JP2005328749A - New mucin gene and diagnosis of mucosa-associated disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a gene related to a mucosa-associated disease, to provide a method for evaluating disease sensitivity to the mucosa-associated disease, and to obtain a medicine therefor. <P>SOLUTION: A kit for diagnosing contraction risk of the mucosa-associated disease comprises a means for detecting polymorphism of mucin G4 gene having a specific base sequence. The medicine for preventing and treating the mucosa-associated disease comprises inhibiting expression of a mucin gene having a polymorphism evaluated to have high contraction risk of the mucosa-associated disease or inhibiting the function of a mucin glycoprotein having the polymorphism. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel mucin gene and a mucin glycoprotein encoded by the gene. The present invention also relates to diagnosis, prevention and treatment of mucosal-related diseases based on polymorphisms in the mucin gene.

  Diffuse panbronchiolitis (hereinafter abbreviated as DPB), a type of mucosal-related disease, is a chronic respiratory inflammatory disease whose concept of disease was proposed in Japan in the 1960s. Although the onset factor of DPB has not been clarified, it is considered to be a multifactor disease that develops due to the interaction between some environmental factor and an internal factor that is sensitive to the environmental factor. Furthermore, since the onset of this disease is concentrated in East Asia and rarely seen in Westerners, and half of the few reports from Western countries are Asian, etc. It has been assumed for some time that some genetic factor is involved.

  Sugiyama et al. Found that the retention rate of HLA-B54 antigen, which is characteristic for East Asians such as Japan, Korea, and China, is significantly higher in the Japanese DPB patient population (Non-patent Document 1). Later, in Japan, the association of DPB with HLA-B54 (B * 5401) was reconfirmed, and B * 5504 was also shown to be associated with disease (Non-patent Document 2). On the other hand, in Korea, there was no clear association with HLA-B54, and instead HLA-A11 showed a strong association with DPB (Non-patent Document 3). Therefore, HLA-B54 itself or HLA-A11 itself does not regulate disease susceptibility, but between the HLA-A locus and the HLA-B locus located on chromosome 6p21.1 to 6p21.33. It was considered more appropriate to consider that one of the genes that regulate the disease susceptibility of DPB exists.

  Identification between the HLA-A locus and the HLA-B locus based on the hypothesis that one of the genes that regulates DPB disease susceptibility exists between the HLA-A locus and the HLA-B locus As a result of narrowing down the disease susceptibility candidate genetic region using 14 kinds of genetic markers (mainly microsatellite markers), the disease was found in the region of about 200 kb on the HLA-A locus side by about 300 kb from the HLA-B locus. It is estimated that one of the susceptibility genes exists (Non-patent Document 4). In addition to DPCR1 (Non-Patent Document 5) recently reported by Matsuzaka et al., There is almost no known gene in this 200 kb region, and “GenscanGenes” (a gene predicted using the gene prediction program Genscan) Are registered in the gene data bank (for example, GenBank registration numbers NT_007592.643, NT_007592.644, etc.).

  Therefore, it has been desired to identify a disease susceptibility gene for DPB and use it for research and treatment of DPB.

Sugiyama et al., Analysis of HLA antigens in patients with diffuse panbronchiolitis, Am Reespir Dis, vol.141, pp.1459-1462, 1990 Keicho N et al., Distribution of HLA genes to genetic predisposition in diffuse panbronchiolitis, Am J Respir Crit Care Med, 158, p.846-850, 1998 Park MH et al., Association of class I antigens with diffuse panbronchiolitis in Korean patients, Am J Respir Crit Care Med, 159, p.526-529, 1999 Keicho N et al., Fine localization of a major disease-susceptibility locus for diffuse panbronchiolitis, Am J Hum Genet, Vol. 66, pp. 501-507, 2000 Matsuzaka et al., Identification of novel candidate genes in the diffuse panbronchiolitis critical region of the class I human MHC, Immunogenetics, 54, p. 301-309, 2002

  Therefore, in view of the above situation, the present invention aims to provide a gene related to a mucosa-related disease including diffuse panbronchiolitis and an evaluation of disease sensitivity (morbidity risk) of the mucosa-related disease.

As a result of intensive studies to solve the above problems, the present inventor succeeded in isolating a novel mucin gene, and the gene polymorphism in this gene has a relationship with the disease susceptibility of DPB. The present inventors have found that the polymorphism can be used for diagnosis, prevention or treatment of DPB and have completed the present invention.
That is, this invention includes the following 1-13.

1. The following protein (a) or (b).
(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and a mucin glycoprotein Is a protein

2. A polynucleotide encoding the following protein (a) or (b).
(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and a mucin glycoprotein Is a protein

3. The polynucleotide of any of the following (a) to (c).
(A) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 (b) hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to all or part of the base sequence shown in SEQ ID NO: 1 A polynucleotide that encodes a mucin glycoprotein (c) and hybridizes under stringent conditions with a polynucleotide comprising a sequence complementary to all or part of the base sequence shown in SEQ ID NO: 1. And a polynucleotide having a function as a primer or a probe

4). The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 27 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 27, and a mucin glycoprotein Is a protein

5). A polynucleotide encoding the following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 27 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 27, and a mucin glycoprotein Is a protein

6). The polynucleotide of any of the following (a) to (c).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 26 (b) hybridizing under stringent conditions with a polynucleotide comprising a sequence complementary to all or part of the nucleotide sequence represented by SEQ ID NO: 26 A polynucleotide encoding soybean mucin glycoprotein (c) and hybridizing under stringent conditions with a polynucleotide comprising a sequence complementary to the whole or a part of the base sequence shown in SEQ ID NO: 26; And a polynucleotide having a function as a primer or a probe

7). A recombinant vector comprising the polynucleotide of any one of 2, 3, 5 and 6 above.
8). A transformant into which the polynucleotide of any of 2, 3, 5 and 6 or the recombinant vector of 7 is introduced.
9. A method for producing a mucin glycoprotein, comprising culturing the transformant according to 8 and collecting a protein from the obtained culture.

10. A kit for diagnosing the risk of suffering from a mucosa-related disease, comprising means for detecting a polymorphism of the mucin G4 gene having the base sequence represented by SEQ ID NO: 1.

  Examples of the polymorphism include polymorphisms of repetitive sequences having different lengths in the second exon of the mucin G4 gene. Specifically, polymorphisms of repetitive sequences having different lengths include, for example, a 4599 bp L polymorphism (SEQ ID NO: 17), a 3735 bp M1 polymorphism (SEQ ID NO: 18), a 3549 bp Ms polymorphism (SEQ ID NO: 19), It is selected from the group consisting of a 3006 bp Sl polymorphism (SEQ ID NO: 20) and a 2709 bp Ss polymorphism (SEQ ID NO: 21). Here, when a subject has an Ss polymorphism, the subject can be evaluated as having a low risk of suffering from a mucosa-related disease.

  Examples of the polymorphism include a 1-base deletion polymorphism or a 3-base deletion polymorphism in the L polymorphism of mucin G4 gene second exon. Specifically, for example, the single nucleotide deletion type polymorphism has a deletion of the 1827th or 1838th C base of the nucleotide sequence shown in SEQ ID NO: 17, and the three nucleotide deletion type polymorphism includes There is a deletion of one ACC sequence in the 3210th to 3224th ACC 5-repeat sequence of the base sequence shown in SEQ ID NO: 17. Here, the subject has L polymorphisms of repetitive sequences having different lengths in the second exon of the mucin G4 gene, and both the 1-base deletion polymorphism and the 3-base deletion polymorphism in the L polymorphism Alternatively, in the case of having either one, the subject can be evaluated as having a high risk of suffering from a mucosa-related disease.

  Furthermore, examples of the polymorphism include a single nucleotide polymorphism in the fourth exon of the mucin G4 gene. Specifically, for example, as a single nucleotide polymorphism, there is a G / A polymorphism at the 5492th base of the base sequence shown in SEQ ID NO: 1. Here, when the 5492th base of the base sequence shown in SEQ ID NO: 1 is G / A heterotype or A / A homotype, the subject can be evaluated as having a high risk of suffering from a mucosa-related disease. .

  In the above diagnostic kit, the detection means is not limited, but comprises a base sequence selected from the group consisting of SEQ ID NOs: 1, 17, 18, 19, 20, 21, 24 and 25 or a partial sequence thereof. An oligonucleotide having an oligonucleotide or its complementary sequence is included.

