U.S. patent application number 16/818337 was filed with the patent office on 2020-09-24 for method for detecting rp2-arhgap6 gene.
The applicant listed for this patent is Astellas Pharma Inc., National Cancer Center. Invention is credited to Hitoshi Ichikawa, Hiroki Sasaki.
Application Number | 20200299782 16/818337 |
Document ID | / |
Family ID | 1000004885258 |
Filed Date | 2020-09-24 |
United States Patent
Application |
20200299782 |
Kind Code |
A1 |
Sasaki; Hiroki ; et
al. |
September 24, 2020 |
METHOD FOR DETECTING RP2-ARHGAP6 GENE
Abstract
The object of the invention is to elucidate a new causative gene
of cancer, polynucleotide, and thereby provide a method for
detecting the polynucleotide or a polypeptide that is encoded by
the polynucleotide, as well as a primer set or a detection kit for
such detection. The detection method detects a fusion gene of a
part of an RP2 gene and a part of an ARHGAP6 gene, or a fusion
protein encoded by such gene. The primer set includes a sense
primer designed from a section encoding RP2 and an antisense primer
designed from a section encoding ARHGAP6.
Inventors: |
Sasaki; Hiroki; (Tokyo,
JP) ; Ichikawa; Hitoshi; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Pharma Inc.
National Cancer Center |
Tokyo
Tokyo |
|
JP
JP |
|
|
Family ID: |
1000004885258 |
Appl. No.: |
16/818337 |
Filed: |
March 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15753994 |
Mar 1, 2018 |
10619216 |
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PCT/JP2016/074442 |
Aug 23, 2016 |
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16818337 |
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62209130 |
Aug 24, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/106 20130101;
C12Q 1/68 20130101; C12Q 1/686 20130101; C12N 15/09 20130101; C12Q
1/6886 20130101; C12Q 1/6853 20130101; C12Q 2600/166 20130101; C12Q
2600/156 20130101; C12Q 2600/16 20130101; C12Q 2600/118
20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/68 20060101 C12Q001/68; C12N 15/09 20060101
C12N015/09; C12Q 1/6853 20060101 C12Q001/6853; C12Q 1/686 20060101
C12Q001/686 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2015 |
JP |
2015/248292 |
Claims
1-14. (canceled)
15. A method of treating cancer in a subject, the method comprising
administering to the subject a pharmaceutical composition
comprising a substance for suppressing the expression of a fusion
protein of retinitis pigmentosa 2 (X-linked recessive) (RP2) and
Rho GTPase activating protein 6 (ARHGAP6), wherein the fusion
protein is a polypeptide according to either (1) or (2): (1) a
polypeptide that comprises an amino acid sequence having no less
than 90% identity with an amino acid sequence of SEQ ID NO:2; (2) a
polypeptide that comprises an amino acid sequence represented by
SEQ ID NO: 2, or a polypeptide that comprises an amino acid
sequence of SEQ ID NO:2, in which 1 to 10 amino acids are deleted,
substituted, inserted and/or added, thereby treating the cancer in
the subject.
16. The method of claim 15, wherein the fusion protein is a
polypeptide comprising an amino acid sequence having no less than
90% identity with an amino acid sequence of SEQ ID NO:2.
17. The method of claim 15, wherein the fusion protein is a
polypeptide comprising an amino acid sequence of SEQ ID NO:2, or
the polypeptide comprises an amino acid sequence of SEQ ID NO:2, in
which 1 to 10 amino acids are deleted, substituted, inserted and/or
added.
18. The method of claim 15, wherein the fusion protein is a
polypeptide consisting of an amino acid sequence of SEQ ID
NO:2.
19. The method of claim 15, wherein the cancer is treated by
inhibiting the growth of cancer cells and/or decreasing the
survival of cancer cells in the subject.
20. The method of claim 15, wherein the substance for suppressing
the expression of the fusion protein of RP2 and ARHGAP6 is an siRNA
targeting ARGHAP6.
21. The method of claim 15, wherein the cancer is stomach cancer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/753,994, filed Mar. 1, 2018, which is a
national stage filing under 35 U.S.C. .sctn. 371 of
PCT/JP2016/074442, filed Aug. 23, 2016, which claims priority to
U.S. Provisional Patent Application No. 62/209,130, filed Aug. 24,
2015, and Japanese Patent Application No. 248292/2015, filed Dec.
21, 2015, the contents of which are each incorporated herein by
reference in their entireties.
[0002] This application incorporates-by-reference the sequence
listing contained in the text file named
127206_05803_Sequence_Listing.txt, which was created Mar. 11, 2020
and is 20,360 bytes in size.
TECHNICAL FIELD
[0003] The present invention relates to a method for detecting a
novel fusion gene.
BACKGROUND ART
[0004] The retinitis pigmentosa 2 (X-linked recessive) (RP2) gene
exists on the short arm of the human chromosome X, and it is known
as a causal gene of retinitis pigmentosa (Nat Genet. 1998; 19(4):
327-332). RP2 is known to have a function to bind to ARL3 (ADP
ribosylation factor like GTPase 3), which is one of the small
GTPase protein family members, to enhance GTP hydrolase activity of
ARL3, i.e. it is a GTPase activating protein (Nat Struct Mol Biol.
2008; 15(4): 373-380). Its association with cancer is currently
unknown.
[0005] Rho GTPase activating protein 6 (ARHGAP6) gene, which has
GTPase activating function, exists on the short arm of human
chromosome X, same as RP2, and the protein encoded by this gene is
a GTPase activating protein possessing a Rho-GAP domain at the
center. The ARHGAP6 gene is known to have a function of enhancing
the GTP hydrolase activity of the small GTPase protein family,
particularly RhoA (Hum Mol Genet. 2000; 9(4): 477-488). With
regards to cancer, it is reported that the gene has decreased its
expression in the African American population, which has a high
incidence rate and mortality rate of colon cancer (PLoS One. 2012;
7(1): e30168), and also that a fusion gene thereof with a claudin
18 (CLDN18) gene was found in patients suffered from diffuse type
gastric cancer (Nature 2014; 513(7517): 202-209).
[0006] There are no reports so far of a fusion gene composed of RP2
and ARHGAP6.
SUMMARY OF INVENTION
Problem to be Solved by Invention
[0007] The object of the present invention is to elucidate a new
causative gene of cancer, polynucleotide, and thereby provide a
method for detecting a polynucleotide or a polypeptide that is
encoded by the polynucleotide, as well as a primer set or a
detection kit for such detection.
Means for Solving the Problems
[0008] The present inventors isolated and identified from stomach
cancer cell line, a novel fusion gene in which a part of the
ARHGAP6 gene and a part of the RP2 gene are fused together (Example
1), and found that this fusion gene was the causative gene of
cancer, since the survival ratio of the stomach cancer cell line
declined with the suppression of expression of fusion genes in
stomach cancer cell lines that endogenously express such fusion
genes (Example 2). The present inventors constructed a detection
method of a fusion gene based on these findings, and provided
primer sets for such purpose, thereby using the detection of such
fusion gene made it possible to select cancer patients
(particularly, stomach cancer patients) that test positive for a
fusion gene composed of an RP2 gene and an ARHGAP6 gene (Example
3).
[0009] In other words, the present invention relates to [1] to [24]
shown below.
[0010] [1] A method for detecting a fusion gene composed of a
retinitis pigmentosa 2 (X-linked recessive) (RP2) gene and a Rho
GTPase activating protein 6 (ARHGAP6) gene, the method comprising a
step of detecting whether a polynucleotide that encodes a
polypeptide described by either (1) or (2) shown below exists in a
sample obtained from a subject:
[0011] (1) a polypeptide that comprises an amino acid sequence
having no less than 90% identity with an amino acid sequence
represented by SEQ ID NO: 2;
[0012] (2) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2, or a polypeptide that comprises an
amino acid sequence represented by SEQ ID NO: 2, in which 1 to 10
amino acids are deleted, substituted, inserted and/or added.
[0013] [2] The method according to [1], wherein the polypeptide
comprises an amino acid sequence having no less than 90% identity
with an amino acid sequence represented by SEQ ID NO: 2, and has an
ability to develop tumor
[0014] [3] The method according to [1], wherein the polypeptide
comprises an amino acid sequence represented by SEQ ID NO: 2 and
has an ability to develop tumor, or the polypeptide comprises an
amino acid sequence represented by SEQ ID NO: 2, in which 1 to 10
amino acids are deleted, substituted, inserted and/or added, and
has an ability to develop tumor.
[0015] [4] The method according to [1], wherein the polypeptide
consists of an amino acid sequence represented by SEQ ID NO: 2.
[0016] [5] A method for detecting a fusion gene composed of an RP2
gene and an ARHGAP6 gene comprising a step of detecting whether a
polynucleotide that encodes a polypeptide consisting of an amino
acid sequence represented by SEQ ID NO: 2 exists in a sample
obtained from a subject.
[0017] [6] The method according to any one of [1] to [5] further
comprising a step in which it is judged when a polynucleotide
targeted in detection is detected, that a fusion gene composed of
an RP2 gene and an ARHGAP6 gene exists.
[0018] [7] The method according to any one of [1] to [6], further
comprising a step of amplifying a nucleic acid existing in a sample
obtained from a subject, or a step of hybridizing a probe with a
nucleic acid existing in a sample obtained from a subject to detect
a polynucleotide targeted in detection.
[0019] [8] The method according to [7] comprising a step of
amplifying the nucleic acid existing in a sample obtained from a
subject using a primer set shown below:
[0020] a primer set for detecting a fusion gene composed of an RP2
gene and an ARHGAP6 gene, the primer set comprising a sense primer
designed from a section encoding RP2 and an antisense primer
designed from a section encoding ARHGAP6, wherein the antisense
primer consists of an oligonucleotide that hybridizes under a
stringent condition with a polynucleotide targeted in detection,
and the sense primer consists of an oligonucleotide that hybridizes
under a stringent condition with a complementary strand of a
polynucleotide targeted in detection.
[0021] [9] The method according to [8], wherein the sense primer
consists of an oligonucleotide that hybridizes under a stringent
condition with a complementary strand of a polynucleotide
consisting of base no. 1 to 102 of SEQ ID NO: 1, and the antisense
primer consists of an oligonucleotide that hybridizes under a
stringent condition with a polynucleotide consisting of base no.
103 to 2439 of SEQ ID NO: 1.
[0022] [10] The method according to any one of [7] to [9]
comprising a step of amplifying the nucleic acid existing in a
sample obtained from a subject using a primer set shown below:
[0023] a primer set for detecting a fusion gene composed of an RP2
gene and an ARHGAP6 gene, wherein a sense primer consists of an
oligonucleotide of at least 16 random consecutive bases between
base no. 1 to 102 of SEQ ID NO: 1, and an antisense primer consists
of an oligonucleotide complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 103 to 2439 of
SEQ ID NO: 1.
[0024] [11] The method according to any one of [7] to [10] further
comprising a step of detecting whether an amplified nucleic acid
fragment of a target size was obtained.
