WO2015129655A1

WO2015129655A1 - Method for detecting dnajb1-prkaca gene

Info

Publication number: WO2015129655A1
Application number: PCT/JP2015/055123
Authority: WO
Inventors: 泰介中澤; 真浅海; 収池田; 綾小林; 和久角山
Original assignee: アステラス製薬株式会社
Priority date: 2014-02-25
Filing date: 2015-02-24
Publication date: 2015-09-03
Also published as: JP2017077183A

Abstract

[Problem] The problem is to elucidate a polynucleotide that is a novel causative gene of cancer and thereby provide a method for detecting this polynucleotide or a polypeptide encoded thereby, as well as a primer set and detection kit for this purpose. [Means of solution] A fusion gene of part of the DNAJB1 gene and part of the PRKACA gene, or a fusion protein encoded thereby, is detected in this detection method. The primer set includes a sense primer designed from a portion that encodes DNAJB1 and an antisense primer designed from a portion that encodes PRKACA.

Description

Method for detecting DNAJB1-PRKACA gene

The present invention relates to a novel fusion gene containing a PRKACA kinase region or a method for detecting a fusion protein encoded by the fusion gene.

The DNJ homolog subfamily B member 1 (DnaJ homolog, subfamily B, member 1; DNAJB1) gene is present in the short arm of human chromosome 19 and the encoding protein is a heat shock protein known as Hsp40, It has a J domain on the amino terminal side. Similarly, DNAJB1 controls its function as a chaperone through controlling the ATP hydrolysis activity of Hsc70, which is a heat shock protein (Journal of Biological Chemistry, 1996, Vol. 271, p. 19617-19624). Regarding the relationship with cancer, it is known that the malignancy of colorectal cancer and the expression level of DNAJA3, which is one of the Hsp40 family, are correlated (International Journal of Molecular Medicine, 2008, 21, p. 19-). 31).

The protein kinase cyclic MP catalytic alpha (protein kinase, cAMP-dependent, catalytic, alpha; PRKACA) gene is present in the short arm of human chromosome 19 as the DNAJB1 gene, and the encoded protein is located in the center. Serine threonine kinase having a kinase domain, which functions as an active domain of protein kinase A (PKA) complex. Specifically, when cAMP binds to the control subunit of the PKA complex, PRKACA released from the control subunit is activated, phosphorylates downstream proteins, and transmits various signals. One of them is phosphorylation of the transcription factor CREB. As a result, it induces binding to CREB-binding protein (CBP) and activates transcription of the target gene (Molecular Biology of the Cell, Fourth Edition, 2002, p.856-858). In relation to cancer, an inactivating mutation in PRKAAR1A, a regulatory subunit of the PKA complex, is often found in the Carney Complex, a disease that causes tumors with a high probability (Human Molecular Genetics). 2000, 9, p. 3037-3046), the lack of CREB function suppresses proliferation and metastasis of melanoma cell lines (Oncogene, 1997, 15, p. 2069-2075), It has been reported that activation of PKA and CREB is involved in drug resistance in melanoma (Nature, 2013, 504, 138-142). In addition, when PRKACA is knocked down, migration and invasion of tumor cells in hypoxia are inhibited. On the other hand, when PRKACA is overexpressed in tumor cells lacking PRKACA, cell migration and invasion are restored (non-patented). Reference 1) has been reported.

However, there have been no reports on DNAJB1 fusion gene or PRKACA fusion gene.

It is an object of the present invention to elucidate a polynucleotide that is a new causative gene of cancer, thereby providing a method for detecting the polynucleotide or a polypeptide encoded thereby, and a primer set or a detection kit therefor.

In the present invention, a novel fusion gene in which a part of a PRKACA gene that is a kinase and a part of a DNAJB1 gene are isolated and identified from a specimen obtained from a liver cancer patient (Example 1), and the fusion gene is partly A retrovirus for expressing the fusion gene (Example 3), and the retrovirus-infected cells have tumorigenicity, and the fusion gene Was found to be a causative gene for cancer (Example 4). Based on these findings, the present inventor constructed a method for detecting the fusion gene or a fusion protein encoded thereby, provides a primer set for the method, and detects the fusion gene or the fusion protein encoded thereby. It was made possible to select cancer patients (especially liver cancer patients) positive for the fusion gene of DNAJB1 gene and PRKACA gene.

That is, the present invention relates to the following [1] to [22].
[1] A DNJ homolog subfamily B member 1 (DNAJB1) gene and a protein kinase cyclic mp dippen comprising the step of detecting the presence of a polynucleotide encoding the following polypeptide in a sample obtained from a subject Method for detecting a fusion gene with a dent catalytic alpha (PRKACA) gene:
A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity.
[2] The polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2 and has a tumorigenicity, or the amino acid sequence shown in SEQ ID NO: 2 has 1 to 10 amino acids deleted, [1] The method according to [1], which is a polypeptide comprising an amino acid sequence substituted and / or added and having tumorigenicity.
[3] The method according to [1], wherein the polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
[4] The method according to any one of [1] to [3], further comprising a step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when the polynucleotide is detected.
[5] The method includes a step of amplifying a nucleic acid in a sample obtained from a subject in order to detect the polynucleotide, or a step of hybridizing a probe to a nucleic acid in a sample obtained from the subject. The method according to any one of [4].
[6] The method according to [5], comprising a step of amplifying a nucleic acid in a sample obtained from a subject using the following primer set:
A primer set for detecting a fusion gene of DNAJB1 gene and PRKACA gene, comprising a sense primer designed from a portion encoding DNAJB1 and an antisense primer designed from a portion encoding PRKACA, The primer set is composed of an oligonucleotide that hybridizes to the polynucleotide under stringent conditions, and the sense primer is composed of an oligonucleotide that hybridizes to the complementary strand of the polynucleotide under stringent conditions.
[7] The sense primer is composed of an oligonucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of base numbers 1 to 211 of SEQ ID NO: 1 or 3, and the antisense primer is SEQ ID NO: 1 or 3 The method according to [6], comprising an oligonucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base numbers 212 to 1221.
[8] The sense primer is composed of any continuous oligonucleotide of at least 16 bases between base numbers 1 to 211 of SEQ ID NO: 1 or 3, and the antisense primer is between base numbers 212 to 1221 of SEQ ID NO: 1 or 3 The method according to [6] or [7], comprising an oligonucleotide that is complementary to any continuous oligonucleotide of at least 16 bases.
[9] The method according to any one of [6] to [8], further comprising a step of detecting whether or not an amplified nucleic acid fragment having a target size is obtained.
[10] The method according to [9], further comprising the step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when an amplified nucleic acid fragment of a target size is obtained.
[11] The method according to any one of [6] to [8], further comprising the step of determining the base sequence of the amplified nucleic acid fragment.
[12] A step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when the amplified nucleic acid fragment contains the base sequence of the portion encoding DNAJB1 and the base sequence of the portion encoding PRKACA in the same fragment The method according to [11], further comprising.
[13] The method according to [5], comprising a step of hybridizing a probe containing an oligonucleotide that hybridizes to the polynucleotide under stringent conditions to a nucleic acid in a sample obtained from a subject.
[14] including a step of performing in situ hybridization using a sample obtained from a subject, a probe designed from a portion encoding DNAJB1 of the polynucleotide, and a probe designed from a portion encoding PRKACA of the polynucleotide The method according to [13].
[15] The method according to [14], wherein a plurality of types of probes designed from a portion encoding DNAJB1 and a plurality of types of probes designed from a portion encoding the PRKACA are used.
[16] Plural kinds of adjacent probe pairs including an oligonucleotide complementary to any continuous oligonucleotide of at least 16 bases of base numbers 1 to 211 of SEQ ID NO: 1 or 3, and SEQ ID NO: 1 or 3 The method according to [14] or [15], wherein a plurality of adjacent probe pairs containing an oligonucleotide that is complementary to any consecutive oligonucleotide having at least 16 bases of nucleotide numbers 212 to 1221
[17] The method according to any one of [14] to [16], further comprising a step of amplifying a hybridization signal.
[18] The method further includes the step of detecting an overlap of a signal from a probe designed from a portion encoding DNAJB1 and a signal from a probe designed from a portion encoding PRKACA. [14] to [17 ] The method in any one of.
[19] The method according to [18], further comprising the step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when it is detected that the two signals are in the same place.
[20] The method according to any one of [1] to [19], comprising a step of obtaining a sample from a subject.
[21] The method according to any one of [1] to [20], wherein the subject is a cancer patient.
[22] The method according to [21], wherein the cancer is liver cancer.

