U.S. patent application number 17/054340 was filed with the patent office on 2021-06-03 for nucleic acid for treating mite allergy.
This patent application is currently assigned to Astellas Pharma Inc.. The applicant listed for this patent is Astellas Pharma Inc.. Invention is credited to Takanori MARUI, Masao UCHIDA.
Application Number | 20210163957 17/054340 |
Document ID | / |
Family ID | 1000005443645 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210163957 |
Kind Code |
A1 |
MARUI; Takanori ; et
al. |
June 3, 2021 |
NUCLEIC ACID FOR TREATING MITE ALLERGY
Abstract
[Problem] To provide a nucleic acid expected to be useful for
treating mite allergy. [Means to be solved] Provided is a nucleic
acid comprising a nucleotide sequence encoding a chimeric protein,
wherein the nucleic acid comprises a nucleotide sequence encoding a
signal peptide, a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP, a nucleotide sequence encoding an
allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7,
a nucleotide sequence encoding a transmembrane domain and a
nucleotide sequence encoding an endosomal/lysosomal targeting
domain of LAMP in this order.
Inventors: |
MARUI; Takanori; (Tokyo,
JP) ; UCHIDA; Masao; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Pharma Inc. |
Chuo-ku, Tokyo |
|
JP |
|
|
Assignee: |
Astellas Pharma Inc.
Chuo-ku, Tokyo
JP
|
Family ID: |
1000005443645 |
Appl. No.: |
17/054340 |
Filed: |
May 10, 2019 |
PCT Filed: |
May 10, 2019 |
PCT NO: |
PCT/JP2019/018657 |
371 Date: |
November 10, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/5256 20130101;
C07K 2319/02 20130101; C12N 15/62 20130101; A61K 39/35 20130101;
A61P 37/08 20180101; A61K 2039/53 20130101; A61K 2039/57 20130101;
C12N 9/50 20130101; C07K 2319/03 20130101; C07K 14/70596 20130101;
C12Y 304/22065 20130101 |
International
Class: |
C12N 15/62 20060101
C12N015/62; C07K 14/705 20060101 C07K014/705; C12N 9/50 20060101
C12N009/50; A61K 39/35 20060101 A61K039/35; A61P 37/08 20060101
A61P037/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2018 |
JP |
2018-091963 |
Claims
1. (canceled)
2. A nucleic acid encoding a chimeric protein, comprising the
following nucleotide sequences in this order: a nucleotide sequence
encoding a signal peptide; a nucleotide sequence encoding an
intra-organelle stabilizing domain of LAMP; a nucleotide sequence
encoding an allergen domain comprising Der p 1, Der p 2, Der p 23,
and Der p 7 in this order; a nucleotide sequence encoding a
transmembrane domain; and a nucleotide sequence encoding an
endosomal/lysosomal targeting domain of LAMP.
3. The nucleic acid according to claim 2, wherein the signal
peptide is a signal peptide of LAMP.
4. The nucleic acid according to claim 2, wherein the transmembrane
domain is a transmembrane domain of LAMP.
5. The nucleic acid according to claim 2, wherein the signal
peptide consists of amino acid numbers 1 to 27 of SEQ ID NO: 2, the
intra-organelle stabilizing domain consists of an amino acid
sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2, Der p 1
consists of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2
consists of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consists of amino acid numbers 732 to 800 of SEQ ID NO: 2, Der p 7
consists of amino acid numbers 805 to 1002 of SEQ ID NO: 2, the
transmembrane domain consists of amino acid numbers 1006 to 1028 of
SEQ ID NO: 2, and the endosomal/lysosomal targeting domain consists
of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
6. A nucleic acid encoding a chimeric protein consisting of an
amino acid sequence having at least 90% identity to SEQ ID NO: 2,
wherein the chimeric protein encoded by the nucleic acid induces
Th1-type immunity to an allergen selected from the group consisting
of Der p 1, Der p 2, Der p 23, and Der p 7.
7. A nucleic acid comprising: a) a nucleotide sequence encoding a
chimeric protein consisting of SEQ ID NO: 2; or b) a nucleotide
sequence encoding a chimeric protein consisting of an amino acid
sequence of SEQ ID NO: 2 in which 1 to 10 amino acids are deleted,
substituted, inserted and/or added, wherein the chimeric protein
encoded by the nucleic acid induces Th1-type immunity to an
allergen selected from the group consisting of Der p 1, Der p 2,
Der p 23, and Der p 7.
8. A nucleic acid comprising: a nucleotide sequence encoding a
chimeric protein consisting of SEQ ID NO: 2.
9. An expression vector comprising: the nucleic acid according to
claim 2.
10. An expression vector comprising: the nucleic acid according to
claim 8.
11. A host cell transformed with the nucleic acid according to
claim 2.
12. A method for producing a nucleic acid, comprising: culturing a
host cell transformed with the nucleic acid according to claim
2.
13. A pharmaceutical composition comprising: the expression vector
according to claim 10 and a pharmaceutically acceptable
excipient.
14. A pharmaceutical composition comprising: the expression
according to claim 9 and a pharmaceutically acceptable
excipient.
15. A method for preventing or treating mite allergy, comprising
administering to a subject in need thereof the expression vector
according to claim 10.
16-17. (canceled)
18. The method of claim 15, wherein the subject is a human.
19. A method for preventing or treating mite allergy, comprising
administering to a subject in need thereof the expression vector
according to claim 9.
20. The method of claim 19, wherein the subject is a human.
21. A method of inducing a TH1-type immune response in a subject,
comprising administering to a subject a nucleic acid according to
claim 2.
22. A method of inducing a TH1-type immune response in a subject,
comprising administering to a subject a nucleic acid according to
claim 8.
Description
TECHNICAL FIELD
[0001] The present invention relates to a nucleic acid which is
expected to be useful as an active ingredient of a pharmaceutical
composition, for example, a nucleic acid which is expected to be
useful for treating mite allergy.
BACKGROUND ART
[0002] Mite allergy is an allergic disease that occurs in response
to mite-derived allergens. The allergic disease is caused by the
following steps: 1) allergens taken into a body are phagocytosed by
antigen-presenting cells and presented to naive T cells, 2) the
naive T cells are differentiated into Th2 cells, 3) cytokine such
as IL-4 is produced from an immune cell such as the Th2 cell, 4) B
cells produce IgE by IL-4, and 5) IgE binding to the allergens
binds to mast cells. It has been known that in allergic disease
patients, antagonism between Th1-type immunity involving Th1 cells
producing IFN-.gamma. or the like and Th2-type immunity involving
Th2 cells producing IL-4 or the like shifts to Th2-type dominant
which results in Th2-type inflammatory immune response (Middleton's
Allergy Seventh edition Principles & Practice, 2009). Thus,
IFN-.gamma. can be used as an indicator of Th1-type immunity, and
IL-4 can be used as an indicator of Th2-type immunity. Further, in
mice, IFN-.gamma. causes a preferential class switch to IgG2a
isotype in activated B cells, while suppresses responses to all the
other isotypes. That is, IgG2a can also be used as an indicator of
Th1-type immunity. For example, it has been known that production
of IgG2a is promoted in IL-4-deficient mice and that IgG2a
production is suppressed in IFN-.gamma.-deficient mice (Arthritis
Res., 2002, Vol. 4, p. 54-58). There is also a report that
antibodies produced from B cells are involved in the mechanism of
action of allergen immunotherapy. For example, it has been known
that in humans, IgG antagonizes IgE binding to an allergen to
inhibit formation of allergen-IgE complex and thereby inhibit
histamine release from mast cells (J Allergy Clin Immunol., 2017,
Vol. 140, p. 1485-1498).
[0003] Until now, development of multiple immunotherapies for mite
allergy has been advanced (J Allergy Clin Immunol., 2013, Vol. 132,
p. 1322-1336; WO 2014/195803; Expert Rev Vaccines., 2014, Vol. 13,
p. 1427-1438). Further, Der p 1, Der p 2, Der p 7, Der p 23, and
the like have been known as allergens related to the mite allergy
(Patent Documents 1 and 2, and Non-Patent Document 1). However, for
example, subcutaneous immunotherapy (SCIT) and sublingual
immunotherapy (SLIT) have problems such as possibility of
anaphylaxis and long treatment period over several years.
[0004] As one of the techniques for nucleic acid vaccines, nucleic
acid vaccines for treating allergy using lysosome-associated
membrane proteins (LAMP) have been studied.
[0005] Further, a plasmid comprising a nucleic acid encoding a
chimeric protein comprising LAMP-1, which is a member of LAMP
family, and Cry J1 and/or Cry J2, which are allergens of
Cryptomeria japonica, was constructed (Patent Document 3 and
Non-Patent Document 2). It has been reported that such a plasmid
does not cause systemic release of free allergen which causes
anaphylaxis, but induces a Th1-type immune response. Furthermore,
it has been reported that a plasmid comprising a nucleic acid
encoding a chimeric protein comprising LAMP-1 and peanut allergens
Ara H1, Ara H2 and Ara H3 reduced production of IgE in a mouse
model (Patent Document 4). In a field of mite allergy, a vaccine
comprising a nucleic acid encoding a chimeric protein comprising
Der p 1 and a transmembrane domain of LAMP-1 and an
endosomal/lysosomal targeting domain has been constructed (Patent
Document 5 and Non-Patent Document 3). However, a nucleic acid
vaccine for treating mite allergy comprising multiple mite allergen
antigens, and an intra-organelle stabilizing domain of LAMP-1 and
an endosomal/lysosomal targeting domain has not been reported.
RELATED ART
Patent Document
[0006] [Patent Document 1] WO 1988/010297 [0007] [Patent Document
2] WO 2007/124524 [0008] [Patent Document 3] WO 2013/187906 [0009]
[Patent Document 4] WO 2015/200357 [0010] [Patent Document 5] WO
2004/019978
Non-Patent Document
[0010] [0011] [Non-Patent Document 1] "Clinical & Experimental
Allergy", (UK), 1995; 25: 416-422 [0012] [Non-Patent Document 2]
"Journal of Immunology Research", (Egypt), 2016; Article ID 4857869
[0013] [Non-Patent Document 3] "Vaccine", (Netherlands), 2006; 24
(29-30): 5762-5771
SUMMARY OF INVENTION
Problems to be Solved by the Invention
[0014] An object of the present invention is to provide a nucleic
acid which is expected to be useful for treating mite allergy.
Means for Solving the Problems
[0015] As a result of repeated investigation with considerable
creativity in the preparation of nucleic acids for treating mite
allergy, the present inventors have prepared LAMP-Der p 1-Der p
2-Der p 23-Der p 7 plasmid (Example 1), confirmed that a chimeric
protein is expressed from the plasmid (Example 2), and found that a
Th1-type immune response is induced in mice to which the plasmid is
administered (Examples 3 and 4). As a result, a nucleic acid which
is expected to be useful for treating mite allergy is provided, and
thereby the present invention has been completed.
[0016] That is, the present invention relates to the following [1]
to [17].
[1]
[0017] A nucleic acid comprising:
[0018] a nucleotide sequence encoding a chimeric protein,
[0019] wherein the nucleotide sequence is a nucleotide sequence
comprising the following nucleotide sequences in this order:
[0020] a nucleotide sequence encoding a signal peptide;
[0021] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP;
[0022] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7;
[0023] a nucleotide sequence encoding a transmembrane domain;
and
[0024] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[2]
[0025] A nucleic acid comprising:
[0026] a nucleotide sequence encoding a chimeric protein,
[0027] wherein the nucleotide sequence is a nucleotide sequence
comprising the following nucleotide sequences in this order:
[0028] a nucleotide sequence encoding a signal peptide;
[0029] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP;
[0030] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7 in this order;
[0031] a nucleotide sequence encoding a transmembrane domain;
and
[0032] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[3]
[0033] The nucleic acid described in [1] or [2], wherein the signal
peptide is a signal peptide of LAMP.
