JPWO2005056597A1 - Immunoglobulin Fc receptor protein - Google Patents

Abstract

An object of the present invention is to provide a novel immunoglobulin Fc receptor protein. According to the present invention, the following protein (a) or (b) is provided. (A) Protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 in the sequence listing (b) SEQ ID NO: 2, 4, 6, 8, 10, 12 in the sequence listing Or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by 14 and having binding activity to IgA and IgM Fc

Description

  The present invention relates to a novel immunoglobulin Fc receptor protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, a method for producing the protein, and the like.

The binding between immunoglobulin (Ig) and immunoglobulin receptor (IgR) elicits various immune responses and plays an important role in biological defense mechanisms. The FC receptor (FcR) binds to the Fc region of immunoglobulin and is an important factor involved in various immune responses such as allergy, autoimmunity and inflammation. Therefore, the Fc receptor is an important clue in understanding and controlling the molecular mechanism of the immune response. The Fc regions of immunoglobulins bind to specific Fc receptors, respectively, and act on activation of cells having Fc receptors and intercellular transport of antibodies. A number of FcRs for IgG and IgE have been identified so far (DH Conrad, Fc epsilon RII / CD23: the low affinity receptor for IgE, Annu Rev Immunol 8 (1990) 623-645 .; J.P. Kinet, Antibody-cell interactions: Fc receptors, Cell 57 (1989) 351-354 .; JV Ravetech, and JP Kinet, Fc receptors, Annu Rev Immunol 9 (1991) 457-492. C. Unkeless, E. Scigliano, and V. H. Freedman, Structure and function of human and murine receptor for IgG, Annu Rev Immunol 6 (1988) 251-281.), FcR for IgA or IgM is Fcα / μR (A. Shibuya, N. Sakamoto, Y. Shimizu, K. ShiMiwa, K. ShiMib. Hiroyama, HJ Eyre, G. R. Sutherland, Y. Endo, T. Fujita, T. Miyabayashi, S. Sakurano, T. Tsuji, E. Nakayama, J. H. Phillips, L. L. Laner and H. Nakauchi, Fc alpha / mu receptor mediators endocytosis of IgM-coated microbes, Nat Immunol 1 (20 0) 441-446.), PIgR (P. Krajci, D. Kvale, K. Tasken, and P. Brandtzaeg, Molecular cloning and exon-in-the-ramp regenerative ensemble humming Eur J Immunol 22 (1992) 2309-2315 .; JF Piskurich, MH Blankard, KR Youngman, JA France, and C. S. Kaetzel, Molecular cloning of the molecule. acceptor. Functional regions of the molecular area conserved ammonia five million specials, J Immunol 154 (1995) 1735-1747. ), And FcαRI (CD89), which has been identified only in humans (CR Maliszewski, CJ March, MA Schoenborn, S. Gimpel, and L. Shen, Expression cloning of a human Fc receptor Only three types are known: IgA, J Exp Med 172 (1990) 1665-1672. A receptor on a cell that binds IgA or IgM activates the cell, or takes the bound antibody into the cell, transports it through the cell, and releases it from the opposite side of the cell (transcytosis). To control the immune response.
IgM is an antibody that is first formed when a pathogen (antigen) enters the body, and is known to bind so as to cover the pathogen. Recently, it has been clarified that this receptor for IgM is involved in the uptake of pathogens covered with IgM into B lymphocytes (A. Shibuya, N. Sakamoto, Y. Shimizu, K. Shibuya, M. Osawa, T. Hiroyama, H. J. Eyre, GR Sutherland, Y. Endo, T. Fujita, T. Miyabayashi, S. Sakurano, T. Tsuji, E. Nakayama, J. H. Ph. L. L. Lanier, and H. Nakauchi, Fc alpha / mu receptor mediates endocysis of IgM-coated microbes, Nat Immunol 1 (2000) 441-446.). On the other hand, a lot of IgA exists in the intestinal mucosa. The mucosal tissue such as the intestinal tract is the place where antigens first enter the living body, and is the largest immune tissue in which 60 to 70% of peripheral lymphocytes are present. M cells, which are specially differentiated epithelial cells, are present in the mucosa covering these immune tissues represented by the Bayer plate. M cells are important for the immune response as the main route of intestinal antigen sampling, and their artificial manipulation leads to manipulation of the immune response itself. However, details of the development and differentiation of mucosal epithelial cells including M cells and the intestinal immunity mechanism are not clear.
Therefore, exploration of FcR molecules against IgM and IgA and their functional studies lead to the elucidation of the above-described immune response mechanism involving IgM and IgA.
Accordingly, an object of the present invention is to obtain a novel Fc receptor and elucidate the molecular and functional properties of the receptor.

In order to solve the above problems, the present inventors searched a database for genes having homology with Ig domains of known Fc receptors for IgA. As a result, humans and mice identified unknown genes having homology with the receptors. Identified. Furthermore, the inventors have found that the proteins encoded by these genes are expressed exclusively in capillary endothelial cells, bind to IgA and IgM, and are taken up into the cells, thereby completing the present invention.
That is, the present invention includes the following inventions.
(1) The following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 in the sequence listing (b) SEQ ID NO: 2, 4, 6, 8, 10, 12 in the sequence listing Or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by 14 and having binding activity to IgA and IgM Fc (2) (1) A protein comprising the partial amino acid sequence of the protein described in 1.
(3) A fusion protein comprising the protein according to (1) and another peptide.
(4) A gene encoding the protein according to any one of (1) to (3).
(5) A gene comprising the following DNA (c) or (d):
(C) DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 in the sequence listing
(D) DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 in the sequence listing
DNA encoding a protein that hybridizes with DNA comprising a complementary base sequence under stringent conditions and has binding activity to IgA and IgM Fc
(6) A recombinant vector containing the gene according to (4) or (5).
(7) A transformant transformed with the gene according to (4) or (5).
(8) The method for producing a protein according to (1) to (3), wherein the transformant according to (7) is cultured in a medium, and the protein expressed from the obtained culture is collected. .
(9) An antibody that specifically recognizes the protein according to any one of (1) to (3).
(10) The antibody according to (9) is reacted with a test sample that is predicted to contain the protein according to any one of (1) to (3), and an immune complex of the antibody and the protein The method for detecting a protein according to any one of (1) to (3), comprising detecting production.
(11) A reagent for detecting a protein according to any one of (1) to (3), comprising the antibody according to (9).
(12) A pharmaceutical composition for controlling immune response comprising the protein according to any one of (1) to (3) or a gene encoding the protein as an active ingredient.
(13) The protein according to any one of (1) to (3) or a cell that expresses the protein is allowed to act on a test sample that is expected to contain a ligand, and has a binding ability to the protein. A method for screening a ligand for the protein, comprising selecting a substance.
(14) The substance according to any one of (1) to (3) is allowed to act on IgM or IgA in the presence of a test sample, and a substance that promotes or inhibits the binding between the protein and IgM or IgA is selected. A method for screening an antagonist or agonist for the protein.
(15) A pharmaceutical composition for controlling immune response comprising a ligand obtained by the method according to (13) or an antagonist or agonist obtained by the method according to (14) as an active ingredient.