The present invention also relates to the following [23] to [25].
[23] A method for detecting the presence of cancer in a subject, comprising the step according to any one of [1] to [19].
[24] The method according to [23], comprising a step of obtaining a sample from a subject.
[25] The method according to [23] or [24], wherein the cancer is liver cancer.

The present invention also relates to the following [26] to [30].
[26] A method for diagnosing cancer in a subject, comprising the steps according to any one of [1] to [19].
[27] The method according to [26], comprising the step of obtaining a sample from the subject.
[28] The method further includes the step of determining that the subject is highly likely to have cancer when a fusion gene of the DNAJB1 gene and the PRKACA gene is detected from a sample obtained from the subject, [26] or The method according to [27].
[29] The method according to [26] or [27], wherein the cancer is liver cancer.
[30] The method further includes a step of determining that the subject is highly likely to have liver cancer when a fusion gene of the DNAJB1 gene and the PRKACA gene is detected from a sample obtained from the subject. [29] The method described in 1.

The present invention also relates to the following [31] to [34].
[31] A method for identifying a subject to be treated with a PRKACA inhibitor and / or a drug that blocks an abnormal signal caused by a fusion gene of DNAJB1 gene and PRKACA gene, the subject being a cancer patient A method comprising the steps according to any one of [1] to [19].
[32] The method according to [31], comprising a step of obtaining a sample from a subject.
[33] When a fusion gene of the DNAJB1 gene and the PRKACA gene is detected from a sample obtained from the subject, the subject has an abnormal signal caused by the PRKACA inhibitor and / or the fusion gene of the DNAJB1 gene and the PRKACA gene. The method according to [31] or [32], further comprising the step of determining that it is an indication for treatment with an agent to be blocked.
[34] The method according to any of [31] to [33], wherein the cancer is liver cancer.

The present invention also relates to the following [35] to [39].
[35] A primer set for detecting a fusion gene of DNAJB1 gene and PRKACA gene in a sample obtained from a subject, which is designed from a sense primer designed from a portion encoding DNAJB1 and a portion encoding PRKACA The antisense primer comprises an oligonucleotide that hybridizes under stringent conditions to the polynucleotide according to any one of [1] to [3], and the sense primer comprises A primer set comprising oligonucleotides that hybridize under stringent conditions to a complementary strand of nucleotides.
[36] The sense primer comprises an oligonucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of base numbers 1 to 211 of SEQ ID NO: 1 or 3, and the antisense primer is SEQ ID NO: 1 or 3 The primer set according to [35], which comprises an oligonucleotide that hybridizes under stringent conditions to a polynucleotide comprising the base numbers 212 to 1221.
[37] The sense primer consists of any continuous oligonucleotide of at least 16 bases between base numbers 1 to 211 of SEQ ID NO: 1 or 3, and the antisense primer is between base numbers 212 to 1221 of SEQ ID NO: 1 or 3 The primer set according to [35] or [36], comprising an oligonucleotide that is complementary to any continuous oligonucleotide of at least 16 bases.
[38] The primer set according to any one of [35] to [37], wherein the subject is a cancer patient.
[39] The primer set according to [38], wherein the cancer is liver cancer.

The present invention also relates to the following [40] to [45].
[40] A probe for detecting a fusion gene of a DNAJB1 gene and a PRKACA gene in a sample obtained from a subject under conditions stringent to the polynucleotide according to any one of [1] to [3] A probe comprising an oligonucleotide that hybridizes with.
[41] A probe set including a plurality of probes according to [40], which encodes a probe designed from the portion encoding DNAJB1 of the polynucleotide according to any one of [1] to [3] and PRKACA A probe set comprising probes designed from parts.
[42] The probe set according to [41], comprising a plurality of types of probes designed from a portion encoding DNAJB1, and a plurality of types of probes designed from a portion encoding PRKACA.
[43] Plural kinds of adjacent probe pairs containing an oligonucleotide complementary to any continuous oligonucleotide of at least 16 bases of base numbers 1 to 211 of SEQ ID NO: 1 or 3, and SEQ ID NO: 1 or 3 The probe set according to [41] or [42], comprising a plurality of adjacent probe pairs including an oligonucleotide that is complementary to any continuous oligonucleotide having at least 16 bases of base numbers 212 to 1221.
[44] The probe or probe set according to any of [40] to [43], wherein the subject is a cancer patient.
[45] The probe or probe set according to [44], wherein the cancer is liver cancer.

The present invention also relates to the following [46] to [50].
[46] A detection kit for detecting a fusion gene of DNAJB1 gene and PRKACA gene in a sample obtained from a subject, comprising the primer set according to any of [35] to [37] Gene detection kit.
[47] A detection kit for detecting a fusion gene of DNAJB1 gene and PRKACA gene in a sample obtained from a subject, comprising the probe or probe set according to any of [40] to [43] Detection kit.
[48] The detection kit according to [47], further comprising a reagent for amplifying a hybridization signal.
[49] The detection kit according to any of [46] to [48], wherein the subject is a cancer patient.
[50] The detection kit according to [49], wherein the cancer is liver cancer.