[4]
[0034] The nucleic acid described in any one of [1] to [3], wherein
the transmembrane domain is a transmembrane domain of LAMP.
[5]
[0035] The nucleic acid described in any one of [1] to [4], wherein
the signal peptide consists of the amino acid sequence of amino
acid numbers 1 to 27 of SEQ ID NO: 2, the intra-organelle
stabilizing domain consists of an amino acid sequence of amino acid
numbers 28 to 380 of SEQ ID NO: 2, the allergen domain is an
allergen domain comprising Der p 1 consisting of the amino acid
sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2
consisting of the amino acid sequence of amino acid numbers 599 to
727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence
of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7
consisting of the amino acid sequence of amino acid numbers 805 to
1002 of SEQ ID NO: 2, the transmembrane domain consists of the
amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID
NO: 2, and the endosomal/lysosomal targeting domain consists of the
an amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID
NO: 2.
[6]
[0036] A nucleic acid comprising:
[0037] a nucleotide sequence encoding a chimeric protein consisting
of an amino acid sequence having at least 90% identity to the amino
acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has
an action of inducing Th1-type immunity to an allergen selected
from the group consisting of Der p 1, Der p 2, Der p 23, and Der p
7.
[7]
[0038] A nucleic acid comprising:
[0039] a) a nucleotide sequence encoding a chimeric protein
consisting of the amino acid sequence shown by SEQ ID NO: 2; or
[0040] b) a nucleotide sequence encoding a chimeric protein
consisting of an amino acid sequence in which 1 to 10 amino acids
are deleted, substituted, inserted and/or added in the amino acid
sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an
action of inducing Th1-type immunity to an allergen selected from
the group consisting of Der p 1, Der p 2, Der p 23, and Der p
7.
[8]
[0041] A nucleic acid comprising:
[0042] a nucleotide sequence encoding a chimeric protein consisting
of an amino acid sequence shown by SEQ ID NO: 2.
[9]
[0043] An expression vector comprising:
[0044] the nucleic described in any one of [1] to [8].
[10]
[0045] An expression vector comprising:
[0046] the nucleic acid described in [8].
[11]
[0047] A host cell transformed with the nucleic acid described in
any one of [1] to [8].
[12]
[0048] A method for producing a nucleic acid, comprising:
[0049] culturing a host cell transformed with the nucleic acid
described in any one of [1] to [8].
[13]
[0050] A pharmaceutical composition comprising:
[0051] the expression vector described in [10] and a
pharmaceutically acceptable excipient.
[14]
[0052] The pharmaceutical composition described in [13], which is a
pharmaceutical composition for preventing or treating mite
allergy.
[15]
[0053] A method for preventing or treating mite allergy,
comprising:
[0054] administering a prophylactically effective or
therapeutically effective amount of the expression vector described
in [10].
[16]
[0055] The expression vector described in [10], for use in
preventing or treating mite allergy.
[17]
[0056] Use of the expression vector described in [10] for the
manufacture of a pharmaceutical composition for preventing or
treating mite allergy.
Effects of the Invention
[0057] The nucleic acid of the present invention can be used for
preventing or treating mite allergy.
BRIEF DESCRIPTION OF DRAWINGS
[0058] FIG. 1 is a diagram illustrating production of IgG2a
specific to Der p 1, Der p 2, Der p 23, and Der p 7, which is
induced when the nucleic acid of the present invention is
administered to a mouse. The vertical axis indicates absorbance at
450 nm, and the horizontal axis indicates each administration
group. The horizontal lines indicate arithmetic mean values.
[0059] FIG. 2 illustrates IFN-.gamma. production when spleen cells
of mice to which the nucleic acid of the present invention has been
administered were stimulated with Der p 1 protein, Der p 2 protein,
Der p 7 protein, or Der p 23 protein. The vertical axis indicates
the concentration of IFN-.gamma. in the culture supernatant
(pg/mL), and the horizontal axis indicates each administration
group. The horizontal lines indicate arithmetic mean values. The
dotted line indicates the value of lower limit of detection
(LLOD).
[0060] FIG. 3 illustrates IL-4 production when spleen cells of mice
to which the nucleic acid of the present invention has been
administered were stimulated with Der p 1 protein, Der p 2 protein,
Der p 7 protein, or Der p 23 protein. The vertical axis indicates
the concentration of IL-4 in the culture supernatant (pg/mL), and
the horizontal axis indicates each administration group. The
horizontal lines indicate arithmetic mean values. The dotted line
indicates the value of lower limit of detection (LLOD).
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0061] Hereinafter, the present invention will be described in
detail.
[0062] <Nucleic Acid of the Present Invention>
[0063] Examples of the nucleic acid of the present invention
include a nucleic acid having the following features:
[0064] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0065] a nucleotide sequence encoding a signal peptide,
[0066] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP,
[0067] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7,
[0068] a nucleotide sequence encoding a transmembrane domain,
and
[0069] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[0070] In the present invention, the nucleic acid is a polymer
which is formed by polymerization of nucleotides and consists of a
nucleotide sequence with an arbitrary length. The nucleotides can
include deoxyribonucleotides, ribonucleotides, and/or their
analogs. The nucleic acid of the present invention is DNA, RNA or
modified a nucleic acid thereof. In one embodiment, the nucleic
acid of the present invention is DNA.
[0071] In one embodiment, the nucleic acid of the present invention
is a nucleic acid introduced into an expression vector. In one
embodiment, the nucleic acid of the present invention is a nucleic
acid introduced into a plasmid vector.
[0072] In the specification, "chimeric protein" means a protein
encoded by a nucleotide sequence in which two or more genes are
fused by using genetic recombination technology. The nucleic acid
of the present invention includes a nucleotide sequence encoding
chimeric protein comprising a signal peptide, an intra-organelle
stabilizing domain of LAMP, an allergen domain comprising Der p 1,
Der p 2, Der p 23, and Der p 7, a transmembrane domain, and an
endosomal/lysosomal targeting domain of LAMP in this order
(hereinafter, referred to as "chimeric protein relating to the
present invention").
[0073] LAMP is well-known protein to those skilled in the art (J
Biol Chem., 1991, Vol. 266, p. 21327-21330). In the present
specification, LAMP is not particularly limited, but examples
thereof include LAMP-1, LAMP-2, CD63/LAMP-3, DC-LAMP, and LIMP II,
and homologs, orthologs, paralogs, variants, and modified proteins
thereof. In one embodiment of the present invention, LAMP is
LAMP-1. In the present invention, an animal from which LAMP is
derived is not particularly limited, but in one embodiment, LAMP is
human LAMP. In one embodiment, human LAMP is human LAMP-1. Examples
of an amino acid sequence of human LAMP-1 include an amino acid
sequence in which the amino acid sequence shown by amino acid
numbers 1005 to 1040 of SEQ ID NO: 2 is bound to a C-terminal of
the amino acid sequence shown by amino acid numbers 1 to 380 of SEQ
ID NO: 2.
[0074] The general structure of the signal peptide is well known to
those skilled in the art (Annu Rev Biochem., 2003, Vol. 72, p. 395
to 447). The signal peptide has a function of directing transport
and localization of a protein. As the signal peptide used in the
present invention, any suitable signal peptide can be selected as
long as it has a function of directing transport and localization
of the protein. In one embodiment, the signal peptide used in the
present invention is a signal peptide of LAMP. In one embodiment,
the signal peptide of LAMP used in the present invention is a
signal peptide of LAMP-1.
[0075] In one embodiment, the signal peptide used in the present
invention consists of the following amino acid sequence of (a) or
(b):
[0076] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO:
2; or
[0077] (b) the amino acid sequence of amino acid numbers 1 to 27 of
SEQ ID NO: 2, or an amino acid sequence in which 1 to 3 amino acids
are deleted, substituted, inserted and/or added in the amino acid
sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2.
[0078] The term of "identity" in the present specification means a
value of Identity obtained by using an EMBOSS Needle (Nucleic Acids
Res., 2015, Vol. 43, p. W580-W584;
https://www.ebi.ac.uk/Tools/psa/embossneedle/) with a parameter
prepared by default. The above parameters are as follows.
[0079] Gap Open Penalty=10
[0080] Gap Extend Penalty=0.5
[0081] Matrix=EBLOSUM62
[0082] End Gap Penalty=false
[0083] In one embodiment, the signal peptide used in the present
invention consists of the amino acid sequence of amino acid numbers
1 to 27 of SEQ ID NO: 2.
[0084] The sequence of the intra-organelle stabilizing domain of
LAMP is well known to those skilled in the art (WO 2013/187906).
The intra-organelle stabilizing domain of
[0085] LAMP has a function of protecting the allergen domain from
proteases, low pH, and other substances and conditions that
destabilize a protein. As the intra-organelle stabilizing domain of
LAMP used in the present invention, any suitable intra-organelle
stabilizing domain of LAMP can be selected as long as it has a
function of protecting the allergen domain from proteases, low pH,
and other substances and conditions that destabilize a protein. In
one embodiment, the intra-organelle stabilizing domain of LAMP used
in the present invention is an intra-organelle stabilizing domain
of LAMP-1.
[0086] In one embodiment, the intra-organelle stabilizing domain of
LAMP used in the present invention consists of the following amino
acid sequence of (a) or (b):
[0087] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID
NO: 2; or
[0088] (b) the amino acid sequence of amino acid numbers 28 to 380
of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino
acids are deleted, substituted, inserted and/or added in the amino
acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2.
[0089] In one embodiment, the intra-organelle stabilizing domain of
LAMP used in the present invention consists of the amino acid
sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2.
[0090] The allergen domain used in the present invention includes
Der p 1, Der p 2, Der p 23, and Der p 7 as allergens. Der p 1, Der
p 2, Der p 23, and Der p 7 are allergens that can be observed in
mites (WO 1988/010297; WO 2007/124524; and Clin Exp Allergy., 1995,
Vol. 25, p. 416-422). Der p 1, Der p 2, Der p 23, and Der p 7 used
in the present invention may be variants thereof as long as they
have antigenicity. The antigenicity of any protein can be
confirmed, for example, by observing that administration to an
animal elicits antibody production or T cell response to that
protein (Bioanalysis., 2012, Vol. 4, p. 397-406). In one
embodiment, Der p 1, Der p 2, Der p 23, and Der p 7 used in the
present invention lack the signal peptide.
[0091] In one embodiment, Der p 1 consists of the following amino
acid sequence of (a) or (b):
[0092] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID
NO: 2; or
[0093] (b) the amino acid sequence of amino acid numbers 383 to 594
of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino
acids are deleted, substituted, inserted and/or added in the amino
acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2.
[0094] In one embodiment, Der p 1 consists of the amino acid
sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2.
[0095] In one embodiment, Der p 2 consists of the following amino
acid sequence of (a) or (b):
[0096] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID
NO: 2; or
[0097] (b) the amino acid sequence of amino acid numbers 599 to 727
of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino
acids are deleted, substituted, inserted and/or added in the amino
acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2.
[0098] In one embodiment, Der p 2 consists of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2.
