JP2005287316A - LIVER X RECEPTOR beta SPLICING MUTANT PROTEIN WHICH IS ISOFORM OF LIVER X RECEPTOR beta, ITS GENE AND THEIR UTILIZATION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for evaluating cancerization degree of a specimen derived from mammal based on detection of genetic expression abnormality suitable for evaluation for diagnostic method or treating method, etc., for early discovering leukemia. <P>SOLUTION: The liver X receptor β splicing mutant protein which is an isoform of liver X receptor β has either one amino acid sequence selected from (1) a specific amino acid sequence related to the isoform of liver X receptor β, (2) a substantially same amino acid sequence as the specific amino acid sequence and (3) an amino acid sequence having ≥95% amino acid identity to the specific amino acid sequence. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a liver X receptor β splicing mutant protein, which is an isoform of liver X receptor β, a gene thereof, and use thereof.

  Although it is becoming increasingly clear that cancer is a disease caused by genetic abnormalities, the mortality rate of cancer patients is still high, and the evaluation of currently available diagnostic methods and treatment methods etc. is not always satisfactory It is not. As one of the causes, diversity based on the type of cancer tissue, low accuracy such as a gene serving as a marker, low detection sensitivity, and the like are conceivable (for example, see Non-Patent Document 1).

"Cancer as a genetic disease" (ISBN4-89553-447-2 C3047), edited by Toshio Kuroki and Tadao Kakizoe, published by Medical View Inc. (May 10, 1994)

  Therefore, development of a method for evaluating the degree of canceration of a mammal-derived specimen based on detection of abnormal gene expression, which is suitable for evaluation of diagnostic methods and therapeutic methods for early detection of leukemia, is eagerly desired.

As a result of intensive studies under these circumstances, the present inventors have isolated 10 types of novel liver X receptor β splicing mutant proteins that are specifically expressed in normal leukocyte samples. The present inventors have found that the gene expression of the liver X receptor β splicing mutant protein is significantly reduced in leukemia-derived cell lines as compared with normal leukocyte specimens, leading to the present invention.
That is, the present invention
1. A liver X receptor β splicing mutant protein (hereinafter, referred to as a splicing mutant protein of the present invention), which is an isoform of liver X receptor β and has any of the following amino acid sequences. )
(1) Amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 (2) Substantially the amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 Identical amino acid sequence (3) amino acid sequence having 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8;
2. A polynucleotide having a base sequence encoding the amino acid sequence of the liver X receptor β-splicing mutant protein according to item 1 or a base sequence complementary to the base sequence;
3. A polynucleotide comprising the base sequence represented by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, or 18, or a base sequence complementary to the base sequence;
4). A partial polypeptide of the liver X receptor β splicing mutant protein according to item 1 above, wherein the polypeptide has one of the following amino acid sequences: (1) present in normal liver X receptor β (2) a sequence comprising a region comprising two amino acid residues newly linked by deletion of an exon (hereinafter also referred to as the polypeptide of the present invention);
5). 5. The polypeptide according to item 4 above, which has an amino acid sequence of any one of SEQ ID NOs: 19, 20, 21, or 22;
6). A polynucleotide having a base sequence encoding the amino acid sequence of the polypeptide according to item 5 or a base sequence complementary to the base sequence;
7). A polynucleotide comprising the base sequence represented by SEQ ID NO: 23, 24, 25, 26, 27, 28 or 29, or a base sequence complementary to the base sequence;
8). A recombinant vector comprising the polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to item 1 or the polynucleotide according to any one of items 2 to 3 (hereinafter referred to as “recombinant vector”) , Sometimes referred to as the vector of the present invention);
9. The polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β-splicing mutant protein according to the above 1 or the polynucleotide according to any one of the above 2 to 3 or the recombinant vector according to the above 8 is a host cell. A transformant (hereinafter sometimes referred to as the transformant of the present invention),
10. The polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β-splicing mutant protein according to item 1 or the polynucleotide according to any one of items 2 to 3 is introduced into a vector capable of autonomous replication in a host cell. A method for producing a vector comprising the step of:
11. A method for producing a liver X receptor β splicing mutant protein, comprising culturing the transformant according to item 9 to produce a liver X receptor β splicing mutant protein;
12 Characterized in that it lacks the ability to express a polynucleotide having a base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein of item 1 or the polynucleotide of any one of items 2 to 3 above. Genetically deficient animals
13. A recombinant vector comprising a polynucleotide having a base sequence encoding the amino acid sequence of the polypeptide according to any one of 4 to 5 above or the polynucleotide according to any one of 6 to 7 above;
14 A polynucleotide having a base sequence encoding the amino acid sequence of any one of the polypeptides described in the above 4 to 5, the polynucleotide according to any one of the above 6 to 7 or the recombinant vector according to the above 13 is introduced into a host cell. A transformant characterized in that
15. An antibody or a part of an antibody, which specifically recognizes the liver X receptor β-splicing mutant protein according to item 1;
16. A primer designed on the basis of any one of the polynucleotides described in 2 to 3 above;
17. A method for detecting the presence or absence of a liver X receptor β-splicing mutant protein according to item 1 above, the first step of synthesizing complementary DNA (cDNA) from messenger RNA (mRNA) in a biological sample, item 1 above Or a polynucleotide having the base sequence encoding the amino acid sequence of the polypeptide of any one of 4 to 5 above or the polynucleotide of any one of 6 to 7 above A second step of amplifying a partial region of the complementary DNA (cDNA), and a third step of detecting the amplified partial region;
18. In the second step, amplification is performed by a PCR method using one or more selected from the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 30, 31, 32 or 33 as a primer, Method;
19. A method for detecting the presence or absence of the liver X receptor β-splicing mutant protein according to item 1, wherein the liver X receptor β-splicing mutant protein according to item 1 or the polypeptide according to any one of items 4 to 5 is detected. A method comprising detecting the liver X receptor β splicing mutant protein based on an antigen-antibody reaction using an antibody that can be specifically detected;
20. A method for detecting the presence or absence of the liver X receptor β-splicing mutant protein according to item 1, wherein the liver X receptor β-splicing mutant protein according to item 1 or the polypeptide according to any one of items 4 to 5 is detected. A first step of binding a first antibody capable of being specifically detected to a microtiter well, wherein the liver X receptor β splicing mutant protein according to item 1 in the biological sample is the first antibody on the microtiter well The second step of binding to the first step, after the second step, the third step of removing all unbound biological samples in the microtiter well, the first antibody in the liver X receptor β splicing variant protein, Labeled second antibody capable of binding to different epitopes, liver X receptor β splicing bound to the first antibody After the fourth step and the fourth step for binding to the foreign protein, after the fifth step and the fifth step for removing all unbound second antibodies in the microtiter well, the presence or absence of the second antibody is determined. A method comprising a sixth step of detecting using the label possessed by the two antibodies as an indicator;
21. Use of any of the following liver X receptor β splicing mutant protein as a marker for examining the degree of canceration of leukocytes (1) Liver X receptor β splicing mutant protein according to item 1 above (2) SEQ ID NO: Liver X receptor β splicing mutant protein having the amino acid sequence of 34. Use of a polynucleotide having a base sequence encoding the amino acid sequence of any of the following proteins as a marker for examining the degree of canceration of leukocytes (1) The liver X receptor β splicing mutant protein according to item 1 ( 2) Liver X receptor β splicing mutant protein having the amino acid sequence of SEQ ID NO: 34 The polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein is any one of the polynucleotide described in the above items 2 to 3, the polynucleotide described in any one of the above items 6 to 7, or SEQ ID NO: 35. 23. Use according to item 22 above, which is a polynucleotide having the base sequence represented by
Etc. are provided.

Hereinafter, the present invention will be described in detail.
The present invention relates to a liver X receptor β splicing mutant protein, which is an isoform of liver X receptor β, a gene thereof, and use thereof.

  The liver X receptor β referred to in the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 1. For example, in the case of humans, the gene of the protein is a gene having the base sequence represented by SEQ ID NO: 9.

  The splicing variant protein of the present invention is an isoform of liver X receptor β, (1) an amino acid sequence represented by any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 8. (2) An amino acid sequence substantially identical to the amino acid sequence represented by any of SEQ ID NOs: 2, 3, 4, 5, 6, 7 or 8, or (3) SEQ ID NOs: 2, 3, 4, 5, 6, 7 or Examples include those having an amino acid sequence having 95% or more amino acid identity with respect to the amino acid sequence represented by any of 8 above.