The present invention also relates to the following [51] to [58].
[51] A method for detecting a fusion protein of DNAJB1 and PRKACA, comprising the step of detecting the presence of the following polypeptide in a sample obtained from a subject:
A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity.
[52] The polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2 and has a tumorigenicity, or the amino acid sequence shown in SEQ ID NO: 2 has 1 to 10 amino acids deleted, [51] The method according to [51], which is a polypeptide comprising a substituted and / or added amino acid sequence and having tumorigenicity.
[53] The method according to [51], wherein the polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
[54] In the step of detecting the presence of the polypeptide, an antibody that recognizes a DNAJB1 gene-derived portion of the polypeptide (primary antibody) and an antibody that recognizes a PRKACA gene-derived portion of the polypeptide in a sample obtained from a subject ( The method according to any one of [51] to [53], further comprising a step of contacting a primary antibody).
[55] The method according to [54], further comprising the steps described in i) to v) below:
i) a step of adding a secondary antibody linked to an oligonucleotide, each binding to the primary antibody, ii) two types of oligonucleotides partially complementary to the oligonucleotide linked to the secondary antibody, and A step of adding a ligation solution containing ligase that can form a circular structure by ligating the two types of oligonucleotides when they are close to each other, and ligation reaction, iii) extending the nucleic acid along the formed circular structure Iv) hybridizing a labeled oligonucleotide probe capable of hybridizing to the elongated nucleic acid; and v) detecting the label signal.
[56] The method according to any one of [51] to [55], comprising a step of obtaining a sample from a subject.
[57] The method according to any of [51] to [56], wherein the subject is a cancer patient.
[58] The method of [57], wherein the cancer is liver cancer.

The present invention also relates to the following [59] to [61].
[59] A method for detecting the presence of cancer in a subject comprising the steps according to any of [51] to [55].
[60] The method of [59], comprising obtaining a sample from a subject.
[61] The method of [59] or [60], wherein the cancer is liver cancer.

The present invention also relates to the following [62] to [66].
[62] A method for diagnosing cancer in a subject, comprising the steps according to any of [51] to [55].
[63] The method of [62], comprising obtaining a sample from a subject.
[64] The method further includes the step of determining that the subject is highly likely to have cancer when a fusion protein of DNAJB1 and PRKACA is detected from a sample obtained from the subject, [62] or [63 ] Method.
[65] The method of [62] or [63], wherein the cancer is liver cancer.
[66] The method according to [65], further comprising the step of determining that the subject is highly likely to have liver cancer when a fusion protein of DNAJB1 and PRKACA is detected from a sample obtained from the subject. the method of.

The present invention also relates to the following [67] to [70].
[67] A method for identifying a subject to be treated with a PRKACA inhibitor and / or a drug that blocks an abnormal signal caused by a fusion gene of a DNAJB1 gene and a PRKACA gene, the subject being a cancer patient A method comprising the steps according to any one of [51] to [55].
[68] The method of [67], comprising the step of obtaining a sample from a subject.
[69] When a fusion protein of DNAJB1 and PRKACA is detected from a sample obtained from a subject, the subject blocks an abnormal signal caused by a PRKACA inhibitor and / or a fusion gene of DNAJB1 gene and PRKACA gene The method according to [67] or [68], further comprising the step of determining that it is an indication for treatment with a drug.
[70] The method according to any of [67] to [69], wherein the cancer is liver cancer.

The present invention also relates to the following [71] to [74].
[71] A detection kit for detecting a fusion protein of DNAJB1 and PRKACA in a sample obtained from a subject, wherein the DNAJB1 gene-derived portion of the polypeptide according to any of [51] to [53] A kit for detecting a fusion protein, comprising an antibody that recognizes (primary antibody) and an antibody that recognizes a PRKACA gene-derived portion of the polypeptide (primary antibody).
[72] A secondary antibody linked to the primary antibody and linked to the primary antibody, two types of oligonucleotides partially complementary to the oligonucleotide linked to the secondary antibody, and when they approach each other The detection kit according to [71], comprising a ligase capable of forming a circular structure by ligating the two kinds of oligonucleotides, and a labeled oligonucleotide probe.
[73] The detection kit according to [71] or [72], wherein the subject is a cancer patient.
[74] The detection kit according to [73], wherein the cancer is liver cancer.

The present invention also relates to the following [75].
[75] The polypeptide according to the following (1) to (3) or a polynucleotide encoding the polypeptide:
(1) a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having tumorigenicity,
(2) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity, or in the amino acid sequence shown in SEQ ID NO: 2, 1 to 10 amino acids are deleted, substituted, and / or A polypeptide comprising an added amino acid sequence and having tumorigenicity, or
(3) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

The detection method of the present invention can be used as a method for detecting cancer that is positive for a fusion gene of DNAJB1 gene and PRKACA gene (particularly liver cancer). The primer set, probe, probe set and detection kit of the present invention can be used in the detection method of the present invention.

<< Detection Method of the Present Invention >>
The detection method of the present invention includes a detection method for a fusion gene and a detection method for a fusion protein encoded by 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 the presence of a specific polynucleotide or polypeptide in a sample obtained from a subject.

Samples obtained from the subject include samples collected from the subject (samples separated from the living body), specifically, any collected cells, tissues, body fluids (blood, oral mucus, circulating tumor cells). , Exosomes, etc.), biopsied samples, etc. are used, but preferably biopsied samples are used. Genomic DNA can be extracted from the collected sample and used, or its transcription product (product resulting from transcription and translation of the genome; for example, mRNA, protein) or cDNA prepared from mRNA can be used. In particular, it is preferable to prepare and use mRNA or cDNA. In addition, a specimen (Formalin-Fixed Paraffin-Embedded; FFPE) that is stabilized by formalin fixation and embedded in paraffin can be used. You may use the FFPE section | slice which sliced FFPE thinly. If an FFPE section is used, a polynucleotide or polypeptide present therein can also be directly detected.

The method for detecting a fusion gene of the present invention is a method for detecting a “fusion gene of DNAJB1 gene and PRKACA gene”, which is a fusion gene comprising a part of DNAJB1 gene and a part of PRKACA gene. is there. As an exemplary fusion gene of DNAJB1 gene and PRKACA gene, a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3 can be mentioned. The polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 consists of DNAJB1 gene (GenBank accession number: NM_006145.1) from base number 41 (corresponding to the 5 ′ end of the coding sequence (hereinafter referred to as CDS)) to 251 and the PRKACA gene (GenBank registration number: NM_002730.3) is a polynucleotide having a base sequence from base number 247 to 1256 (corresponding to the 3 ′ end of CDS). The polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 is a polynucleotide consisting of the base sequence in which cytosine of base number 12 of SEQ ID NO: 1 is substituted with thymine. Among the nucleotide sequences shown in SEQ ID NO: 1 or 3, the nucleotide numbers 1-211 are derived from the DNAJB1 gene, and the nucleotide numbers 212-1221 are derived from the PRKACA gene. A polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3 is also referred to as “fusion polynucleotide”. The amino acid sequence encoded by nucleotide numbers 1 to 1221 of SEQ ID NO: 1 or 3 is shown in SEQ ID NO: 2.

In the “detecting the presence of polynucleotide” in the method for detecting a fusion gene of the present invention, the polynucleotide to be detected (referred to as “detected polynucleotide” in the present specification) is described below. A polynucleotide encoding a polypeptide may be mentioned.
A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity.