[0099] In one embodiment, Der p 23 consists of the following amino
acid sequence of (a) or (b):
[0100] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID
NO: 2; or
[0101] (b) the amino acid sequence of amino acid numbers 732 to 800
of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino
acids are deleted, substituted, inserted and/or added in the amino
acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2.
[0102] In one embodiment, Der p 23 consists of the amino acid
sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2.
[0103] In one embodiment, Der p 7 consists of the following amino
acid sequence of (a) or (b):
[0104] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID
NO: 2; or
[0105] (b) the amino acid sequence of amino acid numbers 805 to
1002 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10
amino acids are deleted, substituted, inserted and/or added in the
amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO:
2.
[0106] In one embodiment, Der p 7 consists of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2.
[0107] In one embodiment, the allergen domain used in the present
invention comprises Der p 1, Der p 2, Der p 23, and Der p 7 in any
order. In addition, in one embodiment, the allergen domain used in
the present invention comprises Der p 1, Der p 2, Der p 23, and Der
p 7 in this order.
[0108] In one embodiment, the allergen domain used in the present
invention consists of the amino acid sequence of amino acid numbers
383 to 1002 of SEQ ID NO: 2.
[0109] The general structure of the transmembrane domain is well
known to those skilled in the art (Annu Rev Biochem., 2007, Vol.
76, p. 125 to 140). The transmembrane domain has a function of
anchoring proteins to biological membranes. As the transmembrane
domain used in the present invention, any suitable transmembrane
domain protein can be selected as long as it has a function of
anchoring proteins to biological membranes. In one embodiment, the
transmembrane domain used in the present invention is a
transmembrane domain of LAMP. In one embodiment, the transmembrane
domain of LAMP used in the present invention is a transmembrane
domain of LAMP-1.
[0110] In one embodiment, the transmembrane domain used in the
present invention consists of the following amino acid sequence of
(a) or (b):
[0111] (a) an amino acid sequence having at least 90% identity to
the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ
ID NO: 2; or
[0112] (b) the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 2
amino acids are deleted, substituted, inserted and/or added in the
amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID
NO: 2.
[0113] In one embodiment, the transmembrane domain used in the
present invention consists of the amino acid sequence of amino acid
numbers 1006 to 1028 of SEQ ID NO: 2.
[0114] The structure of the endosomal/lysosomal targeting domain of
LAMP is well known to those skilled in the art (WO 1994/017192).
The endosomal/lysosomal targeting domain of LAMP has a function of
transporting a protein to lysosome. As the endosomal/lysosomal
targeting domain of LAMP used in the present invention, any
suitable endosomal/lysosomal targeting domain of LAMP can be
selected as long as it has a function of transporting the protein
to lysosome. In one embodiment, endosomal/lysosomal targeting
domain of LAMP used in the present invention is an
endosomal/lysosomal targeting domain of LAMP-1.
[0115] In one embodiment, the endosomal/lysosomal targeting domain
of LAMP used in the present invention consists of the amino acid
sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2, or an
amino acid sequence in which 1 amino acid is deleted, substituted,
inserted and/or added in the amino acid sequence of amino acid
numbers 1037 to 1040 of SEQ ID NO: 2.
[0116] In one embodiment, the endosomal/lysosomal targeting domain
of LAMP used in the present invention consists of an amino acid
sequence in a range of amino acid numbers 1037 to 1040 of SEQ ID
NO: 2.
[0117] In the chimeric protein relating to the present invention,
the signal peptide, the intra-organelle stabilizing domain of LAMP,
each allergen comprised in the allergen domain, the transmembrane
domain, and the endosomal/lysosomal targeting domain of LAMP may be
directly linked or may be indirectly linked via a linker peptide.
The linker peptide to be used can be appropriately selected by
those skilled in the art. In one embodiment, the linker peptide
consists of 10 or less amino acids. In one embodiment, a linker
peptide used between the intra-organelle stabilizing domain of LAMP
and the allergen domain, between allergens, and between the
allergen domain and the transmembrane domain is a linker peptide
selected from the group consisting of LeuGlu, GlyGlyGlyGly, and
GluPheThr. In one embodiment, the linker peptide used between the
transmembrane domain and the endosomal/lysosomal targeting domain
of LAMP is a linker peptide consisting of the amino acid sequence
of amino acid numbers 1029 to 1036 of SEQ ID NO: 2.
[0118] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0119] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0120] a nucleotide sequence encoding a signal peptide of LAMP,
[0121] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP,
[0122] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7,
[0123] a nucleotide sequence encoding a transmembrane domain of
LAMP, and
[0124] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[0125] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0126] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0127] a nucleotide sequence encoding a signal peptide of
LAMP-1,
[0128] a nucleotide sequence encoding the intra-organelle
stabilizing domain of LAMP-1,
[0129] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7,
[0130] a nucleotide sequence encoding a transmembrane domain of
LAMP-1, and
[0131] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain the of LAMP-1.
[0132] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0133] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0134] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0135] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0136] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2,
[0137] Der p 23 consisting of the amino acid sequence of amino acid
numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the
amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO:
2,
[0138] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0139] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0140] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0141] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0142] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0143] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0144] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
[0145] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2,
[0146] a nucleotide sequence encoding a peptide linker consisting
of the amino acid sequence of amino acid numbers 1029 to 1036 of
SEQ ID NO: 2, and
[0147] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0148] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0149] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0150] a nucleotide sequence encoding a signal peptide,
[0151] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP,
[0152] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
[0153] a nucleotide sequence encoding a transmembrane domain,
and
[0154] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[0155] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0156] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0157] a nucleotide sequence encoding a signal peptide of LAMP,
[0158] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP,
[0159] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
[0160] a nucleotide sequence encoding a transmembrane domain of
LAMP, and
[0161] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP.
[0162] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0163] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0164] a nucleotide sequence encoding a signal peptide of
LAMP-1,
[0165] a nucleotide sequence encoding the intra-organelle
stabilizing domain of LAMP-1,
[0166] a nucleotide sequence encoding an allergen domain comprising
Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
[0167] a nucleotide sequence encoding a transmembrane domain of
LAMP-1, and
[0168] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP-1.
[0169] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0170] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0171] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0172] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0173] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0174] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0175] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0176] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0177] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0178] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0179] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0180] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0181] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2,
[0182] a nucleotide sequence encoding a peptide linker consisting
of the amino acid sequence of amino acid numbers 1029 to 1036 of
SEQ ID NO: 2, and
[0183] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0184] The nucleic acid of the present invention is not
particularly limited as long as it encodes the chimeric protein
relating to the present invention, and has an action of inducing
Th1-type immunity with respect to the allergen selected from the
group consisting of Der p 1, Der p 2, Der p 23, and Der p 7, when
the nucleic acid is administered to a human or an animal. One can
confirm whether or not a certain nucleic acid has an action of
inducing Th1-type immunity, when the nucleic acid is administered
to a human or an animal, by the method described in, for example,
Example 3 and/or Example 4. In addition, the nucleic acid of the
present invention may be a nucleic acid having an action of
inducing Th1 cell dominant immune response, when the nucleic acid
is administered to a human or an animal. One can confirm whether or
not a certain nucleic acid has an action of inducing Th1 cell
dominant immune response, when the nucleic acid is administered to
human or animal, by the method described in, for example, Example
4.
[0185] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0186] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of an amino acid sequence having at
least 90%, 92%, 94%, 96%, 98%, or 99% identity to the amino acid
sequence shown by SEQ ID NO: 2.
[0187] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0188] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of an amino acid sequence having at
least 90% identity to the amino acid sequence shown by SEQ ID NO:
2.
[0189] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0190] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of the amino acid sequence shown by SEQ
ID NO: 2, or a chimeric protein consisting of an amino acid
sequence in which 1 to 10 amino acids are deleted, substituted,
inserted and/or added in the amino acid sequence shown by SEQ ID
NO: 2.
[0191] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0192] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of an amino acid sequence having at
least 90% identity to the amino acid sequence shown by SEQ ID NO:
2,
[0193] wherein the nucleic acid has an action of inducing Th1-type
immunity to the allergen selected from the group consisting of Der
p 1, Der p 2, Der p 23, and Der p 7.
[0194] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0195] a) a nucleic acid comprising a nucleotide sequence encoding
a chimeric protein consisting of the amino acid sequence shown by
SEQ ID NO: 2 or
[0196] b) a nucleic acid comprising a nucleotide sequence encoding
a chimeric protein consisting of an amino acid sequence in which 1
to 10 amino acids are deleted, substituted, inserted and/or added
in the amino acid sequence shown by SEQ ID NO: 2, wherein the
nucleic acid has an action of inducing Th1-type immunity to an
allergen selected from the group consisting of Der p 1, Der p 2,
Der p 23, and Der p 7.
[0197] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0198] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of an amino acid sequence having at
least 90% identity to the amino acid sequence shown by SEQ ID NO:
2, wherein the nucleic acid has an action of inducing Th1-type
immunity to Der p 1, Der p 2, Der p 23, and Der p 7.
[0199] In one embodiment, the nucleic acid of the present invention
is the following nucleic acid:
[0200] a) a nucleic acid comprising a nucleotide sequence encoding
a chimeric protein consisting of the amino acid sequence shown by
SEQ ID NO: 2 or
[0201] b) a nucleic acid comprising a nucleotide sequence encoding
a chimeric protein consisting of an amino acid sequence in which 1
to 10 amino acids are deleted, substituted, inserted and/or added
in the amino acid sequence shown by SEQ ID NO: 2, wherein the
nucleic acid has an action of inducing Th1-type immunity to Der p
1, Der p 2, Der p 23, and Der p 7.
[0202] In one embodiment, the nucleic acid of the present invention
is the following nucleic acids
[0203] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein consisting of the amino acid sequence shown by SEQ
ID NO: 2.
[0204] In one embodiment, the nucleotide sequence encoding the
chimeric protein consisting of the amino acid sequence shown by SEQ
ID NO: 2 means the nucleotide sequence shown by SEQ ID NO: 1.
[0205] Based on the nucleotide sequence, the nucleic acid of the
present invention can be easily prepared by those skilled in the
art by using methods known in the art. For example, the nucleic
acid of the present invention can be synthesized by using gene
synthesis methods known in the art. As such a gene synthesis
method, various methods known to those skilled in the art such as a
method for synthesizing an antibody gene described in WO 90/07861
can be used.
[0206] Once being synthesized, the nucleic acid of the present
invention can be easily replicated by those skilled in the art
using methods known in the art. For example, the nucleic acid of
the present invention can be replicated by the method described
later in <Method for producing the nucleic acid of the present
invention and nucleic acid which can be produced by the
method>.
[0207] <Expression Vector of the Present Invention>
[0208] The expression vector of the present invention includes an
expression vector comprising the nucleic acid of the present
invention.
[0209] The expression vector used to express a chimeric protein
from the nucleic acid of the present invention is not particularly
limited as long as it can express the chimeric protein from the
nucleic acid of the present invention in the animal cells. In one
embodiment, the expression vector used to express a chimeric
protein from the nucleic acid of the present invention is an
expression vector which can be used for expressing the chimeric
protein in a human body. Examples of the expression vector used in
the present invention include a plasmid vector, a viral vector (for
example, adenovirus, retrovirus, adeno-associated virus) and the
like. In one embodiment, the expression vector of the present
invention is a plasmid vector. In the present specification,
"plasmid" means the plasmid vector.