FIG. 1 shows the amino acid sequences of human and mouse eIgR (black shaded portion: homologous amino acid, region surrounded by dotted box: transmembrane domain, region surrounded by thin line box is a portion missing due to alternative splicing) .
FIG. 2 shows a schematic diagram of human and mouse eIgR (bars: portions missing due to alternative splicing, Δ numbers: amino acids in the missing portion relative to L, TM: transmembrane domain).
FIG. 3 shows the state of uptake of various immunoglobulins in HeLa cells expressing eIgR [A to E: HeLa cells expressing mouse eIgR-S; F to H, I to J: human eIgR-L and HeLa cells expressing human eIgR-M. Upper row: stained images of various incorporated immunoglobulins (green, AJ), middle row: stained images of expressed eIgR (red, A′-J ′), lower row: superimposed images (A ″ -J) ″)].
FIG. 4 shows immunostained images (green) of eIgR in mouse myocardium (A: 200 times, B: 400 times, A ′ and B ′: superposed images of nuclear stained images (blue), arrows: capillaries Thicker blood vessels).
FIG. 5 shows the structure of the eIgR-Fc fusion protein expression vector pcDNA3-Fc.
FIG. 6 shows immunostained images of HeLa cells using the prepared rat monoclonal antibody and rabbit antiserum [A: control HeLa cells, BD: HeLa cells expressing mouse eIgR-S. C and D are double-stained images of rabbit antiserum and rat monoclonal antibody. Photographed with a confocal microscope (A, B; 400 times, C, D; 630 times)].
Hereinafter, the present invention will be described in detail. This application claims the priority of the Japan patent application 2003-410136 for which it applied on December 9, 2003, and includes the content described in the specification and / or drawing of this patent application.
1. Fc receptor protein and gene encoding it
The Fc receptor protein of the present invention is a novel Fc receptor protein that specifically binds to IgA and IgM, and is expressed exclusively in capillary endothelial cells. Therefore, the Fc receptor protein was named endogenous immunoglobulin receptor (eIgR). .
The gene encoding the Fc receptor protein identified in the present invention includes four types of human-derived genes (h-eIgR-L, h-eIgR-M, h-eIgR-Sα, h) generated by alternative splicing. -EIgR-Sβ) and three types of genes derived from mice (m-eIgR-L, m-eIgR-M, m-eIgR-S). The cDNA base sequences of h-eIgR-L, h-eIgR-M, h-eIgR-Sα, and h-eIgR-Sβ are represented by SEQ ID NOs: 1, 3, 5, and 7, respectively, and the amino acid sequences encoded by these cDNAs Are shown in SEQ ID NOs: 2, 4, 6 and 8, respectively. Further, the base sequences of cDNAs of m-eIgR-L, m-eIgR-M, and m-eIgR-S are respectively shown in SEQ ID NOs: 9, 11, and 13, and the amino acid sequences encoded by these cDNAs are shown in SEQ ID NO: 10 respectively. , 12 and 14.
The Fc receptor protein of the present invention is expressed in capillary endothelial cells of various tissues and organs, and is involved in the transfer of IgM and IgA antibodies from blood to tissue.
Therefore, a substance that binds to the Fc receptor protein of the present invention and inhibits the function of the protein, for example, an antagonist, a specific antibody that binds to the Fc receptor protein and inhibits its function, can suppress the immune response. it can. Further, a substance that binds to the Fc receptor protein of the present invention and promotes the function of the protein, such as a ligand, an agonist, or a specific antibody that binds to and promotes the function of the Fc receptor protein has an effect on the tissue. It can function as a carrier for drug delivery to be transferred.
In the present invention, the “ligand” refers to a substance that binds to the Fc receptor protein of the present invention. In the present invention, “agonist” refers to a substance that can bind to and activate the Fc receptor protein of the present invention, and “antagonist” refers to a ligand for the Fc receptor protein of the present invention. Or a substance that inhibits the action of an agonist. These ligands, agonists and antagonists include both naturally-derived and non-naturally-occurring substances.
The gene encoding the Fc receptor protein of the present invention is screened as a candidate gene by performing a homology search by BLAST using a known Fc receptor for IgA, as shown in Examples below. It can be obtained by receiving (or purchasing) the relevant EST clone.
The gene encoding the Fc receptor protein of the present invention can also be obtained by screening a cDNA library derived from the following cells and tissues using a DNA probe synthesized based on the gene fragment obtained by the above method. It can be isolated. The mRNA supply source for preparing the cDNA library is not particularly limited as long as it is a cell expressing Fc receptor mRNA, and is not limited to humans or other mammals (eg, mouse, rat, guinea pig, rabbit, Any capillary endothelial cells of pigs, sheep, cows, monkeys, etc.) or any tissue in which those cells are present (eg heart, kidney, liver, etc.).
Preparation of mRNA can be performed by a method usually used in the art. For example, the above cells or tissues are treated with a guanidine reagent, a phenol reagent or the like to obtain total RNA, and then by an affinity column method using an oligo (dT) cellulose column or poly U-sepharose using Sepharose 2B as a carrier. Alternatively, poly (A) + RNA (mRNA) is obtained by a batch method. Furthermore, poly (A +) RNA may be further fractionated by sucrose density gradient centrifugation or the like.
Next, using the obtained mRNA as a template, single-stranded cDNA was synthesized using oligo dT primer and reverse transcriptase, and double-stranded using DNA synthase I, DNA ligase, RnaseH, etc. from the single-stranded cDNA. Synthesize cDNA. The synthesized double-stranded cDNA is smoothed with T4 DNA synthase, then ligated with an adapter (for example, EcoRI adapter), phosphorylated, etc., incorporated into λ phage such as λgt11 and packaged in vivo to prepare a cDNA library. Make it. In addition to λ phage, a cDNA library can be prepared using a plasmid vector.
As a screening method for selecting a strain (positive clone) having a target DNA from a cDNA library, for example, sense corresponding to the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 Examples include a method of synthesizing a primer and an antisense primer and using them to perform a polymerase chain reaction (PCR).
As a template DNA for PCR reaction, cDNA synthesized from the mRNA by reverse transcription reaction may be used. In addition, the primer can be designed based on the above amino acid sequence information while appropriately examining the expected size of the DNA fragment when amplified or the combination of degenerate codons.
The amplified DNA fragment thus obtained is labeled with 32P, 35S, biotin or the like to be used as a probe, and this is hybridized with a nitrocellulose filter to which the transformant DNA is denatured and immobilized to search for positive clones.
The base sequence of the obtained positive clone is determined. 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 (eg, 373A DNA manufactured by PERKIN-ELMER) is used. (Sequencer, TAKARA BcaBEST dideoxy sequencing kit, etc.). The determined base sequence is analyzed by DNA analysis software such as DNASIS (Hitachi Software Engineering Co., Ltd.), and the protein coding portion encoded in the obtained DNA strand can be found.
The Fc receptor protein of the present invention comprises (a) a protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 in the sequence listing, and (b) SEQ ID NO: 2 in the sequence listing. Has an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by 4, 6, 8, 10, 12, or 14, and against IgA and IgM Fc It is a protein having binding activity.
“1 to several” in the above “amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14” Although the range is not particularly limited, for example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, particularly preferably about 1 to 3.
The amino acid deletion, addition and substitution can be performed by modifying the gene encoding the Fc receptor protein by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit (for example, Mutant-K) using site-directed mutagenesis. (Manufactured by TAKARA) or Mutant-G (manufactured by TAKARA)) or the like, or a TAKARA LA PCR in vitro Mutagenesis series kit is used to introduce a mutation.
The above “having binding activity to IgA and IgM Fc” means that the binding activity of IgA and IgM to Fc is the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. It is substantially equivalent to the activity of a protein having
More specifically, as long as the activity is equivalent (for example, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times) The element may be different from the original protein.
The Fc receptor protein of the present invention includes a protein that is functionally equivalent to the Fc receptor protein and has homology with the amino acid sequence of the protein. The protein having homology is about 70% or more, preferably about 80% or more, more preferably about 90% or more with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. Most preferably, it means a protein having a homology of about 95% or more. To determine protein homology, the algorithm described in the literature (Wilbur, WJ and Lipman, DJ, Proc. Natl. Acad., Sci. USA (1983) 80, 726-730) is used. Just follow.
The Fc receptor protein of the present invention includes a protein (partial peptide) that is functionally equivalent to the Fc receptor protein and has a partial sequence of the amino acid sequence of the protein.
The Fc receptor protein of the present invention includes a fusion protein in which the protein is fused with another peptide or protein. A method for producing a fusion protein is a method in which DNA encoding an Fc receptor protein and DNA encoding another peptide or protein are ligated so that the frame matches and introduced into an expression vector and expressed in a host. Any known method can be used. Other peptides or proteins to be subjected to fusion are not particularly limited. For example, peptides include FLAG, 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, T7-tag, HSV-tag, E-tag, etc. Known peptides are mentioned. Examples of the protein include GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein), GFP (green fluorescent protein) and the like.
If necessary, the Fc receptor protein of the present invention may be in the form of a salt, preferably a physiologically acceptable acid addition salt. Such salts include salts of inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, Citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
The Fc receptor protein of the present invention can be obtained by extracting or separating from cultured cells or tissues of humans or mammals expressing the protein, or cultivating a transformant containing DNA encoding the protein as described below. Can also be manufactured. When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and extracted with acid or the like, and the resulting extract is subjected to hydrophobic chromatography, reverse phase chromatography, ion exchange chromatography. Isolation and purification can be achieved by combining various types of chromatography such as chromatography.