In the polypeptide, the “identity with the amino acid sequence shown in SEQ ID NO: 2” is preferably 95% or more, more preferably 98% or more.

The “identity” in this specification means a value Identity obtained by using a parameter prepared as a default by NEEDLE program (J Mol Biol 1970; 48: 443-453) search. The parameters are as follows:
Gap penalty = 10
Extended penalty = 0.5
Matrix = EBLOSUM62

It can be confirmed by the method described in Example 4 below that a certain polypeptide has “tumor forming ability”. As a specific method, there is a method in which a polynucleotide encoding the polypeptide is introduced into 184A1 cells (ATCC; CRL-8798) and an anchorage-independent cell proliferation action is confirmed using an ultra-low adhesion flat bottom plate. Can be mentioned. Moreover, after inoculating the cell which introduce | transduced the polynucleotide which codes the said polypeptide subcutaneously of a nude mouse, after observing for a fixed period and confirming tumor formation, the method of evaluating can also be used.

In one embodiment, the polynucleotide to be detected is a polynucleotide encoding a polypeptide of any one of (1) to (3) below.
(1) a polypeptide comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added in the amino acid represented by SEQ ID NO: 2 and having tumorigenic potential;
(2) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity; and (3) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

In the polypeptide (1), the number of amino acids substituted, deleted and / or added to the amino acid sequence shown in SEQ ID NO: 2 is preferably 1 to several, more preferably 1 to 7, The number is preferably 1 to 5.

Examples of the polynucleotide encoding “polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2” include “polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 3”.

The method for detecting a fusion gene of the present invention can further include a step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when the polynucleotide is detected.

The method for detecting a fusion gene of the present invention comprises a step of amplifying a nucleic acid in a sample obtained from a subject, or a step of hybridizing a probe to the nucleic acid in a sample obtained from the subject in order to detect a polynucleotide to be detected. Can be included.

The nucleic acid to be used may be genomic DNA, mRNA, or cDNA prepared from mRNA. Methods for extracting genomic DNA, extracting mRNA, and preparing cDNA from mRNA are known in the art, and can be easily performed using a commercially available DNA extraction kit, RNA extraction kit, or cDNA synthesis kit.

The step of amplifying nucleic acid in a sample obtained from a subject can be performed using a known nucleic acid amplification method. Such methods include, for example, PCR (Polymerase chain reaction, for example, real-time PCR), LCR (Ligase chain reaction), SDA (Strand displacement amplification), NASBA (Nuclideic acidIssessment-basic IC). Primer-initiated amplification of nucleic acid), LAMP (Loop-mediated isometric amplification), TMA (Gen-Probe's TMA system), etc. As preferable methods include PCR.

Specifically, using a primer set designed to specifically amplify a polynucleotide to be detected with a nucleic acid (eg, genomic DNA, mRNA, or cDNA prepared from mRNA) in a sample obtained from a subject. The nucleic acid is amplified for the nucleic acid amplification reaction. The primer set used is not particularly limited as long as it can specifically amplify the polynucleotide to be detected. For example, the primer set software (for example, Primer Express; PE Biosystems) is used to detect the target. It can be easily designed by those skilled in the art based on the nucleotide sequence of the polynucleotide. More specifically, the primer set is a sense primer (5) designed from a part encoding DNAJB1 of the polynucleotide to be detected (for example, any part in the DNAJB1 gene region of the fusion polynucleotide (especially cDNA)). '-Primer) and an antisense primer (3'-primer) designed from the PRKACA-encoding portion of the polynucleotide to be detected (for example, any portion within the PRKACA gene region of the fusion polynucleotide (especially cDNA)) And the antisense primer comprises an oligonucleotide that hybridizes to the polynucleotide to be detected under stringent conditions (preferably under more stringent conditions), and the sense primer is a complementary strand of the polynucleotide to be detected. Under stringent conditions (preferably, more stringent conditions) consisting of an oligonucleotide that hybridizes under. Alternatively, one of the sense primer and the antisense primer may be designed to correspond to a region including the fusion point in the detection target polynucleotide.

In the present specification, “stringent conditions” means “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL salmon sperm DNA” , Overnight at 42 ° C. ”,“ 0.5 × SSC, 0.1% SDS, 42 ° C. ”as the conditions for washing. “More stringent conditions” refer to “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL salmon sperm DNA, 42 ° C.” The condition for “overnight” is “0.2 × SSC, 0.1% SDS, 65 ° C.”.

In the present specification, the “fusion point” in the polynucleotide to be detected means the point where the DNAJB1 gene-derived portion and the PRKACA gene-derived portion in the polynucleotide to be detected are fused. For example, when the polynucleotide to be detected is a polynucleotide having the base sequence shown in SEQ ID NO: 1 or 3, it means a region containing bases of base numbers 211 and 212.

In one embodiment, the sense primer comprises an oligonucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of base numbers 1 to 211 of SEQ ID NO: 1 or 3. It consists of an oligonucleotide that hybridizes under stringent conditions to the polynucleotide consisting of base numbers 212 to 1221 of SEQ ID NO: 1 or 3.

In a specific embodiment, the sense primer consists of any continuous oligonucleotide of at least 16 bases between base numbers 1 to 211 of SEQ ID NO: 1 or 3, and the antisense primer includes SEQ ID NO: 1 or 3 It consists of an oligonucleotide that is complementary to any contiguous at least 16 base oligonucleotide between base numbers 212 to 1221.

In the step of amplifying the nucleic acid, the amplification efficiency deteriorates when the size of the nucleic acid fragment to be amplified becomes large. Therefore, the sense primer and the antisense primer are preferably set so that the size of the nucleic acid fragment to be amplified is 1 kb or less. . Each primer used usually has a chain length of 15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases, and still more preferably 20 to 24 bases.

The primer is not particularly limited, but can be produced by, for example, a chemical synthesis method.

In a preferred embodiment, the method for detecting a fusion gene of the present invention comprises a step of detecting whether or not an amplified nucleic acid fragment of a target size is obtained in addition to the step of amplifying a nucleic acid in a sample obtained from a subject. Is further included. The step of detecting whether or not an amplified nucleic acid fragment of the desired size has been obtained can be performed using, for example, electrophoresis. In the electrophoresis method, for example, a nucleic acid fragment is analyzed by agarose gel electrophoresis, and it can be confirmed whether or not an amplified nucleic acid fragment of a desired size is obtained by ethidium bromide staining or the like.

In addition, by carrying out the PCR amplification monitor (real-time PCR) method (Genome Res., 6 (10), 986, 1996) in the gene amplification process, the amplified nucleic acid fragments should be analyzed more quantitatively. Is possible. As the PCR amplification monitoring method, for example, ABI PRISM 7900 (Life Technologies) can be used.