[0025] [12] The method according to [11] further comprising a step
in which it is judged when an amplified nucleic acid fragment of a
target size is obtained, that a fusion gene composed of an RP2 gene
and an ARHGAP6 gene exists.
[0026] [13] The method according to any one of [7] to [10] further
comprising a step of determining a base sequence of an amplified
nucleic acid fragment.
[0027] [14] The method according to [13] further comprising a step
in which it is judged when an amplified nucleic acid fragment
includes a base sequence of a section encoding RP2 and a base
sequence of a section encoding ARHGAP6 in a same fragment, that a
fusion gene composed of an RP2 gene and an ARHGAP6 gene exists.
[0028] [15] The method according to [7] comprising a step of
hybridizing a probe with the nucleic acid existing in a sample
obtained from a subject, wherein the probe comprises an
oligonucleotide that hybridizes with the polynucleotide under a
stringent condition.
[0029] [16] The method according to [15] comprising a step of
performing in situ hybridization using a sample obtained from a
subject, a probe designed from a section encoding RP2 of the
polynucleotide, and a probe designed from a section encoding
ARHGAP6 of the polynucleotide.
[0030] [17] The method according to [16] using multiple types of
probes designed from a section encoding RP2, and multiple types of
probes designed from a section encoding ARHGAP6.
[0031] [18] The method according to [7], [16] or [17] using
multiple types of adjacent probe pairs comprising an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 1 to 102 of SEQ
ID NO: 1, and multiple types of adjacent probe pairs comprising an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 103 to 2439 of
SEQ ID NO: 1, in a step of hybridizing a probe with the nucleic
acid existing in a sample obtained from a subject.
[0032] [19] The method according to any one of [16] to [18] further
comprising a step of amplifying hybridization signals.
[0033] [20] The method according to any one of [16] to [19] further
comprising a step of detecting a signal overlap of a signal from a
probe designed from a section encoding RP2 and a signal from a
probe designed from a section encoding ARHGAP6.
[0034] [21] The method according to [20] further comprising a step
in which it is judged when two signals are detected at a same area,
that a fusion gene composed of an RP2 gene and an ARHGAP6 gene
exists.
[0035] [22] The method according to any one of [1] to [21]
comprising a step of obtaining a sample from a subject.
[0036] [23] The method according to any one of [1] to [22], wherein
the subject is a cancer patient.
[0037] [24] The method according to [23], wherein cancer is stomach
cancer.
[0038] Further, the present invention relates to [25] to [27] shown
below.
[0039] [25] A method for detecting whether cancer exists in a
subject comprising the step according to any one of [1] to
[21].
[0040] [26] The method according to [25] comprising a step of
obtaining a sample from a subject.
[0041] [27] The method according to [25] or [26], wherein cancer is
stomach cancer.
[0042] Further, the present invention relates to [28] to [32] shown
below.
[0043] [28] The method for diagnosing cancer in a subject
comprising a step according to any one of [1] to [21].
[0044] [29] The method according to [28] comprising a step of
obtaining a sample from a subject.
[0045] [30] The method according to [28] or [29] further comprising
a step in which it is judged when a fusion gene composed of an RP2
gene and an ARHGAP6 gene is detected in a sample obtained from a
subject, that there is a high possibility of the subject having
cancer.
[0046] [31] The method according to [28] or [29], wherein cancer is
stomach cancer.
[0047] [32] The method according to [29] further comprising a step
in which it is judged when a fusion gene composed of an RP2 gene
and an ARHGAP6 gene is detected in a sample obtained from a
subject, that there is a high possibility of a subject having
stomach cancer.
[0048] Further, the present invention relates to [33] to [36] shown
below.
[0049] [33] A method for identifying a subject that is a candidate
for receiving a treatment by an ARHGAP6 function inhibitor and/or a
pharmaceutical agent for blocking an abnormal signal induced by a
fusion gene composed of an RP2 gene and an ARHGAP6 gene, comprising
a step according to any one of [1] to [21], wherein the subject is
a cancer patient.
[0050] [34] The method according to [33] comprising a step of
obtaining a sample from a subject.
[0051] [35] The method according to [33] or [34] further comprising
a step in which it is judged when a fusion gene composed of an RP2
gene and an ARHGAP6 gene is detected in a sample obtained from a
subject, that the subject is a candidate for receiving a treatment
by an ARHGAP6 inhibitor and/or a pharmaceutical agent for blocking
an abnormal signal induced by a fusion gene composed of an RP2 gene
and an ARHGAP6 gene.
[0052] [36] The method according to any one of [33] or [35],
wherein cancer is stomach cancer.
[0053] Further, the present invention relates to [37] to [42] shown
below.
[0054] [37] A primer set for detecting a fusion gene composed of an
RP2 gene and an ARHGAP6 gene existing in a sample obtained from a
subject, the primer set comprising a sense primer designed from a
section encoding RP2 and an antisense primer designed from a
section encoding ARHGAP6, wherein the antisense primer consists of
an oligonucleotide that hybridizes under a stringent condition with
the polynucleotide according to any one of [1] to [5], and the
sense primer consists of an oligonucleotide that hybridizes under a
stringent condition with a complementary strand of the
polynucleotide.
[0055] [38] The primer set according to [37], wherein the sense
primer consists of an oligonucleotide that hybridizes under a
stringent condition with a complementary strand of a polynucleotide
consisting of base no. 1 to 102 of SEQ ID NO: 1, and the antisense
primer consists of an oligonucleotide that hybridizes under a
stringent condition with a polynucleotide consisting of base no.
103 to 2439 of SEQ ID NO: 1.
[0056] [39] A primer set for detecting a fusion gene composed of an
RP2 gene and an ARHGAP6 gene existing in a sample obtained from a
subject, the primer set comprising a sense primer designed from a
section encoding RP2 or an antisense primer designed from a section
encoding ARHGAP6 of the polynucleotide according to any one of [1]
to [5].
[0057] [40] The primer set according to any one of [37] to [39],
wherein the sense primer consists of an oligonucleotide of at least
16 random consecutive bases between base no. 1 to 102 of SEQ ID NO:
1, and the antisense primer consists of an oligonucleotide that is
complementary to an oligonucleotide of at least 16 random
consecutive bases between base no. 103 to 2439 of SEQ ID NO: 1.
[0058] [41] The primer set according to any one of [37] to [40],
wherein the subject is a cancer patient.
[0059] [42] The primer set according to [41], wherein cancer is
stomach cancer.
[0060] Further, the present invention relates to [43] to [48] shown
below.
[0061] [43] A probe for detecting a fusion gene composed of an RP2
gene and an ARHGAP6 gene existing in a sample obtained from a
subject, the probe comprising an oligonucleotide that hybridizes
under a stringent condition with the polynucleotide according to
any one of [1] to [5].
[0062] [44] The probe set comprising multiple probes according to
[43], the probe set comprising a probe designed from a section
encoding RP2 and a probe designed from a section encoding ARHGAP6
of the polynucleotide according to any one of [1] to [5].
[0063] [45] The probe set according to [44] comprising multiple
types of probes designed from a section encoding RP2 and multiple
types of probes designed from a section encoding ARHGAP6.
[0064] [46] The probe set according to [44] or [45] comprising
multiple types of adjacent probe pairs comprising an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 1 to 102 of SEQ
ID NO: 1 and multiple types of adjacent probe pairs comprising an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 103 to 2439 of
SEQ ID NO: 1.
[0065] [47] The probe or a probe set according to any one of [43]
to [46], wherein the subject is a cancer patient.
[0066] [48] The method according to [47], wherein cancer is stomach
cancer.
[0067] Further, the present invention relates to [49] to [53] shown
below.
[0068] [49] A detection kit for detecting a fusion gene composed of
an RP2 gene and an ARHGAP6 gene existing in a sample obtained from
the subject, the detection set comprising a primer set according to
any one of [37] to [40].
[0069] [50] A detection kit for detecting a fusion gene composed of
an RP2 gene and an ARHGAP6 gene existing in a sample obtained from
a subject, the detection kit comprising a probe or a probe set
according to any one of [43] to [46].
[0070] [51] The detection kit according to [50] further comprising
a reagent for amplifying a signal of hybridization.
[0071] [52] The detection kit according to any one of [49] to [51],
wherein the subject is a cancer patient.
[0072] [53] The detection kit according to [52], wherein cancer is
stomach cancer.
[0073] Further, the present invention relates to [54] to [63] shown
below.
[0074] [54] A detection method of a fusion protein of RP2 and
ARHGAP6 comprising a step of detecting whether a polypeptide
according to either (1) or (2) exists in a sample obtained from a
subject:
[0075] (1) a polypeptide that comprises an amino acid sequence
having no less than 90% identity with an amino acid sequence
represented by SEQ ID NO: 2;
[0076] (2) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2, or a polypeptide that comprises an
amino acid sequence represented by SEQ ID NO: 2, in which 1 to 10
amino acids are deleted, substituted, inserted and/or added.
[0077] [55] The method according to [54], wherein the polypeptide
comprises an amino acid sequence having no less than 90% identity
with an amino acid sequence represented by SEQ ID NO: 2, and has an
ability to develop tumor.
[0078] [56] The method according to [54], wherein the polypeptide
comprises an amino acid sequence represented by SEQ ID NO: 2 and
has an ability to develop tumor, or the polypeptide comprises an
amino acid sequence represented by SEQ ID NO: 2, in which 1 to 10
amino acids are deleted, substituted, inserted and/or added, and
has an ability to develop tumor.
[0079] [57] The method according to [54], wherein the polypeptide
consists of an amino acid sequence represented by SEQ ID NO: 2.
[0080] [58] A detection method of a fusion protein of RP2 and
ARHGAP6 comprising a step of detecting whether a polypeptide
consisting of an amino acid sequence represented by SEQ ID NO: 2
exists in a sample obtained from a subject.
[0081] [59] The method according to any one of [54] to [58],
wherein the step for detecting whether the polypeptide exists
comprises a step of bringing an antibody (primary antibody) that
recognizes a section derived from an RP2 gene in the polypeptide
and an antibody (primary antibody) that recognizes a section
derived from an ARHGAP6 gene in the polypeptide in contact with a
sample obtained from a subject.
[0082] [60] The method according to [59] further comprising steps
of i) to v) described below:
[0083] i) a step of adding secondary antibodies that are connected
to oligonucleotides and that respectively bind to primary
antibodies; ii) a step of adding a ligation solution that contains
two types of oligonucleotides that are partially complementary to
oligonucleotides connected to the secondary antibodies and a ligase
that can ligate the two types of oligonucleotides to form a
circular structure when the oligonucleotides approach each other,
thereby inducing a ligation reaction; iii) a step of elongating a
nucleic acid along a circular structure that is formed; and iv) a
step of hybridizing a labeled oligonucleotide probe that can
hybridize with an elongated nucleic acid, and v) a step of
detecting a labeled signal.
[0084] [61] The method according to any one of [54] to [60]
comprising a step of obtaining a sample from a subject.
[0085] [62] The method according to any one of [54] to [61],
wherein the subject is a cancer patient.