  As the detection means, for example, means for detecting polymorphisms of repetitive sequences having different lengths in the second exon of the mucin G4 gene are shown in the oligonucleotide having the base sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12. There is a primer set including an oligonucleotide having a base sequence.

  Further, as the above-mentioned detection means, means for detecting the 1-base deletion polymorphism in the L polymorphism of mucin G4 gene second exon includes an oligonucleotide having the base sequence shown in SEQ ID NO: 22.

  As the detection means, means for detecting the 3-base deletion polymorphism in the L polymorphism of the mucin G4 gene second exon includes an oligonucleotide having the base sequence shown in SEQ ID NO: 23.

11. A kit for diagnosing the risk of suffering from a mucosa-related disease, comprising means for detecting a polymorphism of the mucin G2 gene represented by SEQ ID NO: 26.

  Examples of the polymorphism include a 4-base insertion / deletion polymorphism in the third exon of the mucin G2 gene.

  In the above diagnostic kits 10 and 11, mucosal-related diseases include inflammation (for example, diffuse panbronchiolitis, chronic bronchitis, cystic fibrosis, bronchiectasis and chronic sinusitis), and malignant tumors (respiratory tract) , Malignant tumors in the placenta, testis, brain and spleen).

  Although the diagnostic object of the said diagnostic kit is not limited, It is preferable that it is an Asian race.

12 A mucosa-related disease comprising a compound that suppresses the expression of a mucin gene having a polymorphism that is evaluated to have a high risk of suffering from a mucosa-related disease, or a compound that suppresses the function of a mucin glycoprotein having the polymorphism. A preventive or therapeutic drug.

  Polymorphisms that are evaluated to have a high risk of suffering from the above-mentioned mucosa-related diseases include the 1 base deletion polymorphism and the 3 base deletion polymorphism in the L polymorphism of mucin G4 gene second exon, and SEQ ID NO: 1. Examples include G / A heterotype and A / A homotype at the 5492th base of the base sequence shown.

  Moreover, as said compound, although it is not limited, antisense oligonucleotide, double stranded RNA, and an antibody are mentioned.

13. A compound for increasing the expression of a mucin gene having a polymorphism that is evaluated to have a low risk of suffering from a mucosa-related disease, or a mucosal-related disease for prevention or treatment comprising a mucin glycoprotein having the polymorphism Medicine.

  Examples of the polymorphism evaluated as having a low risk of suffering from the mucosa-related disease include the Ss polymorphism in the second exon of the mucin G4 gene.

  The diagnostic kit of the present invention makes it possible to diagnose the risk of suffering from a mucosa-related disease. In addition, the preventive or therapeutic drug of the present invention makes it possible to effectively prevent or treat mucosa-related diseases.

  Furthermore, the present invention provides a novel mucin gene and mucin glycoprotein. Mucin glycoprotein is known to play an important role in the living body, and thus is useful for elucidating many in vivo mechanisms including mucosa-related diseases.

1. Mucin gene and mucin glycoprotein (1) Identification of novel mucin gene The mucin G4 gene and mucin G2 gene of the present invention were isolated from human bronchial epithelial cells, bronchial epithelium (G4 and G2), placenta (G4), testis ( It was confirmed to be expressed in G4), brain (G2) and spleen (G2). The mucin gene is a general term for genes encoding mucin glycoproteins. Mucin glycoproteins are mucinous proteins with a sugar chain structure that exist in the epithelium (for example, mucous membranes) and are It has the function of physically covering the epithelium against the stimulus and protecting the living body. Therefore, it is considered that the mucin glycoprotein and mucin gene of the present invention also have functions in the epithelium and mucous membrane.

  The mucin gene of the present invention is screened from reverse transcription polymerase chain reaction (RT-PCR) or cDNA library using mRNA purified from RNA extracted from bronchial epithelium, placenta, testis, brain or spleen tissue or cells. Can be obtained.

  Extraction of mRNA from tissues or cells and preparation of a cDNA library can be performed according to conventional methods. For example, after extracting total RNA from the above-mentioned source by guanidinium thiocyanate-cesium trifluoroacetate method or the like, poly (( A) + RNA (mRNA) can be obtained. Furthermore, poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.

  After synthesizing single-stranded cDNA using the thus obtained mRNA as a template using oligo dT primer and reverse transcriptase, double-stranded cDNA is synthesized from the single-stranded cDNA. Next, the obtained double-stranded cDNA is incorporated into an appropriate cloning vector to prepare a recombinant vector. A cDNA library can be obtained by transforming Escherichia coli and the like using this recombinant vector, and selecting transformants using tetracycline resistance, ampicillin resistance and the like as indices.

  In order to select a strain having the target DNA from the transformant obtained as described above, for example, a primer designed based on the base sequence of the mucin gene is synthesized, and this is used for polymerase chain reaction (PCR). And a method of screening from a cDNA library using the obtained fragment as a probe can be employed.

The amplified DNA fragment thus obtained is labeled with ³² P, ³⁵ S, biotin or the like as a probe, and this is hybridized with a nitrocellulose filter on which the transformant DNA is denatured and immobilized. Screening can be done by searching for strains.

  It is desirable to prepare a probe by the PCR method based on the obtained sequence and screen the gene from a cDNA library.

  Next, a full-length cDNA is cloned from the obtained clone. For example, the RACE (Rapid Amplification of cDNA ends) method is used for cloning of cDNA. The RACE method is a method for rapidly recovering a 5 'or 3' deletion site of cDNA by PCR. The RACE method can also be performed using a commercially available kit (First Choice RLM RACE Kit, Ambion).

  In the present invention, the cDNA base sequence of the cDNA isolated clone obtained in the above screening is determined using the PCR product as a template. The base sequence can be determined by a known technique such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage. Usually, an automatic base sequencer (for example, ABI Prism 310 manufactured by Applied Biosystems) is used. Sequencing is performed using Genetic Analyzer or the like.

  SEQ ID NO: 1 exemplifies the base sequence of the mucin G4 gene, and SEQ ID NO: 2 exemplifies the amino acid sequence of the mucin glycoprotein encoded by the mucin G4 gene. Here, the 359th to 5679th base sequence of SEQ ID NO: 1 is CDS. Furthermore, SEQ ID NO: 26 exemplifies the base sequence of the mucin G2 gene, and SEQ ID NO: 27 exemplifies the amino acid sequence of the mucin glycoprotein encoded by the mucin G2 gene. Here, the 313th to 1056th base sequence of SEQ ID NO: 26 is CDS.

  However, as long as the protein containing the amino acid sequence shown in SEQ ID NO: 2 or 27 is a mucin glycoprotein, a plurality of, preferably one or several amino acids in the amino acid sequence have mutations such as deletion, substitution, addition, etc. May occur. Mucin glycoprotein refers to a mucin-type (serine / threonine-linked) glycoprotein. Whether a protein is a mucin glycoprotein is analyzed by analyzing the amino acid sequence or base sequence of the obtained protein. The presence of a signal peptide, the presence of a repetitive sequence of an amino acid sequence containing a large amount of serine / threonine, the serine and threonine content, and the like can be confirmed.

  For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2 or 27 may be deleted, and 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2, preferably 1 to 5 amino acids may be added, or 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted with other amino acids of the present invention. Included in mucin glycoprotein. Specifically, a protein having an amino acid sequence in which the 1712th amino acid residue in the amino acid sequence shown in SEQ ID NO: 2 is substituted from asparagine (Asp) to aspartic acid (Asn) can be mentioned. This protein is due to the presence of a single nucleotide polymorphism (SNP) in the mucin G4 gene.

  The above mucin glycoprotein is expressed in bronchial epithelium (G4 and G2), placenta (G4), testis (G4), brain (G2) and spleen (G2), and mucin glycoprotein has a sugar chain structure. Since it is a mucinous protein and its roles such as cell protection and cell-cell interaction in the epithelium (for example, mucous membrane) are known, the mucin glycoprotein of the present invention is, for example, cell protection or cell-cell interaction in the tissue It can be used to screen for drugs that modulate the action. Furthermore, a drug for preventing or treating a disease associated with the function of such mucin glycoprotein can be developed by screening a drug that enhances or reduces such cytoprotection or cell-cell interaction.