  Here, regarding the “substantially identical amino acid sequence”, in general, when the amino acid sequence of a protein having physiological activity is slightly changed, for example, one or more amino acids in the amino acid sequence are deleted, substituted or added. It is a well-known fact that even if there is such a change, the physiological activity of the protein may be maintained. Therefore, the “substantially identical amino acid sequence” in the present invention means As long as a biological activity substantially equivalent to a specific amino acid sequence (ie, the amino acid sequence represented by SEQ ID NO: 2 to 8 or 34) is maintained, one or more amino acids in the amino acid sequence are deleted or substituted Alternatively, an added human liver X receptor β splicing mutant protein is meant to be included in the scope of the present invention. The number of amino acids to be modified is at least one residue, specifically 1 or several (here, “several” is about 2 to about 10). The number of such modifications may be in a range where the physiological activity of the protein is maintained. More specifically, 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids in the amino acid sequence represented by the above specific amino acid sequence are deleted. Substituted or added human liver X receptor β splicing variant protein. Such a mutation may be a naturally occurring mutation due to, for example, the processing that a protein undergoes in a cell, the species difference of an organism from which the protein is derived, an individual difference, an organ, a tissue difference, or the like. Amino acid mutations (for example, amino acid mutations present in the amino acid sequences of proteins produced by introducing and expressing mutations in DNA encoding natural proteins by site-specific mutagenesis or mutation treatment) ).

  Such a mutant protein resulting from deletion, substitution or addition of amino acids may contain a conservatively substituted amino acid sequence. This means that certain amino acid residues may be replaced by residues having physicochemical similarity (eg, similar properties such as hydrophobicity, charge, pK, conformational features, etc.) Yes. Non-limiting examples of such conservative substitutions include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; (5) Substitution between aliphatic chain-containing amino acid residues, substitution between polar groups, and the like, such as substitution within the group of lysine and arginine; (6) phenylalanine and tyrosine.

  Mutant proteins by amino acid deletion, substitution, or addition can be obtained by, for example, site-directed mutagenesis that is a known technique for genes having a base sequence encoding the amino acid sequence (for example, Nelson and McClelland, Methods Enzymol, 216; 1992, etc., and a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456, 1984), a method using PCR using a primer for mutagenesis).

  Site-directed mutagenesis can be performed using a synthetic primer containing the mutation to be introduced. That is, using the synthetic oligonucleotide and a primer having a base sequence complementary to the base sequence as a primer, an amplification reaction is performed using a plasmid containing the gene for normal liver X receptor β as a template. Next, treatment with Dpn I, which is a methylation-sensitive restriction enzyme, leaves only DNA having a newly formed mutation. Using this reaction solution, E. coli XLI-Blue strain is transformed and plated on LB agar medium containing ampicillin. Incubate overnight at 37 ° C and isolate the plasmid from the grown colonies. Thereby, a plasmid containing the mutated DNA can be obtained. As a kit based on the above method, for example, QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) is sold, and these may be used. The introduction of the target mutation can be confirmed by determining the base sequence.

  Furthermore, as a method for deleting, substituting or adding amino acid sequences, in addition to the above-mentioned site-directed mutagenesis, a method of treating a gene with a mutagen or a gene selected by cleaving a gene with a restriction enzyme Examples thereof include a method of removing, adding or replacing fragments, and then ligating.

  Here, “normal liver X receptor β” means liver X receptor β consisting of the amino acid sequence that occurs most frequently in nature in the amino acid sequence of the receptor protein derived from the same species of organism. . For example, examples of the human-derived normal liver X receptor β include liver X receptor β consisting of an amino acid sequence (GenBank Accession No. NM_007121) registered in a public database.

  In the present invention, “amino acid identity” and “base identity” refer to sequence identity and homology between two proteins or two DNAs. The “identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, the protein or DNA to be compared may have an addition or a deletion (for example, a gap) in an optimal alignment of the two sequences. Such identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The identity is measured using sequence analysis software, specifically analysis tools provided by Vector NTI, GENETYX-MAC, and public databases, for example, the homepage address http: // Generally available at www.ddbj.nig.ac.jp.

  “Amino acid identity” in the present invention is based on amino acid sequence, and is preferably about 95% or more, for example. On the other hand, “base identity” is a base sequence standard, and is preferably about 95% or more, for example, corresponding to the amino acid identity.

The splicing mutant protein of the present invention can be obtained by a hybridization method, a PCR method or the like.
For example, it is described in Sambrook and Russell; Molecular Cloning 3rd Edition, Cold Spring Harbor Laboratory (2001), etc. from animal tissues such as mammals such as humans, monkeys, rabbits, rats and mice, or cultured cells derived from these animals. RNA is extracted according to genetic engineering methods to synthesize single-stranded cDNA. Specifically, for example, tissue such as liver is crushed in a solution containing a protein denaturant such as guanidine thiocyanate, and protein is denatured by adding chloroform or the like to the crushed material. After removing the denatured protein by centrifugation or the like, total RNA is extracted from the collected supernatant fraction using phenol, chloroform or the like. Examples of commercially available kits based on these methods include ISOGEN (manufactured by Nippon Gene) and TRIZOL reagent (manufactured by Invitrogen).
Using the total RNA obtained as a template, oligo dT primer was bound to mRNA polyA sequence, and using reverse transcriptase, for example, RNaseH-Superscript II Reverse Transcriptase (Invitrogen) and attached buffer and oligo dT primer Then, the reverse transcriptase is inactivated by reacting at 42 ° C. for 1 hour and then heating at 99 ° C. for 5 minutes. Next, the mRNA strand is nicked with RNase H, and double-stranded cDNA is synthesized with E. coli DNA polymerase I using the single-stranded cDNA as a template. The ends of the obtained double-stranded cDNA are smoothed with T4 DNA polymerase. The smoothed double-stranded cDNA is inserted into a pBluescript II vector or a bacteriophage such as a vector such as λgt11 or EMBL3 by T4 ligase to create a cDNA library. Examples of commercially available kits based on these methods include cDNA synthesis system plus (Amersham Biosciences) and TimeSaver cDNA synthesis kit (Amersham Biosciences). Hybridization is performed from the cDNA library thus prepared using, for example, a DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 23 as a probe. Examples of hybridization conditions include those that hybridize under stringent conditions. Hybridization is a conventional method described in, for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press. In addition, “stringent conditions” means, for example, a solution containing 6 × SSC (a solution containing 1.5M NaCl and 0.15M trisodium citrate is 10 × SSC) In which the hybrid was formed at 45 ° C. and then washed with 2 × SSC at 50 ° C. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), [Α- ³² P] by random priming method in a solution containing 50% formamide, 6 x SSC, 5 X Denhardt's solution, 0.5% (w / v) SDS and heat-denatured salmon sperm DNA (100 μg / ml) The probe labeled with dCTP (10 X 10 ⁶ cpm / ml), the mixture was incubated overnight at 42 ° C to form a hybrid, then washed in 2 x SSC containing 0.1% (w / v) SDS at room temperature for 10 minutes, and further 0.1% (w / v) Conditions such as washing in 0.2 × SSC containing SDS twice at 55 ° C. for 10 minutes can be exemplified. The salt concentration in the washing step is, for example, from the condition of about 50 ° C. at 2 × SSC (low stringency condition) to about 50 ° C. at 0.2 × SSC (high stringency condition). You can choose. The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
Next, a signal containing a vector having a base sequence that binds to the probe is detected by detecting the signal with an X-ray film (for example, Hyperfilm-MP; manufactured by Amersham Bioscience) or a bioimaging system (BAS-2000; manufactured by Fuji Film). You can get a body.

When designing a primer for the PCR method, for example, the base represented by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 23, 24, 25, 26, 27, 28, or 29. From the array, for example, two may be selected so as to satisfy the following conditions.
1) Primer length is 15 to 40 bases, preferably 20 to 30 bases.
2) The ratio of guanine to cytosine in the primer is 40% to 60%, preferably 45% to 55%, more preferably 50% to 55%.
3) The distribution of adenine, thymine, guanine, and cytosine is not partially biased in the primer sequence. For example, a region where guanine and cytosine repeat is not appropriate.
4) The distance on the base sequence of the gene corresponding to the selected primer is preferably 100 to 3000 bases, more preferably 100 to 500 bases.
5) No complementary sequence exists between each primer itself or two primers.
If the base sequence of the primer is selected, it may be chemically synthesized by a commercially available DNA synthesizer.
For example, the combination which uses the base sequence shown by sequence number 30 as a sense primer and the base sequence shown by sequence number 31 as an antisense primer is mentioned. As PCR conditions, for example, primers are added to the reaction solution at 200 nM, and the single-stranded cDNA synthesized above is used as a template. For example, LA Taq DNA polymerase (Takara Shuzo) and PCR is performed using the reaction buffer attached to the enzyme. As such PCR, for example, after heat denaturation at 95 ° C. for 3 minutes, about 35 cycles of 94 ° C., 30 seconds, 55 ° C., 30 seconds, 72 ° C. and 1 minute are performed. Here, instead of the cDNA synthesized as described above, commercially available cDNAs derived from various animals such as Clontech Quick Clone cDNA may be used. A part of the obtained reaction solution is analyzed by agarose gel electrophoresis, and a target band is cut out directly or from a gel, and then cloned into a pGEM-T Easy vector (manufactured by Promega) using a TA cloning system. The base sequence of the inserted DNA fragment can be determined and confirmed by the dye terminator method.