[0086] [63] The method according to [62], wherein cancer is stomach
cancer.
[0087] Further, the present invention relates to [64] to [66] shown
below.
[0088] [64] A method for detecting whether cancer exists in a
subject comprising the step according to any one of [54] to
[60].
[0089] [65] The method according to [64] comprising a step of
obtaining a sample from a subject.
[0090] [66] The method according to [64] or [65], wherein cancer is
stomach cancer.
[0091] Further, the present invention relates to [67] to [71] shown
below.
[0092] [67] A method for diagnosing cancer in a subject comprising
a step according to any one of [54] to [60].
[0093] [68] The method according to [67] comprising a step of
obtaining a sample from a subject.
[0094] [69] The method according to [67] or [68] further comprising
a step in which it is judged when a fusion protein of RP2 and
ARHGAP6 is detected in a sample obtained from a subject, that there
is a high possibility of the subject having cancer.
[0095] [70] The method according to [67] or [68], wherein cancer is
stomach cancer.
[0096] [71] The method according to [68] further comprising a step
in which it is judged when a fusion protein of RP2 and ARHGAP6 is
detected in a sample obtained from a subject, that there is a high
possibility of the subject having stomach cancer.
[0097] Further, the present invention relates to [72] to [75] shown
below.
[0098] [72] A method for identifying a subject that is a candidate
for receiving a treatment by an ARHGAP6 function inhibitor and/or a
pharmaceutical agent for blocking an abnormal signal induced by a
fusion gene composed of an RP2 gene and an ARHGAP6 gene, the method
comprising a step according to any one of [54] to [60], wherein the
subject is a cancer patient.
[0099] [73] The method according to [72] comprising a step of
obtaining a sample from a subject.
[0100] [74] The method according to [72] or [73] further comprising
a step in which it is judged when a fusion protein of RP2 and
ARHGAP6 is detected in a sample obtained from a subject, that the
subject is a candidate for receiving a treatment by an ARHGAP6
inhibitor and/or a pharmaceutical agent for blocking an abnormal
signal induced by a fusion gene composed of an RP2 gene and an
ARHGAP6 gene.
[0101] [75] The method according to [72] or [74], wherein cancer is
stomach cancer.
[0102] Further, the present invention relates to [76] to [79] shown
below.
[0103] [76] A detection kit for detecting a fusion protein of RP2
and ARHGAP6 existing in a sample obtained from a subject, the
detection kit comprising an antibody (primary antibody) that
recognizes a section derived from an RP2 gene in the polypeptide
according to any one of [54] to [58], and an antibody (primary
antibody) that recognizes a section derived from an ARHGAP6 gene in
said polypeptide.
[0104] [77] The detection kit according to [76] comprising
secondary antibodies that are connected to oligonucleotides and
that respectively bind to primary antibodies, two types of
oligonucleotides that are partially complementary to the
oligonucleotides connected to the secondary antibodies, a ligase
that can ligate the two types of oligonucleotides to form a
circular structure when the oligonucleotides approach each other,
and a labeled oligonucleotide probe.
[0105] [78] The detection kit according to [76] or [77], wherein
the subject is a cancer patient.
[0106] [79] The detection kit according to [78], wherein cancer is
stomach cancer.
[0107] Further, the present invention relates to [80] to [81] shown
below.
[0108] [80] A polypeptide according to any one of (1) to (3) shown
below or a polynucleotide encoding said polypeptide:
[0109] (1) a polypeptide that comprises an amino acid sequence
having no less than 90% identity with an amino acid sequence
represented by SEQ ID NO: 2;
[0110] (2) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2, in which 1 to 10 amino acids are
deleted, substituted, inserted and/or added;
[0111] (3) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2.
[0112] [81] The polypeptide or a polynucleotide encoding said
polypeptide according to [80] that has an ability to develop
tumor.
Advantageous Effect of Invention
[0113] The detection method of the present invention may be used as
a method for detecting cancer (particularly, stomach cancer) that
tests positive for a fusion gene composed of an RP2 gene and an
ARHGAP6 gene (hereinafter referred to as RP2-ARHGAP6 fusion gene).
The primer set, probe, probe set and detection kit of the present
invention may be used in a detection method of the present
invention.
BRIEF DESCRIPTION OF DRAWINGS
[0114] FIG. 1 shows the result of the Western blot. It shows the
change in the amount of protein expression of the RP2-ARHGAP6
fusion protein by the ARHGAP6 siRNA treatment.
[0115] FIG. 2 shows the change in the number of viable cells in the
stomach cancer cell line caused by the ARHGAP6 siRNA treatment.
FIG. 2 compares the number of viable cells cultured in a 0.5%
bovine serum containing RPMI-1640 medium after introduction of
siRNA with that of the control.
[0116] FIG. 3 shows a result of amplification by PCR of a region
containing a fusion point of a RP2-ARHGAP6 fusion gene.
DESCRIPTION OF EMBODIMENTS
[0117] Detection Method of the Present Invention
[0118] The detection method of the present invention includes a
method for detecting a fusion gene, and a method for detecting a
fusion protein encoded in the fusion gene. The method for detecting
a fusion gene of the present invention or the method for detecting
a fusion protein of the present invention includes a step of
detecting whether a specific polynucleotide or polypeptide exists
in a sample obtained from a subject.
[0119] Items collected from the subject (samples separated from a
living body) are used as the sample obtained from the subject,
specifically, any cells, tissues, or body fluids that were
collected (blood, oral mucus, circulating tumor cells, exosome,
etc.), biopsied samples (samples from the primary focus, cancer
cells in the peritoneal lavage solution, cancer cells in ascites,
etc.), of which the biopsied samples are preferred. It is possible
to use genome DNAs extracted from the collected samples or to use
transcription products thereof (products that are obtained by
transcription and translation of a genome; e.g. RNA, protein) or
cDNA prepared from RNA. It is particularly preferable to use RNA or
cDNA that had been formulated. It is also possible to use a
stabilized sample fixed in formalin and embedded in paraffin
(Formalin-Fixed Paraffin-Embedded sample; FFPE Sample). A FFPE
sample sliced into a thin FFPE slice may also be used. A use of a
FFPE slice enables a direct detection of a polynucleotide existing
in the slice.
[0120] The method for detecting a fusion gene in the present
invention is a method for detecting "a fusion gene composed of an
RP2 gene and an ARHGAP6 gene," wherein the fusion gene is a fusion
gene comprising a part of an RP2 gene and a part of an ARHGAP6
gene. An exemplary fusion gene composed of an RP2 gene and an
ARHGAP6 gene includes a polynucleotide consisting of a base
sequence represented by SEQ ID NO: 1. The polynucleotide consisting
of a base sequence represented by SEQ ID NO: 1 is a polynucleotide
with a base sequence of base no. 190 (corresponding to the 5'
terminal of the coding sequence (hereinafter referred to as CDS))
to 291 of an RP2 gene (GenBank registration no: NM_006915.2) and
base no. 1462 to 3798 (corresponding to the 3' terminal of CDS) of
an ARHGAP6 gene (GenBank registration no: NM_013427.2). Of the base
sequence represented by SEQ ID NO: 1, the sequence from base no. 1
to 102 is derived from an RP2 gene, and the sequence from base no.
103 to 2439 is derived from an ARHGAP6 gene. The polynucleotide
consisting of a base sequence represented by SEQ ID NO: 1 is also
referred to as a "fusion polynucleotide." The amino acid sequence
encoded in base no. 1 to 2439 of SEQ ID NO: 1 is shown in SEQ ID
NO: 2.
[0121] In the "step of detecting whether a polynucleotide exists"
in the detection method of a fusion gene of the present invention,
the polynucleotide that is the target of detection (referred to in
the present specification as the "polynucleotide targeted in
detection") includes, for example, a polynucleotide encoding a
polypeptide described in (1) or (2) shown below:
[0122] (1) a polypeptide that comprises an amino acid sequence
having no less than 90% identity with an amino acid sequence
represented by SEQ ID NO: 2;
[0123] (2) a polypeptide that comprises an amino acid sequence
having no less than 90% identity with an amino acid sequence
represented by SEQ ID NO: 2, and has an ability to develop
tumor.
[0124] In the aforementioned polypeptide, the "identity with an
amino acid sequence represented by SEQ ID NO: 2" is preferably 95%
or higher, and more preferably 98% or higher.
[0125] Note that the "identity" as used in the present
specification is a value of "Identity" obtained by using a
parameter prepared by default by the NEEDLE program (J Mol Biol
1970; 48: 443-453) search. The aforementioned parameter is shown
below.
[0126] Gap penalty=10
[0127] Extend penalty=0.5
[0128] Matrix=EBLOSUM62
[0129] Whether a polypeptide "has an ability to develop tumor" or
not may be confirmed by a method shown below in Example 2. One
specific method is to introduce siRNA that suppresses the
expression of a polynucleotide encoding the polypeptide to a cell
expressing the polypeptide (NSC-10C), and to verify that the
viability of the cell decreases.
[0130] In one embodiment of the present invention, the
polynucleotide targeted in detection is a polynucleotide encoding a
polypeptide according to any one of (1) to (4) shown below:
[0131] (1) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2, in which 1 to 10 amino acids are
deleted, substituted, inserted and/or added;
[0132] (2) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2, in which 1 to 10 amino acids are
deleted, substituted, inserted and/or added, and has an ability to
develop tumor;
[0133] (3) a polypeptide that comprises an amino acid sequence
represented by SEQ ID NO: 2 and has an ability to develop tumor;
and
[0134] (4) a polypeptide that consists of an amino acid sequence
represented by SEQ ID NO: 2.
[0135] In the polypeptide of (1) and (2), the number of amino acids
that had been deleted, substituted, inserted and/or added in the
amino acid sequence represented by SEQ ID NO: 2 is preferably one
to a few, more preferably 1 to 7, and even more preferably 1 to
5.
[0136] An example of a polynucleotide that encodes "a polypeptide
that consists of an amino acid sequence represented by SEQ ID NO:
2" includes "a polynucleotide that consists of a base sequence
represented by SEQ ID NO: 1."
[0137] The method for detecting a fusion gene of the present
invention may comprise a step in which it is judged whether the
polynucleotide targeted in detection exists by whether said
polynucleotide was detected.
[0138] The method for detecting a fusion gene of the present
invention may further comprise a step in which it is judged when a
polynucleotide targeted in detection is detected, that a fusion
gene composed of an RP2 gene and an ARHGAP6 gene exists.
[0139] The method for detecting a fusion gene of the present
invention may comprise a step of amplifying the nucleic acid
existing in the sample obtained from a subject or a step of
hybridizing a probe with the nucleic acid existing in the sample
obtained from a subject to detect the polynucleotide targeted in
detection.
[0140] The nucleic acid to be used may be a genome DNA, RNA or a
cDNA prepared from RNA. The methods of extracting DNA, extracting
RNA or preparing cDNA from RNA is commonly known in the field, and
it may be performed easily by using a commercially available DNA
extraction kit, RNA extraction kit or a cDNA synthesis kit.