  The polynucleotide of the above mucin gene is prepared from a DNA prepared from a tissue or cell such as bronchial epithelium, placenta, testis, brain or spleen using a primer designed based on the base sequence (SEQ ID NO: 1 or 26) of the above gene. It can be prepared by performing an amplification reaction, such as the polymerase chain reaction (PCR).

  Moreover, since the base sequence of the mucin gene is determined, a polynucleotide can be prepared by chemical synthesis. Alternatively, a polynucleotide encoding a mucin glycoprotein which is a mutant form of the mucin gene and can be synthesized by site-directed mutagenesis. In addition, in order to introduce | transduce a variation | mutation into a gene, well-known methods, such as Kunkel method and Gapped duplex method, or the method according to this can be employ | adopted.

  Whether or not the recombinant protein produced by the mutated gene is a mucin glycoprotein is confirmed by analyzing the amino acid sequence or base sequence of the obtained protein and examining the characteristics of the mucin glycoprotein, as described above. be able to.

  Furthermore, the present invention also includes a polynucleotide that hybridizes under stringent conditions with a sequence complementary to the whole or a part of the polynucleotide comprising the above base sequence and has a function as a primer or a probe. included. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, it refers to conditions under which polynucleotides having high homology (homology is 60% or more, preferably 80% or more) hybridize. More specifically, the sodium concentration is 0.3 to 1.5M, preferably 0.7 to 0.9M, and the temperature is 50 to 80 ° C, preferably 68 ° C.

  In addition, “having a function as a primer or a probe” means a length and a base composition (melting temperature) that allow specific annealing or hybridization, for example, satisfying conditions allowing specific annealing or hybridization. It means to have. That is, the polynucleotide must contain a sequence that specifically anneals or hybridizes to the polynucleotide (DNA or RNA) of the mucin gene of the present invention, and the base composition is short because the length of the sequence is short. It is intended to exclude polynucleotides that are not suitable and thus frequently cause non-specific annealing or hybridization.

  For example, when a polynucleotide is used as a primer or probe, the length having a function as a primer or probe is preferably 10 bases or more, more preferably 16 to 50 bases, and further preferably 20 to 30 bases. .

  In designing, it is preferable to confirm the melting temperature (Tm) of the primer or probe. For confirmation of Tm, known primer or probe design software can be used. Other conditions that allow specific annealing or hybridization as a primer or probe include GC content, and such conditions are well known to those skilled in the art.

  In the present invention, the “polynucleotide” may be either DNA or RNA, or may be a hybrid nucleotide of DNA and RNA.

(2) Production of Recombinant Vector and Transformant The recombinant vector of the present invention can be obtained by ligating the above mucin gene (polynucleotide) to an appropriate vector. Polynucleotide) or the recombinant vector of the present invention can be obtained by introducing it into a host so that the gene of interest can be expressed.

  As the vector, a phagemid or a plasmid capable of growing in a host is used. Examples of plasmid DNA include bacterial-derived plasmids and yeast-derived plasmids, and examples of phagemid DNA include λ phage (λgt10, λZAP, etc.). Furthermore, transformants can also be prepared using animal virus vectors such as retrovirus or vaccinia virus, insect virus vectors such as baculovirus, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and the like.

  In order to insert the polynucleotide (mucin gene) of the present invention into a vector, first, the purified polynucleotide is cleaved with a suitable restriction enzyme and inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA. A method of linking to is adopted.

  The mucin gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, in addition to the promoter and the mucin gene of the present invention, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like may be linked to the vector of the present invention. Can do. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.

  A known DNA ligase is used to link the above various sequences and the vector DNA. Then, the above various sequences and vector DNA are annealed and then ligated to prepare a recombinant vector.

  The host used for transformation is not particularly limited as long as it can express the introduced gene. Examples thereof include yeast, animal cells (NCIH292 cells, COS cells, CHO cells, etc.) and insect cells.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. The method for introducing a polynucleotide or a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, Chinese hamster ovary cells (CHO cells) and the like are used. As the promoter, SV40 promoter, LTR promoter, CMV promoter and the like are used. Examples of methods for introducing a polynucleotide or a recombinant vector into animal cells include electroporation, calcium phosphate, and lipofection.

  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a polynucleotide or a recombinant vector into insect cells include a calcium phosphate method, a lipofection method, and an electroporation method.

  A transformant is selected by utilizing the property of a marker gene constructed in a gene to be introduced. For example, when a neomycin resistance gene is used, cells showing resistance to the G418 drug are selected.

(3) Production of Mucin Glycoprotein In the present invention, mucin glycoprotein can be obtained by culturing the transformant introduced with a mucin gene and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, or cell lysate. The method of culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.

  As a medium for culturing a transformant obtained using yeast as a host, a natural medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like and can efficiently culture the transformant. Any of synthetic media may be used. The culture is usually performed at about 20 to 40 ° C. for about 1 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is kept near neutral. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

As a medium for cultivating a transformant obtained using animal cells as a host, a generally used RPMI 1640 medium, DMEM medium, a medium obtained by adding fetal calf serum or the like to these mediums, or the like is used. The culture is usually performed at about 37 ° C. for about 1 to 7 days in the presence of 5% by volume CO ₂ . During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  When mucin glycoprotein is produced intracellularly after culturing, the protein is extracted by disrupting the cell. When mucin glycoprotein is produced extracellularly, the culture solution is used as it is or the cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, Mucin glycoprotein can be isolated and purified from

  Whether mucin glycoprotein is obtained can be confirmed by polyacrylamide gel electrophoresis or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or the like.

2. Diagnosis of mucosa-related diseases (1) Mucin gene polymorphisms The mucin genes (G4 and G2) of the present invention were found to have multiple gene polymorphisms associated with diffuse panbronchiolitis (DPB). . As mentioned above, mucin glycoprotein is known to have a role in the epithelium (for example, mucosa), and it has been reported that other mucin genes are associated with inflammatory diseases and malignant tumors. The mucin gene polymorphism of the invention is associated with inflammation in the airway mucosa (eg, diffuse panbronchiolitis (DPB), chronic bronchitis, cystic fibrosis, bronchiectasis and chronic sinusitis), airway, placenta, testis It is thought to be associated with malignant tumors in the brain and spleen. In the present invention, these diseases considered to be related to the polymorphism of the mucin gene of the present invention are referred to as “mucosal-related diseases”.

  Here, “gene polymorphism” or “polymorphism” refers to a difference in genes that causes diversity in traits and morphology among individuals. Single Nucleotide Polymorphism (SNP), insertion / deletion Type polymorphisms and polymorphisms caused by different numbers of repeated sequences are included. A single nucleotide polymorphism (SNP) generally means a polymorphism resulting from substitution of one specific base in a gene or region with another base. The insertion / deletion type polymorphism refers to a polymorphism resulting from deletion or insertion of one or more bases (for example, 1 to several tens of bases). Some have deletions or insertions of ~ several thousand bases. Furthermore, the polymorphism caused by the difference in the number of repetitions of the repetitive sequence refers to a sequence in which a sequence of 2 to several tens of bases is repeated and the number of repetitions varies among individuals. A unit having a repeating unit (repetitive sequence) of several bases to several tens of bases is particularly referred to as VNTR (variable number of tandem repeat). In VNTR, variations are obtained by the difference in the number of repetitions among individual alleles (alleles).

  In the present invention, by utilizing a relationship between a gene polymorphism in the mucin gene (G4 and G2) of the present invention and a mucosa-related disease, a specific gene polymorphism in the mucin gene is detected, thereby causing mucosal association in the subject. Diagnose the risk of disease. Here, “detection” of a polymorphism refers to determining the type of a specific polymorphism or determining the presence or absence of a polymorphism, and is also referred to as “typing”. The “morbidity risk” means the susceptibility (susceptibility) to the disease, and the “polymorphism with a high morbidity risk” means that the subject having the polymorphism compared to the subject having another polymorphism. Mean that a subject with a polymorphism has a lower probability of getting the disease compared to a subject with another polymorphism. Means.