Incidentally, in order to amplify a DNA fragment encoding normal liver X receptor β protein, the present inventors designed and synthesized primers described in SEQ ID NOs: 30 and 31. PCR was performed using the primer and cDNA derived from human leukocytes as a template to obtain an amplified DNA fragment. As a result of analyzing the obtained amplified DNA fragment, 10 types of DNA fragments corresponding to the entire protein coding region of the liver X receptor β splicing mutant protein, which is an isoform of liver X receptor β generated by alternative splicing, are obtained. I was able to.
From comparison with the normal liver X receptor β gene structure based on the nucleotide sequence and amino acid sequence, the isolated liver X receptor β splicing mutant protein has one exon 3 as a whole and the 5 ′ end of exon 5 Deletion of 132 bases (liver X receptor β splicing mutant protein D3D5, SEQ ID NOs: 2 and 10), one deletion of the full length of exon 2 and exon 3 and 132 bases of exon 5 5 ′ end (Liver X receptor β splicing mutant protein D2D3D5, SEQ ID NOS: 3 and 11), one having exon 3 deleted and intron 7 inserted (liver X receptor β splicing mutant protein D3A7, sequence No. 4 and 12), one with exon 3 deleted and intron 4 and intron 7 inserted (liver X receptor β splicing mutation) Protein D3A4A7, SEQ ID NOS: 5 and 13), one with intron 3 inserted (liver X receptor β splicing mutant protein A3, SEQ ID NO: 6 and 14), one with intron 3 inserted entirely, exon 5 In which 5 'terminal 132 bases are deleted (liver X receptor β splicing mutant protein A3D5, SEQ ID NOs: 6 and 15), one in which intron 2 is inserted (liver X receptor β splicing mutant protein A2 · SEQ ID NO: 7 and 16), one with intron 1 and intron 7 inserted and exon 3 deleted (liver X receptor β splicing mutant protein A1D3A7 · SEQ ID NO: 8 and 17), one intron 2 inserted (liver X receptor β splicing mutant protein A2), one inserted by intron 1 And exon 3 deleted (liver X receptor β splicing mutant protein A1D3, SEQ ID NOs: 8 and 18), one exon 3 deleted (liver X receptor β splicing mutant protein D3, sequence Nos. 34 and 35) (hereinafter sometimes collectively referred to as the present splicing mutant protein). The above liver X receptor β splicing mutant protein is substantially the same as normal liver X receptor β except for the insertion or deletion of the above exon or intron.
Incidentally, the liver X receptor β splicing mutant protein D3 has the same structure as the liver X receptor splicing mutant protein (GenBank Accession No. AR035537) already registered in the public database, and is disclosed in WO96 / 09324. It is named as an ECDN small molecule protein and is specifically expressed in colon cancer, esophageal cancer and breast cancer tissue. In addition, the liver X receptor splicing variant protein (GenBank Accession No. AK091535) already registered in the public database lacks exon 5 at 5 'end 132 bases and exon 3 at 3' end 127 bases. The liver X receptor β splicing variant protein D3D5, liver X receptor β splicing variant protein D2D3D5 and liver X receptor β splicing variant protein A3D5 were different in structure.
Liver X receptor β splicing mutant protein D3, liver X receptor β splicing mutant protein D3D5, and liver X receptor β splicing mutant protein D2D3D5 are normal liver X because the number of deleted bases is a multiple of 3. The same stop codon as the receptor β protein is used and consists of 417, 364, 320 and 274 amino acids, respectively.
Liver X receptor β splicing variant protein D3A7, liver X receptor β splicing variant protein D3A4A7, liver X receptor β splicing variant protein A3, liver X receptor β splicing variant protein A3D5, liver X receptor β splicing Mutant protein A2, liver X receptor β splicing mutant protein A1D3A7 and liver X receptor β splicing mutant protein A1D3 are shifted in the frame coding for amino acids by insertion or deletion, and normal liver X receptor β protein Since translation ends with different stop codons, it consists of 409, 160, 188, 188, 67, 16, and 16 amino acids, respectively. In addition, liver X receptor β splicing variant protein A3 and liver X receptor β splicing variant protein A3D5, liver X receptor β splicing variant protein A1D3A7, liver X receptor β splicing variant protein A1D3, and Are proteins having the same amino acid sequence.

  Next, a vector that can be used in a host cell that transforms a polynucleotide having a base sequence encoding the amino acid sequence of the splicing variant protein of the present invention obtained as described above (that is, the splicing variant protein gene of the present invention). Can be introduced. For example, a vector capable of autonomous replication in a host cell, which can be isolated and purified from the host cell, and incorporated into a vector having a detectable marker according to a normal genetic engineering technique, A vector having the splicing mutant protein gene of the present invention can be constructed. Specific examples of the vector having the splicing mutant protein gene of the present invention include plasmid pUC19 (Takara Shuzo), pBluescript II (Stratagene), etc., when Escherichia coli is used as a host cell. . When budding yeast is used as a host cell, plasmids pACT2 (Clontech), pYES2 (Invitrogen) and the like can be mentioned. When mammalian cells are used as host cells, plasmids such as pRc / RSV and pRc / CMV (Invitrogen) can be used.

A promoter capable of functioning in the host cell is linked upstream of the splicing mutant protein gene of the present invention in a functional form, and this is incorporated into a vector as described above, whereby the splicing mutant protein gene of the present invention is incorporated in the host cell. A vector that can be expressed can be constructed. Here, “to bind in a functional manner” means that the splicing mutant protein gene of the present invention is expressed under the control of a promoter in a host cell into which the splicing mutant protein gene of the present invention is introduced. It means that the promoter and the splicing variant protein gene of the present invention are combined. The promoter used exhibits promoter activity in the host cell to be transformed. For example, when the host cell is E. coli, a promoter such as the lactose operon promoter of E. coli, tac promoter, T7 promoter, etc. Can be mentioned. When the host cell is an animal cell, examples thereof include Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) promoter, and the like. When the host cell is budding yeast, examples include alcohol dehydrogenase (ADH) 1 gene promoter and galactose metabolizing enzyme (GAL) 1 gene promoter.
In addition, when using a vector that has a promoter that functions in advance in the host cell, the vector promoter and the splicing variant protein gene of the present invention are linked downstream of the promoter so that they can function in a functional manner. The invention splicing variant protein gene may be inserted. For example, the plasmids pRc / RSV, pRc / CMV, etc. described above have a cloning site downstream of a promoter that can function in animal cells, and the splicing variant protein gene of the present invention is inserted into the cloning site to the animal cells. When introduced, the splicing mutant protein gene of the present invention can be expressed. The budding yeast plasmid pACT2 has an ADH1 promoter, and if the splicing mutant protein gene of the present invention is inserted downstream of the ADH1 promoter of the plasmid or a derivative thereof, the splicing mutant protein gene of the present invention can be expressed, for example, The vector of the present invention that can be expressed in large quantities in budding yeast such as CG1945 strain (Clontech) can be constructed.

The transformant of the present invention can be obtained by introducing the splicing mutant protein gene of the present invention or the vector of the present invention into a host cell. As a method for introducing the splicing mutant protein gene of the present invention or the vector of the present invention into a host cell, a normal introduction method according to the host cell to be transformed can be applied.
For example, when the host cell is Escherichia coli, which is a microorganism, the calcium chloride method and electroporation method described in Molecular Cloning, 3rd edition (Sambrook and Russell, Cold Spring Harbor Laboratory, 2001), etc. A usual method such as can be used. When mammalian cells or insect cells are used as host cells, they can be introduced into the cells by a general gene transfer method such as a calcium phosphate method, an electroporation method or a lipofection method. Further, the splicing mutant protein gene of the present invention may be expressed using yeast as a host cell. In this case, budding yeast (eg, Saccharomyces cerevisiae) is preferably used, but yeast such as Pichia may also be used. Methods for transforming yeast are described, for example, in the method of Ito et al. (J. Bacteriol. 153; 163-168, 1983). In order to incorporate the splicing mutant protein gene of the present invention into a virus such as baculovirus or vaccinia virus, a transfer vector containing a base sequence homologous to the genome of the virus to be used can be used. Specific examples of such transfer vectors include plasmids such as pVL1392 and pVL1393 (Invitrogen). When the splicing mutant protein gene of the present invention is inserted into a transfer vector as described above, and the transfer vector and the viral genome are simultaneously introduced into a host cell, homologous recombination occurs between the transfer vector and the viral genome. A recombinant virus in which the splicing mutant protein gene is integrated on the genome can be obtained. As the viral genome, genomes such as baculovirus and adenovirus can be used. When a virus is used as a vector, viral DNA can be introduced into a host cell by a general gene transfer method as described above. Alternatively, viral DNA can be introduced into a host cell by directly infecting the host cell with a recombinant virus. Can be introduced.