[0210] The expression vector of the present invention may comprise
a promoter operably linked to the nucleic acid of the present
invention. Examples of the promoter for expressing the chimeric
protein from the nucleic acid of the present invention in animal
cells include a virus-derived promoter such as CMV
(cytomegalovirus), RSV (respiratory syncytial virus), and SV40
(simian virus 40), an actin promoter, EF (elongation factor)
1.alpha. promoter, a heat shock promoter and the like. In one
embodiment, the promoter comprised in the expression vector of the
present invention is a CMV promoter. The expression vector of the
present invention may comprise a start codon and a stop codon. In
this case, an enhancer sequence, an untranslated region, a splicing
junction, a polyadenylation site, or a replicable unit may be
comprised.
[0211] In one embodiment, the expression vector of the present
invention is an expression vector comprising the following nucleic
acid:
[0212] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0213] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0214] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0215] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
[0216] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0217] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0218] In one embodiment, the expression vector of the present
invention is an expression vector comprising the following nucleic
acid:
[0219] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0220] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0221] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0222] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
[0223] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2,
[0224] a nucleotide sequence encoding a peptide linker consisting
of the amino acid sequence of amino acid numbers 1029 to 1036 of
SEQ ID NO: 2, and
[0225] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0226] In one embodiment, the expression vector of the present
invention is an expression vector comprising the following nucleic
acid:
[0227] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0228] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0229] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0230] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0231] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0232] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0233] In one embodiment, the expression vector of the present
invention is an expression vector comprising the following nucleic
acid:
[0234] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0235] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0236] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0237] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0238] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2,
[0239] a nucleotide sequence encoding a peptide linker consisting
of the amino acid sequence of amino acid numbers 1029 to 1036 of
SEQ ID NO: 2, and
[0240] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0241] In one embodiment, the expression vector of the present
invention is an expression vector comprising a nucleic acid
comprising a nucleotide sequence encoding a chimeric protein
consisting of the amino acid sequence shown by SEQ ID NO: 2.
[0242] In one embodiment, the expression vector of the present
invention is an expression vector comprising a nucleic acid
comprising the nucleotide sequence shown by SEQ ID NO: 1.
[0243] In one embodiment, the expression vector of the present
invention is an expression vector comprising a nucleic acid
consisting of the nucleotide sequence shown by SEQ ID NO: 3.
[0244] <Host Cell of the Present Invention>
[0245] The host cell of the present invention includes a host cell
transformed with the nucleic acid of the present invention. In one
embodiment, the host cell of the present invention is a host cell
transformed with the expression vector of the present invention. In
one embodiment, the host cell of the present invention is a host
cell transformed with the expression vector of the present
invention which is a plasmid vector.
[0246] The host cell transformed with the nucleic acid of the
present invention is not particularly limited, and any cell known
in the art can be selected as long as it is a cell that can be used
for nucleic acid replication.
[0247] Examples of the host cell that can be used for nucleic acid
replication include various cells such as natural cells or
artificially established cells commonly used in the technical field
of the present invention (for example, animal cells (for example,
CHOK1SV cells), insect cells (for example, Sf9), bacteria (for
example, E. coli), and yeasts (for example, Saccharomyces and
Pichia)). In one embodiment, E. coli can be used as a host cell.
Transformation itself can be carried out by known methods.
[0248] <Method for Producing the Nucleic Acid of the Present
Invention and Nucleic Acid which can be Produced by the
Method>
[0249] Examples of the method for producing the nucleic acid of the
present invention include a method for producing a nucleic acid or
an expression vector, which comprises a step of culturing host
cells transformed with the nucleic acid or the expression vector of
the present invention. In one embodiment, the method for producing
the nucleic acid of the present invention comprises a step of
culturing the host cell transformed with the nucleic acid of the
present invention, and replicating the nucleic acid of the present
invention. In one embodiment, the method for producing the nucleic
acid of the present invention comprises a step of culturing the
host cell transformed with the expression vector of the present
invention, and replicating the expression vector of the present
invention.
[0250] In one embodiment, the host cell used in the method for
producing the nucleic acid of the present invention is E. coli. For
culture of E. coli, an appropriate culture medium such as LB
medium, M9 medium, Terrific Broth medium, SOB medium, SOC medium,
or 2.times.YT medium can be selected. In addition, the culturing of
E. coli can be carried out in an environment where carbon (it is
not particularly limited as long as it is an assimilable carbon
compound; for example, polyols such as glycerin, or organic acids
such as pyruvic acid, succinic acid, or citric acid), nitrogen (it
is not particularly limited as long as it is a nitrogen compound
that can be used by E. coli; for example, peptone, meat extract,
yeast extract, casein hydrolysate, soybean meal alkaline extract,
or ammonia or a salt thereof), inorganics and inorganic ions (it is
not particularly limited, and examples thereof include phosphate,
carbonate, sulfate, magnesium, calcium, potassium, iron, manganese
and zinc), a vitamin source, and an antifoaming agent are
controlled to an appropriate concentration. In addition, the
control of culturing includes control of parameters such as pH,
temperature, stir, air flow and dissolved oxygen. In one
embodiment, the conditions of culturing include pH of 6.7 to 7.5,
temperature of 20.degree. C. to 37.degree. C., and a stirring speed
of 200 to 300 rpm.
[0251] The method for producing the nucleic acid of the present
invention may comprise a step of obtaining lysate from collected
culture solutions. The lysate can be obtained, for example, by
treating the collected culture solutions with an alkaline lysis
method or boiling method. Also, the step of obtaining the lysate
may include a step of sterile filtration of a final lysate
material.
[0252] The method for producing the nucleic acid of the present
invention may further comprise a step of purifying nucleic acid or
an expression vector from lysate. Ion exchange chromatography
and/or hydrophobic interaction chromatography can be used to purify
the nucleic acid or the expression vector from the lysate. The step
of purifying the nucleic acid or the expression vector from the
lysate may include a step of ultrafiltration and/or diafiltration.
In addition, as a final treatment of the purification step, a
sterile filtration step may be comprised.
[0253] In one embodiment, the nucleic acid of the present invention
is a nucleic acid produced by the method for producing the nucleic
acid of the present invention.
[0254] In one embodiment, the expression vector of the present
invention is an expression vector produced by the method for
producing the nucleic acid of the present invention.
[0255] <Pharmaceutical Composition of the Present
Invention>
[0256] The pharmaceutical composition of the present invention
includes a pharmaceutical composition comprising the nucleic acid
of the present invention and a pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition comprising the
vector of the present invention and the pharmaceutically acceptable
excipient. The pharmaceutical composition of the present invention
can be prepared by a generally used method with an excipient
generally used in the field, that is, a pharmaceutical excipient, a
pharmaceutical carrier or the like. Examples of dosage forms of
these pharmaceutical compositions include, for example, parenteral
agents such as injections and drip agents, which can be
administered by intravenous administration, subcutaneous
administration, intradermal administration, and intramuscular
administration. In formulating, excipients, carriers, additives,
and the like can be used according to these dosage forms within the
pharmaceutically acceptable range.
[0257] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition comprising the
nucleic acid or the expression vector of the present invention and
the pharmaceutically acceptable excipient.
[0258] While the administration amount of the nucleic acid of the
present invention or the expression vector varies depending on the
degree of symptoms and age of the patient, and the dosage form of
the preparation used, for example, the amount in a range of 0.001
mg/kg to 100 mg/kg can be used. Further, it is possible to prepare
a formulation by adding the nucleic acid or the expression vector
of the present invention in an amount corresponding to such
administration amount.
[0259] The pharmaceutical composition of the present invention can
be used as an agent for preventing or treating allergy caused by an
allergen selected from Der p 1, Der p 2, Der p 23, and Der p 7.
Further, the pharmaceutical composition of the present invention
can be used as an agent for prevention or treating the mite
allergy.
[0260] The present invention includes a pharmaceutical composition
for preventing or treating allergy, comprising the nucleic acid of
the present invention. In addition, the present invention includes
a method for preventing or treating allergy, comprising
administering a prophylactically effective or therapeutically
effective amount of the nucleic acid of the present invention. The
present invention also includes the nucleic acid of the present
invention for use in preventing or treating allergy. In addition,
the present invention includes use of the nucleic acid of the
present invention for the manufacture of a pharmaceutical
composition for preventing or treating allergy. In one embodiment,
the above-described allergy is allergy caused by an allergen
selected from the group consisting of Der p 1, Der p 2, Der p 23,
and Der p 7. In addition, in one embodiment, the above-described
allergy is allergy affecting an allergy patient having an antibody
that responds to an allergen selected from the group consisting of
Der p 1, Der p 2, Der p 23, and Der p 7. Further, in one
embodiment, the above-described allergy is mite allergy.
[0261] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising the following nucleic acid and a
pharmaceutically acceptable excipient:
[0262] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0263] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0264] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0265] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
[0266] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0267] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0268] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising the following nucleic acid and a
pharmaceutically acceptable excipient:
[0269] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0270] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0271] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0272] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0273] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0274] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0275] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising a nucleic acid comprising a nucleotide
sequence encoding a chimeric protein consisting of the amino acid
sequence shown by SEQ ID NO: 2 and a pharmaceutically acceptable
excipient.
[0276] The present invention includes a pharmaceutical composition
for preventing or treating allergy, comprising the expression
vector of the present invention. In addition, the present invention
includes a method for preventing or treating allergy, comprising
administering a prophylactically effective or therapeutically
effective amount of the expression vector of the present invention.
The present invention also includes the expression vector of the
present invention for use in preventing or treating allergy. In
addition, the present invention includes use of the expression
vector of the present invention for the manufacture of a
pharmaceutical composition for preventing or treating allergy. In
one embodiment, the above-described allergy is allergy caused by an
allergen selected from the group consisting of Der p 1, Der p 2,
Der p 23, and Der p 7. In addition, in one embodiment, the
above-described allergy is allergy affecting an allergy patient
having an antibody that responds to an allergen selected from the
group consisting of Der p 1, Der p 2, Der p 23, and Der p 7.
Further, in one embodiment, the above-described allergy is mite
allergy.
[0277] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising an expression vector comprising the
following nucleic acid and a pharmaceutically acceptable
excipient:
[0278] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0279] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0280] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0281] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
[0282] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0283] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0284] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising an expression vector comprising the
following nucleic acid and a pharmaceutically acceptable
excipient:
[0285] a nucleic acid comprising a nucleotide sequence encoding a
chimeric protein, wherein the nucleotide sequence is a nucleotide
sequence comprising the following nucleotide sequences in this
order:
[0286] a nucleotide sequence encoding a signal peptide consisting
of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID
NO: 2,
[0287] a nucleotide sequence encoding an intra-organelle
stabilizing domain of LAMP consisting of the amino acid sequence of
amino acid numbers 28 to 380 of SEQ ID NO: 2,
[0288] a nucleotide sequence encoding an allergen domain comprising
Der p 1 consisting of the amino acid sequence of amino acid numbers
383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid
sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23
consisting of the amino acid sequence of amino acid numbers 732 to
800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid
sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this
order,
[0289] a nucleotide sequence encoding a transmembrane domain
consisting of the amino acid sequence of amino acid numbers 1006 to
1028 of SEQ ID NO: 2, and
[0290] a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP consisting of the amino acid sequence of
amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[0291] In one embodiment, the pharmaceutical composition of the
present invention is a pharmaceutical composition for preventing or
treating allergy, comprising an expression vector comprising a
nucleic acid comprising a nucleotide sequence encoding a chimeric
protein consisting of the amino acid sequence shown by SEQ ID NO: 2
and a pharmaceutically acceptable excipient.
[0292] Specific examples are provided herein for reference in order
to obtain further understanding of the present invention; however,
these examples are for the purpose of illustration and the present
invention is not limited thereto.