The polymorphisms of the mucin genes (G4 and G2) of the present invention used for diagnosis of mucosa-related diseases include the following:
(A) Five polymorphisms (VNTR) of repetitive sequences having different lengths in the second exon of the mucin G4 gene
-4599 bp L polymorphism (SEQ ID NO: 17): the most frequent polymorphism, repeated about 10 amino acids (TTTASTEGSE as an example) about 150 times-3735 bp Ml polymorphism (SEQ ID NO: 18) ): It has a deletion of 1074 bases and an insertion of 210 bases compared to the L type, and the number of repetitions of the repetitive sequence is reduced by about 29 times. 3549 bp Ms polymorphism (SEQ ID NO: 19): Compared with deletion of 1050 bases, the number of repetitive sequences is reduced by about 35 times. • 3006 bp S1 polymorphism (SEQ ID NO: 20): deletion of 1593 bases compared to L type The number of repeats of the repetitive sequence is reduced by about 53. 2709 bp Ss polymorphism (SEQ ID NO: 21): It has a deletion of 1890 bases compared to the L type, and the number of repeats of the repetitive sequence is about 63. Reduction There is a little Among the above five polymorphisms, a subject having the Ss polymorphism can be evaluated as having a low risk of suffering from a mucosa-related disease.

(B) Single base deletion polymorphism in L polymorphism of mucin G4 gene second exon Lack of C base at 1827th or 1838 th of L polymorphism of the second exon (base sequence shown in SEQ ID NO: 17) Loss (SEQ ID NO: 24)

(C) 3 base deletion type polymorphism in L polymorphism of mucin G4 gene second exon 3210th to 3224th ACC 5th repetitive sequence of L polymorphism of the second exon (base sequence shown in SEQ ID NO: 17) Deletion of one ACC sequence (3 bases) in SEQ ID NO: 25
The mucin G4 gene second exon has an L polymorphism of a repetitive sequence having different lengths, and the L polymorphism is a single nucleotide deletion polymorphism and / or a three nucleotide deletion polymorphism. A subject having a high risk of suffering from a mucosa-related disease can be evaluated.

(D) Single nucleotide polymorphism (SNP) in exon 4 of mucin G4 gene
Polymorphism at the 5492th base of the base sequence shown in SEQ ID NO: 1 (G / A)
A subject whose base is G / A heterotype or A / A homotype can be evaluated as having a high risk of suffering from a mucosa-related disease. Here, “G / A heterotype” means that one allele of the 5492th base has a base G and the other allele has a base A, which is heterozygous. “Homo” means that both alleles have the base A for the 5492th base and are homo-type. As a result of the presence of this single nucleotide polymorphism, the asparagine at position 1712 is substituted with aspartic acid in the amino acid sequence of the mucin glycoprotein. When simply indicated as “G / A”, it represents a single nucleotide polymorphism of the base G or A.

(E) Insertion / deletion polymorphism of four bases in the third exon of the mucin G2 gene Missing “CACT” between the 795th base T and the 795th base G of the base sequence shown in SEQ ID NO: 26 Loss or insertion This polymorphism is associated with mucosal-related diseases.

(2) Diagnosis subject In the present invention, the risk of mucosal-related diseases is diagnosed using a mucin gene polymorphism. The subject to be diagnosed is a mammal including a human (for example, human, monkey, The mouse is not particularly limited. In particular, the subject to be diagnosed is preferably a human, more preferably an Asian race.

  In order to detect a gene polymorphism, it is necessary to prepare DNA or RNA from a subject to be diagnosed. This preparation can be performed using a known method. For example, genomic DNA can be prepared by a phenol / chloroform method, a cetyltrimethylammonium bromide (CTAB) method, or the like. RNA can be prepared by the guanidine isothiocyanate method.

  The source for preparing DNA or RNA is not particularly limited, and cells or tissues scraped from blood, saliva, nasal mucosa, or the like can be used.

(3) Method for detecting gene polymorphism As described above, the mucin gene polymorphism of the present invention has a certain relationship with mucosal-related diseases. Therefore, by detecting this mucin gene polymorphism, mucosal-related diseases are detected. The risk of morbidity can be diagnosed.

  Detection of gene polymorphism can be performed by means of polymorphism detection known in the art, and includes, but is not limited to, direct sequencing, methods utilizing polymerase chain reaction (PCR), restriction enzyme fragment length Examples include a method using a polymorphism (RFLP), a method using a hybridization method, a method using a primer extension reaction, and mass spectrometry. Both of these methods are well known to those skilled in the art. The outline will be described below.

(3-1) Direct sequencing method A gene polymorphism can be detected by a direct sequencing method. In direct sequencing, DNA is first prepared from a subject. A region containing the polymorphism to be detected is cloned into a vector and amplified in a host cell (eg, a bacterium). Alternatively, DNA in the region containing the polymorphism to be detected can be amplified by PCR. After amplification, the DNA in the detection target region is sequenced. Sequencing methods include, but are not limited to, manual sequencing methods or automated sequencing methods. Examples of the manual sequencing method include a method using a radioactive marker nucleotide, and examples of the automatic sequencing method include a method using a dye terminator. Based on the result of sequencing, the polymorphism of the subject is detected.

(3-2) PCR method A gene polymorphism can also be detected using the PCR method. PCR is performed using oligonucleotide primers that hybridize only to sequences having a particular polymorphism or wild type sequence. The polymorphic and wild type primer sets are used to amplify DNA from the subject. If only the polymorphic primer produces a PCR product, the subject will have that particular polymorphism. If only the wild-type primer produces a PCR product, the subject will have the wild-type sequence.

  In the case of VNTR, a primer set capable of amplifying a region including the polymorphic site is designed, and an amplification reaction is performed using DNA derived from a subject as a template. Since the obtained amplified fragment varies in length depending on the number of repeats of the VNTR repeat sequence, polymorphism (VNTR) can be detected using electrophoresis.

(3-3) RFLP method A gene polymorphism can also be detected using restriction fragment length polymorphism (RFLP). First, the region containing the polymorphism to be detected is amplified by PCR. This PCR product is then cleaved with a restriction enzyme known to produce fragments of a length unique to a given polymorphism. PCR products digested with restriction enzymes are generally separated by gel electrophoresis and visualized by ethidium bromide staining. A subject's polymorphism can be detected by comparing the length of the fragment to the molecular weight marker and the length of the fragment produced by the wild-type and specific polymorphic controls.

(3-4) Hybridization method A gene polymorphism can also be detected by utilizing hybridization. The hybridization method is a method for determining the presence or absence of a predetermined polymorphism (SNP or the like) based on the ability of a subject-derived DNA to hybridize with a complementary DNA molecule (for example, an oligonucleotide probe). This hybridization method can be performed using various techniques for hybridization and detection.

  Whether or not the probe is hybridized to a polymorphic sequence to be detected (for example, SNP) can be directly detected by visualizing the bound probe. This is known, for example, as the Northern or Southern assay (see, eg, Ausabel et al. (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1991)). For the Southern assay, genomic DNA is isolated from the subject, and for the Northern assay, RNA is isolated from the subject. Subsequently, the isolated DNA or RNA is cleaved with a series of restriction enzymes. Next, DNA or RNA is separated and transferred to a membrane. This separation operation can be performed, for example, on an agarose gel. The transfer to the membrane is performed by transferring a labeled probe or a probe specific to the polymorphism to be detected to the membrane under suitable stringency conditions. Labeling can be performed, for example, by introducing radioactive nucleotides. After removing the unbound probe, the labeled probe can be visualized to detect the presence of the binding, and whether or not the specific polymorphism is present can be determined based on the presence or absence of the binding.

  Alternatively, hybridization can be detected using a DNA chip. In this method, an oligonucleotide probe is attached to a solid support. This probe is designed to be specific for a given polymorphism. A DNA sample derived from a subject is brought into contact with a DNA chip, and hybridization is detected.

(3-5) Method Using Primer Extension Reaction A gene polymorphism can also be detected using a primer extension reaction. An example of a method using a primer extension reaction is the SniPer method. The SniPer method is based on a technique called a rolling circle amplification (RCA) method, which uses a circular single-stranded DNA as a template and a DNA polymerase moves on the complementary strand DNA. Are synthesized continuously (see, for example, US Pat. Nos. 6,210,884 and 6,183,960). According to this method, polymorphism can be detected by measuring the presence or absence of a color reaction that occurs when DNA amplification occurs (Lizardi, PM et al., Nature Genet., 19, 225-232 (1998). Piated, AS et al., Nature Biotech., 16, 359-363 (1998)).