When an amplified nucleic acid fragment of the desired size is obtained, the detection target polynucleotide is present in the sample obtained from the subject. The method for detecting a fusion gene of the present invention may further include a step of determining that a fusion gene of the DNAJB1 gene and the PRKACA gene is present when an amplified nucleic acid fragment of a desired size is obtained.

In another preferred embodiment, the method for detecting a fusion gene of the present invention further comprises a step of determining the base sequence of the amplified nucleic acid fragment in addition to the step of amplifying the nucleic acid in the sample obtained from the subject. The step of determining the base sequence of the nucleic acid fragment is, for example, a next-generation sequencing method including a Sanger sequencing method (for example, ABI PRISM 3100 (Life Technologies) can be used) and a single base synthesis reaction method (sequence by synthesis method). (Nature Biotechnology, 2008, Vol. 26, p.1135-1145) (for example, HiSeq2000 (Illumina) can be used), and other known sequencing methods can be used.

The step of determining the base sequence of the nucleic acid fragment includes not only a step of determining the full-length sequence of the nucleic acid fragment but also a step of determining partial sequences at both ends of the nucleic acid fragment.

When the sequenced nucleic acid fragment contains the base sequence of the portion encoding DNAJB1 of the polynucleotide to be detected and the base sequence of the portion encoding PRKACA in the same fragment, the polynucleotide to be detected is present in the sample obtained from the subject. It would have been. In the method for detecting a fusion gene of the present invention, when the amplified nucleic acid fragment contains the base sequence of the portion encoding DNAJB1 and the base sequence of the portion encoding PRKACA in the same fragment, the DNAJB1 gene and PRKACA It may further include a step of determining that a fusion gene with the gene exists.

The step of hybridizing a probe to nucleic acid in a sample obtained from a subject uses a probe containing an oligonucleotide that hybridizes to a polynucleotide to be detected under stringent conditions (preferably under more stringent conditions). It can be carried out using a known hybridization method. Examples of such a method include Northern hybridization, dot blot method, DNA microarray method, RNA protection method, in situ hybridization and the like, and a preferable method includes in situ hybridization. The detection using the in situ hybridization technique can be performed, for example, by a known fluorescence in situ hybridization (FISH) method, chromogenic in situ hybridization (CISH) method, or silver in situ hybridization (SISH) method. The chain length of the probe used for hybridization can be appropriately selected by those skilled in the art depending on the hybridization method to be used, but the probe preferably has a chain length of at least 16 bases.

In one embodiment, a probe used for hybridization is an oligonucleotide that hybridizes to a polynucleotide to be detected or a complementary strand thereof under stringent conditions (preferably under more stringent conditions), and is used for detection. An oligonucleotide having at least 16 bases upstream and downstream of the fusion point in the target polynucleotide (specifically, a sequence of base numbers 196 to 227 of SEQ ID NO: 1 or 3) or an oligonucleotide complementary thereto Including.

In one embodiment, the step of hybridizing a probe to a nucleic acid in a sample obtained from a subject is a known RNA FISH method (J. Mol. Diagn. 2012, Vol. 14, No. 1, p. 22-29). Can be implemented according to More specifically, a probe designed from a sample obtained from a subject (for example, FFPE section) and a portion encoding DNAJB1 of the polynucleotide to be detected (for example, an arbitrary portion in the DNAJB1 gene region of the fusion polynucleotide). And in situ hybridization using a probe designed from a portion of the polynucleotide to be detected that encodes PRKACA (for example, any portion in the PRKACA gene region of the fusion polynucleotide). Each probe includes an oligonucleotide that hybridizes to a polynucleotide to be detected under stringent conditions (preferably under more stringent conditions).

In one embodiment, a plurality of types of detection probes designed from a portion encoding DNAJB1 and a plurality of types of detection probes designed from a portion encoding PRKACA are used in in situ hybridization.

In one embodiment, the following probes are used in in situ hybridization:
Plural types of adjacent probe pairs (preferably 15 to 25 types, more preferably, including oligonucleotides complementary to any consecutive at least 16 base oligonucleotides of base numbers 1 to 211 of SEQ ID NO: 1 or 3) 18 to 22, more preferably 20 probe pairs), and an oligonucleotide that is complementary to any contiguous at least 16 base oligonucleotides of base numbers 212 to 1221 of SEQ ID NO: 1 or 3 Plural kinds of adjacent probe pairs (preferably 15 to 25 kinds, more preferably 18 to 22 kinds, further preferably 20 kinds of probe pairs).

In the present specification, “adjacent probe pairs” consist of two types of probes that hybridize adjacent to the polynucleotide to be detected. Each probe includes an oligonucleotide complementary to the polynucleotide to be detected, and the length of the oligonucleotide is at least 16 bases, preferably 16 to 30 bases, more preferably 18 to 25 bases.

In a preferred embodiment, the method for detecting a fusion gene of the present invention further includes a step of amplifying a hybridization signal in addition to the step of performing in situ hybridization. The step of amplifying the hybridization signal can be performed, for example, by hybridizing a probe that hybridizes to the nucleic acid in the sample with a reagent that amplifies the hybridization signal.

Reagents for amplifying hybridization signals used in in situ hybridization include PreAmplifier Mix QT, Amplifier Mix QT, Label Probe Mix, and Label Probe Diluent QF, which are available from Affymetrix.

In a more preferred embodiment, the method for detecting a fusion gene of the present invention detects an overlap of a signal from a probe designed from a portion encoding DNAJB1 and a signal from a probe designed from a portion encoding PRKACA. Further comprising the step of: By separating the probe designed from the portion encoding DNAJB1 and the fluorescent reagent or coloring reagent detecting the probe designed from the portion encoding PRKACA, the signals from the two different probes can be located at the same place (in the same molecule). ) Can be observed. When it is detected that the two signals are in the same place (in the same molecule), the detection target polynucleotide is present in the sample obtained from the subject. The method for detecting a fusion gene of the present invention may further include a step of determining that a fusion gene between the DNAJB1 gene and the PRKACA gene is present when two signals are detected at the same place (in the same molecule). .

Each probe is not particularly limited, but can be produced, for example, by a chemical synthesis method.

The method for detecting a fusion protein of the present invention is a method for detecting “a fusion protein of DNAJB1 and PRKACA”, and the fusion protein is a fusion protein encoded by a fusion gene of a DNAJB1 gene and a PRKACA gene.

In the “detecting the presence of polypeptide” in the method for detecting a fusion protein of the present invention, a polypeptide to be detected (referred to as “detected polypeptide” in the present specification) is a polynucleotide to be detected. The polypeptide encoded by can be mentioned.