[0141] The step of amplifying a nucleic acid in the sample obtained
from a subject may be performed by a commonly known method of
amplifying a nucleic acid. Such method includes PCR (Polymerase
chain reaction, e.g. realtime PCR), LCR (Ligase chain reaction),
SDA (Strand displacement amplification), NASBA (Nucleic acid
sequence-based amplification), ICAN (Isothermal and chimeric
primer-initiated amplification of nucleic acids), LAMP
(Loop-mediated isothermal amplification), TMA
(Transcription-mediated amplification, e.g. Gen-Probe's TMA
system), and a preferable method is PCR.
[0142] Specifically, the nucleic acid (e.g. genome DNA, RNA, or
cDNA prepared from RNA, etc.) in the sample obtained from a subject
is subjected to a nucleic acid amplification reaction using a
primer set designed to specifically amplify a polynucleotide
targeted in detection. The primer set to be used is not
particularly limited as long as it can specifically amplify the
polynucleotide targeted in detection. For example, a use of a
primer design software (e.g. Primer Express; Applied Biosystems)
allows a person skilled in the art to easily design the primer set
based on the base sequence of the polynucleotide targeted in
detection. More specifically, a primer set includes a sense primer
(5'-primer) designed from a section that encodes the RP2 of a
polynucleotide targeted in detection (e.g. any section in an RP2
gene region of the fusion polynucleotide (particularly, cDNA)) and
an antisense primer (3'-primer) designed from a section encoding
ARHGAP6 of a polynucleotide targeted in detection (e.g. any section
in an ARHGAP6 gene region of the fusion polynucleotide
(particularly, cDNA)), and the antisense primer consists of an
oligonucleotide that hybridizes with a polynucleotide targeted in
detection under a stringent condition (preferably, under a highly
stringent condition), and the sense primer consists of an
oligonucleotide that hybridizes with a complementary strand of a
polynucleotide targeted in detection under a stringent condition
(preferably, under a highly stringent condition). Otherwise, either
the sense primer or the antisense primer may be designed so that it
corresponds to the region comprising the fusion point of the
polynucleotide targeted in detection.
[0143] The "stringent condition" in the present specification
refers to a hybridization condition of "5.times.SSPE,
5.times.Denhardt's solution, 0.5% SDS, 50% formaldehyde, 200
.mu.g/mL salmon sperm DNA, at 42.degree. C. overnight" and a
washing condition of "0.5.times.SSC, 0.1% SDS, 42.degree. C." "A
highly stringent condition" refers to a hybridization condition of
"5.times.SSPE, 5.times.Denhardt's solution, 0.5% SDS, 50%
formaldehyde, 200 .mu.g/mL salmon sperm DNA, at 42.degree. C.
overnight" and a washing condition of "0.2.times.SSC, 0.1% SDS,
65.degree. C."
[0144] The "fusion point" of a polynucleotide targeted in detection
in the present specification is a point in which a section derived
from an RP2 gene and a section derived from an ARHGAP6 gene in the
polynucleotide targeted in detection are fused together, and the
"region comprising the fusion point" in the polynucleotide targeted
in detection is, for example, the region comprising bases of base
no. 102 and 103 when the polynucleotide targeted in detection is a
polynucleotide consisting of a base sequence represented by SEQ ID
NO: 1.
[0145] In an embodiment of the present invention, the sense primer
consists of an oligonucleotide hybridizing with a complementary
strand of a polynucleotide that consists of base no. 1 to 102 of
SEQ ID NO: 1 under a stringent condition, and the antisense primer
consists of an oligonucleotide hybridizing with a polynucleotide
that consists of base no. 103 to 2439 of SEQ ID NO: 1 under a
stringent condition.
[0146] In an embodiment of the present invention, the sense primer
consists of at least 16 consecutive bases of an oligonucleotide
between base no. 1 to 102 of SEQ ID NO: 1, and the antisense primer
consists of an oligonucleotide that is complementary with at least
16 consecutive bases of an oligonucleotide that consists of base
no. 103 to 2439 of SEQ ID NO: 1.
[0147] In a step to amplify nucleic acid, the sense primer and the
antisense primer should preferably be set so that the fragment size
of the nucleic acid to be amplified is 1 kb or lower, since a large
fragment size of the nucleic acid to be amplified leads to poor
amplification efficiency. The primers to be used generally have a
chain length of at least 15 bases, preferably at least 16 bases,
more preferably at least 18 bases, even more preferably at least 20
bases. In one embodiment of the present invention, the primer has
15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24
bases, even more preferably 20 to 24 bases.
[0148] The primer may be produced by chemical synthesis without
being particularly limited thereby.
[0149] In a preferable embodiment, the detection method of a fusion
gene of the present invention further encompasses a step of
detecting whether an amplified nucleic acid fragment of a desired
size was obtained in addition to a step of amplifying nucleic acid
in the sample obtained from a subject. The step of detecting
whether an amplified nucleic acid fragment of a desired size was
obtained may be performed using electrophoresis, for example. By
using electrophoresis, the nucleic acid fragment may be analyzed by
agarose gel electrophoresis to confirm whether amplified nucleic
acid fragments were produced in the desired size by using ethidium
bromide dye, etc.
[0150] Further, by performing a PCR amplification monitor in the
amplification process of the gene (real time PCR) (Genome Res.
1996; 6(10): 986-994), it is possible to perform a quantified
analysis of amplified nucleic acid fragments. A possible candidate
to be used in the PCR amplification monitoring method is ABI
PRISM7900 (Applied Biosystems).
[0151] When an amplified nucleic acid fragment of the desired size
is obtained, that means that a polynucleotide targeted in detection
existed in the sample obtained from a subject. The detection method
of a fusion gene of the present invention may further include a
step in which it is judged when an amplified nucleic acid fragment
of the desired size is obtained, that a fusion gene composed of an
RP2 gene and an ARHGAP6 gene exists.
[0152] In a separate preferable embodiment, the detection method of
the fusion gene of the present invention further encompasses a step
of determining the base sequence of the amplified nucleic acid in
addition to a step of amplifying the nucleic acid of the sample
obtained from a subject. The step of determining the base sequence
of the nucleic acid fragment may use sequencing methods commonly
known in the field of art including next generation sequencing
methods (Nature Biotechnology 2008; 26: 1135-1145) (e.g. HiSeq2500
(Illumina)), such as the Sanger sequencing (e.g. ABI PRISM3100
(Applied Biosystems) may be used), or sequencing by synthesis,
etc.
[0153] The step of determining the base sequence of the nucleic
acid fragment includes not just a step of sequencing the full
length of a nucleic acid fragment, but a step of sequencing partial
sequences corresponding to both ends of the nucleic acid
fragment.
[0154] When the sequenced nucleic acid fragment includes a base
sequence of a section encoding RP2 and a base sequence of a section
encoding ARHGAP6 of the polynucleotide targeted in detection in the
same fragment, that means that the polynucleotide targeted in
detection existed in the sample obtained from a subject. The
detection method of the fusion gene of the present invention may
further include a step in which it is judged when the amplified
nucleic acid fragment includes a base sequence of a section
encoding RP2 and a base sequence of a section encoding ARHGAP6 of
the polynucleotide targeted in detection in the same fragment, that
a fusion gene composed of an RP2 gene and an ARHGAP6 gene
exists.
[0155] The step of hybridizing a probe with a nucleic acid in the
sample obtained from a subject may be performed using a probe
including an oligonucleotide that hybridizes under a stringent
condition (preferably, under a highly stringent condition) with a
polynucleotide targeted in detection, and using a commonly known
hybridization method. Such methods include, for example, Northern
hybridization, dot blot method, DNA micro array method, RNA
protection method, in situ hybridization, etc. A preferable method
is the in situ hybridization. Detection using the in situ
hybridization may be performed by a commonly known fluorescent in
situ hybridization (FISH), chromogenic in situ hybridization
(CISH), or silver in situ hybridization (SISH). The chain length of
the probe used in hybridization may be selected as necessary by a
person skilled in the art according to the hybridization method to
be used, but the probe preferably has a chain length of at least 16
bases.
[0156] In one embodiment of the present invention, the probe used
in hybridization is an oligonucleotide that hybridizes under a
stringent condition (preferably, under a highly stringent
condition) with a polynucleotide targeted in detection, or a
complementary strand thereof, and it includes an oligonucleotide of
at least 16 bases upstream and at least 16 bases downstream of the
fusion point on the polynucleotide targeted in detection (a
specific example being a sequence of base no. 87 to 118 in SEQ ID
NO: 1) or an oligonucleotide that is complementary to said
oligonucleotide.
[0157] In one embodiment of the present invention, the step of
hybridizing a probe with a nucleic acid existing in a sample
obtained from a subject may be performed according to the commonly
known RNA FISH method (J. Mol. Diagn. 2012; 14(1): 22-29). More
specifically, in situ hybridization is performed using a sample
obtained from a subject (e.g. FFPE fragment), a probe designed from
a section encoding RP2 of the polynucleotide targeted in detection
(e.g. any section in an RP2 gene region of the fusion
polynucleotide), and a probe designed from a section encoding
ARHGAP6 of the polynucleotide targeted in detection (e.g. any
section in an ARHGAP6 gene region of the fusion polynucleotide).
The probes include oligonucleotides that hybridize under a
stringent condition (preferably, under a highly stringent
condition) with the polynucleotide targeted in detection.
[0158] In one embodiment of the present invention, the in situ
hybridization is performed using multiple detection probes designed
from a section encoding RP2 and multiple detection probes designed
from a section encoding ARHGAP6.
[0159] In one embodiment of the present invention, the in situ
hybridization is performed using the following probes:
[0160] multiple types of adjacent probe pairs including
oligonucleotides that are complementary to at least 16 random
consecutive oligonucleotides in base no. 1 to 102 of SEQ ID NO: 1
(preferably 10 to 25 types, more preferably 18 to 22 types, even
more preferably 20 types of probe pairs), and multiple types of
adjacent probe pairs including oligonucleotides that are
complementary to at least 16 random consecutive oligonucleotides in
base no. 103 to 2439 of SEQ ID NO: 1 (preferably 10 to 25 types,
more preferably 18 to 22 types, even more preferably 20 types of
probe pairs).
[0161] In a further embodiment, the probe pair to be used in the in
situ hybridization may include an adjacent probe pair including an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases in the nontranslating region on
the 5' side of the RP2 gene (base no. 1 to 189 of GenBank
registration no: NM_006915.2), and/or a probe pair including an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases in the nontranslating region on
the 3' side of the ARHGAP6 gene (base no. 3799 to 5118 of GenBank
registration no: NM_013427.2).
[0162] The "adjacent probe pairs" in the present specification
consist of two types of probes that are arranged next to each other
when they hybridize with the polynucleotide targeted in detection.
The probes include an oligonucleotide that is complementary to the
polynucleotide targeted in detection, and the length of the
oligonucleotide is generally at least 16 bases, preferably at least
18 bases. In one embodiment of the present invention, the length of
the oligonucleotide is 16 to 30 bases, preferably 18 to 25
bases.