  In the present invention, the polymorphism of the mucin gene (G4 and G2) is detected using the polymorphism detection method described above. Here, examples of the polymorphism detection means include oligonucleotide probes or primers for use in the above-described method.

  In the present invention, the oligonucleotide used as a primer or probe is based on the nucleotide sequence shown in SEQ ID NO: 1, 17, 18, 19, 20, 21, 24 or 25, and may be used as the whole of these sequences. Alternatively, it may be designed and synthesized to include a portion of these sequences (eg, to include a polymorphic site or to a position where the polymorphic site can be amplified). In addition, an oligonucleotide having a sequence complementary to the above sequence may be designed.

  The design of primers and probes is well known in the art, and can be appropriately designed according to the amplification reaction or hybridization used. For the specific design method, refer to the description in the section “(1) Identification of novel mucin gene” in “1. Mucin gene and mucin glycoprotein”.

  The primer or probe designed as described above can be chemically synthesized by a known oligonucleotide synthesis method, but is usually synthesized using a commercially available chemical synthesizer.

  As means for detecting a polymorphism of a mucin gene that can be used in the present invention, oligonucleotides having the following sequences can be exemplified.

(A) Oligonucleotide (primer) for detecting five polymorphisms (VNTRs) present in repetitive sequences having different lengths in the second exon of the mucin G4 gene
Sense primer: 5'-CCC ACC ACC TTA TTT GTT CTC CAC-3 '(SEQ ID NO: 11)
Antisense primer: 5'-CAG GGA AAT GAG GAT GAT AGC CCA-3 '(SEQ ID NO: 12)

  Using the primer, an amplification reaction is performed using DNA derived from a subject as a template. As the conditions for the amplification reaction, since the fragments obtained by amplification differ in length depending on the type of the five polymorphisms, the polymorphism can be detected by using electrophoresis. Specifically, the L polymorphism is a fragment of about 7058 bp, the Ml polymorphism is a fragment of about 6194 bp, the Ms polymorphism is a fragment of about 6008 bp, the Sl polymorphism is a fragment of about 5465 bp, and the Ss polymorphism is It is amplified as a fragment of about 5168 bp. Here, the electrophoresis method is not particularly limited, and agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis and the like can be used.

(B) an oligonucleotide for detecting a single nucleotide deletion polymorphism in the L type of mucin G4 gene second exon, ie, deletion of C base at 1827th position or 1838 position of the nucleotide sequence shown in SEQ ID NO: 17 ( Primer)
Sequence primer: 5'- TGT GGT GGT CTC CAA GCC TGT ATT-3 '(SEQ ID NO: 22)

  The resulting PCR product is purified to remove the primer (for example, using ExoSapIT (Amersham Pharmacia)) and, for example, a BigDyeTerminator kit (ABI) cycle sequence reaction is performed. Then, the polymorphism can be detected by removing the die terminator with Sephadex G50, analyzing with ABI3100 Genetic Analyzer, and confirming the waveform with a sequencer.

(C) Deletion of one ACC sequence in the 3 base deletion polymorphism in the L type of mucin G4 gene second exon, that is, in the 3210th to 3224th ACC 5th repeat of the nucleotide sequence shown in SEQ ID NO: 17 Oligonucleotide (primer) for detection
Sequence primer: 5'-TAA AAT GAC CAC AGT CTT CAC TGA AAA CTC-3 '(SEQ ID NO: 23)

  This sequence primer is used in the same manner as described above, and a polymorphism can be detected by confirming the waveform with a sequencer.

(4) Diagnostic kit The means (oligonucleotide) for detecting the polymorphism of the mucin gene mentioned above is provided as a kit for diagnosing the risk of suffering from a mucosa-related disease.

  The diagnostic kit of the present invention comprises means for detecting at least one mucin gene polymorphism. Furthermore, one or more kinds of components necessary for carrying out polymorphism detection may be included. Such components include, but are not limited to, oligonucleotides, polymerases, buffers (eg, Tris buffer), dNTPs, labeling and detection reagents (such as fluorescent dyes), and instructions.

  The kit for diagnosing the morbidity risk of the mucosa-related disease of the present invention makes it possible to easily and quickly diagnose the morbidity risk of the subject, and appropriate treatment can be performed according to the diagnosis result.

3. Prevention and treatment of mucosal-related diseases The mucin glycoprotein and mucin gene of the present invention are associated with the risk of suffering from mucosal-related diseases. That is, a subject having a specific gene polymorphism in the mucin gene (G4 and G2) is evaluated as having a high or low risk of suffering from a mucosa-related disease. Therefore, by using the mucin glycoprotein or the mucin gene of the present invention, it is possible to reduce the morbidity risk of a subject who is evaluated as having a high morbidity risk, and to prevent and treat mucosa-related diseases.

  More specifically, when there is a single nucleotide deletion polymorphism and / or a triple nucleotide deletion polymorphism in the L type of mucin G4 gene second exon, there is a high risk of suffering from a mucosa-related disease. Therefore, mucosal-related diseases can be prevented or treated by suppressing the expression of a mucin gene having such a polymorphism or by suppressing the function of a mucin glycoprotein having such a polymorphism.

  In addition, in the case of having the G / A heterotype or A / A homotype at the 5492th base of the base sequence shown in SEQ ID NO: 1, since the risk of suffering from mucosa-related diseases is high, mucin having such polymorphism By suppressing the expression of a gene or by suppressing the function of a mucin glycoprotein having such a polymorphism, a mucosa-related disease can be prevented or treated.

  Therefore, the medicament for preventing or treating mucosa-related diseases of the present invention is a compound that suppresses the expression of a mucin gene having a polymorphism that is evaluated to have a high risk of suffering from a mucosa-related disease, or a mucin sugar having the polymorphism. It contains a compound that suppresses the function of the protein.

  When the purpose is to suppress the expression of a mucin gene having a specific polymorphism, a drug that directly suppresses the expression of the gene is designed. Common methods include antisense methods and RNA interference. Alternatively, it is possible to design a medicine so as to suppress the function of mucin glycoprotein. In this case, an antibody against the protein can be used.

  The antisense method is to specifically bind an antisense sequence to a target mucin gene sequence to suppress the expression of the target gene. An antisense sequence is an oligonucleotide that can specifically hybridize to at least a portion of a target sequence. Antisense sequences bind to cellular mRNA or genomic DNA, block translation or transcription, and inhibit target mucin gene expression. As the antisense sequence, any oligonucleotide can be used as long as it blocks the translation or transcription of the target mucin gene, and examples thereof include DNA or RNA. Therefore, the antisense oligonucleotide sequence is designed to be complementary to a partial sequence of the target mucin gene.

  Although the length of the antisense oligonucleotide which should be designed is not specifically limited as long as the expression of a target mucin gene can be suppressed, For example, it is 10-50 bases, Preferably it is 15-25 bases. Oligonucleotides can be easily chemically synthesized by known methods.

  It is also possible to use RNA interference in order to suppress the translation of the mucin gene, which has a high risk of suffering from mucosa-related diseases. Specifically, when a double-stranded RNA complementary to the base sequence of the target mucin gene is introduced into the cell, the mRNA of the gene is degraded, resulting in specific suppression of gene expression in the cell. Will be. This technique has also been confirmed in mammalian cells and the like (Hannon, GJ., Nature (2002) 418,244-251 (review); JP-T 2002-516062; JP-T 8-506734).

  Antisense oligonucleotides and double-stranded RNAs can reach the target location (such as cancer cells) by various administration methods using expression vectors. Administration can be performed by any known method, for example, a method using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system, or a method using various virus vectors including a retrovirus vector or an adeno-associated virus vector. it can.

  Vectors that can be used to deliver antisense oligonucleotides and double-stranded RNA include, but are not limited to, RNA viruses such as adenovirus, herpes virus, vaccinia virus, retrovirus and the like.

  Other gene delivery mechanisms that can be used to administer antisense oligonucleotides and double-stranded RNA to a tissue or cell of interest include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like. For example, delivery systems can utilize macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, and the like. In addition, as the administration form of antisense oligonucleotide and double-stranded RNA, systemic administration such as normal intravenous and intraarterial administration can be performed. The dose varies depending on age, sex, symptom, route of administration, number of doses, and dosage form, but can be determined as appropriate using a method routine in the art.