For example, in the step of detecting the presence of a polypeptide, a solubilized solution derived from a sample obtained from a subject (for example, cancer tissue or cells obtained from the subject) is prepared, and the detection target polypeptide contained therein is fused. It can be carried out by an immunological measurement method or an enzyme activity measurement method by combining antibodies against each protein constituting the protein, or a detection method combining these. In this step, the polypeptide to be detected contained in a sample (eg, FFPE section) obtained from a subject that has been appropriately pretreated (eg, removal of paraffin) is combined with antibodies against each protein constituting the fusion protein. The detection may be performed by an immunohistological staining technique. Alternatively, this step may be performed using an antibody that recognizes the fusion part of the fusion protein in place of the antibody against each protein constituting the fusion protein in each of the detection methods described above. As these techniques, an enzyme immunoassay method, a two-antibody sandwich ELISA method, a fluorescence immunoassay method, a radioimmunoassay method, a Western blotting method, an immune tissue, using a monoclonal antibody or a polyclonal antibody specific for the polypeptide to be detected Examples of the method include dyeing.

In the present specification, the “fusion part” of the fusion protein means a part where the part derived from the DNAJB1 gene and the part derived from the PRKACA gene in the polypeptide to be detected are fused.

Detection using an immunohistochemical staining technique can be performed according to, for example, Proximity Ligation Assay (Nat. Methods. 2006, Vol. 3, No. 12, p. 995-1000). More specifically, using the antibody that recognizes the DNAJB1 gene-derived portion of the polypeptide to be detected and the antibody that recognizes the PRKACA gene-derived portion of the polypeptide to be detected, the two antibodies recognize the same molecule. By detecting this by the above technique, the presence of the polypeptide to be detected can be detected. More specifically, the detection includes i) an antibody that recognizes a DNAJB1 gene-derived portion of a polypeptide to be detected (primary antibody) and an antibody that recognizes a PRKACA gene-derived portion of the polypeptide to be detected (primary) in a sample obtained from a subject. Antibody), ii) adding an oligonucleotide-linked secondary antibody that binds to each of the primary antibodies, iii) partially complementary to the oligonucleotide linked to the secondary antibody A step of adding a ligation solution containing a ligase that can form a circular structure by ligating the two types of oligonucleotides and the two types of oligonucleotides when they are close to each other, and iv) formed. A step of extending the nucleic acid along the circular structure, v) a hybrida to the extended nucleic acid Step of hybridizing a labeled oligonucleotide probe capable of's, vi) the step of detecting the label signal, can be performed. Such detection can be carried out using a PLA probe and reagents included in the Duolink II reagent kit or Duolink II Bright field reagent kit (Olink).

In one embodiment, the detection method of the present invention includes a step of obtaining a sample from a subject.

In one embodiment, the subject in the detection method of the present invention is a cancer patient, and in a more specific embodiment, the cancer is liver cancer.

In the detection method of the present invention, when a detection target polynucleotide or a detection target polypeptide is detected from a sample obtained from a subject, it is determined that the subject is highly likely to have cancer (particularly liver cancer). it can.

The detection step in the detection method of the present invention can be used for a method for detecting the presence of cancer (particularly liver cancer) in a subject or a method for diagnosing cancer (particularly liver cancer) in a subject. In the diagnostic method of the present invention, in addition to the detection step, the subject suffers from cancer (particularly liver cancer) when the detection target polynucleotide or the detection target polypeptide is detected from a sample obtained from the subject. A step of determining that the possibility is high may be included. In addition, the detection step involves subjecting a subject (cancer patient such as liver cancer) who is a target for treatment with a PRKACA inhibitor and / or a drug that blocks an abnormal signal caused by a fusion gene of DNAJB1 gene and PRKACA gene. It can also be used for identification methods. In the identification method of the present invention, in addition to the detection step, when the polynucleotide or polypeptide is detected from a sample obtained from the subject, the subject is a PRKACA inhibitor and / or a fusion gene of the DNAJB1 gene and the PRKACA gene. The method may include a step of determining that the target of treatment with a drug that blocks an abnormal signal caused by.

<< Primer set, probe, probe set and detection kit of the present invention >>
The present invention includes primer sets, probes, and probe sets used in the detection method of the present invention.

The primer set of the present invention includes a sense primer designed from a portion encoding DNAJB1 and an antisense primer designed from a portion encoding PRKACA, and the antisense primer is subjected to stringent conditions ( Preferably, it consists of an oligonucleotide that hybridizes under more stringent conditions, and the sense primer hybridizes under stringent conditions (preferably under more stringent conditions) to the complementary strand of the polynucleotide to be detected. Consisting of oligonucleotides.

In the primer set of the present invention, one of the sense primer and the antisense primer may be designed to correspond to a region including the fusion point of the detection target polynucleotide.

Specific embodiments of the primer set of the present invention include the following primer sets:
A sense primer comprising an oligonucleotide that hybridizes under stringent conditions to a complementary strand of a polynucleotide comprising base numbers 1 to 211 of SEQ ID NO: 1 or 3, and a polynucleotide comprising base numbers 212 to 1221 of SEQ ID NO: 1 or 3 A primer set of antisense primers consisting of oligonucleotides that hybridize under stringent conditions.

More specific embodiments of the primer set of the present invention include the following primer sets:
Sense primer comprising any continuous at least 16 base oligonucleotide between base numbers 1 to 211 of SEQ ID NO: 1 or 3 and any continuous at least 16 base oligo between base numbers 212 to 1221 of SEQ ID NO: 1 or 3 A primer set of antisense primers consisting of oligonucleotides that are complementary to nucleotides.

In the primer set, the interval between the selected positions of the sense primer and the antisense primer is preferably 1 kb or less, or the size of the nucleic acid fragment amplified by the sense primer and the antisense primer is preferably 1 kb or less. The primer of the present invention usually has a chain length of 15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases, and further preferably 20 to 24 bases.

Each primer included in the primer set of the present invention is not particularly limited, but can be produced by, for example, a chemical synthesis method.

Each probe included in the probe of the present invention and the probe set of the present invention includes an oligonucleotide that hybridizes to the polynucleotide to be detected under stringent conditions (preferably under more stringent conditions). The chain length of each probe included in the probe of the present invention and the probe set of the present invention can be appropriately selected by those skilled in the art depending on the hybridization method used, but the probe preferably has a chain length of at least 16 bases. .

In one embodiment, the probe of the present invention is an oligonucleotide having at least 16 bases each upstream and downstream of a fusion point in a polynucleotide to be detected (as a specific example, base number 196 of SEQ ID NO: 1 or 3). ˜227 sequences) or oligonucleotides complementary thereto.

In one embodiment, the probe set of the present invention comprises a probe designed from a portion encoding DNAJB1 (eg, any portion within the DNAJB1 gene region of the fusion polynucleotide) and a portion encoding PRKACA (eg, It is a probe set including a probe designed from any part in the PRKACA gene region of the fusion polynucleotide.

In one embodiment, the probe set of the present invention includes a plurality of types of probes designed from a portion encoding DNAJB1 and a plurality of types of probes designed from a portion encoding PRKACA.