[0163] In a preferable embodiment of the present invention, the
detection method of the fusion gene of the present invention
further encompasses a step of amplifying a hybridization signal in
addition to a step of performing in situ hybridization. To perform
a step of amplifying a hybridization signal, a reagent that
amplifies a hybridization signal may be hybridized with a probe
that hybridizes with a nucleic acid contained in the sample.
[0164] Reagents that amplify a hybridization signal used in in situ
hybridization include PreAmplifier Mix QT, Amplifier Mix QT, Label
Probe Mix, and Label Probe Diluent QF, which may be obtained from
Affymetrix.
[0165] In a more preferable embodiment, the detection method of the
fusion gene of the present invention further encompasses a step of
detecting a signal overlap between a signal from a probe designed
from a section encoding RP2 and a signal from a probe designed from
a section encoding ARHGAP6. By separating the fluorescent reagent
or the color reagent that detects a probe designed from a section
encoding RP2 and a probe designed from a section encoding ARHGAP6,
it is possible to observe whether the signals from the two
different probes are in the same area (inside the same molecule).
When it is observed that the signals from the two different probes
are in the same area (inside the same molecule), that would mean
that the polynucleotide targeted in detection existed in the sample
obtained from a subject. The detection method of the fusion gene of
the present invention may further include a step in which it is
judged when the two signals are in the same area (inside the same
molecule), that a fusion gene composed of an RP2 gene and an
ARHGAP6 gene exists.
[0166] The probes are not particularly limited, but they may be
produced by a chemical synthesis method.
[0167] The detection method of the fusion protein of the present
invention is a method for detecting "a fusion protein of RP2 and
ARHGAP6" and the fusion protein is a fusion protein encoded by the
fusion gene of the RP2 gene and the ARHGAP6 gene.
[0168] In the "step of detecting whether polypeptide exists" in the
detection method of the fusion protein of the present invention,
the polypeptide targeted in detection includes a polypeptide that
is encoded by a polynucleotide targeted in detection.
[0169] The detection method of the fusion protein of the present
invention may encompass a step in which it is judged whether a
polynucleotide exists by whether the polypeptide targeted in
detection is detected.
[0170] The detection method of the fusion protein of the present
invention may further encompass a step in which it is judged when
the polypeptide targeted in detection is detected, that a fusion
protein of RP2 and ARHGAP6 exists.
[0171] The step of detecting whether a polypeptide exists may be
performed by preparing a lysate derived from a sample obtained from
a subject (e.g. cancer tissue or cell obtained from a subject) and
measuring the polypeptide targeted in detection, contained in the
sample by an immunological measurement method or an enzyme active
measurement method, which combine antibodies against proteins that
constitute the fusion protein, or a detection method that combines
these methods, or by mass spectrometry. Further, this step may be
performed by a detection method using an immunological tissue
staining technology performed by combining the polypeptide targeted
in detection, included in the sample (e.g. FFPE fragment) obtained
from a subject, that had undergone appropriate pretreatment (such
as, removal of paraffin), with the antibodies against proteins
constituting the fusion protein. Otherwise, this step may be
performed by exchanging the antibodies against proteins
constituting the fusion protein to antibodies that recognize the
fusion section of the fusion protein. Exemplary approaches to these
methods include the following methods using monoclonal antibodies
and polyclonal antibodies specific to the polypeptide targeted in
detection: enzyme immunizing measurement, double antibody sandwich
ELISA method, fluorescent immunological measurement method,
radioimmunological measurement method, Western blot,
immunohistologic staining, a detection method combining immune
precipitation and mass spectrometry, etc.
[0172] The "fusion section" of the fusion protein of the present
specification refers to a section in the polypeptide targeted in
detection, in which the section derived from an RP2 gene and a
section derived from an ARHGAP6 gene are fused.
[0173] The detection using an immunohistologic staining technology
may be performed according to Proximity Ligation Assay (Nat.
Methods. 2006; 3(12): 995-1000). More specifically, whether the
polypeptide targeted in detection exists or not may be detected by
using an antibody that recognizes a section derived from the RP2
gene of the polypeptide targeted in detection, and an antibody that
recognizes a section derived from an ARHGAP6 gene of a polypeptide
targeted in detection, and by detecting that the two antibodies
recognize the same molecule by the aforementioned technologies.
More specifically, the detection may be performed by i) a step of
bringing an antibody (primary antibody) that recognizes a section
derived from an RP2 gene of the polypeptide targeted in detection,
and the antibody (primary antibody) that recognizes a section
derived from an ARHGAP6 gene of the polypeptide targeted in
detection, in contact with the sample obtained from the subject;
ii) a step of adding secondary antibodies that are connected to
oligonucleotides, and binds to the respective primary antibodies,
iii) a step of inducing ligation by adding two types of
oligonucleotides that are partly complementary to the
oligonucleotides connected to the secondary antibodies, and a
ligation solution containing ligase that can form a circular
structure by ligation of the two types of oligonucleotides when
they approach each other; iv) a step of elongating a nucleic acid
along the circular structure that was formed, v) a step of
hybridizing a labeled oligonucleotide probe that can hybridize with
the elongated nucleic acid; and vi) a step of detecting the
labeling signal. Such detection may be performed using a PLA probe
and reagents included in the Duolink II reagent kit or the Duolink
II Bright field reagent kit (Olink).
[0174] In one embodiment of the present invention, the detection
method of the present invention encompasses a step of obtaining a
sample from the subject.
[0175] In one embodiment of the present invention, the subject of
the detection method of the present invention is a cancer patient,
and in a more specific embodiment, the cancer is stomach cancer.
The type of stomach cancer is not particularly limited, but it may
be a diffuse type, an intestinal type, or a mix type in the Lauren
classification. Further, without being limited thereby, the stomach
cancer may be any of papillary adenocarcinoma, tubular
adenocarcinoma, poorly differentiated adenocarcinoma, signet ring
cell carcinoma, or carcinoma mucoides, etc.
[0176] In the detection method of the present invention, it is
possible to judge when the polynucleotide targeted in detection, or
the polypeptide targeted in detection is detected in the sample
obtained from the subject, that the subject has cancer
(particularly, stomach cancer).
[0177] The detection step in the detection method of the present
invention may be used as a method for detecting whether cancer
(particularly, stomach cancer) exists in a subject or a method for
diagnosing cancer (particularly, stomach cancer) in the subject.
The diagnosis method of the present invention may include, in
addition to the aforementioned detection step, a step in which it
is judged when the polynucleotide targeted in detection, or the
polypeptide targeted in detection is detected in the sample
obtained from the subject, that there is a high possibility that
the subject has cancer (particularly, stomach cancer). Further, the
detection step may be used in a method for identifying a subject
(cancer patients of stomach cancer, etc.) that is a candidate for
receiving a treatment by an ARHGAP6 function inhibitor and/or a
pharmaceutical agent that blocks abnormality signal induced by a
fusion gene composed of an RP2 gene and an ARHGAP6 gene. The
identification method of the present invention may include, in
addition to the detection step, a step in which it is judged when a
polynucleotide is detected in a sample obtained from the subject,
that the subject is a candidate for receiving a treatment by an
ARHGAP6 function inhibitor and/or a pharmaceutical agent that
blocks abnormality signal induced by a fusion gene composed of an
RP2 gene and an ARHGAP6 gene.
[0178] The Primer Set, Probe, Probe Set and Detection Kit of the
Present Invention
[0179] The present invention encompasses a primer set, probe, probe
set and a detection kit used in the detection method of the present
invention.
[0180] The primer set of the present invention includes a sense
primer designed from a section encoding RP2 and an antisense primer
designed from a section encoding ARHGAP6, and the antisense primer
consists of an oligonucleotide that hybridizes with the
polynucleotide targeted in detection under a stringent condition
(preferably, under a highly stringent condition), and the sense
primer consists of an oligonucleotide that hybridizes with a
complementary strand of a polynucleotide targeted in detection
under a stringent condition (preferably, under a highly stringent
condition).
[0181] In the primer set of the present invention, either the sense
primer or the antisense primer may be designed so that it
corresponds to a region in a polynucleotide targeted in detection
that comprises a fusion point.
[0182] A specific embodiment of the primer set of the present
invention includes the following primer set:
[0183] a primer set consisting of a sense primer consisting of an
oligonucleotide that hybridizes under a stringent condition with a
complementary strand of a polynucleotide consisting of base no. 1
to 102 of SEQ ID NO: 1 and an antisense primer consisting of an
oligonucleotide that hybridizes under a stringent condition with a
polynucleotide consisting of base no. 103 to 2439 of SEQ ID NO:
1.
[0184] A more specific embodiment of the primer set of the present
invention includes the following primer set:
[0185] a primer set consisting of a sense primer consisting of an
oligonucleotide of at least 16 random consecutive bases between
base no. 1 to 102 of SEQ ID NO: 1 and an antisense primer
consisting of an oligonucleotide that is complementary with at
least 16 random consecutive bases between base no. 103 to 2439 of
SEQ ID NO: 1.
[0186] It is preferable for the primer set to have a space of 1 kb
or lower between the selected positions of the sense primer and the
antisense primer, or a nucleic acid fragment amplified by the sense
primer and the antisense primer with a size of 1 kb or lower.
Further, the primer of the present invention normally has a chain
length of at least 15 bases, preferably at least 16 bases, more
preferably at least 18 bases, even more preferably at least 20
bases. In one embodiment of the present invention, the primer has a
chain length of 15 to 40 bases, preferably 16 to 24 bases, more
preferably 18 to 24 bases, and even more preferably 20 to 24
bases.
[0187] The primers included in the primer set of the present
invention, without being particularly limited, may be produced by a
chemical synthesis method.
[0188] The probes included in the probe of the present invention
and the probe set of the present invention includes an
oligonucleotide that hybridizes with the polynucleotide targeted in
detection under a stringent condition (preferably, under a highly
stringent condition). The chain length of the probes included in
the probe of the present invention or the probe set of the present
invention may be selected as necessary by a person skilled in the
art according to the applied hybridization method, but the probe
preferably has a chain length of at least 16 bases.
[0189] In one embodiment of the present invention, the probe of the
present invention includes an oligonucleotide of at least 16 bases
upstream and at least 16 bases downstream of the fusion point in
the polynucleotide targeted in detection (specifically, the
sequence between base no. 87 to 118 of SEQ ID NO: 1), or an
oligonucleotide that is complementary thereto.
[0190] In one embodiment of the present invention, the probe set of
the present invention includes a probe designed from a section
encoding RP2 (e.g. any section in the RP2 gene region of the fusion
polynucleotide) and a probe designed from a section encoding
ARHGAP6 (e.g. any section in the ARHGAP6 gene region of the fusion
polynucleotide).
[0191] In one embodiment of the present invention, the probe set of
the present invention includes multiple types of probes designed
from a section encoding RP2 and multiple types of probes designed
from a section encoding ARHGAP6.