EXAMPLES
Example 1: Construction of LAMP-Der p 1-Der p 2-Der p 23-Der p 7
Plasmid
[0293] LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid consisting of
the nucleotide sequence shown by SEQ ID NO: 3 (an expression vector
comprising a nucleic acid comprising a nucleotide sequence
comprising the following nucleotide sequences in this order (that
is, a nucleotide sequence encoding a chimeric protein consisting of
the amino acid sequence shown by SEQ ID NO: 2): a nucleotide
sequence encoding a signal peptide of LAMP-1 (the amino acid
sequence of 1 to 27 of SEQ ID NO: 2), a nucleotide sequence
encoding an intra-organelle stabilizing domain of LAMP-1 (the amino
acid sequence of 28 to 380 of SEQ ID NO: 2), a nucleotide sequence
encoding an allergen domain comprising Der p 1, Der p 2, Der p 23,
and Der p 7 in this order (the amino acid sequence of 383 to 1002
of SEQ ID NO: 2), a nucleotide sequence encoding a transmembrane
domain of LAMP-1 (the amino acid sequence of 1006 to 1028 of SEQ ID
NO: 2), and a nucleotide sequence encoding an endosomal/lysosomal
targeting domain of LAMP-1 (the amino acid sequence of 1037 to 1040
of SEQ ID NO: 2)) was constructed. The plasmid can be constructed
by inserting synthetic DNA, in which Xho I recognition sequence is
added to 5' end of the nucleotide sequence of 1147 to 3006 of SEQ
ID NO: 1 (a nucleotide sequence encoding an allergen domain
comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order)
and Eco RI recognition sequence is added to the 3' end of the
nucleic acid sequence, into Eco RI-Xho I site of the plasmid shown
by SEQ ID NO: 6 of Japanese Patent No. 5807994. E. coli was
transformed with the constructed LAMP-Der p 1-Der p 2-Der p 23-Der
p 7 plasmid and cultured in a liquid medium. The amplified LAMP-Der
p 1-Der p 2-Der p 23-Der p 7 plasmid was obtained by a method of
centrifuging the culture solution and collecting the cells based on
a general plasmid extraction and purification method (miniprep
method).
Example 2: Expression of LAMP-Der p 1-Der p 2-Der p 23-Der p 7
Chimeric Protein
[0294] In vitro expression of the LAMP-Der p 1-Der p 2-Der p 23-Der
p 7 chimeric protein (a chimeric protein consisting of an amino
acid sequence encoded by the nucleotide sequence shown by SEQ ID
NO: 1 (that is, the amino acid sequence shown by SEQ ID NO: 2)) by
using human fetal kidney-derived 293T cell line was evaluated.
[0295] (1) Cell Culture and Plasmid Introduction
[0296] Human fetal kidney-derived 293T cells (Thermo Fisher
Scientific, Cat. HCL4517) were seeded in 6-well plates (Cat.
3810-006 manufactured by IWAKI) at 3.times.10.sup.5 cells/well in
D-MEM medium (Sigma-Aldrich, Cat. D5796) containing 10% fetal
bovine serum (Hyclone, Cat. SH30070.03) and 100-fold diluted
penicillin-streptomycin (Thermo Fisher Scientific, Cat. 15070063).
After overnight culture of the seeded cells at 37.degree. C. in the
presence of 5% CO.sub.2, a mixed solution having a ratio of
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid:Lipofectamine 2000
(Thermo Fisher Scientific, Cat. 11668027)=2.5 (.mu.g):10 (.mu.L)
was added. After overnight culture of the seeded cells at
37.degree. C. in the presence of 5% CO.sub.2 again, the medium was
removed and washed once with PBS, and then western blotting was
performed.
[0297] (2) Western Blotting
[0298] Pretreatment: Cells were lysed in RIPA buffer (Pierce, Cat.
89900) containing a protease inhibitor (Sigma-Aldrich, Cat.
1873580), and the protein concentration of the supernatant after
centrifugation at 20,000.times.g for 5 minutes was measured. To 5
.mu.L of the cell lysate diluted with PBS containing protease
inhibitor, 5 .mu.L of LDS sample buffer (Thermo Fisher Scientific,
Cat. NP0007) containing 100 mM DTT was added so that the protein
concentration would be 200 .mu.g/mL, and heat-treated at 70.degree.
C. for 10 minutes.
[0299] SDS-PAGE: Using NuPAGE (Registered trademark) MOPS SDS
Running buffer (Thermo Fisher Scientific, Cat. NP0001) and NuPAGE
(Registered trademark) 4%-12% Bis-Tris Gel (Thermo Fisher
Scientific, Cat. NP0323), the above-mentioned pretreated cell
lysate was applied to the gel and electrophoresis was performed at
a constant voltage of 200 V.
[0300] Blotting: Blotting was performed by bringing PVDF membrane
(Thermo Fisher Scientific, Cat. LC2005) into contact with the gel
after SDS-PAGE, and electrifying for 90 minutes at 180 mA in XCell
II Blot Module (Thermo Fisher Scientific, Cat. EI9051) filled with
NuPAGE (Registered trademark) Transfer buffer (Thermo Fisher
Scientific, Cat. NP0006) containing 20% of methanol.
[0301] Blocking: The membrane after electrification was immersed in
Blocking One (Nacalai Tesque, Cat. 03953-95) and shaken at room
temperature for one hour.
[0302] Primary antibody: Anti-human LAMP-1 antibody (Sino
biological, Cat. 11215-RP01) was added at 1000-fold dilution in TBS
Tween-20 buffer (Thermo Fisher Scientific, Cat. 28360) containing
10% of Blocking One. The membrane was immersed in this buffer and
shaken overnight at 4.degree. C.
[0303] Secondary antibody: The membrane was washed with TBS
Tween-20 buffer. Anti-rabbit IgG (H+L chain) pAb-HRP (MBL, Cat.
458) was added at 3000-fold dilution in TBS Tween-20 buffer
containing 10% of Blocking One. The membrane was immersed in this
buffer and shaken at room temperature for one hour.
[0304] Detection: The membrane was washed with TBS Tween-20 buffer.
The membrane was immersed in ECL prime western blotting detection
reagent (GE Healthcare, Cat. RPN2232), and an image was detected
with LumiVision PRO 400EX (Aisin Seiki Co., Ltd.). In the image, a
band responsive to the anti-human LAMP-1 antibody corresponding to
the chimeric protein was detected.
[0305] As the result of the above-mentioned tests, it was confirmed
that LAMP-Der p 1-Der p 2-Der p 23-Der p 7 chimeric protein in the
cell was expressed by introducing the LAMP-Der p 1-Der p 2-Der p
23-Der p 7 plasmid to the human fetal kidney-derived 293T cell
line.
Example 3: Induction of IgG2a Production by Administration of
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 Plasmid
[0306] Evaluation of induction of antibody production in vivo was
performed. In eight examples in each group, 25 .mu.L of a PBS
solution containing 50 .mu.g of LAMP-Der p 1-Der p 2-Der p 23-Der p
7 plasmid was administered in the ear of 7-week-old BALB/c female
mice (Charles River Laboratories Japan, Inc.) at the start of
administration intradermally three times every week (Day 0, 7 and
14). One week after the final administration, blood was collected
and plasma samples were obtained (Day 21). As a control, LAMP-Der p
23-Der p 7-Der p 2-Der p 1 plasmid (an expression vector comprising
a nucleic acid comprising a nucleotide sequence comprising the
following nucleotide sequences in this order: a nucleotide sequence
encoding the amino acid sequence of amino acid numbers 1 to 380 of
SEQ ID NO: 2 (hereinafter, refer to as N-terminal of LAMP-1 in
Examples 3 and 4), a nucleotide sequence encoding an allergen
domain comprising Der p 23 consisting of the amino acid sequence of
amino acid numbers 732 to 800 of SEQ ID NO: 2 (hereinafter, refer
to as Der p 23 domain in Examples 3 and 4), Der p 7 consisting of
the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID
NO: 2 (hereinafter, refer to as Der p 7 domain in Examples 3 and
4), Der p 2 consisting of the amino acid sequence of amino acid
numbers 599 to 727 of SEQ ID NO: 2 (hereinafter, refer to as Der p
2 domain in Examples 3 and 4), and Der p 1 consisting of the amino
acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2
(hereinafter, refer to as Der p 1 domain in Examples 3 and 4) in
this order, and a nucleotide sequence encoding the amino acid
sequence of amino acid numbers 1006 to 1040 of SEQ ID NO: 2
(hereinafter, refer to as C-terminal of LAMP-1 in Examples 3 and
4)); a mixture of LAMP-Der p 1-Der p 2 plasmid (an expression
vector comprising a nucleic acid comprising a nucleotide sequence
comprising the following nucleotide sequences in this order: a
nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide
sequence encoding an allergen domain comprising Der p 1 domain and
Der p 2 domain in this order, and a nucleotide sequence encoding
C-terminal of LAMP-1) and LAMP-Der p 23-Der p 7 plasmid (an
expression vector comprising a nucleic acid comprising a nucleotide
sequence comprising the following nucleotide sequences in this
order: a nucleotide sequence encoding N-terminal of LAMP-1, a
nucleotide sequence encoding allergen domain containing Der p 23
domain and Der p 7 domain in this order, and a nucleotide sequence
encoding C-terminal of LAMP-1); and a mixture of LAMP-Der p 1
plasmid (an expression vector comprising a nucleic acid comprising
a nucleotide sequences comprising the following nucleotide
sequences in this order: a nucleotide sequence encoding N-terminal
of LAMP-1, a nucleotide sequence encoding an allergen domain
comprising Der p 1 domain, and a nucleotide sequence encoding
C-terminal of LAMP-1), LAMP-Der p 2 plasmid (an expression vector
comprising a nucleic acid comprising a nucleotide sequence
comprising the following nucleotide sequences in this order: a
nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide
sequence encoding an allergen domain comprising Der p 2 domain, and
a nucleotide sequence encoding C-terminal of LAMP-1), LAMP-Der p 7
plasmid (an expression vector comprising a nucleic acid comprising
a nucleotide sequence comprising the following nucleotide sequences
in this order: a nucleotide sequence encoding N-terminal of LAMP-1,
a nucleotide sequence encoding an allergen domain comprising Der p
7 domain, and a nucleotide sequence encoding C-terminal of LAMP-1),
and LAMP-Der p 23 plasmid (an expression vector comprising a
nucleic acid comprising a nucleotide sequence comprising the
following nucleotide sequences in this order: a nucleotide sequence
encoding N-terminal of LAMP-1, a nucleotide sequence encoding an
allergen domain comprising Der p 23 domain, and a nucleotide
sequence encoding C-terminal of LAMP-1) were prepared. Each control
plasmid can be prepared by the same method as the method described
in Example 1. To mice, 25 .mu.L of PBS solution containing 50 .mu.g
of the above plasmid or the above plasmid mixture or 25 .mu.L of
PBS was administered. An antibody titer was measured by ELISA using
a 100-fold or 1000-fold diluted plasma sample, and the absorbance
at 450 nm was measured. ELISA measurement was performed based on a
general ELISA method using F96 MAXISORP NUNC-IMMUNO PLATE (Nunc,
Cat. 439454) as a test plate. Der p 1 which is a purified protein
(Indoor biotechnologies, NA-DP1-1, lot: 38052), Der p 2 which is a
purified protein (Indoor biotechnologies, NA-DP2-1, lot: 36118),
Der p 7 which is a recombinant purified protein (Indoor
biotechnologies, RP-DP7-1, lot: 34033), or Der p 23 which is a
recombinant purified protein (Sysmex, UniProtKB: A0A0K2DQU8) is
prepared to 1 .mu.g/mL with PBS, added at 50 .mu.L/well and allowed
to stand overnight at 4.degree. C. After washing a test plate three
times with a washing buffer (PBS Tween-20 buffer; Thermo Fisher
Scientific, Cat. 28352), 100 .mu.L/well of PBS containing 1% of BSA
(Sigma-Aldrich, Cat. A8022) was added and allowed to stand at room
temperature for one hour. After washing three times with the
washing buffer, 50 .mu.L/well of a 100-fold or 1000-fold diluted
plasma sample in PBS containing 1% of BSA was added and allowed to
stand at room temperature for one hour. After washing three times
with the washing buffer, 50 .mu.L/well of a 50000-fold diluted
secondary antibody, Goat anti-mouse IgG2a HRP Conjugated (Bethyl
Laboratories, Cat. A90-107P), in PBS containing 1% of BSA was
added, and the test plate was allowed to stand at room temperature
for one hour. After washing three times with the washing buffer, 50
.mu.L/well of TMB Microwell Peroxidase Substrate System (SeraCare
Life Sciences, Inc., Cat. 50-76-03) which is a substrate solution
was added and the plate was allowed to stand at room temperature
for 15 minutes with blocking light. A reaction stop solution (2N
H.sub.2SO.sub.4) was added at 50 .mu.L/well and absorbance at 450
nm was measured.
