ACC AGC CTG 667 Trp Gly Ile His His Pro Ser Thr Asn Gln Glu Gln Thr Ser Leu 180 185 190 TAT GTT CAA GCA TCA GGG AGA GTC ACA GTC TCT ACC AGG AGA AGC 712 Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg Ser 195 200 205 CAG CAA ACT ATA ATC CCG AAT ATC GGG TCC AGA CCC TGG GTA AGG 757 Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg 210 215 220 GGT CTG TCT AGT AGA ATA AGC ATC TAT TGG ACA ATA GTT AAG CCG 802 Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro 225 230 235 GGA GAC GTA CTG GTA ATT AAT AGT AAT GGG AAC CTA ATC GCT CCT 847 Gly Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro 240 245 250 CGG GGT TAT TTC AAA ATG CGC ACT GGG AAA AGC TCA ATA ATG AGG 892 Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg 255 260 265 TCA GAT GCA CCT ATT GAT ACC TGT ATT TCT GAA TGC ATC ACT CCA 937 Ser Asp Ala Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro 270 275 280 AAT GGA AGC ATT CCC AAT GAC AAG CCC TTT CAA AAC GTA AAC AAG 982 Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys 285 290 295 ATC ACA TAT GGA GCA TGC CCC AAG TAT GTT AAG CAA AAC ACC CTG 1027 Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Va l Lys Gln Asn Thr Leu 300 305 310 AAG TTG GCA ACA GGG ATG CGG AAT GTA CCA GAG AAA CAA ACT AGA 1072 Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg 315 320 325 GGC CTA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG 1117 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu 330 335 340 GGA ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCT GAG 1162 Gly Met Ile Asp Gly Trp Gly Phe Arg His Gln Asn Ser Glu 345 350 355 GGC ACA GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC 1207 Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile 360 365 370 GAC CAA ATC AAT GGG AAA TTG AAC AGG GTA ATC GAG AAG ACG AAC 1252 Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 375 380 385 GAG AAA TTC CAT CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG 1297 Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly 390 395 400 AGA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT 1342 Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp 405 410 415 CTC TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG AAT CAA 1387 Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln 420 425 430 CAT ACA ATT GAC CTG ACT GAC TCG GAA ATG AAC AAG CTG TTT GAA 1432 His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu 435 440 445 AAA ACA AGG AGG CAA CTG AGG GAA AAT GCT GAA GAG ATG GGC AAT 1477 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Glu Met Gly Asn 450 455 460 GGT TGC TTC AAA ATA TAC CAC AAA TGT GAC AAC GCT TGC ATA GAG 1522 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu 465 470 475 TCA ATC AGA AAT GGT ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA 1567 Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu 480 485 490 GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT GAA CTG AAG TCT 1612 Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser 495 500 505 GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC ATA TCA TGC 1657 Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys 510 515 520 TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG GCC TGC 1702 Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys 525 530 535 CAG AGA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGAGTGTATT AGTAATTAAA 1755 Gln Arg Gly Asn Ile Arg Cys Asn Ile Cys Asn Ile Cys Ile AACACCCTTG TTTCTGCTAG CCG 1778

SEQ ID NO: 55 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ATTGTTGCAT ATTTTCCCCG 20

SEQ ID NO: 56 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ATTGATACCT GTATTTCTGA 20