[0192] In one embodiment of the present invention, the probe set of
the present invention includes the following:
[0193] multiple types of adjacent probe pairs including an
oligonucleotide that is complementary to an oligonucleotide of at
least 16 random consecutive bases between base no. 1 to 102 of SEQ
ID NO: 1 (preferably 10 to 25 types, more preferably 18 to 22
types, even more preferably 20 types of probe pairs), and multiple
types of adjacent probe pairs including an oligonucleotide that is
complementary with an oligonucleotide of at least 16 random
consecutive bases between base no. 103 to 2439 of SEQ ID NO: 1
(preferably 10 to 25 types, more preferably 18 to 22 types, even
more preferably 20 types of probe pairs).
[0194] The probes of the probe pair include an oligonucleotide that
is complementary with the polynucleotide targeted in detection, and
the length of the oligonucleotide is normally at least 16 bases,
preferably at least 18 bases. In one embodiment of the present
invention, the length of the oligonucleotide is 16 to 30 bases,
preferably 18 to 25 bases.
[0195] The probe of the present invention and the probe included in
the probe set of the present invention, without being limited
thereby, may be produced by chemical synthesis.
[0196] The present invention encompasses a detection kit including
a primer set of the present invention, a probe of the present
invention or the probe set of the present invention. The detection
kit of the present invention may include in addition to the primer
set of the present invention, the probe of the present invention or
the probe set of the present invention, components that may be used
together with the primer set, the probe or the probe set for the
detection of a polynucleotide targeted in detection such as
reagents to amplify the signal of hybridization.
[0197] The present invention also encompasses a detection kit for
detecting a polypeptide targeted in detection. Preferably, the
detection kit includes an antibody (primary antibody) that
recognizes a section derived from an RP2 gene of polypeptide
targeted in detection, and an antibody (primary antibody) that
recognizes a section derived from an ARHGAP6 gene of polypeptide
targeted in detection. More preferably, the present invention may
include secondary antibodies connected with oligonucleotides, that
are respectively bound to primary antibodies, two types of
oligonucleotides that are partially complementary to the
oligonucleotides connected to the secondary antibodies, ligase that
forms a circular structure by ligation of the two types of
oligonucleotides when they approach each other, and labeled
oligonucleotide probes.
[0198] The primer set, probe, probe set, and detection kit of the
present invention may be used for the detection method, diagnosis
method, identification method of a patient, and identification
method of a subject of the present invention. In one embodiment of
the present invention, with respect to the primer set, probe, probe
set and detection kit of the present invention, the subject is a
cancer patient and more specifically, the cancer is stomach cancer.
The stomach cancer is not particularly limited, but it may be a
diffuse type, an intestinal type, or a mix type in the Lauren
classification. Further, without being limited thereby, the stomach
cancer may be any of papillary adenocarcinoma, tubular
adenocarcinoma, poorly differentiated adenocarcinoma, signet ring
cell carcinoma, or carcinoma mucoid.
EXAMPLES
[0199] The Examples may be performed by commonly known methods
unless otherwise indicated. When using commercially available
reagents or kits, the Examples may be performed according to the
manuals of the commercial products.
Example 1 Isolation of RP2-ARHGAP6 Fusion Gene
[0200] Total RNA was prepared from stomach cancer cell line NSC-10C
established at the Division of Translational Oncology, National
Cancer Center Research Institute, and reverse-transcribed into cDNA
with reverse transcriptase (SuperScriptIII; Life Technologies) and
Oligo(dT) Primer (Oligo(dT)20 Primer; Life Technologies) according
to the standard protocol of the reagent.
[0201] Next, primers of RP2_full fwd01 represented by SEQ ID NO: 3
and ARHGAP6_full rev01 represented by SEQ ID NO: 4 were used to
perform PCR (10 sec. at 98.degree. C., 15 sec. at 55.degree. C.,
and 3 min at 68.degree. C., 30 cycles, followed by 5 min at
68.degree. C.) using DNA polymerase (PrimeSTAR GXL; TAKARA BIO
INC.) with cDNA obtained in the above step as a template. Then,
using the aforementioned PCR product diluted by 10-fold as a
template, primers of RP2_full fwd02 represented by SEQ ID NO: 5 and
ARHGAP6_full rev02 represented by SEQ ID NO: 6 were subjected to
PCR (10 sec. at 98.degree. C., 15 sec. at 55.degree. C., and 3 min
at 68.degree. C., 30 cycles, followed by 5 min at 68.degree. C.)
using the same DNA polymerase. Electrophoresis was performed after
the PCR to obtain PCR product of about 2.5 kbp. After adding A to
the 3'-end of the PCR product using Takara Taq (TAKARA BIO INC.),
it was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life
Technologies), and sequenced by dideoxy sequencing method (BigDye
Terminator v3.1 Cycle Sequencing Kit; Life Technologies).
Consequently, the PCR product about 2.5 kbp derived from the
NSC-10C cell line was found to be a transcription product (SEQ ID
NO: 1) in which nucleotide sequence of base no. 190 (corresponding
to the 5' terminal of CDS) to 291 of RP2 (NM_006915.2) registered
in NCBI is fused to a nucleotide sequence of base no. 1462 to 3798
of ARHGAP6 (NM_013427.2) (corresponding to the 3' terminal of CDS).
The amino acid sequence of a polypeptide encoded in SEQ ID NO: 1 is
represented by SEQ ID NO: 2.
Example 2 Evaluation of Ability to Suppress Expression of
RP2-ARHGAP6 Fusion Protein in Stomach Cancer Cell Lines Expressing
RP2-ARHGAP6 Fusion Gene Using ARHGAP6 siRNA, and Evaluation of
Viability of Those Cell Lines Under the Same Condition
[0202] After culturing the cell line NSC-10C that expresses
RP2-ARHGAP6 fusion gene, as shown in Example 1, and two substrains,
NSC-10X1aA and NSC-10X1aF, established at the Division of
Translational Oncology, National Cancer Center Research Institute,
in RPMI-1640 medium (Wako Pure Chemical Industries, Ltd.)
containing 10% bovine serum (Gibco), siRNA was introduced into the
cells according to the standard protocol of the transfection
reagent DharmaFECT1 (GE Healthcare). Specifically, the above cancer
cells were seeded at 2.times.10.sup.5 cells per well to a 6 well
plate (140675, Nunc), 75 pmol of siRNA that targets ARHGAP6
(288604, Life Technologies) and control siRNA (AM4611, Life
Technologies) were added to the cells (final concentration 75 nM),
and the cells were cultured at 37.degree. C. under an environment
of 5% CO.sub.2 for 120 h. (hereinafter, the group in which control
siRNA was transfected is referred to as the Control siRNA group,
and the group in which siRNA that targets ARHGAP6 is transfected is
referred to as the ARHGAP6 siRNA group).
[0203] The suppressive effect of RP2-ARHGAP6 fusion protein by
siRNA treatment was evaluated by the Western blot analysis.
Specifically, the cultured cells were dissolved in 350 mM
dithiothreitol (Fermentas)-containing Laemmli Sample Buffer
(Bio-Rad) to extract protein. Protein concentration was measured by
Protein Quantification Assay (MACHEREY-NAGEL GmbH & Co. KG). 5
ug or 20 ug of the protein extract was loaded onto a 8% or 12%
Poly-Acrylamide gel (Serva) containing SDS (Wako Pure Chemical
Industries, Ltd.) and gel electrophoresis was performed for 1 h.
under a condition of 40 mA. After 80 min of transfer to a PVDF
membrane (Millipore Corporation) under a 60 mA condition using
TRANS-BLOT SD SEMI-DRY TRANSFER CELL (Bio-Rad), blocking was
performed for 2 h. at room temperature using PBS containing 5%
Membrane Blocking Agent (GE Healthcare) (hereinafter referred to as
the blocking buffer). The membrane was shaken in a primary antibody
solution of anti-ARHGAP6 antibody (HPA036779, Sigma-Aldrich)
diluted with a blocking buffer to a rate of 1:250 and
anti-.beta.-Actin antibody (4967, Cell Signaling Technology)
diluted with a blocking buffer to a rate of 1:3000, and incubated
overnight at 4.degree. C. After washing with PBS containing 0.05%
Tween 20 (Wako Pure Chemical Industries, Ltd.) (hereinafter
referred to as the washing buffer), the membrane was shaken in a
secondary antibody solution of HRP labeled anti-rabbit antibody
(P0399, Daco) diluted with a blocking buffer to a rate of 1:3000,
and incubated for 1 h. at room temperature. After washing with a
washing buffer, Pierce Western blot Substrate Plus (Thermo Fisher
Scientific Inc.) was added onto the membrane, and the
chemiluminescence on a membrane was detected using LAS-4000R (Fuji
Film). Western blotting analysis confirmed that the expression of
RP2-ARHGAP6 fusion protein was suppressed by the siRNA for ARHGAP6
in all cell lines that endogenously expressed RP2-ARHGAP6 fusion
gene (FIG. 1).
[0204] In order to evaluate the effect of the RP2-ARHGAP6 fusion
gene on the viability of the cancer cells, siRNA that targets
ARHGAP6 and the control siRNA were transfected into the NSC-10C and
the substrain thereof under the same conditions as shown above.
After 24 h., the medium was changed to RPMI-1640 medium containing
0.5% bovine serum, and the cells were seeded at 1.times.10.sup.3
cells per well to a 96 well plate (167008, Nunc), at 100 .mu.L
each, so that cells of each group were seeded to 6 wells, and
cultured for additional 96 h. at 37.degree. C. under a 5% CO.sub.2
environment. Wells containing only RPMI-1640 medium containing 10%
bovine serum without cells was prepared as a control (hereinafter
referred to as the medium group). The number of living cells was
measured according to the standard protocol of Cell Counting Kit-8
(DOJINDO LABORATORIES). Specifically, 10 .mu.L of the reagent was
added per well and the cells were cultured for 4 h. at 37.degree.
C. under a 5% CO.sub.2 environment, then, the number of living
cells was determined by measuring an absorbance of 450 nm by a
micro plate reader (BioTek). Total 4 wells excluding the maximum
and the minimum absorbance values of each group were adopted for
the analysis. Viability of the Control siRNA group and the ARHGAP6
siRNA group was determined by subtracting the absorbance of the
medium group from the absorbance of each group (hereinafter
referred to as the correction value), and setting the correction
value of the Control siRNA group as 100%. Student's t-test was used
for the significance test between Control siRNA group and ARHGAP6
siRNA group.
[0205] Consequently, the viability of cells decreased (FIG. 2) when
the expression level of RP2-ARHGAP6 fusion protein was suppressed
by transfecting siRNA that targets ARHGAP6 into all cell lines that
endogenously express RP2-ARHGAP6 fusion gene. Since the p value is
less than 0.05 even performing the correction by the multiple test
method (by Bonferroni method), it was judged that a significant
difference exists. It was thus found that suppressing the
expression of the fusion gene in the cancer cells that endogenously
express the RP2-ARHGAP6 fusion gene has an effect to suppress the
growth of cancer cells and/or to decrease the survival of those
cells.
[0206] Thus, it was found that the RP2-ARHGAP6 defined the tumor
advancing capacity of cancer cells.