[0307] As the result of the above-mentioned tests, the production
of Der p 1, Der p 2, Der p 23 and Der p 7 specific IgG2a was
detected by administering LAMP-Der p 1-Der p 2-Der p 23-Der p 7
plasmid (Der p1-p2-p23-p7) to mice (FIG. 1). On the other hand,
even when LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid (Der
p23-p7-p2-p1), a mixture of LAMP-Der p 1-Der p 2 plasmid and
LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7), and a mixture
of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid
and LAMP-Der p 23 plasmid (4 plasmid mix) were administered to the
mice, the production of Der p 1-specific IgG2a was not detected. In
addition, when a mixture of LAMP-Der p 1-Der p 2 plasmid and
LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7) is
administered to mice, the production of Der p 2 specific IgG 2a was
not detected as well. That is, it was only the LAMP-Der p 1-Der p
2-Der p 23-Der p 7 plasmid that the production of IgG2a specific
for all allergens encoded in the plasmid was detected. From the
above results, it has been suggested that among the tested
plasmids, only LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid
induces Th1 immune responses for all the allergens and causes class
switch of activated B cells to the IgG2a isotype.
Example 4: Induction of IFN-.gamma. and IL-4 Production by LAMP-Der
p 1-Der p 2-Der p 23-Der p 7 Plasmid
[0308] Evaluation of cytokine production induction upon stimulation
with allergen was performed on splenocytes collected from mice
administered with LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid.
Splenocytes were prepared according to a general method from the
mice used in Example 3 and the mice administered with a control
plasmid (an expression vector comprising a nucleic acid comprising
a nucleotide sequence comprising the following nucleotide sequences
in this order: a nucleotide sequence encoding N-terminal of LAMP-1
and a nucleotide sequence encoding C-terminal of LAMP-1) with the
same protocol as in Example 3 on Day 63. The control plasmid can be
prepared by deleting Eco RI-Xho I site of the plasmid shown by SEQ
ID NO: 6 of Japanese Patent No. 5807994. Splenocytes were seeded in
96-well plates (Cat. 3860-096 manufactured by IWAKI) at
8.times.10.sup.5 cells/well in RPMI-1640 medium (Sigma-Aldrich,
Cat. R8758) containing 10% fetal bovine serum (Hyclone, Cat.
SH30070.03) and 100-fold diluted penicillin-streptomycin
(ThermoFisher Scientific, Cat. 15070063). Der p 1 (Indoor
biotechnologies, NA-DP1-1, lot: 38052), Der p 2 (Indoor
biotechnologies, NA-DP2-1, lot: 36118), Der p 7 (Indoor
biotechnologies, RP-DP7-1, lot: 34033), or Der p 23 (Sysmex,
UniProtKB: A0A0K2DQU8) were added such that the final
concentrations thereof were respectively 3, 3, 3, and 1.3 .mu.g/mL.
Culturing was performed at 37.degree. C. under 5% of CO.sub.2 for
72 hours. The concentrations of IFN-.gamma. and IL-4 in the culture
supernatant were measured by ELISA method. A supernatant sample
diluted 10-fold with TBS containing 0.1% BSA and 0.05% Tween 20 was
used for the measurement of IFN-.gamma., and a supernatant
undiluted sample was used for the measurement of IL-4. As a test
plate for ELISA measurement, F96 MAXISORP NUNC-IMMUNO PLATE (Nunc,
Cat. 439454) was used. The measurement was carried out using mouse
IFN-.gamma. DuoSet ELISA (R&D Systems, Cat. DY485) and mouse
IL-4 DuoSet ELISA (R&D Systems, Cat. DY 404) according to
attached protocol. As the result of the above-mentioned test, a
mite-derived allergen-specific IFN-.gamma. production was induced
by administering 50 .mu.g of LAMP-Der p 1-Der p 2-Der p 23-Der p 7
plasmid (Der p 1-p2-p 23-p 7) to mice three times (FIG. 2). Also,
even in a case where LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid
(Der p23-p7-p2-p1), a mixture of LAMP-Der p 1-Der p 2 plasmid and
LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7), and a mixture
of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7
plasmid, and LAMP-Der p 23 plasmid (4 plasmid mix) were
administered to the mice three times, comparable mite-derived
allergen-specific IFN-.gamma. production was induced. On the other
hand, in the mice administered three times with 50 .mu.g of
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid (Der p 1-p2-p23-p'7),
the mite-derived allergen-specific IL-4 production was the lower
limit of detection (FIG. 3). Even in a case where LAMP-Der p 23-Der
p 7-Der p 2-Der p 1 plasmid (Der p23-p7-p2-p1), a mixture of
LAMP-Der p 1-Der p 2 plasmid and LAMP-Der p 23-Der p 7 plasmid (Der
p1-p2+Der p23-p'7), and a mixture of LAMP-Der p 1 plasmid, LAMP-Der
p 2 plasmid, LAMP-Der p 7 plasmid, and LAMP-Der p 23 plasmid (4
plasmid mix) were administered to the mice three times, the
mite-derived allergen-specific IL-4 production was below the lower
limit of detection.
[0309] As the result of the above-mentioned tests, a mixture of
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid, LAMP-Der p 23-Der p
7-Der p 2-Der p 1 plasmid, LAMP-Der p 1-Der p 2 plasmid, and
LAMP-Der p 23-Der p 7 plasmid, and a mixture of LAMP-Der p 1
plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid, and LAMP-Der p
23 plasmid have been shown to induce Th1 cell dominant immune
responses.
INDUSTRIAL APPLICABILITY
[0310] The nucleic acid of the present invention is expected to be
useful for the prevention or treatment of mite allergy. In
addition, the method for producing the nucleic acid of the present
invention is useful for producing the nucleic acid.
Sequence Listing Free Text
[0311] The numerical heading <223> in the following sequence
listing describes the description of "Artificial Sequence".
Specifically, the nucleotide sequence shown by SEQ ID NO: 1 in the
sequence listing is a nucleotide sequence encoding LAMP-Der p 1-Der
p 2-Der p 23-Der p 7 chimeric protein, and the amino acid sequence
shown by SEQ ID NO: 2 in the sequence listing is the amino acid
sequence encoded by SEQ ID NO: 1. In addition, the nucleotide
sequence shown by SEQ ID NO: 3 is the nucleotide sequence of
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid.
Sequence CWU 1
1
313123DNAArtificial SequenceNucleic acid encoding a chimeric
proteinCDS(1)..(3123) 1atg gcg ccc cgc agc gcc cgg cga ccc ctg ctg
ctg cta ctg ctg ttg 48Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu
Leu Leu Leu Leu Leu1 5 10 15ctg ctg ctc ggc ctc atg cat tgt gcg tca
gca gca atg ttt atg gtg 96Leu Leu Leu Gly Leu Met His Cys Ala Ser
Ala Ala Met Phe Met Val 20 25 30aaa aat ggc aac ggg acc gcg tgc ata
atg gcc aac ttc tct gct gcc 144Lys Asn Gly Asn Gly Thr Ala Cys Ile
Met Ala Asn Phe Ser Ala Ala 35 40 45ttc tca gtg aac tac gac acc aag
agt ggc cct aag aac atg acc ctt 192Phe Ser Val Asn Tyr Asp Thr Lys
Ser Gly Pro Lys Asn Met Thr Leu 50 55 60gac ctg cca tca gat gcc aca
gtg gtg ctc aac cgc agc tcc tgt gga 240Asp Leu Pro Ser Asp Ala Thr
Val Val Leu Asn Arg Ser Ser Cys Gly65 70 75 80aaa gag aac act tct
gac ccc agt ctc gtg att gct ttt gga aga gga 288Lys Glu Asn Thr Ser
Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly 85 90 95cat aca ctc act
ctc aat ttc acg aga aat gca aca cgt tac agc gtc 336His Thr Leu Thr
Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val 100 105 110cag ctc
atg agt ttt gtt tat aac ttg tca gac aca cac ctt ttc ccc 384Gln Leu
Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro 115 120
125aat gcg agc tcc aaa gaa atc aag act gtg gaa tct ata act gac atc
432Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile
130 135 140agg gca gat ata gat aaa aaa tac aga tgt gtt agt ggc acc
cag gtc 480Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr
Gln Val145 150 155 160cac atg aac aac gtg acc gta acg ctc cat gat
gcc acc atc cag gcg 528His Met Asn Asn Val Thr Val Thr Leu His Asp
Ala Thr Ile Gln Ala 165 170 175tac ctt tcc aac agc agc ttc agc cgg
gga gag aca cgc tgt gaa caa 576Tyr Leu Ser Asn Ser Ser Phe Ser Arg
Gly Glu Thr Arg Cys Glu Gln 180 185 190gac agg cct tcc cca acc aca
gcg ccc cct gcg cca ccc agc ccc tcg 624Asp Arg Pro Ser Pro Thr Thr
Ala Pro Pro Ala Pro Pro Ser Pro Ser 195 200 205ccc tca ccc gtg ccc
aag agc ccc tct gtg gac aag tac aac gtg agc 672Pro Ser Pro Val Pro
Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser 210 215 220ggc acc aac
ggg acc tgc ctg ctg gcc agc atg ggg ctg cag ctg aac 720Gly Thr Asn
Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn225 230 235
240ctc acc tat gag agg aag gac aac acg acg gtg aca agg ctt ctc aac
768Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn
245 250 255atc aac ccc aac aag acc tcg gcc agc ggg agc tgc ggc gcc
cac ctg 816Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala
His Leu 260 265 270gtg act ctg gag ctg cac agc gag ggc acc acc gtc
ctg ctc ttc cag 864Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val
Leu Leu Phe Gln 275 280 285ttc ggg atg aat gca agt tct agc cgg ttt
ttc cta caa gga atc cag 912Phe Gly Met Asn Ala Ser Ser Ser Arg Phe
Phe Leu Gln Gly Ile Gln 290 295 300ttg aat aca att ctt cct gac gcc
aga gac cct gcc ttt aaa gct gcc 960Leu Asn Thr Ile Leu Pro Asp Ala
Arg Asp Pro Ala Phe Lys Ala Ala305 310 315 320aac ggc tcc ctg cga
gcg ctg cag gcc aca gtc ggc aat tcc tac aag 1008Asn Gly Ser Leu Arg
Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys 325 330 335tgc aac gcg
gag gag cac gtc cgt gtc acg aag gcg ttt tca gtc aat 1056Cys Asn Ala
Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn 340 345 350ata