  When using an antibody, it may be a polyclonal antibody or a monoclonal antibody. Moreover, an antibody fragment (for example, Fab fragment etc.) can be used. The antibody can be prepared by a method known in the art (for example, a method using a hybridoma).

  The dose of the antibody varies depending on age, sex, symptom, administration route, number of administrations, and dosage form, but can be appropriately determined using a method routine in the art.

  In addition, when administering an antibody (parenteral administration), formulation forms such as intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. can be selected. In the case of unit dose ampoules or multi-dose containers.

  On the other hand, when the polymorphism of the repetitive sequence having different lengths in the second exon of the mucin G4 gene is the Ss polymorphism, the risk of suffering from a mucosa-related disease is low, and therefore the expression of the mucin gene having such polymorphism is increased. Or by administering mucin glycoproteins having such polymorphisms, mucosal-related diseases can be prevented or treated.

  Therefore, the medicament for preventing or treating mucosa-related diseases of the present invention is a compound that increases the expression of a mucin gene having a polymorphism that is evaluated to have a low risk of suffering from a mucosa-related disease, or a mucin sugar having the polymorphism. Proteins can be included.

  When the purpose is to increase the expression of a mucin gene having a specific polymorphism, a drug that directly increases the expression of the gene is designed. General methods include the use of a vector (targeting vector) incorporating a mucin gene having a specific polymorphism to be targeted. A targeting vector means a nucleic acid sequence of an expressed gene linked to a promoter sequence.

  One method of increasing gene expression is to increase mucin gene expression by linking a strong promoter to the target mucin gene sequence. First, a targeting vector capable of highly expressing the mucin gene in the host is constructed by linking a promoter capable of functioning in the host upstream of the mucin gene and incorporating it into a vector such as a viral vector. Can be built. Here, “to be linked in a functional manner” means that the promoter and the gene are linked so that the mucin gene is expressed under the control of the promoter in the host into which the mucin gene is introduced. To do. That is, mucin gene expression is increased by the action of a strong promoter.

  Strong promoters that can function in the host include, for example, when the host is a mammal, the Rous sarcoma virus (RSV) promoter, the cytomegalovirus (CMV) promoter, the simian virus (SV40) early or late promoter, Examples thereof include, but are not limited to, mouse papilloma virus (MMTV) promoter and CAG promoter.

  A vector into which a mucin gene and a promoter are incorporated is a vector that can be used in a host, for example, contains genetic information that can be replicated in a host cell, can be propagated autonomously, and can be isolated and purified from the host cell. A vector that is capable and has a detectable marker. Therefore, in addition to the target mucin gene and promoter, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like can be linked to the vector as desired. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like. As examples of vectors, when mammalian cells are used as host cells, plasmids such as pRC / RSV and pRC / CMV (Invitrogen), bovine papillomavirus plasmid pBPV (Amersham Pharmacia), EB virus plasmid pCEP4 (Invitrogen) Examples of such vectors include, but are not limited to, viral vectors such as vaccinia viruses, retroviruses and adenoviruses.

  The targeting vector thus prepared may be directly administered to the subject (in vivo method) or may be introduced into a cell collected from the subject, preferably a stem cell, to express a cell expressing the target mucin gene. The cells may be administered after selection (ex vivo method). Direct administration of the targeting vector can be performed, for example, by intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. Moreover, cell introduction | transduction of a targeting vector can be performed using general gene transfer methods, such as a calcium phosphate method, DEAE dextran method, electroporation method, or lipofection method, for example. Selection of cells expressing the target mucin gene can be performed using a selectable marker, and this method is well known in the art. Administration of cells expressing the mucin gene can also be administered in the same formulation as in the direct administration of the targeting vector.

  The dosage of the targeting vector varies depending on age, sex, symptom, administration route, number of administrations, and dosage form, but can be appropriately determined using a technique routine in this technical field.

  Alternatively, a mucin gene expression product having a specific polymorphism (ie, a mucin glycoprotein) may be directly administered, and in that case, the mucin glycoprotein is obtained in large quantities using conventional recombinant protein production methods. can do. The mucin glycoprotein can be administered in the same manner as the above-described targeting vector formulation, and the dosage varies depending on age, sex, symptom, route of administration, frequency of administration, and dosage form, but is customary in the art. It can be determined appropriately using a typical method.

  The above-mentioned various preparations include excipients, disintegrating agents, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, stabilizers, tonicity agents, etc. Etc. are appropriately selected, and the ratio with the active ingredient is also appropriately adjusted (about 1 to 90%), and can be produced by a conventional method.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[Example 1] Identification and expression of a novel mucin G4 gene Total RNA was extracted from human bronchial epithelial cell line NCI-H292 cell (ATCC accession number CRL-1848) derived from pulmonary mucinous epidermoid carcinoma, and 5 'Race and 3 'Race (First Choice RLM RACE Kit, Ambion) was performed. In order to connect the 5 ′ end fragment and the 3 ′ end fragment of the gene obtained by the RACE method, cDNA was synthesized from total RNA using SuperScript III RT (Invitrogen) using Oligo dT primer, and TaKaRa LA Taq Long PCR using (TaKaRa) was performed to perform full-length cloning of the gene.

  For analysis of protein properties predicted from genes, SOSUI (Classification and Secondary Structure Prediction of Membrane Protein; Internet homepage http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html) and SignalIP (Improved prediction of signal peptides-SignalP 3.0. Jannick Dyrlov Bendtsen, Henrik Nielsen, Gunnar von Heijne and Soren Brunak. JMB: under revision 2004; Internet homepage http://www.cbs.dtu.dk/services/SignalP).

  The total length of the G4 gene obtained from NCI-H292 cells was 6,170 bases, and the gene consisted of 4 exons and encoded a protein of 1,773 amino acids. The base sequence of this G4 gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

  A signal peptide consisting of 27 amino acids was predicted on the N-terminal side of the protein, and a transmembrane site having a length of 23 amino acids was predicted on the C-terminal side. In the region encoded by the second exon (second exon), about 10 amino acids (typically having a pattern of TTTASTEGSE) containing a large amount of serine and threonine were repeated about 150 times. The expected serine content of the entire protein was 18.4%, and the threonine content was 33.6%, for a total of 52%. From the above characteristics, the protein encoded by the G4 gene was predicted to be a transmembrane mucin.

  In order to confirm the expression of the G4 gene in human bronchial epithelium, total RNA was extracted from bronchial epithelial cells NCI-H292, reverse transcription (RT) was performed with random primers, and primers that sandwiched introns (SEQ ID NOs: 9 and 10; PCR was performed according to Table 2). In order to examine the expression in other tissues, G4 gene was similarly detected by PCR using 16 kinds of cDNA derived from human tissues (Human MTC panel I & II, CLONTECH).

  Although mRNA expression of G4 gene was detected in normal human bronchial epithelium, the results using MTC Panel were positive not only in the lung but also in placenta and testicles.

[Example 2] Detection of gene polymorphism in mucin G4 gene In this example, gene polymorphism in the mucin G4 gene was detected, and the relationship with the disease was examined.
(1) Detection of VNTR Since the second exon of the mucin G4 gene, which encodes a protein characteristic of mucin, was thought to contain VNTR having a repeat sequence of about 4 kb in length, diffuse panbronchiolitis ( DPB) The lengths of the second exon were compared using DNA prepared from 92 patients and 100 healthy controls, and the missing sites were identified for those with a short length.

  Specifically, a DNA fragment containing the second exon of about 7 kbp of the mucin G4 gene identified in Example 1 was amplified by PCR using the primer set shown in Table 3, and the PCR product was 0.5% agarose gel. The bands were detected by EtBr staining, and the PCR product lengths were compared.

  Next, the DNA sequences of the second exons having different lengths were analyzed. However, since it is impossible to perform sequencing directly by setting primers inside the exons for repetitive sequences, BamHI sites were added to both ends. Amplification was performed with a primer set (Table 4), digested with restriction enzyme MboI, and cloned into BBlueHI-treated pBlueScriptSK +.