  The splicing mutant protein of the present invention can be prepared as a natural protein by operations such as extraction and purification from a naturally occurring organism, or can be prepared as a recombinant protein using genetic engineering techniques. it can. For example, a purified protein can be prepared by preparing a crude extract from human cells and tissues and using various columns. The cells here are not particularly limited as long as they produce and express the splicing mutant protein of the present invention. For example, liver-derived cells, kidney-derived cells and the like can be used. In addition, among the splicing mutant proteins of the present invention, those produced and expressed in non-human organisms can be prepared from the organism.

In order to produce the splicing mutant protein of the present invention, the splicing mutant protein gene of the present invention or the vector of the present invention is transformed into an appropriate host cell as described above, and the transformant (that is, the transformant of the present invention). ) May be cultured to produce liver X receptor β splicing mutant protein. The produced liver X receptor β splicing mutant protein is recovered according to the usual method. The recovered splicing mutant protein of the present invention is purified by an appropriate method according to the purpose. For example, when the transformant of the present invention is a microorganism, the transformant includes various media appropriately containing carbon sources and nitrogen sources, organic salts, inorganic salts and the like used for normal culture in general microorganisms. Cultured. Cultivation is carried out according to the usual method for general microorganisms, and solid culture, liquid culture (test tube shaking culture, reciprocating shaking culture, Jar Fermenter culture, tank culture, etc.) are possible. . The culture temperature can be appropriately changed within the range in which the microorganism grows. For example, the culture is generally performed in a culture temperature of about 15 ° C. to about 40 ° C. and a culture medium having a pH of about 6.0 to about 8.0. The culture time varies depending on the culture conditions, but is usually about 1 hour to about 24 hours. When an inducible promoter is used, the induction time is preferably within one day, usually several hours.
In addition, when the transformant is an animal cell such as a mammal or insect, the transformant can be cultured using a medium used for normal culture in general cultured cells. In the case of animal cells, for example, a liquid medium (Invitrogen Corp.) supplemented with fetal bovine serum (FBS) so that the final concentration is about 5% (v / v) to about 10% (v / v). And the like under the conditions of 37 ° C. and 5% CO ₂ . When the cells grow to confluence, for example, about 0.25% (v / v) trypsin / PBS solution is added to disperse the cells, dilute several times, seed in a new petri dish, and continue culturing. Similarly, in the case of insect cells, for example, a culture temperature of about 25 ° C. to about 30 ° C. using a Grace medium containing 10% (v / v) FBS or a serum-free medium such as SF-900 (manufactured by Invitrogen). Incubate with In addition, when a recombinant viral vector such as baculovirus is used, it is desirable to collect cells within 72 hours after infection.
Recovery of the splicing mutant protein of the present invention produced by the transformant of the present invention may be performed by appropriately combining ordinary isolation and purification methods. For example, after completion of the culture, the cells of the transformant are centrifuged. After suspending the collected cells in a normal buffer, for example, PBS containing an appropriate protease inhibitor, the cells are disrupted by sonication, a Dounce homogenizer, etc., and the disrupted solution at 20,000 X g. By centrifuging for tens of minutes to about 1 hour and collecting the supernatant fraction, a fraction containing the target splicing mutant protein of the present invention can be obtained. Furthermore, the spliced variant protein of the present invention, which is a more purified object, can be recovered from the supernatant fraction by subjecting it to various chromatographies using conventional protein purification techniques. The polypeptide of the present invention can be recovered in the same manner as described above instead of the splicing mutant protein of the present invention.

Next, utilization of the splicing mutant protein of the present invention will be described.
The present invention provides a method for detecting the presence or absence of the splicing mutant protein of the present invention.
As a method for detecting the presence or absence of the splicing mutant protein of the present invention, the first step of synthesizing complementary DNA (cDNA) from messenger RNA (mRNA) in a biological sample, the splicing mutant protein gene of the present invention or the present invention Examples thereof include a method having a second step of amplifying a partial region of the complementary DNA (cDNA) corresponding to a polypeptide gene and the like and a third step of detecting the amplified partial region.

  Specific examples of the splicing mutant protein gene of the present invention or the polypeptide gene of the present invention include SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, 23, 24, 25, 26, Examples thereof include a polynucleotide having a base sequence represented by 27, 28 or 29.

  Preferably, in the second step of the method, a step of amplifying by a PCR method using one or more selected from the polynucleotide consisting of the base sequence represented by SEQ ID NO: 30, 31, 32 or 33 as a primer is used. You can also.

The present invention includes an antibody or a part of an antibody that specifically recognizes the splicing variant protein of the present invention. The antibody or part of the antibody can be prepared by the following method.
For example, an antibody that specifically recognizes the splicing variant protein of the present invention represented by SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8 is the splicing of the present invention obtained by the genetic engineering method as described above. Animals used for normal antibody production such as rabbits, guinea pigs, rats, mice, goats or sheep using the mutant protein or a partial region (polypeptide) thereof or the peptide fragment shown in SEQ ID NO: 19, 20, 21 or 22 It can be obtained by immunizing. A monoclonal antibody can also be obtained by fusing spleen cells and myeloma cells of an immunized mammal.
Examples of a method for detecting the presence or absence of the splicing mutant protein of the present invention using these antibodies include, for example, an antigen using an antibody capable of specifically detecting the splicing mutant protein of the present invention or the polypeptide of the present invention. The method which has the process of detecting this splicing variant protein based on an antibody reaction can be mention | raise | lifted. More specifically, for example, the first step of binding the first antibody capable of specifically detecting the splicing mutant protein or the polypeptide of the present invention to a microtiter well, the splicing mutation of the present invention in a biological sample. A second step in which the body protein binds to the first antibody on the microtiter well, a second step after the second step, a third step in which all unbound biological samples in the microtiter well are removed, the splicing mutation of the present invention A fourth step in which a labeled second antibody capable of binding to an epitope different from that of the first antibody in the body protein is bound to the liver X receptor β splicing variant protein bound to the first antibody; After the step, a fifth step of removing all unbound second antibody in the microtiter well, After step method for detecting the presence or absence of the present invention splicing mutant protein having a sixth step of detecting the presence or absence of the second antibody in the index labels with the secondary antibody and the like.

The present invention also provides a gene-deficient animal in which the ability to express a polynucleotide or the like having a base sequence encoding the amino acid sequence of the splicing variant protein of the present invention is deleted.
Regarding experimental animals such as gene-deficient animals that lack the ability to express a polynucleotide having a base sequence encoding the amino acid sequence of the splicing variant protein of the present invention and its use, the amino acid sequence of the splicing variant protein of the present invention A model animal is created by destroying (inactivating) a gene having a base sequence encoding the amino acid sequence of an endogenous splicing variant derived from an experimental animal having the function of a gene having the base sequence to be encoded. By analyzing the physical, biological, pathological and genetic characteristics of the protein, it is possible to elucidate the relationship between the function of the splicing mutant protein of the present invention and the disease.
In addition, by introducing the human-derived gene of the present invention into a model animal in which the gene having the base sequence encoding the amino acid sequence of the endogenous liver X receptor β splicing mutant protein described above is destroyed (inactivated) It is also possible to create a model animal having only the human-derived gene of the present invention and evaluate the therapeutic effect of the drug by administering a drug (compound, etc.) targeting the introduced human-derived gene It is.

Among the base sequences encoding the amino acid sequence of the splicing mutant protein, the base sequence represented by SEQ ID NO: 23, 24, 25, 26, 27, 28 or 29 is a probe or primer other than the splicing mutant protein of the present invention. It can be used as a search tool for further liver X receptor β splicing mutant protein.
The length is preferably a base sequence containing at least 15 consecutive bases. The probe can be labeled by a conventional method, for example, with a radioisotope, digoxigenin, biotin, a detectable enzyme, or the like. For example, in the case of using radioactively labeled phosphorus, it is convenient to label with a random priming label when using a cDNA fragment, and when using a synthetic primer, it is convenient to label the 5 'end with a phosphorylating enzyme. The probe labeled in this manner is hybridized to a cDNA library for cloning. Hybridization can be performed by a normal method and conditions. For example, washing at 1 XSSC, 0.5% (w / v) SDS, 65 ° C. The cDNA library may be derived from animals including mammals, but is particularly preferably derived from human tissues / cells.

In addition, the splicing mutant protein of the present invention can be used in, for example, a ligand / receptor binding assay for evaluating the binding ability and binding amount between a test substance and the splicing mutant protein of the present invention or a fragment thereof.
Utilizing this method, the ligand (agonist) is first identified for the association with the pathological condition / disease in which the splicing mutant protein of the present invention is directly or indirectly involved, and then the ligand and the splicing mutant of the present invention are identified. It can be elucidated by specifying a group of genes whose transcription is controlled by proteins. The ligand (agonist) is identified by bringing the test substance into contact with the splicing variant protein of the present invention or the polypeptide of the present invention, and the expression level of the gene group transcriptionally regulated by the splicing variant protein of the present invention or the polypeptide of the present invention. This can be done by detecting changes.

