JPH01501206A - DNA expressing Fc receptor protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] DNA that expresses Fc receptor protein! Yumuguchi” The present invention relates to immunoglobulin receptors.The present invention particularly relates to immunoglobulin receptors. The nucleotide sequences that express the molecules, the receptor molecules themselves, and the molecules that produce these molecules. The present invention also relates to transformed cell lines (cell lines) that produce the above-mentioned proteins. 9 The present invention also relates to methods, and the present invention further relates to methods for treating organs related to immunoglobulin receptors. It also concerns analogues.

one The immune system, which has the function of protecting the body from foreign antigens, uses Fc receptors. Antibody-antigen complexing mediated by specific antibody receptors known as receptors between the humoral and III-vesicular pathways through interactions between the body and effector cells. A cooperative effect occurs. These receptor molecules are antibodies against effector cells. plays a crucial role in mediating the binding of antibodies and also participates in the regulation of antibody function .

Fc domain of immunoglobulin G, the most common immunoglobulin The receptors for this are B cells, some TIB cells, and natural killer cells or rNKJ cells. , are known to be present on macrophages and polymorphonuclear leukocytes. to this For example, see Unkeless et al., ^dv, Immuno 1.31:247. -270 (1981); Springer et al., Contea, To, I mmuno io 1.13:1-31 (1984); Dikcler, Mo 1. See Immunol, 19:1301-1308 (1982). .

When immune complexes bind to Fe receptors on neutrophils and macrophages, phagocytosis occurs. Activated oxygen metabolites and inflammatory mesoietors such as leukotrienes and prosthetics A cellular response occurs that includes the release of tagrondin as well as the induction of neutral hydrolases.

See, for example, Nathan et al., N. En, J. Med.

303:822 (1980). Fe receptors are, for example, Uhe n et al., Ce1lular lm5uno1.95:368-379 (1985 ) has also been described on lymphocytes, and in this case antibodies produced by B cells are also described. It is said to play a role in regulating production.

Fc receptors act on all classes of immunoglobulins (Ig^, IgD, IgE , IgG, IgN), but there is almost nothing about the molecules themselves. The best-characterized receptor, which remains unexplored, is probably the “high-affinity” receptor. Basophils/mast cells, 1. known as E receptors (FcE), and Mouse macrophage Fc receptors bind to IgG2b/fgGI immune complexes and IgG2b/fgGI immune complexes. For murine species, the various rgc subcategories may be Competitive binding of lasers (Diamond et al., U■).

150 Near 2l-726 (1979); J, Immunol, 125:63 1-633 (1980); J, Ex, Med, Ward: 514-519 (198 1)) and differences in sensitivity to proteases (tlnkless, mu hL 1gG3 (FcG3R) according to the research of , IgG2a (FcG2aR) and IgG2b/C1 complex (FeC2b/I The existence of a binding site for R) was revealed.

This molecule has four N-linked glycosylation sites and is a 50- to 60-dalton membrane integrant. It has been considered to be a glycoprotein.

For this, see Green et al., J. Biol. Chem, 260:9867. -9874 (1985).

Recent research has created a need to understand the detailed biochemical properties of the receptor. Publicly known 2,4G2, one of the monoclonal antibodies of It acts against an epitope present on both murine FeG receptors.

Unkeless, L-dama, 150:580-596 (1979). However, this recognized epitope is present on macrophages and lymphocytes. For example, J. Immunol. 134:2835-2838 Ph1lips and other research described in (1985); 2-296 (1985) and J. Immunol, 1 34:1774-1779 (1985), Telland et al. 2.4 Isotype specificity of lymphocyte receptors active against G2gG2a It has been demonstrated that this is wider than FeC2b/IR, which does not bind to. Also, FeC2b /IR Mark et al., J. Immunol, 135:2635-241 (1 985) and Hibs et al., Mu. It was identified as an alloantigen related to the known H1 system described in ). chromosome 1 The above locus specifies the product on antigen-presenting cells and strongly induces mixed lymphocyte reactions. Control the non-H-27 cell proliferation response that will be stimulated. Alleles of this locus Four genes have been identified so far (FestensLein, Trans 1a nLaiton Proeeedin s8:339-342 (1976)), Ly-17 antigen (Hole s et al., Proc, Natl, Hecad, Sei, 82 Near 706-7710 (1985)) are tightly bound to or determined by this locus. be done.

Differences in isotype specificity and function between macrophage and lymphocyte receptors Isolation of eDN^ of macrophages and T cell clones and and characteristics analysis. As a result of these experiments, two genes were isolated. That's right One exists in two different allelic forms and has an extracellular domain that binds to igc. The proteins have extremely high homology, but the transmembrane domain and cytoplasmic domain are different. It expresses proteins that are different from each other. These genes are hereinafter referred to as FcαR1Fc7 9R and Fcβ2R.

′, once Figure 1 shows the restriction map and sequencing method (a) of macrophage FeGα-expressed genes. nucleotide sequence (b), UT is untranslated sequence, S is signal sequence, E-C is the extracellular domain, TM is the transmembrane domain, and C is the cytoplasmic domain. represent.