The partial peptide can be produced by a known peptide synthesis method or by cleaving the Fc receptor protein with an appropriate peptidase (eg, trypsin, chymotrypsin, arginyl endopeptidase). As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used.
The gene encoding the Fc receptor protein of the present invention may be any gene as long as it encodes the protein of the present invention described above. Specifically, (a) SEQ ID NOs: 1, 3, 5, DNA consisting of the base sequence shown in 7, 9, 11, or 13; (b) a base complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 in the sequence listing Examples thereof include a gene comprising a DNA that hybridizes with a DNA comprising a sequence under stringent conditions and that encodes a protein having a binding activity to IgA and IgM Fc.
As described above, “DNA capable of hybridizing under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13” About 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology with the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. And DNA having a base sequence having a property. Here, the stringent condition refers to, for example, a condition where the sodium concentration is 600 to 900 mM and the temperature is 60 to 68 ° C., preferably 65 ° C.
Once the base sequence of the gene of the present invention has been determined, the subsequent sequence can be obtained by chemical synthesis, by PCR using the cDNA of the present gene as a template, or by hybridization with a DNA fragment having the base sequence as a probe. The gene of the invention can be obtained.
2. Production of recombinant vectors and transformants
(1) Production of recombinant vector
The recombinant vector of the present invention can be obtained by ligating the gene of the present invention to an appropriate vector. The vector for inserting the gene of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA. As plasmid DNA, plasmids derived from E. coli (eg, pRSET, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.), and examples of phage DNA include λ phage (Charon 4A, Charon 21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
In order to insert the gene of the present invention into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Adopted.
The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the vector of the present invention contains a promoter, the gene of the present invention, and optionally 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. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
Examples of such a vector include a pET vector (manufactured by Novagen), a pTrxFUS vector (manufactured by Invitrogen), a pCYB vector (manufactured by NEW ENGLAMD Bio Labs), etc. In the case of pESP-1 expression vector (manufactured by STRATAGENE), pAUR123 vector (manufactured by Takara Shuzo), pPIC vector (manufactured by Invitrogen) and the like, and in the case where the host cell is an animal cell, for example, pMAM- When the neo expression vector (manufactured by CLONTECH), pCDNA3.1 vector (manufactured by Invitrogen), pBK-CMV vector (manufactured by STRATAGENE) or the like is an insect cell, for example, the pBacPAK vector (C ONTECH Inc.), manufactured by pAcUW31 vector (CLONTECH Inc.), PACP (+) manufactured by IE1 vector (Novagen Co., Ltd.), respectively.
(2) Production of transformant
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia such as Escherichia coli, Bacillus subtilis such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas genus, Rhizobium meriroti such as Rhizobium meriroti Yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, human G cells, human G cells, FL cells Animal cells; or insect cells such as Sf9 and Sf21.
When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and at the same time is composed of a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Moreover, the gene which controls a promoter may be contained.
Examples of Escherichia coli include Escherichia coli K12 and DH1, and examples of Bacillus subtilis include Bacillus subtilis. Any promoter may be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as trp promoter, lac promoter, PL promoter, PR promoter, etc. are used. An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium anion (Cohen, SN et al. (1972) Proc. Natl. Acad. Sci., USA 69, 2110-2114), electroporation method and the like.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. Is mentioned. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, electroporation (Becker, DM et al. (1990) Methods. Enzymol., 194, 182-187), spheroplast method (Hinnen, A. et al. (1978) Proc. Natl. Acad. Sci., USA 75, 1929-1933), lithium acetate method (Itoh, H. (1983) J. Bacteriol.153,163-168) and the like.
When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human HeLa, FL cells and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like may be used, and human cytomegalovirus early gene promoter and the like may be used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.
Moreover, gene transfer into each of the above host cells can be performed by a method not using a recombinant vector, such as a particle gun method.
3. Production of Fc receptor protein of the present invention
The Fc receptor protein of the present invention can be obtained by culturing the transformant and collecting it from the culture. “Culture” means any of cultured cells, cultured cells, or disrupted cells or cells in addition to the culture supernatant.
The method of culturing the transformant of the present invention in a medium can be carried out according to a usual method used for culturing the host cell.
As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Any carbon source may be used as long as the organism can assimilate, and carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.
The culture is usually performed at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is maintained at 7.0 to 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the Lac promoter, a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter is cultured. Sometimes indole acetic acid (IAA) or the like may be added to the medium.
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. Culture is usually 5% CO ₂ 1-30 days at 37 ° C. in the presence. During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.
When the protein of the present invention is produced in cells or cells after culturing, the protein is extracted by disrupting the cells or cells. When the protein of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or 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, From the above, the protein of the present invention can be isolated and purified.
4). Antibody to Fc receptor protein of the present invention
The antibody of the present invention can be obtained by the following general antibody preparation method.
(1) Preparation of antigen
In the present invention, the Fc receptor protein of the present invention isolated or purified as described above or a partial fragment thereof is used as an antigen.
(2) Preparation of polyclonal antibody
Animals are immunized with the antigen prepared as described above. In the case of rabbits, the dose of antigen per animal is, for example, 100 to 500 μg using an adjuvant. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
Immunization is performed by administering to a mammal (eg, rat, mouse, rabbit, etc.). The site of administration is intravenous, subcutaneous or intraperitoneal. Further, the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 3 times at intervals of several days to several weeks, preferably at intervals of 2-3 weeks. Then, the antibody titer is measured 6 to 60 days after the final immunization day, and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. The antibody titer can be measured by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or the like.
When purification of antibody from antiserum is required, it should be purified by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof. Can do.
(3) Production of monoclonal antibodies
(3-1) Collection of immune and antibody-producing cells
Animals are immunized with the antigenic protein prepared as described above. If necessary, in order to effectively immunize, an adjuvant (commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, etc.) may be mixed as described above.
Immunization is performed by administering to a mammal (eg, rat, mouse, rabbit, etc.). One dose of antigen is 50 μg per mouse in the case of mice. The administration site is mainly intravenous, subcutaneous and intraperitoneal. Further, the immunization interval is not particularly limited, and is performed at least 2-3 times at intervals of several days to several weeks, preferably 2-3 weeks. Then, antibody-producing cells are collected after the final immunization. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells being preferred.
(3-2) Cell fusion
In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As myeloma cells to be fused with antibody-producing cells, cell lines derived from animals such as mice and generally available can be used. As a cell line to be used, it has drug selectivity and cannot survive in an unfused state in a HAT selection medium (including hypoxanthine, aminopterin and thymidine), but can only survive in a state fused with antibody-producing cells. What has is preferable. For example, specific examples of myeloma cells include P3X63-Ag. 8). Examples include mouse myeloma cell lines such as U1 (P3U1), P3 / NSI / 1-Ag4-1, Sp2 / 0-Ag14.
Next, the myeloma cell and the antibody-producing cell are fused. In cell fusion, antibody-producing cells and myeloma cells are mixed at a ratio of 15: 1 to 25: 1 in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and cells such as polyethylene glycol are used. The fusion reaction is performed in the presence of a fusion promoter or by electric pulse treatment (for example, electroporation).
(3-3) Selection and cloning of hybridomas
The target hybridoma is selected from the cells after cell fusion treatment. For example, cells can be cultured using a medium containing hypoxanthine, aminopterin and thymidine, and the growing cells can be obtained as hybridomas.
Next, it is screened whether the target antibody exists in the culture supernatant of the grown hybridoma. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme-linked immunosorbent assay (ELISA), RIA (radioimmuno assay), or the like. Cloning of the fused cells is performed by a limiting dilution method or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established.
(3-4) Collection of monoclonal antibody
As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method or the like can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum or MEM medium (for example, 37 ° C., 5% CO 2). ₂ The antibody is obtained from the culture supernatant.
In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, gel chromatography or the like is appropriately selected, or these It can be purified by a combination of methods.
5). Method for detecting Fc receptor protein using antibody of the present invention, detection reagent