The splicing variant protein of the present invention is a method for constructing an expression system of the splicing variant protein and a target gene to which the splicing variant protein binds, and detecting an increase in the protein that is the target gene product due to addition of a ligand. Can also be used.
For example, by using a reporter gene that expresses a reporter protein such as luciferase, chloramphenicol acetyltransferase, β-galactosidase, etc. under the control of the promoter of the target gene, expression of the target gene in the host cell The presence or absence of can be easily detected. Furthermore, instead of the full length of the splicing mutant protein of the present invention, a chimeric gene of the ligand binding region of the protein and the DNA binding protein is expressed, and a minimal reporter gene is downstream of the base sequence to which the DNA binding protein binds. A plasmid in which an active promoter and a gene encoding the aforementioned reporter protein are linked can be used. As the DNA binding protein, for example, GAL4, LeXA and the like can be used. An expression system of the splicing mutant protein of the present invention and a target gene to which the splicing mutant protein binds, and a plasmid for expressing a chimeric gene of the ligand-binding region of the splicing mutant protein and a DNA-binding protein have an ordinary gene set. It can be prepared by substitution techniques.

  In addition, the splicing mutant protein of the present invention is obtained by transferring a labeled DNA from mRNA expressed in a cell and hybridizing the obtained labeled DNA to a DNA library chip. It can also be used to identify genes whose expression is controlled by.

  Furthermore, the relationship with the pathological condition / disease in which the splicing mutant protein of the present invention is directly or indirectly involved acts on the splicing mutant protein of the present invention in cells expressing the splicing mutant protein of the present invention. By comparing the expression level of the target gene when a substance (specifically, an antagonist or agonist) is brought into contact with the expression level of the target gene in the splicing mutant protein-expressing cell not in contact with the test substance The function can be elucidated by specifying the activated gene.

  As a first screening method of a substance that acts on the splicing mutant protein of the present invention, for example, first, a step of contacting the test substance with the splicing mutant protein of the present invention and a change in the liver X receptor β splicing mutant protein are measured. The method (namely, the 1st screening method of this invention) which has a process can be mention | raise | lifted.

  In the above method, the concentration of the test substance to be contacted with the splicing mutant protein of the present invention is usually about 0.1 μM to about 10 μM, and preferably 1 μM to 10 μM. The time for contacting the splicing variant protein of the present invention with the test substance is usually about 18 hours to 60 hours, preferably about 24 hours to 40 hours.

  In the above method, in order to measure the change of the splicing mutant protein of the present invention, for example, in addition to measuring the binding ability of the chemical substance to the splicing mutant protein of the present invention and quantifying the binding amount, analysis of binding specificity and binding force A ligand / receptor binding assay using a test method capable of the above may be used.

Specifically, for example, in the state where the labeled ligand is bound to the splicing mutant protein of the present invention recovered from the transformant of the present invention as described above, when the test substance coexists, Labeled ligand is released from the splicing mutant protein of the present invention and bound to the splicing mutant protein of the present invention in accordance with the affinity of the liver X receptor β splicing mutant protein from the competition between the labeling ligand and the labeled ligand. The amount of ligand is reduced, thus reducing the amount of label bound to the splicing variant protein of the invention. Therefore, the ability of the test substance to bind to the splicing variant protein of the present invention can be indirectly determined by monitoring the labeling amount of the free labeled ligand or the labeling amount of the bound labeled ligand.
As the labeled ligand, for example, tritium-labeled oxycholesterol can be used. The reaction system is roughly divided into three groups. One system is a group in which only the solvent is added to the place where the labeled ligand is bound to the splicing mutant protein of the present invention, which corresponds to a system in which the concentration of the test substance is zero, and the binding type obtained from this system The labeled amount of the labeled ligand indicates the total binding amount of the labeled ligand to the splicing variant protein of the present invention. The other system is where the labeled ligand is bound to the splicing variant protein of the present invention.For example, unlabeled oxycholesterol sufficiently saturates the splicing variant protein of the present invention and the labeled ligand cannot be bound. The amount of labeled labeled ligand obtained from this system was determined to be the amount of nonspecific binding of the labeled ligand to the splicing mutant protein of the present invention. The Therefore, the specific binding amount of the labeled ligand to the splicing variant protein of the present invention is a value obtained by subtracting the latter non-specific binding amount from the former total binding amount. In the third system, the test substance is added to a place where the labeled ligand is bound to the splicing mutant protein of the present invention, for example, at a final concentration of 10 μM (this concentration is arbitrarily changed depending on the purpose). It is a system. When the test substance has the ability to bind to the splicing variant protein of the present invention, the amount of labeled labeled ligand obtained from this system is the amount of the test substance determined as described above when the test substance concentration is zero. It becomes smaller than the specific binding amount of the labeled ligand to the splicing variant protein of the present invention. By conducting the ligand / receptor binding assay in this manner, the binding ability of the test substance to the splicing mutant protein of the present invention can be examined. When the test substance contains a plurality of substances, the splicing mutation of the present invention is included therein. It is also possible to examine whether or not there is a substance showing affinity for a body protein.
Furthermore, in order to evaluate the binding ability of the test substance to the splicing mutant protein of the present invention in more detail, for example, the assay is performed in the same manner while changing the addition concentration of the test substance in the third system, and the bound labeled ligand Measure the amount of labeling. Based on the measured value, the amount of bound and free ligand in each assay can be calculated to evaluate the binding affinity, binding specificity, binding capacity, etc. between the test substance and the splicing variant protein of the present invention. .
In addition, the evaluation of whether the test substance activates transcription of a gene under the control of a transcription control region containing a binding sequence of liver X receptor β includes, for example, a binding sequence of liver X receptor β. By using a reporter gene linked downstream of the transcription control region, it can be evaluated by a test method such as a reporter assay described later.

  As a second screening method for a substance that acts on the splicing mutant protein of the present invention, the step of bringing a test substance into contact with a cell producing the splicing mutant protein of the present invention, expression regulation by the liver X receptor β splicing mutant protein A method (second screening method) comprising a step of measuring a production amount of another protein that receives the protein and a step of evaluating the action of the test substance on the liver X receptor β splicing mutant protein protein based on the production amount. May be.

  The concentration of the test substance to be contacted with the cell producing the splicing variant protein of the present invention may be usually about 0.1 μM to about 10 μM, and preferably 1 μM to 10 μM. The time for contacting the cells producing the invention splicing mutant protein with the test substance is usually about 18 hours to about 60 hours, preferably about 24 hours to 40 hours.

  As a third screening method for a substance that acts on the splicing mutant protein of the present invention, a cell producing the splicing mutant protein of the present invention is contacted with the ligand of the liver X receptor β splicing mutant protein, and the liver X A first step of measuring the production amount of another protein whose expression is regulated by the receptor β splicing mutant protein, the cell producing the liver X receptor β splicing mutant protein according to any one of claims 1 to 3, A second step in which both the ligand of the liver X receptor β splicing mutant protein and the test substance are brought into contact with each other, and the production amount of another protein whose expression is regulated by the liver X receptor β splicing mutant protein is measured; The other tamper measured in the first step and the second step A method (third screening method) having a step of evaluating the antagonistic activity of the test substance against the ligand based on the difference in the production amount of the protein can also be mentioned.

  The concentration of the ligand or test substance to be contacted with the cell producing the splicing variant protein of the present invention may be usually about 0.1 μM to about 10 μM, and preferably 1 μM to 10 μM. The time for contacting the cell producing the splicing mutant protein of the present invention with the ligand or test substance is usually about 18 hours to 60 hours, preferably about 24 hours to 40 hours.

  These screening methods (that is, three screening methods, hereinafter sometimes collectively referred to as the screening method of the present invention) are substances that act on the liver X receptor β splicing mutant protein selected by the method (the present invention). Then, the substance that acts on the liver X receptor β splicing mutant protein has a broad meaning including antagonists to the substance, and includes substances having agonist activity and substances having antagonist activity to the substance. To make these substances available as drug candidates.

  A drug containing a substance selected by the screening method of the present invention as an active ingredient can be orally or parenterally administered to a mammal such as a human or the like in an effective amount. For example, when administered orally, the drug can be used in a normal form such as a tablet, capsule, syrup, suspension or the like. Moreover, when administering parenterally, the said chemical | medical agent can be used with the form of normal liquid agents, such as a solution, an emulsion, and a suspension. Examples of a method for parenterally administering the drug in the form of the present fat accumulation regulator include an injection method and a suppository method for rectal administration.

  The appropriate dosage form described above can be produced by blending a substance selected by the screening method of the present invention with an acceptable normal carrier, excipient, binder, stabilizer, diluent and the like. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.