SEQ ID NO: 57 Sequence length: 1110 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A2 / Aichi / 2/68 SEQ CTAGAAGCAA AGCAGGGGAT AATTCTATTA ATCATGAAGA CCATCATTGC TTTGAGCTAC 60 ATTTTCTGTC TGGCTCTCGG CCAAGACCTT CCAGGAAATG ACAACAGCAC AGCAACGCTG 120 TGCCTGGGAC ATCATGCGGT GCCAAACGGA ACACTAGTGA AAACAATCAC AGATGATCAG 180 ATTGAAGTGA CTAATGCTAC TGAGCTAGTT CAGAGCTCCT CAACGGGGAA AATATGCAAC 240 AATATTGATA CCTGTATTTC TGAATGCATC ACTCCAAATG GAAGCATTCC CAATGACAAG 300 CCCTTTCAAA ACGTAAACAA GATCACATAT GGAGCATGCC CCAAGTATGT TAAGCAAAAC 360 ACCCTGAAGT TGGCAACAGG GATGCGGAAT GTACCAGAGA AACAAACTAG AGGCCTATTC 420 GGCGCAATAG CAGGTTTCAT AGAAAATGGT TGGGAGGGAA TGATAGACGG TTGGTACGGT 480 TTCAGGCATC AAAATTCTGA GGGCACAGGA CAAGCAGCAG ATCTTAAAAG CACTCAAGCA 540 GCCATCGACCAATCAGAGCACATACAAGCA CATAATTCAACGA GACAATTGAACAGGGTAATCG AGAAGACGAA CGAGAAGACGAA CGAGAAGACAA CGAGAAGACAA GA TCTCTGGTCT TACAATGCGG AGCTTCTTGT CGCTCTGGAG 720 AATCAACATA CAATTGACCT GACTGACTCG GAAATGAACA AGCTGTTTGA AAAAACAAGG 780 AGGCAACTGA GGGAAAATGC TGAAGAGATG GGCAATGGTT GCTTCAAAAT ATACCACAAA 840 TGTGACAACG CTTGCATAGA GTCAATCAGA AATGGTACTT ATGACCATGA TGTATACAGA 900 GACGAAGCAT TAAACAACCG GTTTCAGATC AAAGGTGTTG AACTGAAGTC TGGATACAAA 960 GACTGGATCC TGTGGATTTC CTTTGCCATA TCATGCTTTT TGCTTTGTGT TGTTTTGCTG 1020 GGGTTCATCA TGTGGGCCTG CCAGAGAGGC AACATTAGGT GCAACATTTG CATTTGAGTG 1080 TATTAGTAAT TAAAAACACC CTTGTTTCTG 1110

SEQ ID NO: 58 Sequence Length: 346 Sequence Type: Amino Acid Number of Chains: Single Chain Topology: Linear Sequence Type: Peptide Sequence Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu -15 -10 -5 Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys 1 5 10 Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile 15 20 25 Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln 30 35 40 Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Ile Asp Thr Cys Ile 45 50 55 Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro 60 65 70 Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr 75 80 85 Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val 90 95 100 Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 105 110 115 Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe 120 125 130 Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 135 140 145 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg 150 155 160 Val Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu 165 170 175 Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val 180 185 190 Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu 195 200 205 Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu 210 215 220 Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn 225 230 235 Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 240 245 250 Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His 255 260 265 Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys 270 275 280 Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile 285 290 295 Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly 300 305 310 Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile 315 320 325 Cys Ile 330

[Brief description of drawings]

FIG. 1 is a schematic diagram of the tertiary structure of the HA molecule and the position of the common conserved region in the HA molecule between the H1N1 subtype and the H2N2 subtype.

FIG. 2 is a schematic diagram of the tertiary structure of the HA molecule and shows the position of the common conserved region in the HA molecule of the H3N2 subtype.

FIG. 3 is a diagram showing the survival rate of influenza virus-infected groups.

FIG. 4 is a diagram showing the survival rate of influenza virus-infected groups.

FIG. 5 is a diagram showing changes in body weight of influenza virus-infected groups.

FIG. 6 shows the survival rate of influenza virus-infected groups.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 9161-4B (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1: 91) C07K 99:00

Claims (5)

[Claims] 1. An immunogenic artificial polypeptide which has substantially the same antigenicity as the stem region in the hemagglutinin molecule of human influenza A virus and lacks the globular region in the hemagglutinin molecule. 2. SEQ ID NO: 1 in the sequence listing in the molecule
And a GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing, wherein the configurations of both sequences are H1N1 subtype and H2
The immunogenic artificial polypeptide according to claim 1, which has substantially the same antigenicity as the stem region in the N2 subtype hemagglutinin molecule. 3. SEQ ID NO: 3 of the sequence listing in the molecule
And a QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the sequence listing, and the configuration of both sequences is a stem in a hemagglutinin molecule of H3N2 subtype. The immunogenic artificial polypeptide according to claim 1, which has substantially the same antigenicity as the region. 4. The immunogenic artificial polypeptide according to claim 1, which is isolated from a protease-treated product of a hemagglutinin molecule of human influenza A virus. 5. A gene encoding an immunogenic artificial polypeptide which has substantially the same antigenicity as the stem region in the hemagglutinin molecule of human influenza A virus and lacks the globular region in the hemagglutinin molecule.