In one embodiment, the probe set of the present invention comprises:
Adjacent comprising an oligonucleotide complementary to any consecutive at least 16 bases (preferably 16 to 30 bases, more preferably 18 to 25 bases) of base numbers 1 to 211 of SEQ ID NO: 1 or 3 Plural kinds of probe pairs (preferably 15 to 25 kinds, more preferably 18 to 22 kinds, further preferably 20 kinds of probe pairs), and any continuous at least 16 of base numbers 212 to 1221 of SEQ ID NO: 1 or 3 A plurality of adjacent probe pairs (preferably 15 to 25, more preferably 18 to 25) including oligonucleotides complementary to oligonucleotides having bases (preferably 16 to 30 bases, more preferably 18 to 25 bases). 22 types, more preferably 20 types of probe pairs).

The probes of the present invention and the probes included in the probe set of the present invention are not particularly limited, but can be produced by, for example, a chemical synthesis method.

The present invention includes a detection kit comprising the primer set of the present invention, the probe of the present invention or the probe set of the present invention. The detection kit of the present invention includes the primer set for detecting a polynucleotide to be detected, such as a primer set of the present invention, a probe of the present invention or a probe set of the present invention, and a reagent for amplifying a hybridization signal. May include components for use with a probe or probe set.

The present invention also includes a detection kit for detecting the polypeptide to be detected. Preferably, the detection kit includes an antibody that recognizes a DNAJB1 gene-derived portion of the detection target polypeptide (primary antibody) and an antibody that recognizes a PRKACA gene-derived portion of the detection target polypeptide (primary antibody). More preferably, each secondary antibody that binds to the primary antibody is linked to an oligonucleotide, two types of oligonucleotides that are partially complementary to the oligonucleotide linked to the secondary antibody, and close proximity to each other In some cases, the two kinds of oligonucleotides may be ligated to form a circular structure, and a labeled oligonucleotide probe may be included.

The primer set, probe, probe set, and detection kit of the present invention can be used for the detection method, diagnostic method, and patient identification method of the present invention. In one embodiment, with respect to the primer set, probe, probe set, and detection kit of the present invention, the subject is a cancer patient, and in a more specific embodiment, the cancer is liver cancer.

Unless otherwise noted, it can be performed according to a known method. Moreover, when using a commercially available reagent, kit, etc., it can implement according to the instruction manual of a commercial item.

[Example 1] Isolation of DNAJB1-PRKACA-D1P3 Reverse transcriptase (SuperScriptIII; Life Technologies) and oligo (dT) were used for samples 1091071F and 9191B1 derived from liver cancer tissue of hepatocellular carcinoma patients (Asterland, USA). Using a primer (oligo (dT) 20 primer; Life Technologies), reverse transcription was performed according to the protocol of the kit to synthesize cDNA.

Next, using the DNAJ-PRKA_full fwd01 represented by SEQ ID NO: 4 and the DNAJ-PRKA_full rev01 represented by SEQ ID NO: 5 and using the cDNA obtained above as a template, DNA polymerase (PrimeSTAR GXL; Takara Bio Inc.) PCR (98 ° C for 10 seconds, 55 ° C for 15 seconds, 68 ° C for 2 minutes for 30 cycles, followed by 68 ° C for 5 minutes) was performed. Thereafter, using the PCR product diluted 10-fold as a template, DNAJ-PRKA_full fwd02 shown in SEQ ID NO: 6 and DNAJ-PRKA_full rev02 shown in SEQ ID NO: 7 and PCR (98 ° C. 10 ° C.) using the same DNA polymerase Seconds, 55 ° C. for 15 seconds and 68 ° C. for 2 minutes for 30 cycles, followed by 68 ° C. for 5 minutes). Electrophoresis after the PCR reaction revealed that a PCR product of about 1.3 kbp was obtained. A was added to the 3 ′ end of the PCR product using DNA polymerase (rTaq; Takara Bio Inc.) and then cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies). The insert was sequenced by the dideoxy sequencing method (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies). As a result, about 1.3 kbp PCR product derived from 1091071F contains 251 to PRKACA gene (GenBank accession number) from DNAJB1 (NM_006145.1) base number 41 (corresponding to the 5 ′ end of CDS) registered in NCBI. : NM_002730.3) was found to have a transcript (SEQ ID NO: 1) fused with base numbers 247 to 1256 (corresponding to the 3 ′ end of CDS). Further, it was revealed that about 1.3 kbp PCR product derived from 9191B1 had a transcription product (SEQ ID NO: 3) in which cytosine of base number 12 of SEQ ID NO: 1 was substituted with thymine. The polypeptides encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 3 are the same, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2. A fusion gene of the DNAJB1 gene consisting of the base sequence shown in SEQ ID NO: 1 or 3 and the PRKACA gene is also referred to as DNAJB1-PRKACA-D1P3.

DNA polymerase (KOD-plus-Ver.2; Toyobo Co., Ltd.) using primers of DNAJB1-PRKACA_cloning_BamHI_F shown in SEQ ID NO: 8 and DNAJB1-PRKACA_cloning_NotI_R shown in SEQ ID NO: 9 as a template from the above 1091071F-derived PCR product Gradient PCR (98 ° C. for 2 minutes, followed by 98 ° C. for 15 seconds, 55 ° C. to 60 ° C. for 15 seconds, 68 ° C. for 2 minutes, followed by 68 ° C. for 7 minutes) was performed. Electrophoresis after the PCR reaction revealed that a PCR product of about 1.2 kbp was obtained under all conditions in which annealing was performed in the above gradient PCR in the range of 55 ° C. to 60 ° C. A was added to the 3 ′ end of the PCR product using DNA polymerase (Ex Taq; Takara Bio Inc.) and then cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies). The insert was sequenced by the dideoxy sequencing method (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies), and it was confirmed that the transcript shown in SEQ ID NO: 1 was present.

In order to express the full open reading frame (ORF) of DNAJB1-PRKACA-D1P3 as a protein, a DNA fragment treated with the restriction enzyme by purifying the restriction enzyme NotI at 37 ° C. for 2 hours was purified from the above cloning vector, and BamHI was further purified. The DNA fragment treated with the restriction enzyme was purified by carrying out an enzyme reaction at 37 ° C. for 2 hours. The DNA fragment containing this ORF was cloned into the BamHI and NotI sites present in the multiple cloning site of the expression vector (pMXs-puro; Cosmo Bio) to construct an expression plasmid (DNAJB1-PRKACA-D1P3 / pMXs-puro).

[Example 2] Detection of DNAJB1-PRKACA-D1P3 For 18 clinical specimens of liver cancer (Asterland, USA), reverse transcriptase (SuperScriptIII; Life Technologies) and random primers (Random Primers; Life Technologies) The reverse transcription reaction was performed according to the protocol of the kit to synthesize cDNA.

Next, using the DNAJ-PRKA_part fwd01 shown in SEQ ID NO: 10 and the primer of DNAJ-PRKA_part rev01 shown in SEQ ID NO: 11, using the cDNA obtained above as a template, DNA polymerase (PrimeSTAR GXL; Takara Bio Inc.) PCR (98 ° C for 10 seconds, 55 ° C for 15 seconds, 68 ° C for 1 minute for 30 cycles, followed by 68 ° C for 5 minutes) was performed. Thereafter, using the PCR product diluted 10-fold as a template, DNAJ-PRKA_part fwd02 shown in SEQ ID NO: 12 and DNAJ-PRKA_part rev02 shown in SEQ ID NO: 13 and PCR (98 ° C. 10 ° C.) using the same DNA polymerase Second, 55 ° C. for 15 seconds, 68 ° C. for 1 minute for 30 cycles, followed by 68 ° C. for 5 minutes). As a result of electrophoresis after the PCR reaction, it was found that a PCR product of about 300 bp was obtained from a plurality of specimens including the above-mentioned samples 1091071F and 9191B1.