  Each colony plasmid obtained using DH5α was sequenced with T3 primer (SEQ ID NO: 15) and T7 primer (SEQ ID NO: 16). For MboI treatment, if the fragment is too large and the entire sequence of the inserted fragment cannot be obtained using T3 and T7, digest with the restriction enzyme HphI to obtain a fragment of an appropriate size for that site, and TaKaRa BKL Kit The ends were blunted with (TaKaRa) and cloned into pCRScript (Stratagene).

  In the analysis of the length of VNTR of the second exon, the length of the PCR product of about 7 kb was separated into five types on electrophoresis (FIG. 1). In FIG. 1, from the left, lane 1 is a marker, lane 2 is an S1 polymorphism, lane 3: L polymorphism, lane 4: Ms polymorphism, lane 5: M1 polymorphism, lane 6: L polymorphism, lane 7: Ss polymorphism, lane 8: L polymorphism.

  As a result of fragmenting and sequencing four types of PCR products having different lengths from the expected length, 53L9 (GenBank accession number AB023048) showing the full-length sequence of chromosome 6p21.3 registered in the database has a length of the second exon of 4 , 599 bases (corresponding to the L polymorphism), as shown in Table 5, a second exon having a deletion or insertion was observed. Moreover, each allele frequency in DPB and a healthy control was as follows.

  Thus, Ss-type allyl was significantly less in DPB, suggesting that it may be involved in DPB resistance.

  The base sequences of each length of the second exon shown in Table 5 are shown in FIGS. 2 to 6 (SEQ ID NOs: 17 to 21, respectively). In the Ml polymorphism shown in FIG. 3, the part inserted with respect to the base sequence of the 2nd exon of L polymorphism is shown by a small letter.

(2) 1 base and 3 base deletion type polymorphism Next, the polymorphism which exists in VNTR of the 2nd exon of G4 gene was detected. That is, when the VNTR of the second exon of the G4 gene was examined, a 1-base deletion polymorphism and a 3-base deletion polymorphism were found. These mutations are mutations possessed by a part of the above-mentioned “L polymorphism”.

  PCR products amplified using the primer sets (SEQ ID NOs: 11 and 12) shown in Table 3 were directly sequenced. Specifically, the PCR product was purified with ExoSAP, reacted with an ABI cycle sequence kit using the following sequence primers (SEQ ID NOs: 22 and 23; Table 6), purified with Sephadex G-50, and purified with an ABI3100 sequencer. Sequenced.

  7 and 8 show the base sequences of the second exon of the mucin G4 gene in the case of the 1 base deletion polymorphism (SEQ ID NO: 24) and the 3 base deletion polymorphism (SEQ ID NO: 25), respectively.

  The single nucleotide deletion type polymorphism (SEQ ID NO: 24) is the second exon consisting of 4599 bases (L polymorphism; SEQ ID NO: 17). It is a mutation with one deletion.

  The 3-base deletion polymorphism (SEQ ID NO: 25) is a sequence in which the ACC from 3210 to 3224 is repeated 5 times among the second exon (L polymorphism: SEQ ID NO: 17) consisting of 4599 bases (ACC ACC ACC ACC ACC) is a mutation in which ACC is reduced by one and is repeated four times.

  By direct sequencing, in each polymorphic homozygote, the polymorphism can be determined by analyzing the sequence and determining the base sequence of the part. In the heterozygote, PCR is performed using a primer set having the nucleotide sequences shown in SEQ ID NOs: 36 and 37, and a part (about 10 microliters) of the resulting product (about 494 bp) is digested with EcoRI. By performing agarose (3%) electrophoresis and ethidium bromide staining, G type fragments of 320, 138, 24 and 12 bp are obtained, and A type fragments of 320, 150 and 24 bp are obtained. The heterozygote can be determined based on the presence or absence of the band and the band of 150 bp.

These deletion-type polymorphisms have no significant difference in the allele frequency for individual polymorphisms, but have the following relationships when evaluated as genotypes containing both polymorphisms.
(1) 57 patients (62.0%) of DPB patients who have either 1-base deletion type polymorphism or 3-base deletion type polymorphism
41 healthy controls (41.8%)
(2) 35 patients (38.0%) of DPB patients who do not have either 1-base deletion polymorphism or 3-base deletion polymorphism
57 healthy controls (58.2%)

This result has a significant difference of p = 0.0055 by chi-square.
Therefore, these results strongly suggested that this mucin G4 gene is a gene related to DPB resistance and sensitivity.

(3) Single nucleotide polymorphism For the G4 gene, the gene polymorphism was detected using the DNA of 92 DPB patients and 100 healthy controls centering on 4 exons, and the relationship was analyzed.

  As a result, the single nucleotide polymorphism present in the fourth exon of the mucin G4 gene, specifically, the 5492th base (G / A, G404.3) of SEQ ID NO: 1 was most strongly associated with DPB. (A / A + G / A type vs. G / G type with an odds ratio of 2.28 95% CI 1.24-4.18).

  This single nucleotide polymorphism caused an amino acid mutation (asparagine / aspartic acid) on the gene.

  This single nucleotide polymorphism is prepared by preparing DNA from a subject, amplifying the DNA region by PCR, determining the base sequence of the amplified DNA, RFLP (restriction fragment length polymorphism) digested with restriction enzyme EcoRII, etc. (G / A) could be discriminated.

[Example 3] Identification and expression of a novel mucin G2 gene Total RNA was extracted from human bronchial epithelial cells NCI-H292 and subjected to 5 'Race and 3' Race (First Choice RLM RACE Kit, Ambion). In order to connect the 5 ′ end fragment and the 3 ′ end fragment of the gene obtained by the RACE method, cDNA was synthesized from total RNA using SuperScript III RT (Invitrogen) using Oligo dT primer, and TaKaRa LA Taq Long PCR using (TaKaRa) was performed to perform full-length cloning of the gene.

  SOSUI and SignalIP were used for the analysis of protein properties predicted from the gene.

  The total length of the G2 gene obtained from bronchial epithelial cells was about 1,670 bases, and the gene was composed of 4 exons and encoded 251 amino acids. The base sequence of this G2 gene is shown in SEQ ID NO: 26, and the amino acid sequence is shown in SEQ ID NO: 27.

  A signal peptide consisting of 22 amino acids was predicted on the N-terminal side of the protein, and a transmembrane site was not predicted. In the region encoded by the second exon (second exon), 11 amino acids (typically having a pattern of TTTASTEGSE) containing a large amount of serine and threonine were repeated about 14 times. The expected protein content was 8%, the threonine content was 19%, and the proline content was 8%, for a total of 35%. From the above characteristics, the protein encoded by the G2 gene was predicted to be a secretory mucin.

  In order to confirm the expression of the G2 gene in human bronchial epithelium, total RNA was extracted from bronchial epithelial cells NCI-H292, reverse transcription (RT) was performed with random primers, and primers that sandwiched introns (SEQ ID NOs: 34 and 35; PCR was performed in Table 10). Further, in order to examine the expression in other tissues, G2 gene was similarly detected by PCR using 16 kinds of cDNA derived from human tissues (Human MTC panel I & II, CLONTECH).

  Although mRNA expression of the G2 gene was detected in normal human bronchial epithelium, the results using MTC Panel were positive not only in the lung but also in the brain and spleen.

[Example 4] Detection of gene polymorphism in mucin G2 gene Detection of polymorphism existing in the exon region of G2 gene and related analysis revealed that the most strongly related was the 4 bases present in the third exon. Insertion / deletion type polymorphism. That is, 4 bases of “CACT” are deleted or inserted between the 795th base T and the 796th base G of the G2 gene shown in SEQ ID NO: 26. There was a microsatellite (irr) polymorphism in the promoter region of the G2 gene, and alleles 428 and 432 were strongly associated with DPB.

  The diagnostic kit of the present invention makes it possible to diagnose the risk of suffering from a mucosa-related disease. In addition, the preventive or therapeutic drug of the present invention makes it possible to effectively prevent or treat mucosa-related diseases.

  Furthermore, the present invention provides a novel mucin gene and mucin glycoprotein. Mucin glycoprotein is known to play an important role in the living body, and thus is useful for elucidating many in vivo mechanisms including mucosa-related diseases.