ttc aaa gtg tgg gtc cag gct ttc aag gtg gaa ggt ggc cag ttt 1104Ile
Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe 355 360
365ggc tct gtg gag gag tgt ctg ctg gac gag aac agc ctc gag act aac
1152Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Thr Asn
370 375 380gca tgc tcc att aac ggt aat gct ccc gcc gag att gat ctg
cgc caa 1200Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu
Arg Gln385 390 395 400atg aga acc gtt aca ccc ata agg tca gga gta
gcc gct acg gaa tca 1248Met Arg Thr Val Thr Pro Ile Arg Ser Gly Val
Ala Ala Thr Glu Ser 405 410 415gca tac ctg gct tac aga aac cag tct
ctg gac ctt gca gaa cag gaa 1296Ala Tyr Leu Ala Tyr Arg Asn Gln Ser
Leu Asp Leu Ala Glu Gln Glu 420 425 430ctg gtg gac tgc gca tca cag
cac aac gga tgt cac ggg gac acc ata 1344Leu Val Asp Cys Ala Ser Gln
His Asn Gly Cys His Gly Asp Thr Ile 435 440 445ccc aga ggg atc gag
tat atc cag cac aat ggc gta gtg cag gaa agc 1392Pro Arg Gly Ile Glu
Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser 450 455 460tac tat agg
tat gtc gcg cga gag caa agc tgt agg cgc cct aac gca 1440Tyr Tyr Arg
Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala465 470 475
480cag cgt ttc gga atc tcc aat tac tgc cag atc tat ccg caa aac gtt
1488Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Gln Asn Val
485 490 495aac aaa atc cgt gaa gca ctg gcc caa acc cat agc gcc ata
gct gtc 1536Asn Lys Ile Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile
Ala Val 500 505 510atc atc ggt atc aaa gat ctg gat gcc ttt cgg cac
tac gat gga agg 1584Ile Ile Gly Ile Lys Asp Leu Asp Ala Phe Arg His
Tyr Asp Gly Arg 515 520 525act att ata cag cgc gac aat ggg tac cag
ccc aat tat cac gca gtg 1632Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln
Pro Asn Tyr His Ala Val 530 535 540aac att gtg gga tat agc aat gcg
caa ggt gtc gat tac tgg atc gtc 1680Asn Ile Val Gly Tyr Ser Asn Ala
Gln Gly Val Asp Tyr Trp Ile Val545 550 555 560cgc aac tcc tgg gac
aca aat tgg ggc gac aat ggc tat ggc tac ttt 1728Arg Asn Ser Trp Asp
Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe 565 570 575gct gcc aat
att gac ctc atg atg ata gag gag tat ccg tac gtc gtg 1776Ala Ala Asn
Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val 580 585 590atc
ctt ggc ggc ggc gga gat cag gta gac gtt aag gat tgc gct aac 1824Ile
Leu Gly Gly Gly Gly Asp Gln Val Asp Val Lys Asp Cys Ala Asn 595 600
605cat gag atc aag aaa gtc ttg gtg cca ggc tgt cat ggc tct gag cct
1872His Glu Ile Lys Lys Val Leu Val Pro Gly Cys His Gly Ser Glu Pro
610 615 620tgc att atc cat cgc ggg aag cct ttt cag ctt gaa gcc gtc
ttt gaa 1920Cys Ile Ile His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val
Phe Glu625 630 635 640gcc aac cag aac acc aag acg gcc aaa atc gag
atc aag gct agc att 1968Ala Asn Gln Asn Thr Lys Thr Ala Lys Ile Glu
Ile Lys Ala Ser Ile 645 650 655gat ggg ctg gaa gtc gac gtt cca ggc
att gac cca aac gcc tgt cac 2016Asp Gly Leu Glu Val Asp Val Pro Gly
Ile Asp Pro Asn Ala Cys His 660 665 670tac atg aag tgc cct ctg gtg
aaa ggg cag cag tac gac ata aag tac 2064Tyr Met Lys Cys Pro Leu Val
Lys Gly Gln Gln Tyr Asp Ile Lys Tyr 675 680 685acc tgg aat gtg cct
aaa atc gcc cca aaa tct gag aat gtg gtg gtt 2112Thr Trp Asn Val Pro
Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val 690 695 700aca gtg aaa
gtg atg ggc gat gac gga gtt ttg gcc tgt gcc ata gca 2160Thr Val Lys
Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala Ile Ala705 710 715
720acc cat gct aag att agg gac ggc ggt ggt ggg gcg aat gat aac gat
2208Thr His Ala Lys Ile Arg Asp Gly Gly Gly Gly Ala Asn Asp Asn Asp
725 730 735gac gac ccc aca act act gta cat cct acc acc aca gaa caa
ccg gat 2256Asp Asp Pro Thr Thr Thr Val His Pro Thr Thr Thr Glu Gln
Pro Asp 740 745 750gat aag ttt gaa tgc cca agc aga ttc ggt tat ttc
gcg gat cca aag 2304Asp Lys Phe Glu Cys Pro Ser Arg Phe Gly Tyr Phe
Ala Asp Pro Lys 755 760 765gat cct cac aag ttc tac att tgc tca aac
tgg gag gca gtg cat aaa 2352Asp Pro His Lys Phe Tyr Ile Cys Ser Asn
Trp Glu Ala Val His Lys 770 775 780gac tgt ccc ggc aat act cgg tgg
aat gag gac gag gaa act tgt acc 2400Asp Cys Pro Gly Asn Thr Arg Trp
Asn Glu Asp Glu Glu Thr Cys Thr785 790 795 800ggc ggt gga gga gat
cca atc cac tat gac aag ata aca gaa gaa atc 2448Gly Gly Gly Gly Asp
Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile 805 810 815aat aag gct
gtg gat gag gct gtt gcc gcc atc gaa aag tcc gag aca 2496Asn Lys Ala
Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu Thr 820 825 830ttc
gat ccc atg aaa gtc ccc gac cac agc gac aag ttt gaa cgg cac 2544Phe
Asp Pro Met Lys Val Pro Asp His Ser Asp Lys Phe Glu Arg His 835 840
845att gga att atc gac ctg aaa ggg gaa ctc gac atg cgg aac att cag
2592Ile Gly Ile Ile Asp Leu Lys Gly Glu Leu Asp Met Arg Asn Ile Gln
850 855 860gtt aga ggc ctc aaa cag atg aaa cga gtt ggg gat gct aat
gtg aag 2640Val Arg Gly Leu Lys Gln Met Lys Arg Val Gly Asp Ala Asn
Val Lys865 870 875 880agt gag gat ggg gtg gtg aag gca cat ctg ctc
gta ggg gtc cat gac 2688Ser Glu Asp Gly Val Val Lys Ala His Leu Leu
Val Gly Val His Asp 885 890 895gat gtg gtc agt atg gag tat gat ctg
gcc tac aaa ctc ggt gat ttg 2736Asp Val Val Ser Met Glu Tyr Asp Leu
Ala Tyr Lys Leu Gly Asp Leu 900 905 910cac ccc aat aca cac gta atc
agt gac att cag gac ttt gtg gtg gag 2784His Pro Asn Thr His Val Ile
Ser Asp Ile Gln Asp Phe Val Val Glu 915 920 925ctg tct ctg gaa gtt
tcc gag gag gga aac atg acc ctg act tcc ttc 2832Leu Ser Leu Glu Val
Ser Glu Glu Gly Asn Met Thr Leu Thr Ser Phe 930 935 940gag gtg cga
cag ttc gca aac gtc gtg aat cac att gga ggc ctg tct 2880Glu Val Arg
Gln Phe Ala Asn Val Val Asn His Ile Gly Gly Leu Ser945 950 955
960att ctg gat cct atc ttt gcc gtg ttg agt gac gtg ctg aca gcg atc
2928Ile Leu Asp Pro Ile Phe Ala Val Leu Ser Asp Val Leu Thr Ala Ile
965 970 975ttc caa gac acg gtc aga gcc gag atg acc aaa gtg ctc gct
cca gcc 2976Phe Gln Asp Thr Val Arg Ala Glu Met Thr Lys Val Leu Ala
Pro Ala 980 985 990ttc aag aag gag ctt gag cgg aac aac cag gaa ttc
acg ctg atc ccc 3024Phe Lys Lys Glu Leu Glu Arg Asn Asn Gln Glu Phe
Thr Leu Ile Pro 995 1000 1005atc gct gtg ggt ggt gcc ctg gcg ggg
ctg gtc ctc atc gtc ctc 3069Ile Ala Val Gly Gly Ala Leu Ala Gly Leu
Val Leu Ile Val Leu 1010 1015 1020atc gcc tac ctc gtc ggc agg aag
agg agt cac gca ggc tac cag 3114Ile Ala Tyr Leu Val Gly Arg Lys Arg
Ser His Ala Gly Tyr Gln 1025 1030 1035act atc tag 3123Thr Ile
104021040PRTArtificial SequenceSynthetic Construct 2Met Ala Pro Arg
Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Leu
Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val 20 25 30Lys Asn
Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala 35 40 45Phe
Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu 50 55
60Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly65
70 75 80Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg
Gly 85 90 95His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr
Ser Val 100 105 110Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr
His Leu Phe Pro 115 120 125Asn Ala Ser Ser Lys Glu Ile Lys Thr Val
Glu Ser Ile Thr Asp Ile 130 135 140Arg Ala Asp Ile Asp Lys Lys Tyr
Arg Cys Val Ser Gly Thr Gln Val145 150 155 160His Met Asn Asn Val
Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala 165 170 175Tyr Leu Ser
Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln 180 185 190Asp
Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser 195 200
205Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser
210 215 220Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln
Leu Asn225 230 235 240Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val
Thr Arg Leu Leu Asn 245 250 255Ile Asn Pro Asn Lys Thr Ser Ala Ser
Gly Ser Cys Gly Ala His Leu 260 265 270Val Thr Leu Glu Leu His Ser
Glu Gly Thr Thr Val Leu Leu Phe Gln 275 280 285Phe Gly Met Asn Ala
Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln 290 295 300Leu Asn Thr
Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala305 310 315
320Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys
325 330 335Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser
Val Asn 340 345 350Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu
Gly Gly Gln Phe 355 360 365Gly Ser Val Glu Glu Cys Leu Leu Asp Glu
Asn Ser Leu Glu Thr Asn 370 375 380Ala Cys Ser Ile Asn Gly Asn Ala
Pro Ala Glu Ile Asp Leu Arg Gln385 390 395 400Met Arg Thr Val Thr
Pro Ile Arg Ser Gly Val Ala Ala Thr Glu Ser 405 410 415Ala Tyr Leu
Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu 420 425 430Leu
Val Asp Cys Ala Ser Gln His Asn Gly Cys His Gly Asp Thr Ile 435 440
445Pro Arg Gly Ile Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser
450 455 460Tyr Tyr Arg Tyr Val Ala Arg Glu Gln Ser Cys Arg Arg Pro
Asn Ala465 470 475 480Gln Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile
Tyr Pro Gln Asn Val 485 490 495Asn Lys Ile Arg Glu Ala Leu Ala Gln
Thr His Ser Ala Ile Ala Val 500 505 510Ile Ile Gly Ile Lys Asp Leu
Asp Ala Phe Arg His Tyr Asp Gly Arg 515 520 525Thr Ile Ile Gln Arg
Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val 530 535 540Asn Ile Val
Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val545 550 555
560Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe
565 570 575Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr
Val Val 580 585 590Ile Leu Gly Gly Gly Gly Asp Gln Val Asp Val Lys
Asp Cys Ala Asn 595 600 605His Glu Ile Lys Lys Val Leu Val Pro Gly
Cys His Gly Ser Glu Pro 610 615 620Cys Ile Ile His Arg Gly Lys Pro