Thereafter, the PCR product was sequenced by the dideoxy sequencing method (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies). As a result, the PCR product of about 300 bp is the same fragment of DNAJB1-PRKACA-D1P3 identified in Example 1 with the base sequence of the portion encoding DNAJB1 and the base sequence of the portion encoding PRKACA across the fusion point. It became clear that the sequence was included in From the above, it is shown that the detection method of the present invention can detect the presence of a fusion gene of DNAJB1 gene and PRKACA gene in a sample derived from a liver cancer clinical specimen, and select a patient who is positive for the fusion gene. It was.

[Example 3] Preparation of DNAJB1-PRKACA-D1P3 retrovirus solution D-MEM medium (Dulbecco's Modified Eagle's Medium-high glucose medium) containing 10% fetal bovine serum (Sigma-Aldrich); Sigma-Aldrich medium GPJ-293 cells (Clontech; 631458) cultured under conditions of 5% CO ₂ and 37 ° C. with 4 μg of DNAJB1-PRKACA-D1P3 / pMXs-puro (Example 1), 1 μg of p10A1 envelope vector (Clontech) Was transfected using Lipofectamine (registered trademark) 2000 (Life Technologies). 24 hours after transfection, D-MEM medium containing 10% fetal bovine serum was added, and the culture supernatant was further collected for 24 hours at 37 ° C. with 5% CO ₂ to prepare a retrovirus solution. Similarly, a retrovirus solution was prepared using pMXs-puro, which is an empty vector, instead of DNAJB1-PRKACA-D1P3 / pMXs-puro.

[Example 4] Examination of anchorage-independent growth-promoting action of DNAJB1-PRKACA-D1P3 Polybrene (Sigma-Aldrich) was added to the retrovirus solution prepared using DNAJB1-PRKACA-D1P3 / pMXs-puro in Example 3. ) Was added to a final concentration of 16 μg / mL, and then added to 184A1 cells (ATCC; CRL-8798), a human immortalized mammary epithelial cell line, and infected. After infecting with 5% CO ₂ at 37 ° C. for 8 hours, a medium dedicated to mammary epithelial cells (MEGM® BulletKit®; Lonza) containing transferrin (Sigma-Aldrich) at a final concentration of 5 μg / mL. in replacement, and further changed after 16 hours to mammary epithelial cells only medium containing final concentration 5 [mu] g / mL transferrin and final concentration 0.5 [mu] g / mL of puromycin (Sigma-Aldrich, _{Inc.), 5% CO 2, 37} ℃ The culture was continued for 2 weeks to obtain 184A1 cells stably expressing DNAJB1-PRKACA-D1P3 (named as DNAJB1-PRKACA-D1P3 expression / 184A1 cells). Similarly, 184A1 cells (named Mock / 184A1 cells) infected with a retrovirus (Example 3) prepared using pMXs-puro were obtained.

In order to investigate the ability of DNAJB1-PRKACA-D1P3 expression / 184A1 cells to promote anchorage-independent growth, 96JV ultra-low adhesion flat bottom plates (Corning) were used with DNAJB1-PRKACA-D1P3 expression / 184A1 cells and Mock / 184A1 cells. Mammary epithelial cell-dedicated medium containing transferrin (Sigma-Aldrich) at a final concentration of 5 μg / mL and puromycin (Sigma-Aldrich) at a final concentration of 0.5 μg / mL so that 2 × 10 ³ cells are present per well. (Lonza) and seeded with 5% CO ₂ at 37 ° C. On the day of seeding (Day 1), 4 days (Day 4), 7 days (Day 7), 11 days (Day 11) and 14 days (Day 14), the cell number measurement reagent (CELLTITER-Glo ^™ Luminescent Cell Viability Assay) And measured according to the manual method using Promega). A luminescence measuring device (ARVO X3; Perkin Elmer) was used for detection. Mock / 184A1 cells did not increase in cell number from Day1 to Day14, whereas DNAJB1-PRKACA-D1P3 expression / 184A1 cells were confirmed to have an approximately 2.0-fold increase in cell number from Day1 to Day14. . From the above, it became clear that DNAJB1-PRKACA-D1P3 expression / 184A1 cells show anchorage-independent cell proliferation, and that the polypeptide encoded by DNAJB1-PRKACA-D1P3 has tumorigenicity. It was.

The detection method of the present invention is a method for detecting a fusion gene of DNAJB1 gene and PRKACA gene, and is useful as a method for detecting and diagnosing cancer in a subject. Moreover, the primer set and detection kit of the present invention can be used in the method of the present invention.

The description of “Artificial Sequence” is described in the numerical heading <223> of the following sequence listing. Specifically, the base sequences represented by SEQ ID NOs: 8 and 9 in the Sequence Listing are artificially synthesized primer sequences.

Claims

A Donager homolog subfamily B member 1 (DNAJB1) gene and a protein kinase cyclic MP dependent catalytic alpha comprising the step of detecting the presence of a polynucleotide encoding the following polypeptide in a sample obtained from a subject: Method for detecting fusion gene with (PRKACA) gene:
A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having tumorigenicity.
The polypeptide includes the amino acid sequence shown in SEQ ID NO: 2 and has a tumorigenicity, or in the amino acid sequence shown in SEQ ID NO: 2, 1 to 10 amino acids are deleted, substituted, and The method according to claim 1, wherein the polypeptide comprises an added amino acid sequence and has a tumorigenic potential.
The method according to claim 1, wherein the polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
The method of claim 1, further comprising a step of amplifying a nucleic acid in a sample obtained from a subject or a step of hybridizing a probe to the nucleic acid in a sample obtained from the subject in order to detect the polynucleotide. The method according to any one.
The method of Claim 4 including the process of amplifying the nucleic acid in the sample obtained from the test subject using the following primer sets:
A primer set for detecting a fusion gene of DNAJB1 gene and PRKACA gene, comprising a sense primer designed from a portion encoding DNAJB1 and an antisense primer designed from a portion encoding PRKACA, The primer comprises an oligonucleotide that hybridizes to the polynucleotide to be detected under stringent conditions, and the sense primer comprises an oligonucleotide that hybridizes to the complementary strand of the polynucleotide to be detected under stringent conditions. set.
The method according to any one of claims 1 to 5, wherein the subject is a cancer patient.
The method according to claim 6, wherein the cancer is liver cancer.
A method for diagnosing cancer in a subject, comprising the step according to any one of claims 1 to 5.
The method according to claim 8, wherein the cancer is liver cancer.