  The dose varies depending on the age, sex, weight, disease level, type of the drug, dosage form, etc. of the mammal to be administered. 1 mg to about 2 g, preferably about 5 mg to about 1 g may be administered as the amount of active ingredient, and in the case of injection, about 0.1 mg to about 500 mg may be administered as an active ingredient amount for an adult. In addition, the above-mentioned daily dose can be administered once or divided into several times.

The invention further provides for gene therapy methods
(1) the polypeptide of the present invention,
(2) A polynucleotide having the base sequence encoding the amino acid sequence of the polypeptide of (1) or a base sequence having complementarity to the base sequence (3) SEQ ID NO: 19, 20, 21, or 22 A base sequence encoding any of the amino acid sequences (for example, the base sequence represented by SEQ ID NO: 23, 24, 25, 26, 27, 28 or 29) or a base sequence having a complementarity to the base sequence The use of a specific polynucleotide probe (specific RNA probe or specific DNA probe) for the base sequence of the characteristic polynucleotide is also provided.
Normal liver X consisting of all or part of a gene (DNA or RNA) having a base sequence encoding the amino acid sequence of such a splicing variant protein of the present invention (or a base sequence complementary to the base sequence) The present invention also provides a diagnostic probe for a disease involved in the prevention of leukocyte canceration by a receptor and a diagnostic agent for a disease associated with leukocyte canceration comprising the probe. For example, by performing in situ hybridization of the splicing variant protein gene of the present invention on a pathological section, it is possible to detect the presence or the progress of the disease.
Here, the diagnostic probe is the whole or a part of the antisense strand of the gene (DNA, RNA, cDNA) of the splicing variant protein of the present invention, and has a length (at least 20 bases or more) enough to be established as a probe. If it has, it will not restrict | limit in particular. In order to use the probe as an active ingredient of a diagnostic agent, it is preferable to dissolve in an appropriate buffer or sterilized water so that the probe is not decomposed. Examples of the in situ hybridization method include the method described in J. Neurobiol. 29, 1-17 (1996). It is also possible to use an in situ PCR method.
In the diagnosis, not only a probe but also an antibody (see below) that specifically recognizes the splicing mutant protein of the present invention can be used. The condition can be detected (see Dev. Biol. 170, 207-222 (1995); J. Neurobiol. 29, 1-17 (1996)). The antibody used is easily prepared by the method described in, for example, Antibodies; A Laboratory Manual, Lane, H, D. et al., Cold Spring Harber Lboratory Press published New York 1989.

The gene of the splicing mutant protein of the present invention or the gene of the peptide of the present invention is useful as an antisense drug for controlling the function of the splicing mutant protein of the present invention at the gene level in gene therapy. For example, a base sequence of 20 bases or more, preferably a base sequence of about 30 bases, contained in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, or 18 can be used.
The above-mentioned antisense drug includes not only DNA but also RNA. The base sequence may be a sense sequence or an antisense sequence (that is, a base sequence complementary to the sense sequence).

Here, “the peptide of the present invention” and “the gene of the peptide of the present invention” mean, as described above, a partial region (partial polypeptide) of the splicing mutant protein of the present invention or a gene of the splicing mutant protein of the present invention corresponding thereto. A partial region (partial polynucleotide),
(1) an amino acid sequence consisting of a region that does not exist in normal liver X receptor β, or (2) a polymorphism having an amino acid sequence consisting of a region containing two amino acid residues newly linked by deletion of an exon. It means a peptide or a corresponding polynucleotide. Preferably, it contains at least 6 consecutive amino acids or at least 18 consecutive bases.
Specifically, a polypeptide having any amino acid sequence of SEQ ID NO: 19, 20, 21, or 22, or a base sequence encoding the amino acid sequence of the polypeptide or a base sequence having complementarity to the base sequence And a polynucleotide having a base sequence represented by SEQ ID NO: 23, 24, 25, 26, 27, 28 or 29 or a base sequence complementary to the base sequence.
A polynucleotide having a base sequence encoding the amino acid sequence of the above polypeptide or a recombinant vector containing the above polynucleotide may be prepared according to the above-described method for preparing the vector of the present invention. Furthermore, a transformant can also be prepared by introducing the recombinant vector thus prepared into a host cell according to the method for preparing a transformant of the present invention described above.

The present invention also provides use of liver X receptor β splicing mutant protein as a leukemia marker and a method for evaluating the degree of leukocyte canceration.
Expression of liver X receptor β splicing variant protein is lower in leukemia-derived cell lines than in healthy mammal-derived leukocyte specimens. That is, the amount of the expression product of the liver X receptor β splicing variant protein gene contained in the mammal-derived specimen is measured, and the degree of canceration of the specimen is determined based on the difference obtained by comparing with the control. be able to. Therefore, the present invention
A method for evaluating the degree of canceration of leukocytes derived from mammals,
(1) Amount of expression product of a gene of any of the following liver X receptor β splicing mutant protein (ie, the present β splicing mutant protein) contained in a mammal-derived specimen (for example, liver X receptor β The first step of measuring the amount of the transcription product of the splicing variant protein gene, the amount of the translation product of the gene of the liver X receptor β splicing variant protein, and the like (2) the measured liver X receptor β splicing variant protein gene expression product amount (eg, liver X receptor β splicing variant protein gene transcription product amount, liver X receptor β splicing variant protein gene translation product amount, etc. And a second step of determining the degree of canceration of the white blood cells based on the difference obtained by comparing the control with the control Evaluation method (hereinafter, also referred to as the present evaluation method is.)

(A) Liver X receptor β splicing mutant protein according to item 1 above (B) Liver X receptor β splicing mutant protein having the amino acid sequence of SEQ ID NO: 34

Is included.

  Examples of mammal-derived specimens in the evaluation method of the present invention include biological samples such as thymus, spleen, blood, lymph and lymph nodes. These biological samples may be used as they are as specimens, or biological samples prepared by various operations such as separation, fractionation, and immobilization from such biological samples may be used as specimens. When the mammal-derived specimen is blood, it can be expected that the evaluation method of the present invention will be used in periodic health examinations and simple tests.

  In the evaluation method of the present invention, a primer, probe or specific antibody that can be used in various methods for measuring the amount of an expression product of a gene of liver X receptor β splicing mutant protein is a kit for detecting cancerous white blood cells. It is useful as a reagent. The present invention relates to a kit for detecting cancerous white blood cells containing these primers, probes or specific antibodies as reagents, and cancerous white blood cells in which these primers, probes or specific antibodies are immobilized on a carrier. The scope of the rights of the evaluation method of the present invention is that it can be used in the form of a detection kit or a detection chip as described above using the substantial principle of the method. Of course.

  The following examples further illustrate the invention, but the invention is not limited to these specific examples.

Example 1 (Cloning of liver X receptor β splicing mutant protein gene)
Primers capable of amplifying the entire translation region of the liver X receptor β splicing mutant protein gene were designed. Human leukocyte-derived cDNA (Clontech) available as a commercial product using hLXRβ (ORF) -F / H (SEQ ID NO: 30) as a sense primer and hLXRβ (ORF) -R / B (SEQ ID NO: 31) as an antisense primer The DNA fragment was amplified by the PCR method using the produced cDNA) as a template.
The reaction conditions for the PCR method were as follows.
2.5 unit EX Taq DNA Polymerase (Takara Shuzo), 20 pmol sense primer hLXRβ (ORF) -F / H (SEQ ID NO: 30), 20 pmol antisense primer hLXRβ (ORF) -R / B (sequence) No. 31) and 50 μl of a reaction solution (including MgCl ₂ and deoxyribonucleotide mixture) containing single-stranded cDNA (clontech leukocyte-derived cDNA) as a template was prepared. This reaction solution was subjected to 40 cycles of reaction at 96 ° C. for 30 seconds, 59 ° C. for 30 seconds and 72 ° C. for 165 seconds. A part of the obtained PCR product was electrophoresed on a 1% agarose gel containing ethidium bromide to confirm the amplification of the DNA fragment.
The reaction product was ligated to pGEM-T Easy vector (Promega) using a TA cloning system. Ligation was performed according to the method recommended by the kit used. First, competent cells of E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) were transformed with the ligation reaction solution, and then the resulting cells were transformed into X-gal and It was seeded on LB agar medium containing ampicillin coated with isopropyl β-D-galactopyranoside (IPTG) and cultured at 37 ° C. overnight. After the culture, ampicillin resistant white colonies formed on the agar medium were obtained as the above PCR products cloned into the TA vector.
Subsequently, the exon intron structure of the liver X receptor β protein cloned in the plasmid was detected by performing colony PCR using the obtained white transformant. The reaction conditions for the colony PCR method were as follows. A 50 μl reaction solution (containing a mixed solution of MgSO ₄ and deoxyribonucleotides) containing KOD-Plus-DNA Polymerase (manufactured by Toyobo Co., Ltd.) and single-stranded cDNA as a template was prepared. In the reaction solution for amplifying exons 1 to 4, hLXRβ (ORF) -F / H (SEQ ID NO: 30) is used as a sense primer, hLXRβR5 (SEQ ID NO: 32) is used as an antisense primer, and exon 4 to exon 8 is amplified. In the solution, 20 pmol each of hLXRβF5 (SEQ ID NO: 33) as a sense primer and hLXRβ (ORF) -R / B (SEQ ID NO: 31) as an antisense primer were added. A small amount of the above-mentioned white transformant collected from a plate by sterilized toothpick was suspended, and then the reaction solution was heat denatured at 94 ° C. for 2 minutes, then at 94 ° C. for 15 seconds, 59 ° C. A cycle of 30 seconds at 68 ° C and 45 seconds at 68 ° C was used for 35 cycles of reaction. The obtained PCR product was electrophoresed on an agarose gel containing ethidium bromide, and a plasmid was extracted from a transformant in which a DNA fragment having a length different from that of normal liver X receptor β protein was amplified. The base sequence of the DNA fragment inserted therein was confirmed. As a result, a clone containing the entire translation region containing 10 types of liver X receptor β-splicing mutant proteins could be obtained.