Phe Gln Leu Glu Ala Val Phe Glu625 630 635 640Ala Asn Gln Asn Thr
Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile 645 650 655Asp Gly Leu
Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His 660 665 670Tyr
Met Lys Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr 675 680
685Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Val Val Val
690 695 700Thr Val Lys Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala
Ile Ala705 710 715 720Thr His Ala Lys Ile Arg Asp Gly Gly Gly Gly
Ala Asn Asp Asn Asp 725 730 735Asp Asp Pro Thr Thr Thr Val His Pro
Thr Thr Thr Glu Gln Pro Asp 740 745 750Asp Lys Phe Glu Cys Pro Ser
Arg Phe Gly Tyr Phe Ala Asp Pro Lys 755 760 765Asp Pro His Lys Phe
Tyr Ile Cys Ser Asn Trp Glu Ala Val
His Lys 770 775 780Asp Cys Pro Gly Asn Thr Arg Trp Asn Glu Asp Glu
Glu Thr Cys Thr785 790 795 800Gly Gly Gly Gly Asp Pro Ile His Tyr
Asp Lys Ile Thr Glu Glu Ile 805 810 815Asn Lys Ala Val Asp Glu Ala
Val Ala Ala Ile Glu Lys Ser Glu Thr 820 825 830Phe Asp Pro Met Lys
Val Pro Asp His Ser Asp Lys Phe Glu Arg His 835 840 845Ile Gly Ile
Ile Asp Leu Lys Gly Glu Leu Asp Met Arg Asn Ile Gln 850 855 860Val
Arg Gly Leu Lys Gln Met Lys Arg Val Gly Asp Ala Asn Val Lys865 870
875 880Ser Glu Asp Gly Val Val Lys Ala His Leu Leu Val Gly Val His
Asp 885 890 895Asp Val Val Ser Met Glu Tyr Asp Leu Ala Tyr Lys Leu
Gly Asp Leu 900 905 910His Pro Asn Thr His Val Ile Ser Asp Ile Gln
Asp Phe Val Val Glu 915 920 925Leu Ser Leu Glu Val Ser Glu Glu Gly
Asn Met Thr Leu Thr Ser Phe 930 935 940Glu Val Arg Gln Phe Ala Asn
Val Val Asn His Ile Gly Gly Leu Ser945 950 955 960Ile Leu Asp Pro
Ile Phe Ala Val Leu Ser Asp Val Leu Thr Ala Ile 965 970 975Phe Gln
Asp Thr Val Arg Ala Glu Met Thr Lys Val Leu Ala Pro Ala 980 985
990Phe Lys Lys Glu Leu Glu Arg Asn Asn Gln Glu Phe Thr Leu Ile Pro
995 1000 1005Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val Leu Ile
Val Leu 1010 1015 1020Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser His
Ala Gly Tyr Gln 1025 1030 1035Thr Ile 104036127DNAArtificial
SequenceA vector comprising nucleic acid encoding a chimeric
protein 3ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt
aagcgggcac 60tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc
ggggttcgag 120ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg
cccgcgtgtc gaacccaggt 180gtgcgacgtc agacaacggg ggagtgctcc
ttttggcttc cttccccttc ttccgcttcc 240tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc agctcactca 300aaggcggtaa
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
360aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg 420ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg 480acaggactat aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt 540ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 600tctcatagct
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
660tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt 720gagtccaacc cggtaagaca cgacttatcg ccactggcag
cagccactgg taacaggatt 780agcagagcga ggtatgtagg cggtgctaca
gagttcttga agtggtggcc taactacggc 840tacactagaa gaacagtatt
tggtatctgc gctctgctga agccagttac cttcggaaaa 900agagttggta
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
960tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
gatcttttct 1020acggggtctg acgctcagtg gaacgaaaac tcacgttaag
ggattttggt catgagatta 1080tcaaaaagga tcttcaccta gatcctttta
aattaaaaat gaagttttaa atcaatctaa 1140agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga ggcacctatc 1200tcagcgatct
gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg
1260tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt
gtctgattat 1320tgatttttgg cgaaaccatt tgatcatatg acaagatgtg
tatctacctt aacttaatga 1380ttttgataaa aatcattagg taccccggct
ctagatggca tgacattaac ctataaaaat 1440aggcgtatca cgaggccctt
tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500cacatgcagc
tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
1560gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
ctatgcggca 1620tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
aaataccgca cagatgcgta 1680aggagaaaat accgcatcag attggctatt
ggccattgca tacgttgtat ccatatcata 1740atatgtacat ttatattggc
tcatgtccaa cattaccgcc atgttgacat tgattattga 1800ctagttatta
atagtaatca attacggggt cattagttca tagcccatat atggagttcc
1860gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
ccccgcccat 1920tgacgtcaat aatgacgtat gttcccatag taacgccaat
agggactttc cattgacgtc 1980aatgggtgga gtatttacgg taaactgccc
acttggcagt acatcaagtg tatcatatgc 2040caagtacgcc ccctattgac
gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100acatgacctt
atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
2160ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
gactcacggg 2220gatttccaag tctccacccc attgacgtca atgggagttt
gttttggcac caaaatcaac 2280gggactttcc aaaatgtcgt aacaactccg
ccccattgac gcaaatgggc ggtaggcgtg 2340tacggtggga ggtctatata
agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400gccatccacg
ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct
2460cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg
ccggttgagt 2520cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
cgtccgccgt ctaggtaagt 2580ttaaagctca ggtcgagacc gggcctttgt
ccggcgctcc cttggagcct acctagactc 2640agccggctct ccacgctttg
cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700aatgagacag
atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg
2760tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa
gaagacgaag 2820aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc
agcgcccggc gacccctgct 2880gctgctactg ctgttgctgc tgctcggcct
catgcattgt gcgtcagcag caatgtttat 2940ggtgaaaaat ggcaacggga
ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000gaactacgac
accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac
3060agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca
gtctcgtgat 3120tgcttttgga agaggacata cactcactct caatttcacg
agaaatgcaa cacgttacag 3180cgtccagctc atgagttttg tttataactt
gtcagacaca caccttttcc ccaatgcgag 3240ctccaaagaa atcaagactg
tggaatctat aactgacatc agggcagata tagataaaaa 3300atacagatgt
gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga
3360tgccaccatc caggcgtacc tttccaacag cagcttcagc cggggagaga
cacgctgtga 3420acaagacagg ccttccccaa ccacagcgcc ccctgcgcca
cccagcccct cgccctcacc 3480cgtgcccaag agcccctctg tggacaagta
caacgtgagc ggcaccaacg ggacctgcct 3540gctggccagc atggggctgc
agctgaacct cacctatgag aggaaggaca acacgacggt 3600gacaaggctt
ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca
3660cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc
agttcgggat 3720gaatgcaagt tctagccggt ttttcctaca aggaatccag
ttgaatacaa ttcttcctga 3780cgccagagac cctgccttta aagctgccaa
cggctccctg cgagcgctgc aggccacagt 3840cggcaattcc tacaagtgca
acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900caatatattc
aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt
3960ggaggagtgt ctgctggacg agaacagcct cgagactaac gcatgctcca
ttaacggtaa 4020tgctcccgcc gagattgatc tgcgccaaat gagaaccgtt
acacccataa ggtcaggagt 4080agccgctacg gaatcagcat acctggctta
cagaaaccag tctctggacc ttgcagaaca 4140ggaactggtg gactgcgcat
cacagcacaa cggatgtcac ggggacacca tacccagagg 4200gatcgagtat
atccagcaca atggcgtagt gcaggaaagc tactataggt atgtcgcgcg
4260agagcaaagc tgtaggcgcc ctaacgcaca gcgtttcgga atctccaatt
actgccagat 4320ctatccgcaa aacgttaaca aaatccgtga agcactggcc
caaacccata gcgccatagc 4380tgtcatcatc ggtatcaaag atctggatgc
ctttcggcac tacgatggaa ggactattat 4440acagcgcgac aatgggtacc
agcccaatta tcacgcagtg aacattgtgg gatatagcaa 4500tgcgcaaggt
gtcgattact ggatcgtccg caactcctgg gacacaaatt ggggcgacaa
4560tggctatggc tactttgctg ccaatattga cctcatgatg atagaggagt
atccgtacgt 4620cgtgatcctt ggcggcggcg gagatcaggt agacgttaag
gattgcgcta accatgagat 4680caagaaagtc ttggtgccag gctgtcatgg
ctctgagcct tgcattatcc atcgcgggaa 4740gccttttcag cttgaagccg
tctttgaagc caaccagaac accaagacgg ccaaaatcga 4800gatcaaggct
agcattgatg ggctggaagt cgacgttcca ggcattgacc caaacgcctg
4860tcactacatg aagtgccctc tggtgaaagg gcagcagtac gacataaagt
acacctggaa 4920tgtgcctaaa atcgccccaa aatctgagaa tgtggtggtt
acagtgaaag tgatgggcga 4980tgacggagtt ttggcctgtg ccatagcaac
ccatgctaag attagggacg gcggtggtgg 5040ggcgaatgat aacgatgacg
accccacaac tactgtacat cctaccacca cagaacaacc 5100ggatgataag
tttgaatgcc caagcagatt cggttatttc gcggatccaa aggatcctca
5160caagttctac atttgctcaa actgggaggc agtgcataaa gactgtcccg
gcaatactcg 5220gtggaatgag gacgaggaaa cttgtaccgg cggtggagga
gatccaatcc actatgacaa 5280gataacagaa gaaatcaata aggctgtgga
tgaggctgtt gccgccatcg aaaagtccga 5340gacattcgat cccatgaaag
tccccgacca cagcgacaag tttgaacggc acattggaat 5400tatcgacctg
aaaggggaac tcgacatgcg gaacattcag gttagaggcc tcaaacagat
5460gaaacgagtt ggggatgcta atgtgaagag tgaggatggg gtggtgaagg
cacatctgct 5520cgtaggggtc catgacgatg tggtcagtat ggagtatgat
ctggcctaca aactcggtga 5580tttgcacccc aatacacacg taatcagtga
cattcaggac tttgtggtgg agctgtctct 5640ggaagtttcc gaggagggaa
acatgaccct gacttccttc gaggtgcgac agttcgcaaa 5700cgtcgtgaat
cacattggag gcctgtctat tctggatcct atctttgccg tgttgagtga
5760cgtgctgaca gcgatcttcc aagacacggt cagagccgag atgaccaaag
tgctcgctcc 5820agccttcaag aaggagcttg agcggaacaa ccaggaattc
acgctgatcc ccatcgctgt 5880gggtggtgcc ctggcggggc tggtcctcat
cgtcctcatc gcctacctcg tcggcaggaa 5940gaggagtcac gcaggctacc
agactatcta gtaaggatct ttttccctct gccaaaaatt 6000atggggacat
catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
6060tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
taagggcggc 6120cgctagc 6127
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References