Since the antibody of the present invention reacts with the Fc receptor protein of the present invention or a partial fragment thereof, it can be used as a reagent for detecting the receptor protein. The method for detecting the Fc receptor protein is not particularly limited, and for example, Western blotting can be employed. For example, a test sample (cell component or each fraction thereof) is fractionated by electrophoresis etc., and then reacted with the antibody of the present invention that has been previously labeled (radiolabel, fluorescent staining, etc.) to detect a signal. . The antibody used for detecting the Fc receptor protein of the present invention may be an antibody against a protein having the full-length amino acid sequence of the receptor protein, or an antibody against a peptide having a partial amino acid sequence of the protein.
Quantification of the Fc receptor protein using the antibody of the present invention can be performed, for example, by immunoblotting, enzyme antibody method (EIA), radioimmunoassay (RIA), fluorescent antibody method, immune cell staining, and the like. There are, but are not limited to them.
In addition, the antibody may be a fragment thereof, and specifically includes a single chain antibody fragment (scFv) of the antibody.
Specifically, in the case of the ELISA method, it is performed as follows. First, after a diluted sample such as blood is adsorbed on a 96-well microplate, the antibody of the present invention is reacted as a primary antibody. Subsequently, an antiglobulin antibody labeled with a specific enzyme such as POD (peroxidase) necessary for the color development reaction is reacted, and after washing, ABTS2,2′-azino-di- (3-ethyl-benzothiazoline-6 as a color development substrate -Sulphonic acid) or the like is added for color development, and the Fc receptor protein of the present invention in the sample is detected by measuring by a colorimetric method. Alternatively, in the case of the sandwich ELISA method, it is performed as follows. First, a diluted sample such as blood is added to a 96-well microplate to which the antibody of the present invention has been adsorbed in advance, and incubated for a predetermined time. Thereafter, the plate is washed, purified antibody labeled with biotin is added to each well and incubated for a certain period of time, and then the plate is washed, and enzyme-labeled avidin is added and further incubated. After incubation, the plate is washed, color is developed by adding orthophenylenediamine or the like as a color developing substrate, and the colorimetric method is used for measurement.
In addition, the above Fc receptor protein detection reagent can be combined with other reagents and used in an Fc receptor protein detection kit. The kit only needs to contain at least the antibody of the present invention. When the antibody is immobilized on a solid phase, the antibody has an antigen recognition site different from that of the antibody and includes an antibody used as a secondary antibody. You may go out. The antibody used as the secondary antibody may be labeled with an enzyme, for example, and may contain various reagents (for example, an enzyme substrate, a buffer solution, a diluent, etc.) in addition to these two antibodies.
6). Method for screening ligand for Fc receptor protein of the present invention
Screening for a ligand for the Fc receptor protein of the present invention allows the Fc receptor protein of the present invention or a cell expressing the protein to act on a test sample that is expected to contain the ligand, and binds to the protein. This can be done by selecting a substance having a function. As a specific method for selecting a substance having binding ability, a test sample is allowed to act on the Fc receptor protein of the present invention or a cell expressing the receptor protein, and the binding amount of the test sample to the receptor protein is measured. It is done by doing. A substance having a large amount of binding in the measurement can be selected as a candidate substance for a ligand for the receptor protein of the present invention.
Any substance can be used as the test sample, and the type thereof is not particularly limited. Specific examples of test samples include peptides, proteins, non-peptide compounds, synthetic compounds, natural product extracts (plant extracts, animal tissues / animal cell extracts), compound libraries, phage display libraries, or combinatorials. It may be a library. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.
Since the ligand obtained by this screening method can bind to the Fc receptor protein expressed in the tissue, for example, a carrier for drug delivery that allows the immunosuppressant, antiallergic agent, and anti-inflammatory agent to efficiently reach the tissue Can be used as Alternatively, by reacting with Fc receptor protein in the presence of a test sample, it can also be used for screening for agonists or antagonists to Fc receptor protein (see next section 7).
7). Screening system for agonist or antagonist for Fc receptor protein of the present invention
The Fc receptor protein of the present invention comprises a substance (antagonist) that inhibits the binding of the receptor protein to the ligand IgM or IgA, and a substance that binds to the receptor and causes an immune response similar to the ligand (agonist). It is useful as a means for screening (screening method, screening kit).
The method for screening an agonist or antagonist for the Fc receptor protein of the present invention comprises: (a) a case where an Fc receptor protein is allowed to act on IgM or IgA in the absence of a test sample; and (b) an Fc receptor protein which is IgM. Alternatively, the substance is characterized by selecting a substance that affects the binding between the Fc receptor protein and the IgM or IgA by comparing with the case where IgA is allowed to act in the presence of a test sample. If a test sample is found in which the amount of Fc receptor protein binding to IgM or IgA decreases or increases when the test sample is added, the test sample inhibits or antagonizes the binding of the protein of the present invention to IgM or IgA. Therefore, it becomes a candidate substance for an antagonist or an agonist useful for controlling the immune response.
The Fc receptor protein of the present invention can also be used in a screening kit for agonists or antagonists to the protein. The kit only needs to contain at least the Fc receptor protein of the present invention, and may contain a labeled ligand, a ligand standard solution, and various reagents (for example, a buffer solution, a washing solution, and a diluent).
Any substance can be used as the test sample, and the kind thereof is not particularly limited, and is the same as that mentioned in the previous section.
8). Medicament for controlling immune response of the present invention
The Fc receptor protein eIgR of the present invention is expressed in capillary endothelial cells of various tissues and organs, and is involved in the transfer of IgM and IgA antibodies from blood to tissue. Moreover, the ligand, antagonist, and agonist obtained by the above method can bind to the Fc receptor protein of the present invention and have a function of promoting or inhibiting the function of the receptor. Therefore, any of the Fc receptor proteins, genes, ligands, antagonists, and agonists of the present invention can be used as pharmaceuticals for controlling immune responses.
For example, when the medicament of the present invention is used for a disease caused by a failure of the immune response control mechanism, the disease can be treated by controlling the transfer of IgM and IgA antibodies to tissues. Examples of such diseases include autoimmune diseases (eg, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, dermatomyositis, Sjogren's syndrome, Behcet's disease, ankylosing spondylitis, insulin dependence) Diabetes mellitus, pernicious anemia, etc.), tumor (stomach cancer, colon cancer, breast cancer, lung cancer, esophageal cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, skin cancer, uterine cancer, brain tumor, osteosarcoma, bone marrow tumor, etc.), immunity Deficiency diseases (primary immune deficiency syndrome, secondary immune deficiency syndrome, etc.), inflammatory diseases [inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, arthritis, uveitis, SIRS (systemic inflammatory response syndrome) Etc.], allergic diseases (bronchial asthma attack, atopic dermatitis, allergic rhinitis, hay fever, urticaria, etc.), etc., but are not limited thereto.
The medicament of the present invention can be prepared in various preparation forms and administered systemically or locally orally or parenterally. When the medicament of the present invention is orally administered, it is formulated into tablets, capsules, granules, powders, pills, liquids for internal use, suspensions, emulsions, syrups, etc., or redissolved when used. It may be a dry product. When the pharmaceutical of the present invention is administered parenterally, it is formulated into an intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. Are provided in unit dose ampoules or in multi-dose containers.
These various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, A flavoring agent, a soothing agent, a stabilizer, a tonicity agent and the like can be appropriately selected and produced by a conventional method.
The above various preparations may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Examples include gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. The additive to be used is appropriately or in combination selected from the above depending on the dosage form.
The dosage of the pharmaceutical agent of the present invention varies depending on the age of the administration subject, administration route, and number of administrations, and can vary widely. For example, an effective amount administered as a combination of an effective amount of the protein of the present invention with an appropriate diluent and a pharmacologically usable carrier is a dose in the range of 0.01 mg to 1000 mg per kg body weight. It can be selected from 1 to several times a day and administered for 1 day or more.
When the gene of the present invention is used as a gene therapy agent for immune system diseases, in addition to the method of directly administering the gene of the present invention by injection, a method of administering a vector in which the gene is incorporated can be mentioned. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, and the like, and can be efficiently administered by using these virus vectors. Alternatively, a method of introducing the gene of the present invention into a phospholipid vesicle such as a liposome and administering the liposome may be employed.
As a mode of administration of the gene therapy agent, local administration can be performed to the immune system tissue (bone marrow, lymph node, etc.) in addition to the usual systemic administration such as intravenous and intraarterial administration. Furthermore, administration forms combined with catheter techniques, surgical operations and the like can also be employed. The dosage of the gene therapeutic agent varies depending on age, sex, symptoms, administration route, number of administrations, and dosage form, but is usually in the range of 0.1 to 100 mg / body weight per day for an adult when the weight of the gene of the present invention is used. Is appropriate.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