From comparison with the normal liver X receptor β gene structure based on the nucleotide sequence and amino acid sequence, the isolated liver X receptor β splicing mutant protein has one exon 3 as a whole and the 5 ′ end of exon 5 Deletion of 132 bases (liver X receptor β splicing mutant protein D3D5, SEQ ID NOs: 2 and 10), one deletion of the full length of exon 2 and exon 3 and 132 bases of exon 5 5 ′ end (Liver X receptor β splicing mutant protein D2D3D5, SEQ ID NOS: 3 and 11), one having exon 3 deleted and intron 7 inserted (liver X receptor β splicing mutant protein D3A7, sequence No. 4 and 12), one with exon 3 deleted and intron 4 and intron 7 inserted (liver X receptor β splicing mutation) Protein D3A4A7, SEQ ID NOS: 5 and 13), one with intron 3 inserted (liver X receptor β splicing mutant protein A3, SEQ ID NO: 6 and 14), one with intron 3 inserted entirely, exon 5 In which 5 'terminal 132 bases are deleted (liver X receptor β splicing mutant protein A3D5, SEQ ID NOs: 6 and 15), one in which intron 2 is inserted (liver X receptor β splicing mutant protein A2 · SEQ ID NO: 7 and 16), one with intron 1 and intron 7 inserted and exon 3 deleted (liver X receptor β splicing mutant protein A1D3A7 · SEQ ID NO: 8 and 17), one intron 2 inserted (liver X receptor β splicing mutant protein A2), one inserted by intron 1 And exon 3 deleted (liver X receptor β splicing mutant protein A1D3, SEQ ID NOs: 8 and 18), one exon 3 deleted (liver X receptor β splicing mutant protein D3, sequence Nos. 34 and 35) (hereinafter may be collectively referred to as the present splicing mutant protein). The above liver X receptor β splicing mutant protein was substantially the same as normal liver X receptor β except for the insertion or deletion of the above exon or intron.
Liver X receptor β splicing mutant protein D3, liver X receptor β splicing mutant protein D3D5, and liver X receptor β splicing mutant protein D2D3D5 are normal liver X because the number of deleted bases is a multiple of 3. The same stop codon as the receptor β protein was used and consisted of 417, 364, 320 and 274 amino acids, respectively.
Liver X receptor β splicing variant protein D3A7, liver X receptor β splicing variant protein D3A4A7, liver X receptor β splicing variant protein A3, liver X receptor β splicing variant protein A3D5, liver X receptor β splicing Mutant protein A2, liver X receptor β splicing mutant protein A1D3A7 and liver X receptor β splicing mutant protein A1D3 are shifted in the frame coding for amino acids by insertion or deletion, and normal liver X receptor β protein Since the translation was terminated by different stop codons, it consisted of 409, 160, 188, 188, 67, 16, and 16 amino acids, respectively. In addition, liver X receptor β splicing variant protein A3 and liver X receptor β splicing variant protein A3D5, liver X receptor β splicing variant protein A1D3A7, liver X receptor β splicing variant protein A1D3, and Was a protein having the same amino acid sequence.
FIG. 1 collectively shows a comparison of exon intron structures between normal liver X receptor β protein derived from normal human tissue and the obtained liver X receptor β splicing mutant protein.

Example 2 (Examination of Expression Level of Transcript of Liver X Receptor β Splicing Mutant Protein Gene in Human Normal Leukocyte Specimen and Human Leukemia Derived Cell Line)
Whether the liver X receptor β splicing mutant protein found by the present invention is present in RNA of human normal tissue and human leukemia-derived cell line was confirmed by PCR.
A primer that specifically amplifies liver X receptor β protein, and a cDNA derived from Clontech cDNA human normal leukocytes (leukocytes, Killler T cells, Helper T cells, monocytes, B cells) and leukemia cell line K562 ( Chronic myelogenous leukemia), U-937 (histiocytic lymphoma derived), THP-1 (derived from acute monocytic leukemia), HuT 78 (derived from skin lymphoma), Jurkat (derived from acute T cell leukemia) PCR was performed using the template. In the PCR, in the reaction solution for amplifying exons 1 to 4, hLXRβ (ORF) -F / H (SEQ ID NO: 30) is used as the sense primer, hLXRβR5 (SEQ ID NO: 32) is used as the antisense primer, and exon 4 to exon 8 is used. In the reaction solution for amplifying, hLXRβF5 (SEQ ID NO: 33) was used as a sense primer and hLXRβ (ORF) -R / B (SEQ ID NO: 31) was used as an antisense primer. Amplification of a normal liver X receptor β protein-derived DNA fragment results in a DNA fragment having 505 base pairs when exons 1 to 4 are amplified, and 916 base pairs when exons 4 to 8 are amplified. It has a DNA fragment. The results are shown in FIG.
As is clear from these figures,
1) In normal leukocytes, many liver X receptor β splicing mutant proteins were expressed in addition to normal liver X receptor β protein.
2) On the other hand, only normal liver X receptor β was expressed in leukemia-derived cell lines other than THP-1.
Therefore, the presence or absence of liver X receptor β splicing mutant protein is useful for evaluating whether a leukocyte specimen is normal or cancerous.

Example 3 (Construction of Liver X Receptor β Splicing Mutant Protein Expression Vector)
Plasmids in which normal liver X receptor β protein and liver X receptor β splicing mutant protein obtained in Example 1 were respectively cloned were digested with restriction enzymes Hind III and BamHI according to the method recommended by the kit. did. The solution treated with the enzyme was subjected to agarose gel electrophoresis, and a DNA fragment band corresponding to the insert DNA was recovered from the gel. The recovered DNA fragment was cloned into pFLAG-CMV2 (Sigma) which had been cleaved with HindIII and BamHI. After mixing pFLAG-CMV2 (about 300 ng) that had been cleaved with Hind III and BamIH I and the above insert DNA (about 500 ng), T4 ligase was added thereto, and the mixture was mixed at 16 ° C. with 2 ° C. Time ligation was performed. Ligation was performed according to the method recommended by the kit used. First, competent cells of E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) were transformed with the ligation reaction solution, and then the resulting cells were transformed into LB containing ampicillin. It was seeded on an agar medium and cultured overnight at 37 ° C. After the culture, plasmid DNA was prepared from ampicillin resistant colonies formed on the agar medium, and the base sequence of the inserted DNA fragment was determined. The obtained base sequence was compared with the base sequence obtained by the above-mentioned direct sequence, and a plasmid in which the base sequence of the translation region was confirmed to be completely matched was selected, and each plasmid was designated as pCMV2- They were named hLXRβWT, pCMV2-hLXRβD5, pCMV2-hLXRβD3D5, pCMV2-hLXRβD3A7, pCMV2-hLXRβD3A4A7, pCMV2-hLXRβA3, pCMV2-hLXRβD3.

Example 4 (Preparation of a plasmid containing a luciferase reporter gene having a binding sequence of liver X receptor β)
An oligonucleotide consisting of a base sequence containing the binding sequence (DR4) of liver X receptor β and an oligonucleotide consisting of a base sequence complementary to the base sequence were synthesized and annealed to form double-stranded DNA (hereinafter, After that, phosphorylated DNA fragments were obtained by phosphorylating both ends by the action of T4 polynucleotide kinase. Next, the pGL2 promoter vector (Promega) was digested with the restriction enzyme NheI, then E. coli-derived alkaline phosphatase was added, and the mixture was incubated at 65 ° C. for 1 hour. The heat retaining solution is subjected to low melting point agarose gel electrophoresis, and then a DNA fragment is recovered from the detected gel of the band part. About 100 ng of the recovered DNA was mixed with about 1 μg of the above phosphorylated DNA fragment, T4 ligase was added thereto, and the mixture was ligated at 16 ° C. for 30 minutes. Ligation was performed according to the method recommended by the kit used. First, Escherichia coli DH5α competent cells (Takara Shuzo) were transformed with the ligation reaction solution, and then the obtained cells were placed on an LB agar medium containing ampicillin. This was cultured at 37 ° C. overnight. After the culture, plasmid DNA was prepared from ampicillin resistant colonies formed on the agar medium, and the base sequence of the inserted DNA fragment was determined. From the obtained base sequence, a plasmid having a DNA fragment in which two DR4 DNAs were linked in reverse tandem at the Nhe I site of the pGL2 promoter was selected and named plasmid pGL2-DR4.