Example 3 Detection of RP2-ARHGAP6 Fusion Gene
[0207] Total RNA prepared from the substrains of NSC-10C, i.e.
NSC-10X1A, NSC-10X1aA, NSC-10X1F, NSC-10X1aF, NSC-10X1aX1 and
NSC-10X1aX1a, the stomach cancer cell lines KATO-III (JCRB0611,
JCRB cell bank) and HSC-39 (provided from National Cancer Center
Japan, Animal Experiment Section) and stomach cancer cell lines
NSC-9C, NSC-6C and NSC-16C that were established at the Division of
Translational Oncology, National Cancer Center Research Institute,
in addition to the stomach cancer cell line NSC-10C that expresses
an RP2-ARHGAP6 fusion gene shown in Example 1, were
reverse-transcribed into cDNA using reverse transcriptase
(SuperScriptIII; Life Technologies) and oligo(dT) primer (oligo(dT)
20 primer; Life Technologies).
[0208] Next, primers of RP2_CDS_F23 represented by SEQ ID NO: 7 and
ARHGAP6_GCF-R1 represented by SEQ ID NO: 8 were used to perform PCR
(2 min at 94.degree. C., followed by 15 sec. at 94.degree. C., 15
sec. at 55.degree. C., and 1 min at 68.degree. C., 30 cycles) using
DNA polymerase (AccuPrime Taq DNA Polymerase; Life Technologies),
and cDNA obtained above as a template (200 ng when converted to
total RNA). Likewise, to confirm the equal amount of cDNA template
in the reactions, primers of ACTB_F2 represented by SEQ ID NO: 9
and ACTB_R2 represented by SEQ ID NO: 10 were used to perform PCR
(2 min at 94.degree. C., followed by 15 sec. at 94.degree. C., 15
sec. at 55.degree. C., and 1 min at 68.degree. C., 25 cycles) using
the same DNA polymerase as above. Electrophoresis was performed
with 2% agarose gel (Lonza) after PCR reaction, and about 200 bp
PCR product was amplified only at NSC-10C and substrains thereof
which were already confirmed the expression of RP2-ARHGAP6 fusion
gene (FIG. 3). The PCR product amplified by the aforementioned
primer set was 214 bp according to the nucleotide sequence of the
fusion gene identified in Example 1. Therefore, it was showed that
the fusion gene that expresses the cancer cells is detectable with
PCR method.
INDUSTRIAL APPLICABILITY
[0209] The detection method of the present invention is a method
for detecting a fusion gene composed of the RP2 gene and the
ARHGAP6 gene, and it is useful as a method for detecting and
diagnosing cancer in a subject. Further, the primer set and the
detection kit of the present invention may be used in a method of
the present invention.
Sequence CWU 1
1
1012439DNAHomo sapiensCDS(1)..(2439) 1atg ggc tgc ttc ttc tcc aag
aga cgg aag gct gac aag gag tcg cgg 48Met Gly Cys Phe Phe Ser Lys
Arg Arg Lys Ala Asp Lys Glu Ser Arg1 5 10 15ccc gag aac gag gag gag
cgg cca aag cag tac agc tgg gat cag cgc 96Pro Glu Asn Glu Glu Glu
Arg Pro Lys Gln Tyr Ser Trp Asp Gln Arg 20 25 30gag aag ggt gat ttc
acc tgg aac agc atg tca ggc cgc agt gta cgg 144Glu Lys Gly Asp Phe
Thr Trp Asn Ser Met Ser Gly Arg Ser Val Arg 35 40 45ctg agg tca gtc
ccc atc cag agt ctc tca gag ctg gag agg gcc cgg 192Leu Arg Ser Val
Pro Ile Gln Ser Leu Ser Glu Leu Glu Arg Ala Arg 50 55 60ctg cag gaa
gtg gct ttt tat cag ttg caa cag gac tgt gac ctg agc 240Leu Gln Glu
Val Ala Phe Tyr Gln Leu Gln Gln Asp Cys Asp Leu Ser65 70 75 80tgt
cag atc acc att ccc aaa gat gga caa aag aga aag aaa tct tta 288Cys
Gln Ile Thr Ile Pro Lys Asp Gly Gln Lys Arg Lys Lys Ser Leu 85 90
95aga aag aaa ctg gat tca cta gga aag gag aaa aac aaa gac aaa gaa
336Arg Lys Lys Leu Asp Ser Leu Gly Lys Glu Lys Asn Lys Asp Lys Glu
100 105 110ttc atc cca cag gca ttt gga atg ccc tta tcc caa gtc att
gcg aat 384Phe Ile Pro Gln Ala Phe Gly Met Pro Leu Ser Gln Val Ile
Ala Asn 115 120 125gac agg gcc tat aaa ctc aag cag gac ttg cag agg
gac gag cag aaa 432Asp Arg Ala Tyr Lys Leu Lys Gln Asp Leu Gln Arg
Asp Glu Gln Lys 130 135 140gat gca tct gac ttt gtg gct tcc ctc ctc
cca ttt gga aat aaa aga 480Asp Ala Ser Asp Phe Val Ala Ser Leu Leu
Pro Phe Gly Asn Lys Arg145 150 155 160caa aac aaa gaa ctc tca agc
agt aac tca tct ctc agc tca acc tca 528Gln Asn Lys Glu Leu Ser Ser
Ser Asn Ser Ser Leu Ser Ser Thr Ser 165 170 175gaa aca ccg aat gag
tca acg tcc cca aac acc ccg gaa ccg gct cct 576Glu Thr Pro Asn Glu
Ser Thr Ser Pro Asn Thr Pro Glu Pro Ala Pro 180 185 190cgg gct agg
agg agg ggt gcc atg tca gtg gat tct atc acc gat ctt 624Arg Ala Arg
Arg Arg Gly Ala Met Ser Val Asp Ser Ile Thr Asp Leu 195 200 205gat
gac aat cag tct cga cta cta gaa gct tta caa ctt tcc ttg cct 672Asp
Asp Asn Gln Ser Arg Leu Leu Glu Ala Leu Gln Leu Ser Leu Pro 210 215
220gct gag gct caa agt aaa aag gaa aaa gcc aga gat aag aaa ctc agt
720Ala Glu Ala Gln Ser Lys Lys Glu Lys Ala Arg Asp Lys Lys Leu
Ser225 230 235 240ctg aat cct att tac aga cag gtc cct agg ctg gtg
gac agc tgc tgt 768Leu Asn Pro Ile Tyr Arg Gln Val Pro Arg Leu Val
Asp Ser Cys Cys 245 250 255cag cac cta gaa aaa cat ggc ctc cag aca
gtg ggg ata ttc cga gtt 816Gln His Leu Glu Lys His Gly Leu Gln Thr
Val Gly Ile Phe Arg Val 260 265 270gga agc tca aaa aag aga gtg aga
caa tta cgt gag gaa ttt gac cgt 864Gly Ser Ser Lys Lys Arg Val Arg
Gln Leu Arg Glu Glu Phe Asp Arg 275 280 285ggg att gat gtc tct ctg
gag gag gag cac agt gtt cat gat gtg gca 912Gly Ile Asp Val Ser Leu
Glu Glu Glu His Ser Val His Asp Val Ala 290 295 300gcc ttg ctg aaa
gag ttc ctg agg gac atg cca gac ccc ctt ctc acc 960Ala Leu Leu Lys
Glu Phe Leu Arg Asp Met Pro Asp Pro Leu Leu Thr305 310 315 320agg
gag ctg tac aca gct ttc atc aac act ctc ttg ttg gag ccg gag 1008Arg
Glu Leu Tyr Thr Ala Phe Ile Asn Thr Leu Leu Leu Glu Pro Glu 325 330
335gaa cag ctg ggc acc ttg cag ctc ctc ata tac ctt cta cct ccc tgc
1056Glu Gln Leu Gly Thr Leu Gln Leu Leu Ile Tyr Leu Leu Pro Pro Cys
340 345 350aac tgc gac acc ctc cac cgc ctg cta cag ttc ctc tcc atc
gtg gcc 1104Asn Cys Asp Thr Leu His Arg Leu Leu Gln Phe Leu Ser Ile
Val Ala 355 360 365agg cat gcc gat gac aac atc agc aaa gat ggg caa
gag gtc act ggg 1152Arg His Ala Asp Asp Asn Ile Ser Lys Asp Gly Gln
Glu Val Thr Gly 370 375 380aat aaa atg aca tct cta aac tta gcc acc
ata ttt gga ccc aac ctg 1200Asn Lys Met Thr Ser Leu Asn Leu Ala Thr
Ile Phe Gly Pro Asn Leu385 390 395 400ctg cac aag cag aag tca tca
gac aaa gaa ttc tca gtt cag agt tca 1248Leu His Lys Gln Lys Ser Ser
Asp Lys Glu Phe Ser Val Gln Ser Ser 405 410 415gcc cgg gct gag gag
agc acg gcc atc atc gct gtt gtg caa aag atg 1296Ala Arg Ala Glu Glu
Ser Thr Ala Ile Ile Ala Val Val Gln Lys Met 420 425 430att gaa aat
tat gaa gcc ctg ttc atg gtt ccc cca gat ctc cag aac 1344Ile Glu Asn
Tyr Glu Ala Leu Phe Met Val Pro Pro Asp Leu Gln Asn 435 440 445gaa
gtg ctg atc agc ctg tta gag acc gat cct gat gtc gtg gac tat 1392Glu
Val Leu Ile Ser Leu Leu Glu Thr Asp Pro Asp Val Val Asp Tyr 450 455
460tta ctc aga aga aag gct tcc caa tca tca agc cct gac atg ctg cag
1440Leu Leu Arg Arg Lys Ala Ser Gln Ser Ser Ser Pro Asp Met Leu
Gln465 470 475 480tcg gaa gtt tcc ttt tcc gtg gga ggg agg cat tca
tct aca gac tcc 1488Ser Glu Val Ser Phe Ser Val Gly Gly Arg His Ser
Ser Thr Asp Ser 485 490 495aac aag gcc tcc agc gga gac atc tcc cct
tat gac aac aac tcc cca 1536Asn Lys Ala Ser Ser Gly Asp Ile Ser Pro