塩基配列は、ＡＢＩ ＰＲＩＳＭ３１００Ａｖａｎｔシーケンサーを用いて決定した。ｈ−ｅＩｇＲ−Ｌ、ｈ−ｅＩｇＲ−Ｍ、ｈ−ｅＩｇＲ−Ｓα、ｈ−ｅＩｇＲ−Ｓβの塩基配列をそれぞれ配列番号１，３，５，７に、また対応するアミノ酸配列をそれぞれ配列番号２，４，６，８に示す。ｍ−ｅＩｇＲ−Ｌ、ｍ−ｅＩｇＲ−Ｍ、ｍ−ｅＩｇＲ−Ｓの塩基配列をそれぞれ配列番号９，１１，１３に、また対応するアミノ酸配列をそれぞれ配列番号１０，１２，１４に示す。Identification of a novel Fc receptor gene For the purpose of isolating a new Fc receptor gene that binds to IgA / IgM, the BLAST program (http: //www.ncbi.nlm.nih.n) of National Center for Biotechnology Information (NCBI). The sequence of immunoglobulin (Ig) domains with high homology between multimeric Ig receptor (pIgR) and Fcα / μ receptor (Fcα / μR), known Fc receptors for IgA, by gov / BLAST /) Based on the above, human and mouse cDNA databases were searched.
As a result, a candidate gene for a new Fc receptor gene having about 36% homology with a known Fc receptor gene was identified in humans and mice.
From the analysis results of the gene expression mode described later, it was found that the identified gene was expressed exclusively in the capillary endothelial cells. Therefore, the protein encoded by the gene was named endogenous immunoglobulin receptor (eIgR).
In addition, analysis of EST, cDNA, and genomic databases revealed that the identified genes have four types of gene products with different lengths generated by alternative splicing in humans and three types in mice.
FIG. 1 shows a comparison of amino acid sequences of human eIgR (h-eIgR-L) and mouse eIgR (m-eIgR-L). Amino acid sequence homology between human and mouse was about 51%.
FIG. 2 shows a schematic diagram of human eIgR (h-eIgR) and mouse eIgR (m-eIgR). Among gene products with different lengths generated by alternative splicing, L represents a long-type gene product, M represents a middle-type gene product, and S represents a short-type gene product. Human eIgR-S was diversified into two types, Sα and Sβ, with C-terminal diversification (C-terminal (−296) splicing variant).
EST clones (IMAGE Consortium) from which the identified Fc receptor genes are derived are summarized in the table below.