Example 5 (Functional Evaluation of Liver X Receptor β Splicing Mutant Protein D5 by Reporter Assay in Transient Expression System)
Human fetal kidney-derived HEK293 cell line (ATCC; CRL-1573), fetal bovine serum (FBS) 10% from which low molecular weight substances were removed by charcoal dextran, and penicillin (100 unit / ml) and streptomycin (100 μg / and subcultured in Dulbecco's modified Eagle's medium (DMEM) without phenol red at 37 ° C. in the presence of 5% CO ₂ . The subcultured HEK293 cell line is seeded on a 10 cm cell culture dish so that there are about 2 × 10 ⁶ cells. After culturing this at 37 ° C. for 1 day, 1 μg of normal liver X receptor constructed in Example 2 was used for each of the cells according to the method recommended by the kit using Lipofectamine (manufactured by Invitrogen). The β protein and human liver X receptor β splicing mutant protein expression plasmid and the reporter plasmid pGL2-DR4 prepared in Example 4 are introduced. This is cultured at 37 ° C. for 24 hours, and then the cells are collected by pipetting. The collected cells are uniformly suspended and then seeded in a 96-well plate. Further, various compounds dissolved in ethanol are added to the plate. After further incubation at 37 ° C. for about 2 days, add 50 μl / well of cell lysing agent PGC50 (Nippon Gene) diluted 5 times to the plate and leave it at room temperature for 30 minutes with occasional shaking. To lyse the cells. Collect 10 μl of the cell lysate thus prepared in a 96-well white sample plate (Berthold), and add 50 μl / well of enzyme substrate solution PGL100 (Nippon Gene) with a luminometer LB96p (Berthold). Add and immediately measure luminescence for 1 second. A measurement result of a liver X receptor ligand (agonist) for normal liver X receptor β protein or the liver X receptor β splicing mutant protein is obtained. Moreover, the result of co-expressing normal liver X receptor α protein or normal liver X receptor β protein and the liver X receptor β splicing mutant protein is obtained. From these results, the function of the liver X receptor β splicing mutant protein is confirmed.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a liver X receptor β splicing mutant protein that is a novel liver X receptor β isoform that can be used in a method for evaluating the degree of canceration of leukocyte samples, its gene, and use thereof. It was.

FIG. 1 is a diagram schematically showing the domain structure of normal liver X receptor β protein and the splicing mutant protein of the present invention. WT represents the structure of normal liver X receptor β. The translation area is shown in black. FIG. 2 is a graph showing the expression of liver X receptor β protein in cell lines derived from normal leukocytes and leukemia. The upper row shows the results of amplification of exons 1 to 4, and the lower row shows the results of amplification of exons 4 to 8 by PCR. The arrow represents a fragment of a length corresponding to normal liver X receptor β.

SEQ ID NO: 30
Oligonucleotide primer designed for PCR SEQ ID NO: 31
Oligonucleotide primer designed for PCR SEQ ID NO: 32
Oligonucleotide primer designed for PCR SEQ ID NO: 33
Oligonucleotide primers designed for PCR

Claims (23)

A liver X receptor β splicing mutant protein, which is an isoform of liver X receptor β and having any of the following amino acid sequences:
(1) Amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 (2) Substantially the amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 Identical amino acid sequence (3) Amino acid sequence having 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 A polynucleotide comprising a base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to claim 1, or a base sequence complementary to the base sequence. A polynucleotide comprising the base sequence represented by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, or 18, or a base sequence complementary to the base sequence. A partial polypeptide of the liver X receptor β-splicing mutant protein according to claim 1, wherein the polypeptide has any one of the following amino acid sequences.
(1) A sequence consisting of a region not present in normal liver X receptor β (2) A sequence consisting of a region containing two amino acid residues newly linked by deletion of an exon The polypeptide according to claim 4, which has the amino acid sequence of any one of SEQ ID NOs: 19, 20, 21, and 22. A polynucleotide comprising a base sequence encoding the amino acid sequence of the polypeptide according to claim 5 or a base sequence complementary to the base sequence. A polynucleotide comprising the base sequence represented by SEQ ID NO: 23, 24, 25, 26, 27, 28 or 29 or a base sequence complementary to the base sequence. A recombinant vector comprising the polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to claim 1 or the polynucleotide according to any one of claims 2 to 3. . The polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to claim 1, the polynucleotide according to any one of claims 2 to 3, or the recombinant vector according to claim 8. A transformant characterized in that is introduced into a host cell. A vector capable of autonomously replicating in a host cell a polynucleotide having a nucleotide sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to claim 1 or the polynucleotide according to any one of claims 2 to 3. A method for producing a vector, comprising the step of introducing the vector into a vector. A method for producing a liver X receptor β splicing mutant protein, comprising culturing the transformant according to claim 9 to produce a liver X receptor β splicing mutant protein. The ability to express a polynucleotide having a base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to claim 1 or the polynucleotide according to any one of claims 2 to 3 is deleted. Characteristic gene-deficient animal. A recombinant vector comprising a polynucleotide having a base sequence encoding the amino acid sequence of the polypeptide according to any one of claims 4 to 5 or the polynucleotide according to any one of claims 6 to 7. A polynucleotide having a base sequence encoding the amino acid sequence of the polypeptide of any one of claims 4 to 5, the polynucleotide of any one of claims 6 to 7, or the recombinant vector of claim 13 is introduced into a host cell. A transformant that is introduced. An antibody or a part of an antibody, which specifically recognizes the liver X receptor β splicing mutant protein according to claim 1. A primer designed based on the polynucleotide according to any one of claims 2 to 3. A method for detecting the presence or absence of a liver X receptor β splicing mutant protein according to claim 1, wherein the first step synthesizes complementary DNA (cDNA) from messenger RNA (mRNA) in a biological sample, A polynucleotide having the base sequence encoding the amino acid sequence of the liver X receptor β splicing mutant protein according to item 1 or the polypeptide according to any one of claims 4 to 5, or the poly according to any one of claims 6 to 7. A method comprising: a second step of amplifying a partial region of the complementary DNA (cDNA) corresponding to a nucleotide; and a third step of detecting the amplified partial region. 18. In the second step, amplification is performed by a PCR method using one or more selected from a polynucleotide consisting of the base sequence represented by SEQ ID NO: 30, 31, 32 or 33 as a primer. the method of. A method for detecting the presence or absence of the liver X receptor β splicing mutant protein according to claim 1, wherein the liver X receptor β splicing mutant protein according to claim 1 or any one of claims 4 to 5 is used. A method comprising a step of detecting the liver X receptor β splicing mutant protein based on an antigen-antibody reaction using an antibody capable of specifically detecting a polypeptide. A method for detecting the presence or absence of the liver X receptor β splicing mutant protein according to claim 1, wherein the liver X receptor β splicing mutant protein according to claim 1 or any one of claims 4 to 5 is used. A first step of binding a first antibody capable of specifically detecting a polypeptide to a microtiter well, wherein the liver X receptor β splicing mutant protein of claim 1 in a biological sample is on a microtiter well A second step of binding to the first antibody; a second step after the second step; a third step of removing all unbound biological samples in the microtiter well; the first step in the liver X receptor β splicing variant protein; A labeled second antibody capable of binding to an epitope different from that of one antibody is treated with a liver X receptor β-spray conjugated to the first antibody. After the fourth and fourth steps for binding to the mutant protein, the second step after removing all unbound second antibodies in the microtiter well, after the fifth step, the presence or absence of the second antibody A method comprising a sixth step of detecting using the label of the second antibody as an indicator. Use of any of the following liver X receptor β splicing mutant proteins as a marker for examining the degree of canceration of leukocytes.
(1) Liver X receptor β splicing mutant protein according to claim 1 (2) Liver X receptor β splicing mutant protein having the amino acid sequence of SEQ ID NO: 34 Use of a polynucleotide having a base sequence encoding the amino acid sequence of any of the following proteins as a marker for examining the degree of canceration of leukocytes.
(1) The liver X receptor β splicing mutant protein according to claim 1.
(2) Liver X receptor β splicing mutant protein having the amino acid sequence of SEQ ID NO: 34 The polynucleotide according to any one of claims 2 to 3, or the polynucleotide or sequence according to any one of claims 6 to 7, wherein the polynucleotide having a base sequence encoding the amino acid sequence of liver X receptor β splicing mutant protein is used. The use according to claim 22, which is a polynucleotide having the base sequence represented by number 35.

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