Tyr Asp Asn Asn Ser Pro 500 505 510gtg ctg tct gag cgc tcc ctg ctg
gct atg caa gag gac gcg gcc ccg 1584Val Leu Ser Glu Arg Ser Leu Leu
Ala Met Gln Glu Asp Ala Ala Pro 515 520 525ggg ggc tcg gag aag ctt
tac aga gtg cca ggg cag ttt atg ctg gtg 1632Gly Gly Ser Glu Lys Leu
Tyr Arg Val Pro Gly Gln Phe Met Leu Val 530 535 540ggc cac ttg tcg
tcg tca aag tca agg gaa agt tct cct gga cca agg 1680Gly His Leu Ser
Ser Ser Lys Ser Arg Glu Ser Ser Pro Gly Pro Arg545 550 555 560ctt
ggg aaa gat ctg tca gag gag cct ttc gat atc tgg gga act tgg 1728Leu
Gly Lys Asp Leu Ser Glu Glu Pro Phe Asp Ile Trp Gly Thr Trp 565 570
575cat tca aca tta aaa agc gga tcc aaa gac cca gga atg aca ggt tcc
1776His Ser Thr Leu Lys Ser Gly Ser Lys Asp Pro Gly Met Thr Gly Ser
580 585 590tct gga gac att ttt gaa agc agc tcc cta aga gcg ggg ccc
tgc tcc 1824Ser Gly Asp Ile Phe Glu Ser Ser Ser Leu Arg Ala Gly Pro
Cys Ser 595 600 605ctt tct caa ggg aac ctg tcc cca aat tgg cct cgg
tgg cag ggg agc 1872Leu Ser Gln Gly Asn Leu Ser Pro Asn Trp Pro Arg
Trp Gln Gly Ser 610 615 620ccc gca gag ctg gac agc gac acg cag ggg
gct cgg agg act cag gcc 1920Pro Ala Glu Leu Asp Ser Asp Thr Gln Gly
Ala Arg Arg Thr Gln Ala625 630 635 640gca gcc ccc gcg acg gag ggc
agg gcc cac cct gcg gtg tcg cgc gcc 1968Ala Ala Pro Ala Thr Glu Gly
Arg Ala His Pro Ala Val Ser Arg Ala 645 650 655tgc agc acg ccc cac
gtc cag gtg gca ggg aaa gcc gag cgg ccc acg 2016Cys Ser Thr Pro His
Val Gln Val Ala Gly Lys Ala Glu Arg Pro Thr 660 665 670gcc agg tcg
gag cag tac ttg acc ctg agc ggc gcc cac gac ctc agc 2064Ala Arg Ser
Glu Gln Tyr Leu Thr Leu Ser Gly Ala His Asp Leu Ser 675 680 685gag
agt gag ctg gat gtg gcc ggg ctg cag agc cgg gcc aca cct cag 2112Glu
Ser Glu Leu Asp Val Ala Gly Leu Gln Ser Arg Ala Thr Pro Gln 690 695
700tgc caa aga ccc cat ggg agt ggg agg gat gac aag cgg ccc ccg cct
2160Cys Gln Arg Pro His Gly Ser Gly Arg Asp Asp Lys Arg Pro Pro
Pro705 710 715 720cca tac ccg ggc cca ggg aag ccc gcg gca gcg gca
gcc tgg atc cag 2208Pro Tyr Pro Gly Pro Gly Lys Pro Ala Ala Ala Ala
Ala Trp Ile Gln 725 730 735ggg ccc ccg gaa ggc gtg gag aca ccc acg
gac cag gga ggc caa gca 2256Gly Pro Pro Glu Gly Val Glu Thr Pro Thr
Asp Gln Gly Gly Gln Ala 740 745 750gcc gag cga gag cag cag gtc acg
cag aaa aaa ctg agc agc gcc aac 2304Ala Glu Arg Glu Gln Gln Val Thr
Gln Lys Lys Leu Ser Ser Ala Asn 755 760 765tcc ctg cca gcg ggc gag
cag gac agt ccg cgc ctg ggg gac gct ggc 2352Ser Leu Pro Ala Gly Glu
Gln Asp Ser Pro Arg Leu Gly Asp Ala Gly 770 775 780tgg ctc gac tgg
cag aga gag cgc tgg cag atc tgg gag ctc ctg tcg 2400Trp Leu Asp Trp
Gln Arg Glu Arg Trp Gln Ile Trp Glu Leu Leu Ser785 790 795 800acc
gac aac ccc gat gcc ctg ccc gag acg ctg gtc tga 2439Thr Asp Asn Pro
Asp Ala Leu Pro Glu Thr Leu Val 805 8102812PRTHomo sapiens 2Met Gly
Cys Phe Phe Ser Lys Arg Arg Lys Ala Asp Lys Glu Ser Arg1 5 10 15Pro
Glu Asn Glu Glu Glu Arg Pro Lys Gln Tyr Ser Trp Asp Gln Arg 20 25
30Glu Lys Gly Asp Phe Thr Trp Asn Ser Met Ser Gly Arg Ser Val Arg
35 40 45Leu Arg Ser Val Pro Ile Gln Ser Leu Ser Glu Leu Glu Arg Ala
Arg 50 55 60Leu Gln Glu Val Ala Phe Tyr Gln Leu Gln Gln Asp Cys Asp
Leu Ser65 70 75 80Cys Gln Ile Thr Ile Pro Lys Asp Gly Gln Lys Arg
Lys Lys Ser Leu 85 90 95Arg Lys Lys Leu Asp Ser Leu Gly Lys Glu Lys
Asn Lys Asp Lys Glu 100 105 110Phe Ile Pro Gln Ala Phe Gly Met Pro
Leu Ser Gln Val Ile Ala Asn 115 120 125Asp Arg Ala Tyr Lys Leu Lys
Gln Asp Leu Gln Arg Asp Glu Gln Lys 130 135 140Asp Ala Ser Asp Phe
Val Ala Ser Leu Leu Pro Phe Gly Asn Lys Arg145 150 155 160Gln Asn
Lys Glu Leu Ser Ser Ser Asn Ser Ser Leu Ser Ser Thr Ser 165 170
175Glu Thr Pro Asn Glu Ser Thr Ser Pro Asn Thr Pro Glu Pro Ala Pro
180 185 190Arg Ala Arg Arg Arg Gly Ala Met Ser Val Asp Ser Ile Thr
Asp Leu 195 200 205Asp Asp Asn Gln Ser Arg Leu Leu Glu Ala Leu Gln
Leu Ser Leu Pro 210 215 220Ala Glu Ala Gln Ser Lys Lys Glu Lys Ala
Arg Asp Lys Lys Leu Ser225 230 235 240Leu Asn Pro Ile Tyr Arg Gln
Val Pro Arg Leu Val Asp Ser Cys Cys 245 250 255Gln His Leu Glu Lys
His Gly Leu Gln Thr Val Gly Ile Phe Arg Val 260 265 270Gly Ser Ser
Lys Lys Arg Val Arg Gln Leu Arg Glu Glu Phe Asp Arg 275 280 285Gly
Ile Asp Val Ser Leu Glu Glu Glu His Ser Val His Asp Val Ala 290 295
300Ala Leu Leu Lys Glu Phe Leu Arg Asp Met Pro Asp Pro Leu Leu
Thr305 310 315 320Arg Glu Leu Tyr Thr Ala Phe Ile Asn Thr Leu Leu
Leu Glu Pro Glu 325 330 335Glu Gln Leu Gly Thr Leu Gln Leu Leu Ile
Tyr Leu Leu Pro Pro Cys 340 345 350Asn Cys Asp Thr Leu His Arg Leu
Leu Gln Phe Leu Ser Ile Val Ala 355 360 365Arg His Ala Asp Asp Asn
Ile Ser Lys Asp Gly Gln Glu Val Thr Gly 370 375 380Asn Lys Met Thr
Ser Leu Asn Leu Ala Thr Ile Phe Gly Pro Asn Leu385 390 395 400Leu
His Lys Gln Lys Ser Ser Asp Lys Glu Phe Ser Val Gln Ser Ser 405 410
415Ala Arg Ala Glu Glu Ser Thr Ala Ile Ile Ala Val Val Gln Lys Met
420 425 430Ile Glu Asn Tyr Glu Ala Leu Phe Met Val Pro Pro Asp Leu
Gln Asn 435 440 445Glu Val Leu Ile Ser Leu Leu Glu Thr Asp Pro Asp
Val Val Asp Tyr 450 455 460Leu Leu Arg Arg Lys Ala Ser Gln Ser Ser
Ser Pro Asp Met Leu Gln465 470 475 480Ser Glu Val Ser Phe Ser Val
Gly Gly Arg His Ser Ser Thr Asp Ser 485 490 495Asn Lys Ala Ser Ser
Gly Asp Ile Ser Pro Tyr Asp Asn Asn Ser Pro 500 505 510Val Leu Ser
Glu Arg Ser Leu Leu Ala Met Gln Glu Asp Ala Ala Pro 515 520 525Gly
Gly Ser Glu Lys Leu Tyr Arg Val Pro Gly Gln Phe Met Leu Val 530 535
540Gly His Leu Ser Ser Ser Lys Ser Arg Glu Ser Ser Pro Gly Pro
Arg545 550 555 560Leu Gly Lys Asp Leu Ser Glu Glu Pro Phe Asp Ile
Trp Gly Thr Trp 565 570 575His Ser Thr Leu Lys Ser Gly Ser Lys Asp
Pro Gly Met Thr Gly Ser 580 585 590Ser Gly Asp Ile Phe Glu Ser Ser
Ser Leu Arg Ala Gly Pro Cys Ser 595 600 605Leu Ser Gln Gly Asn Leu
Ser Pro Asn Trp Pro Arg Trp Gln Gly Ser 610 615 620Pro Ala Glu Leu
Asp Ser Asp Thr Gln Gly Ala Arg Arg Thr Gln Ala625 630 635 640Ala
Ala Pro Ala Thr Glu Gly Arg Ala His Pro Ala Val Ser Arg Ala 645 650
655Cys Ser Thr Pro His Val Gln Val Ala Gly Lys Ala Glu Arg Pro Thr
660 665 670Ala Arg Ser Glu Gln Tyr Leu Thr Leu Ser Gly Ala His Asp
Leu Ser 675 680 685Glu Ser Glu Leu Asp Val Ala Gly Leu Gln Ser Arg
Ala Thr Pro Gln 690 695 700Cys Gln Arg Pro His Gly Ser Gly Arg Asp
Asp Lys Arg Pro Pro Pro705 710 715 720Pro Tyr Pro Gly Pro Gly Lys
Pro Ala Ala Ala Ala Ala Trp Ile Gln 725 730 735Gly Pro Pro Glu Gly
Val Glu Thr Pro Thr Asp Gln Gly Gly Gln Ala 740 745 750Ala Glu Arg
Glu Gln Gln Val Thr Gln Lys Lys Leu Ser Ser Ala Asn 755 760 765Ser
Leu Pro Ala Gly Glu Gln Asp Ser Pro Arg Leu Gly Asp Ala Gly 770 775
780Trp Leu Asp Trp Gln Arg Glu Arg Trp Gln Ile Trp Glu Leu Leu
Ser785 790 795 800Thr Asp Asn Pro Asp Ala Leu Pro Glu Thr Leu Val
805 810318DNAHomo sapiens 3caaactaagg ctgcggac 18418DNAHomo sapiens
4gtgctctacc tctgtagg 18518DNAHomo sapiens 5gttcacgcca cactctag
18618DNAHomo sapiens 6actaagtgtg ccagtggc 18720DNAHomo sapiens
7gacggaaggc tgacaaggag 20822DNAHomo sapiens 8aggtcacagt cctgttgcaa
ct 22920DNAHomo sapiens 9gaagtccctt gccatcctaa 201020DNAHomo
sapiens 10gcacgaaggc tcatcattca 20
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