The nucleotide sequence was determined using an ABI PRISM 3100 Avant sequencer. The base sequences of h-eIgR-L, h-eIgR-M, h-eIgR-Sα, and h-eIgR-Sβ are respectively SEQ ID NO: 1, 3, 5, and 7, and the corresponding amino acid sequences are SEQ ID NO: 2, respectively. Shown in 4, 6, and 8. The base sequences of m-eIgR-L, m-eIgR-M, and m-eIgR-S are shown in SEQ ID NOs: 9, 11, and 13, respectively, and the corresponding amino acid sequences are shown in SEQ ID NOs: 10, 12, and 14, respectively.

ｍ−ｅＩｇＲ−Ｓ ｃＤＮＡ用ＰＣＲプライマー：

カバーグラス上に培養したＨｅＬａ細胞にＦｕＧＥＮＥ ６ ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔ（Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）を用いてヒト及びマウスのｅＩｇＲ ｃＤＮＡ発現ベクターを導入した。４８時間後に各種Ｉｇ［ヒトＩｇＡ１（λ）及びヒトＩｇＡ２（κ）（Ａｔｈｅｎｓ Ｒｅｓｅａｒｃｈ ＆ Ｔｅｃｈｎｏｌｏｇｙ）、マウスＩｇＡ（λ）及びマウスＩｇＭ（κ）（ＩＣＮ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ．）、マウスＩｇＧ（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）］を含む培地に交換してさらに２時間４℃又は３７℃で培養した。
ＰＢＳで洗浄後、細胞を４％パラフォルムアルデヒドで固定し、０．１％Ｔｒｉｔｏｎ Ｘ−１００で膜を透過させ、ＦＩＴＣ標識した抗イムノグロブリンサブタイプ抗体［抗ヒトＩｇＡ抗体，抗マウスＩｇＡ抗体，抗マウスＩｇＭ抗体（いずれもＩＣＮ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ．），抗マウスＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）］と反応させた。同時に、ラット抗ＨＡ抗体（３Ｆ１０：Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）及びＣｙ３標識抗ラットＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）を段階的に反応させることで発現したｅＩｇＲを検出した。観察は共焦点顕微鏡（ＴＣＳ−ＳＰ２，Ｌｅｉｃａ）を用いて行った。図３のＡ〜Ｅは、マウスｅＩｇＲ−Ｓ（ｍ−ｅＩｇＲ−Ｓ）を発現させたＨｅＬａ細胞、図３のＦ〜Ｈ及びＩ〜Ｊは、それぞれヒトｅＩｇＲ−Ｌ（ｈ−ｅＩｇＲ−Ｌ）およびヒトｅＩｇＲ−Ｍ（ｈ−ｅＩｇＲ−Ｍ）を発現させたＨｅＬａ細胞における各種免疫グロブリンの取りこみの様子を共焦点顕微鏡により撮影した写真（６３０倍）を示す。各種イムノグロブリンの取込みは３７℃、１時間にて行った。図３に示すように、マウスｅＩｇＲ発現細胞（Ａ〜Ｅ）、ヒトｅＩｇＲ発現細胞（Ｆ〜Ｊ）とも、ヒトＩｇＡ１、ヒトＩｇＡ２、マウスＩｇＡ、マウスＩｇＭと結合し、細胞内に取り込むが、マウスＩｇＧとは結合が認められなかった。
（３）免疫組織染色（ｅＩｇＲの発現部位の同定）
マウス組織を１０％ホルマリン又は１％硫酸亜鉛／１０％ホルマリン溶液にて固定後、パラフィン包埋して病理切片を作成し、（１）で調製した抗マウスｅＩｇＲ抗血清と反応させた。抗原特異的な結合は、ビオチン標識抗ウサギＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）、ヒドロペルオキジダーゼ標識ストレプトアビジン、及びＦＩＴＣ標識チラミド（いずれもＰｅｒｋｉｎ Ｅｌｍｅｒ）を段階的に反応させることで検出した。
図４に心筋の免疫染色像を示す。毛細血管内皮細胞にのみ特異的に発現が認められ、それより太い細血管以上の血管内皮細胞には発現は認められなかった（図中矢印）。
同様の毛細血管内皮細胞特異的なｅＩｇＲの発現が舌及び肝臓の組織切片による免疫染色においても認められた。
以上の実験結果から、新たに同定したｅＩｇＲは毛細血管内皮細胞に特異的に発現し、血中のＩｇＭやＩｇＡをトランスサイトーシスによって経毛細血管内皮的に組織に移行させることにより、免疫応答の制御に役割を果たしている可能性が示唆された。Analysis of novel Fc receptor protein (eIgR) (1) Preparation of antibody (preparation of polyclonal antibody)
A peptide antibody (antiserum) against the cytoplasmic region of human eIgR was obtained by combining the peptide MPPLHTSEEEELGFSKFVSA (SEQ ID NO: 16) conjugated with scab shell hemocyanin (KLH: SEQ ID NO: 15) in two rabbits (rabbit, New Zealand white, rabbit). Obtained by subcutaneous immunization (anti-human eIgR antibodies # B3498, # B3499).
On the other hand, peptide antibodies (antisera) against the cytoplasmic region of mouse eIgR were obtained in the same manner as described above (anti-mouse eIgR antibodies # B3496 and # B3497) except that peptide PPPLQMSAEELAFSEFISV (SEQ ID NO: 17) was used.
In addition, immunization was performed a total of 4 times over about 50 days, and CFA (Freund's complete adjuvant) was used only for the first time as an immune adjuvant, and IFA (Freund's incomplete adjuvant) was used for the second and subsequent times. Antibody titer was measured by ELISA using the same peptide as the antigen. Approximately 80 days after the first immunization, whole blood was collected, and about 100 ml of antiserum was obtained from any rabbit.
(2) Antibody uptake eIgR is homologous to known IgA / IgM FcRs, pIgR and Fcα / μR, suggesting that eIgR may bind to IgA or IgM. Therefore, human and mouse eIgR (h-eIgR-L, m-eIgR-S) were expressed in HeLa cells by gene transfer, and the presence or absence of binding activity to various Igs and their uptake into cells was examined.
Restriction enzyme sites were incorporated into human and mouse eIgR (h-eIgR-L, m-eIgR-S) cDNA by PCR using the following PCR primers, and the resulting fragment was inserted into the expression vector pcDNA3-HAC [inserted A vector (M. Hosaka, K. Toda, H. Takatsu, S. Torii,) in which pcDNA3 (Invitrogen) is modified so that an HA epitope sequence (YPYDVPDYA: SEQ ID NO: 18) is added to the C-terminal side of the coding region of the gene. K. Murakami, and K. Nakayama, Structure and intralocalization of mouse ADP-ribosylation factors type 1 to type 6 (ARF1-ARF6) Was constructed EIgR cDNA expression vectors of human and mouse by inserting into iochem (Tokyo) 120 (1996) 813-819)].
PCR primers for h-eIgR-L cDNA and h-elgR-M cDNA:

PCR primers for m-eIgR-S cDNA:

Human and mouse eIgR cDNA expression vectors were introduced into HeLa cells cultured on cover glass using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). After 48 hours, various Ig [human IgA1 (λ) and human IgA2 (κ) (Athens Research & Technology), mouse IgA (λ) and mouse IgM (κ) (ICN Pharmaceuticals, Inc.), mouse IgG (Jackson Immunorear) The medium was replaced with a medium containing 2] and further cultured at 4 ° C or 37 ° C for 2 hours.
After washing with PBS, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and FITC-labeled anti-immunoglobulin subtype antibody [anti-human IgA antibody, anti-mouse IgA antibody, Anti-mouse IgM antibody (both ICN Pharmaceuticals, Inc.) and anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories)]. At the same time, eIgR expressed by stepwise reaction of rat anti-HA antibody (3F10: Roche Molecular Biochemicals) and Cy3 labeled anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) was detected. Observation was performed using a confocal microscope (TCS-SP2, Leica). 3A to 3E are HeLa cells expressing mouse eIgR-S (m-eIgR-S), and FIGS. 3F to 3H are human eIgR-L (h-eIgR-L), respectively. 2 shows a photograph (630 ×) taken with a confocal microscope of the state of uptake of various immunoglobulins in HeLa cells expressing human eIgR-M (h-eIgR-M). Uptake of various immunoglobulins was performed at 37 ° C. for 1 hour. As shown in FIG. 3, mouse eIgR-expressing cells (A to E) and human eIgR-expressing cells (F to J) bind to human IgA1, human IgA2, mouse IgA, and mouse IgM and are taken into the cells. No binding was observed with IgG.
(3) Immunohistochemical staining (identification of eIgR expression site)
The mouse tissue was fixed with 10% formalin or 1% zinc sulfate / 10% formalin solution, embedded in paraffin to prepare a pathological section, and reacted with the anti-mouse eIgR antiserum prepared in (1). Antigen-specific binding was detected by a stepwise reaction with a biotin-labeled anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), hydroperoxidase-labeled streptavidin, and FITC-labeled tyramide (all Perkin Elmer).
FIG. 4 shows an immunostained image of the myocardium. Expression was specifically observed only in capillary endothelial cells, and expression was not observed in vascular endothelial cells larger than that of thick blood vessels (arrows in the figure).
Similar capillary endothelial cell specific eIgR expression was also observed in immunostaining with tissue sections of the tongue and liver.
From the above experimental results, the newly identified eIgR is specifically expressed in capillary endothelial cells, and by translocating IgM and IgA in the blood to the tissue in a transcapillary endothelium by transcytosis, The possibility of playing a role in control was suggested.

ｍ−ｅＩｇＲ−Ｓ ｃＤＮＡ用ＰＣＲプライマー：

構築したｅＩｇＲ−Ｆｃ融合タンパク質発現ベクターを、ＦｕＧＥＮＥ６ ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔ（Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）を用いて２９３Ｔ細胞に導入した。９６時間後に培養上清を回収し、凍結保存した。回収した培養上清が３００ｍｌ程度に達した後、ＨｉＴｒａｐ Ｐｒｏｔｅｉｎ Ａ ＨＰ Ｃｏｌｕｍｎｓ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ）を用いて、ｅＩｇＲ−Ｆｃ融合タンパク質を精製した。精製操作には、高性能液体クロマトグラフィーＡＫＴＡ ｅｘｐｌｏｒｅｒ １０Ｓ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ）を使用した。精製した融合タンパク質は、ＰＤ−１０ Ｄｅｓａｌｔｉｎｇ ｃｏｌｕｍｎｓ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ）で脱塩した後、ＰＢＳで２４時間透析し、抗原タンパク質として用いた。
ヒト及びマウスのｅＩｇＲ細胞外領域に対する抗体（抗血清）は、精製したｅＩｇＲ−Ｆｃ融合タンパク質をそれぞれラット（Ｗｉｓｔｅｒ、♀、４週齢）２匹にＦｏｏｔ Ｐｕｄｓ免疫して得た。免疫は１週間をはさんで２回行い、免疫アジュバントはＴｉｔｅｒ Ｍａｘ Ｇｏｌｄを用いた。１回目の免疫から１０日後に採血した。抗体価測定は、抗原と同じｅＩｇＲ−Ｆｃ融合タンパク質を用いて、ヒト免疫グロブリンＩｇＧ Ｆｃフラグメント（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ）をコントロールとしたＥＬＩＳＡ法によって行った。
採血して得られた抗血清を、マウスＭｙｅｌｏｍａ Ｐ３Ｕ１細胞と融合させハイブリドーマを作製し、限界希釈法とＥＬＩＳＡ法の組合せによりポジティブクローンのスクリーニングを行った。単離されたハイブリドーマの培養上清を用いて、ヒト及びマウスのｅＩｇＲを強制発現させたＨｅＬａ細胞を免疫染色して観察し、ポジティブクローンを確認した。その結果、ヒトｅＩｇＲに対するモノクローナル抗体は７クローン（＃１Ｆ６、＃１Ｃ１２、＃１Ｅ１０、＃５Ｃ８、＃１６Ｈ３、＃ＩＤ９、＃１４Ｃ８）、マウスｅＩｇＲに対するモノクローナル抗体は２クローン（＃Ｂ３Ａ１、＃Ｂ７Ｆ１１）を得た。
（２）モノクローナル抗体によるｅＩｇＲの免疫染色（モノクローナル抗体のｅＩｇＲに対する特異性の確認）
カバーグラス上に培養したＨｅＬａ細胞にＦｕＧＥＮＥ ６ ｔｒａｎｓｆｅｃｔｉｏｎ ｒｅａｇｅｎｔ（Ｒｏｃｈｅ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｃｈｅｍｉｃａｌｓ）を用いてマウスのｅＩｇＲ（ｍ−ｅＩｇＲ−Ｓ）ＣＤＮＡ発現ベクターを一過性に導入した。４８時間後にＰＢＳで洗浄後、細胞を４％パラフォルムアルデヒドで固定し、（１）で調製したモノクローナル抗体と反応させた。ＰＢＳで洗浄後、Ｃｙ３標識抗マウスＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）と反応させることにより、細胞に結合したモノクローナル抗体を検出した。一部のサンプルでは、モノクローナル抗体の特異性を確かめるために、さらに、細胞の膜を０．１％Ｔｒｉｔｏｎ Ｘ−１００で透過させ、実施例２で調製したｅＩｇＲの細胞内領域を認識するポリクローナル抗体と反応させ、ＰＢＳで洗浄後、さらにＡｌｅｘａ Ｆｌｕｏｒ４８８標識抗ウサギＩｇＧ抗体（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ，Ｉｎｃ）と反応させた。観察は共焦点レーザー顕微鏡（ＴＣＳ−ＳＰ２，Ｌｅｉｃａ）を用いて行った。
ｅＩｇＲを導入していないＨｅＬａ細胞を同様に染色した場合（図６のＡ）に比較して、ｍ−ｅＩｇＲ−Ｓを導入した細胞ではモノクローナル抗体（＃Ｂ３Ａ１）による特異的な染色像が見られた（図６のＢ）。図６のＣはＡｌｅｘａ Ｆｌｕｏｒ４８８（緑色）によるポリクローナル抗体（＃Ｂ３４９６）の染色像を、図６のＤは、Ｃと同じ細胞のＣｙ３（赤色）によりモノクローナル抗体の染色像を示す。両者で同一の細胞が染色されていることから、ポリクローナル抗体とモノクローナル抗体はともにｅＩｇＲ特異的に結合していることがわかる。実施例２および３（１）で調製したしたその他のポリクローナル抗体およびモノクローナル抗体でも同様の結果が得られている。
本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書に組み入れるものとする。Production of monoclonal antibody against Fc receptor protein (eIgR) (1) Production of monoclonal antibody Restriction enzyme by PCR using the following PCR primers on cDNA of human and mouse eIgR (h-eIgR-L, m-eIgR-S) The site was incorporated, and the resulting fragment was modified with an expression vector pcDNA3-Fc (a vector obtained by modifying pcDNA3 (Invitrogen) so that a human IgG1 heavy chain Fc region was fused to the C-terminal side of the coding region of the inserted gene): By inserting into FIG. 5), an expression vector was produced that produced a fusion protein (eIgR-Fc fusion protein) of the human and mouse eIgR extracellular region and the heavy chain Fc region of human immunoglobulin IgG1.
PCR primers for h-eIgR-L cDNA:

PCR primers for m-eIgR-S cDNA:

The constructed eIgR-Fc fusion protein expression vector was introduced into 293T cells using FuGENE6 transfection reagent (Roche Molecular Biochemicals). After 96 hours, the culture supernatant was collected and stored frozen. After the collected culture supernatant reached about 300 ml, the eIgR-Fc fusion protein was purified using HiTrap Protein A HP Columns (Amersham Biosciences). High performance liquid chromatography AKTA explorer 10S (Amersham Biosciences) was used for the purification operation. The purified fusion protein was desalted with PD-10 Desalting columns (Amersham Biosciences), dialyzed against PBS for 24 hours, and used as an antigen protein.
Antibodies against human and mouse eIgR extracellular regions (antisera) were obtained by immunizing two rats (Wister, ♀, 4 weeks old) with the purified eIgR-Fc fusion protein, respectively. Immunization was performed twice over a week, and Titer Max Gold was used as an immune adjuvant. Blood was collected 10 days after the first immunization. The antibody titer was measured by ELISA using human immunoglobulin IgG Fc fragment (Jackson ImmunoResearch) as a control using the same eIgR-Fc fusion protein as the antigen.
The antiserum obtained by collecting blood was fused with mouse Myeloma P3U1 cells to produce a hybridoma, and positive clones were screened by a combination of limiting dilution method and ELISA method. Using the culture supernatant of the isolated hybridoma, HeLa cells in which human and mouse eIgR were forcibly expressed were immunostained and observed to confirm positive clones. As a result, 7 clones (# 1F6, # 1C12, # 1E10, # 5C8, # 16H3, # ID9, # 14C8) of monoclonal antibodies against human eIgR and 2 clones (# B3A1, # B7F11) of monoclonal antibodies against mouse eIgR Obtained.
(2) Immunostaining of eIgR with monoclonal antibody (confirmation of specificity of monoclonal antibody for eIgR)
A mouse eIgR (m-eIgR-S) cDNA expression vector was transiently introduced into HeLa cells cultured on a cover glass using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). After 48 hours, the cells were washed with PBS, fixed with 4% paraformaldehyde, and reacted with the monoclonal antibody prepared in (1). After washing with PBS, a monoclonal antibody bound to the cells was detected by reacting with a Cy3-labeled anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories). In some samples, in order to confirm the specificity of the monoclonal antibody, the cell membrane was further permeated with 0.1% Triton X-100, and the polyclonal antibody recognizing the intracellular region of eIgR prepared in Example 2 After washing with PBS, it was further reacted with Alexa Fluor 488-labeled anti-rabbit IgG antibody (Molecular Probes, Inc). Observation was performed using a confocal laser microscope (TCS-SP2, Leica).
Compared with the case where HeLa cells not introduced with eIgR were stained in the same manner (A in FIG. 6), a specific staining image with a monoclonal antibody (# B3A1) was seen in cells into which m-eIgR-S was introduced. (B in FIG. 6). 6C shows a stained image of a polyclonal antibody (# B3496) with Alexa Fluor 488 (green), and FIG. 6D shows a stained image of a monoclonal antibody with Cy3 (red) of the same cells as C. Since both the same cells are stained, it can be seen that both the polyclonal antibody and the monoclonal antibody are specifically bound to eIgR. Similar results were obtained with other polyclonal and monoclonal antibodies prepared in Examples 2 and 3 (1).
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  According to the present invention, a novel Fc receptor protein that specifically binds IgA and IgM is provided. The Fc receptor protein of the present invention is expressed in capillary endothelial cells of various tissues and organs, and induces an antigen-antibody reaction by transferring IgA and IgM from the blood to the tissue, thereby controlling the initiation of the immune response. . Therefore, the Fc receptor protein of the present invention is useful for elucidating immune surveillance mechanisms, searching for immunomodulators, developing drugs such as anti-autoimmune diseases and anti-inflammatory drugs, and developing drug delivery systems.

  Sequence number 19-26: Synthetic DNA

Claims (15)

The following protein (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 in the sequence listing (b) SEQ ID NO: 2, 4, 6, 8, 10, 12 in the sequence listing Or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by 14, and having binding activity to IgA and IgM Fc A protein comprising the partial amino acid sequence of the protein according to claim 1. A fusion protein comprising the protein according to claim 1 and another peptide. A gene encoding the protein according to any one of claims 1 to 3. A gene comprising the following DNA (c) or (d):
(C) DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 in the sequence listing
(D) DNA comprising a base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 in the sequence listing
DNA encoding a protein that hybridizes with DNA comprising a complementary base sequence under stringent conditions and has binding activity to IgA and IgM Fc A recombinant vector containing the gene according to claim 4 or 5. A transformant transformed with the gene according to claim 4 or 5. The method for producing a protein according to any one of claims 1 to 3, wherein the transformant according to claim 7 is cultured in a medium, and a protein expressed from the obtained culture is collected. An antibody that specifically recognizes the protein according to any one of claims 1 to 3. Reacting the antibody according to claim 9 with a test sample expected to contain the protein according to any of claims 1 to 3, and detecting the formation of an immune complex between the antibody and the protein The method for detecting a protein according to claim 1, comprising: A reagent for detecting a protein according to any one of claims 1 to 3, comprising the antibody according to claim 9. The pharmaceutical for immune response control containing the protein in any one of Claims 1-3, or the gene which codes this protein as an active ingredient. The protein according to any one of claims 1 to 3 or a cell expressing the protein is allowed to act on a test sample which is expected to contain a ligand, and a substance having an ability to bind to the protein is selected. A method for screening a ligand for the protein. A protein comprising: allowing the protein according to any one of claims 1 to 3 to act on IgM or IgA in the presence of a test sample, and selecting a substance that promotes or inhibits binding between the protein and IgM or IgA. Screening method for antagonists or agonists for. A medicament for controlling an immune response, comprising as an active ingredient a ligand obtained by the method according to claim 13, or an antagonist or agonist obtained by the method according to claim 14.

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