It is a figure which shows the photograph which electrophoresed the PCR product with the agarose gel and was dye | stained ethidium bromide. From left to right lane 1: marker, 2: S1, 3: L, 4: Ms, 5: Ml, 6: L, 7: Ss, 8: L It is a figure which shows the base sequence (sequence number 17) of L polymorphism (4599bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 17) of L polymorphism (4599bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 18) of Ml polymorphism (3735bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 18) of Ml polymorphism (3735bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 19) of Ms polymorphism (3549bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 19) of Ms polymorphism (3549bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 20) of S1 polymorphism (3006bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 20) of S1 polymorphism (3006bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 21) of Ss polymorphism (2709bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 21) of Ss polymorphism (2709bp) in a mucin gene 2nd exon. It is a figure which shows the base sequence (sequence number 24) of the 1 base deletion type | mold polymorphism in L polymorphism of the mucin G4 gene 2nd exon. It is a figure which shows the base sequence (sequence number 24) of the 1 base deletion type | mold polymorphism in L polymorphism of the mucin G4 gene 2nd exon. It is a figure which shows the base sequence (sequence number 25) of 3 base deletion type polymorphism in L polymorphism of the mucin G4 gene 2nd exon. It is a figure which shows the base sequence (sequence number 25) of 3 base deletion type polymorphism in L polymorphism of the mucin G4 gene 2nd exon.

  Sequence number 3-16, 22, 23, 28-37: Synthetic oligonucleotide

Claims (34)

The following protein (a) or (b).
(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and a mucin glycoprotein Is a protein A polynucleotide encoding the following protein (a) or (b).
(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and a mucin glycoprotein Is a protein The polynucleotide of any of the following (a) to (c).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (b) hybridizing under stringent conditions with a polynucleotide comprising a sequence complementary to all or part of the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a mucin glycoprotein (c) and hybridizes under stringent conditions with a polynucleotide comprising a sequence complementary to the whole or a part of the base sequence shown in SEQ ID NO: 1 And a polynucleotide having a function as a primer or a probe The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 27 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 27, and a mucin glycoprotein Is a protein A polynucleotide encoding the following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 27 (b) a protein containing a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 27, and a mucin glycoprotein Is a protein The polynucleotide of any of the following (a) to (c).
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 26 (b) hybridizing under stringent conditions with a polynucleotide comprising a sequence complementary to all or part of the nucleotide sequence represented by SEQ ID NO: 26 A polynucleotide encoding soybean mucin glycoprotein (c) and hybridizing under stringent conditions with a polynucleotide comprising a sequence complementary to the whole or a part of the base sequence shown in SEQ ID NO: 26; And a polynucleotide having a function as a primer or a probe   A recombinant vector comprising the polynucleotide according to any one of claims 2, 3, 5 and 6.   A transformant into which the polynucleotide according to any one of claims 2, 3, 5, and 6 or the recombinant vector according to claim 7 has been introduced.   A method for producing a mucin glycoprotein, comprising culturing the transformant according to claim 8 and collecting a protein from the obtained culture.   A kit for diagnosing the risk of suffering from a mucosa-related disease, comprising means for detecting a polymorphism of the mucin G4 gene having the base sequence represented by SEQ ID NO: 1.   The diagnostic kit according to claim 10, wherein the polymorphism is a polymorphism of a repetitive sequence having a different length in the second exon of the mucin G4 gene.   The polymorphisms of repetitive sequences with different lengths are 4599 bp L polymorphism (SEQ ID NO: 17), 3735 bp Ml polymorphism (SEQ ID NO: 18), 3549 bp Ms polymorphism (SEQ ID NO: 19), 3006 bp S1 polymorphism ( The diagnostic kit according to claim 11, wherein the diagnostic kit is selected from the group consisting of SEQ ID NO: 20) and S709 polymorphism (SEQ ID NO: 21) of 2709 bp.   The diagnostic kit according to claim 12, wherein when the subject has an Ss polymorphism, the subject is evaluated as having a low risk of suffering from a mucosa-related disease.   The diagnostic kit according to claim 10, wherein the polymorphism is a 1-base deletion polymorphism or a 3-base deletion polymorphism in the L polymorphism of mucin G4 gene second exon.   The diagnostic kit according to claim 14, wherein the single nucleotide deletion type polymorphism is a deletion of C base at 1827th position or 1838th position of the base sequence shown in SEQ ID NO: 17.   The diagnostic kit according to claim 14, wherein the 3-base deletion type polymorphism is a deletion of one ACC sequence in the 3210th to 3224th ACC 5-repeat sequence of the base sequence represented by SEQ ID NO: 17.   The subject has an L polymorphism of a repetitive sequence having different lengths in the second exon of the mucin G4 gene, and / or either the 1-base deletion polymorphism or the 3-base deletion polymorphism in the L polymorphism The diagnostic kit according to any one of claims 14 to 16, wherein the subject is evaluated as having a high risk of suffering from a mucosa-related disease when having one.   The diagnostic kit according to claim 10, wherein the polymorphism is a single nucleotide polymorphism in the fourth exon of the mucin G4 gene.   The diagnostic kit according to claim 18, wherein the single nucleotide polymorphism is a G / A polymorphism at the 5492th base of the nucleotide sequence represented by SEQ ID NO: 1.   20. The subject is evaluated to have a high risk of suffering from a mucosa-related disease when the 5492st base of the base sequence shown in SEQ ID NO: 1 is G / A heterotype or A / A homotype. Diagnostic kit.   The detection means is an oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 17, 18, 19, 20, 21, 24 and 25 or an oligonucleotide consisting of a partial sequence thereof or a complementary sequence thereof. Item 20. The diagnostic kit according to any one of Items 10 to 20.   Primer comprising a means for detecting polymorphisms of repetitive sequences having different lengths in the second exon of the mucin G4 gene, an oligonucleotide having a base sequence represented by SEQ ID NO: 11 and an oligonucleotide having a base sequence represented by SEQ ID NO: 12 The diagnostic kit according to any one of claims 10 to 21, which is a set.   The detection method for a single nucleotide deletion polymorphism in the L polymorphism of the mucin G4 gene second exon is an oligonucleotide having the base sequence represented by SEQ ID NO: 22. Diagnostic kit.   The detection means for the 3-base deletion polymorphism in the L polymorphism of the mucin G4 gene second exon is an oligonucleotide having the base sequence represented by SEQ ID NO: 23, Diagnostic kit.   A kit for diagnosing the risk of suffering from a mucosa-related disease, comprising means for detecting a polymorphism of the mucin G2 gene represented by SEQ ID NO: 26.   26. The diagnostic kit according to claim 25, wherein the polymorphism is a 4-base insertion / deletion polymorphism in the third exon of the mucin G2 gene.   The diagnostic kit according to any one of claims 10 to 26, wherein the mucosa-related disease is inflammation or malignant tumor.   28. The diagnostic kit according to claim 27, wherein the mucosa-related disease is a disease selected from the group consisting of diffuse panbronchiolitis, chronic bronchitis, cystic fibrosis, bronchiectasis and chronic sinusitis.   The diagnostic kit according to any one of claims 10 to 28, wherein the diagnostic object is an Asian race.   A mucosa-related disease comprising a compound that suppresses the expression of a mucin gene having a polymorphism that is evaluated to have a high risk of suffering from a mucosa-related disease, or a compound that suppresses the function of a mucin glycoprotein having the polymorphism. A preventive or therapeutic drug.   Polymorphisms that are evaluated to have a high risk of afflicting mucosal-related diseases are shown in SEQ ID NO: 1, as well as the 1-base deletion polymorphism and the 3-base deletion polymorphism in the L polymorphism of mucin G4 gene second exon. The preventive or therapeutic drug according to claim 30, which is selected from the group consisting of G / A heterotype and A / A homotype at the 5492th base of the base sequence.   32. The preventive or therapeutic medicament according to claim 30 or 31, wherein the compound is selected from the group consisting of an antisense oligonucleotide, a double-stranded RNA and an antibody.   A compound for increasing the expression of a mucin gene having a polymorphism that is evaluated to have a low risk of suffering from a mucosa-related disease, or a mucosal-related disease for prevention or treatment comprising a mucin glycoprotein having the polymorphism Medicine.   34. The preventive or therapeutic drug according to claim 33, wherein the polymorphism evaluated to have a low risk of suffering from a mucosa-related disease is the Ss polymorphism in the second exon of the mucin G4 gene.

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