Figure 2 shows a single dot showing the homology of FcC to MHC class II protein E. represents a conservative mutation and two dots represent identity.

Figure 3 shows the distribution of FcGαmRN^ in various cell lines.

Figure 4 is an explanatory diagram similar to Figure 1, showing the restriction map and Figure 5 shows the sequencing method (a) and nucleotide sequence (b) of FcCα and The amino acid sequence of FcGβ1 protein shows a total homology of 95% in the extracellular part. It is.

Figure 6 shows Southern analysis of the [IN expression] of inbred mice, and shows that it is related to the FCC gene. The polymorphic phenomenon has been clarified.

Figure 7 shows the distribution of β2 transcripts in various cell lines and shows the distribution of β2 transcripts in macrophages. It proves the transcription.

Figure 8 shows restriction map and sequencing method (a) of FcCβ2 eDN^ and nucleotides. Array (b) is shown.

Figure 9 shows transfection activity as shown by restoration of immunoglobulin binding activity. Figure 2 shows the expression of FcCβ2 in mouse melanoma cells analyzed.

Figure 10 shows IgG2b/GIFcRcD obtained from macrophages and TM cell lines. The structure of the N^ gene is summarized.

Maino=f; day Fc R and amino Mellsin described in "L on" μm, 152 + 1048 (1980) Fcγ receptors were purified from the S49.1 cell line using other known methods with slight modifications. Ta. Cells grown in suspension medium (1 x lO”) were treated with 0.27 IU/l of approx. Tinine and 5J diisopropylfluorophosphate did. Supernatant of the lysate 40,000x was added with 2 mg of 2,4G per resin 1-1. 2. Passed through a 5-1 column of Sepbarose 4B coupled with igc.

This column was first washed with 10 column volumes of 1% NP-4070.2% sodium dodecyl sulfate. 10 column volumes of 10 sM octyl-β-D. - Washed with PBS containing thioglucoside.

50mM) ethylamine and 10-M octyl-β-D-thioglucoside Elute the protein with PBS containing p) 111.0 and immediately neutralize with Tris-HCI. Adjusted to. The protein was acidified with trifluoroacetic acid and J. Chromi. to, 297:03-19 (1984) 4. :According to the Pin other method described 5 acetonitrile 0.1% trifluoro through an upeleo C82cm column Gradient elution was performed with acetic acid.

S49.11gf; The 22 amine-terminal amino acids of 2b/γ Lucebuter were ters RPLC system and microsequence equipment (＾pplied Bi ossystems model 470^).

As a result of pro I investigation, TIIDLPKAVVKLEPPWIQVLK It turned out to be ED. In order to synthesize the corresponding oligodeoxynucleotides, The seven amino acids EPPWIQV were selected because of its relatively low degree of polymerization.

Described by Chen et al. in DNA 4:365-374 (1982). Considering the preferred codon usage and the ability of G-T base pairs to form, 20 The cleotide sequence was synthesized. This sequence is the complementary strand of the sequence indicated by the symbol above. Corresponds to This array is It is.

hand! Lofage F-” and 9゜mekure book ± upper l Naga XJ! 2”9 array The aforementioned mixed probe with low degeneracy was end-labeled with 32p-γ^TP to increase its specific activity. J774 mR subdivided in size in plasmid vector pUce Screening of mouse macrophage cDN^ library constructed in NA (Portnoy et al., J. Biochem, (1986)), 5 After screening a library of 0,000 clones, two positive clones were found. identified.

The screening was performed using 6X NETS, IX Denhardts, 10'e p++/-1 32p-end labeled oligonucleotide (10”cpm/pg) The hybridization reaction was carried out at 45°C for 16 hours. 1. The filter was then washed with 6X SSC at 25°C for 5 minutes, and then Wash with buffer for 1 minute at 40°C, and then wash with the same buffer for 1 minute at 45°C. did. Dry the filter and autoradiograph at -70°C for 6 hours using an intensifying plate. It was developed using Positive clones were identified and filtered at 50% in 6X SSC. The plate was washed again at 65°C for 1 minute and then at 65°C for 1 minute. positive clones higher melts differentially at different temperatures (powerful for colony purification, plasmid preparation and restriction enzyme analysis) Kana.

The clone with the largest insert (1300 base pair Pstl fragment) was selected and Further analysis was conducted. The sequencing of this clone is shown in Figure 1a, with the nucleotide sequence and the deduced amino acid sequence is shown in Figure 1b, starting with ^TG at position 64 and ending at position 846. An open reading frame of 782 nucleotides was discovered that spanned the entire length of the study. Estimated signal pep The tidase cleavage site is indicated by an arrow and is based on consensus rules for this type of sequence. (Van Heljne, Eur, J.

Biochee+, 133:17-21 (1983)), nucleotide 160- The 19 amino acids encoded from 210 are amino acids 3 of the S49.1 sequence described above. -22, but the difference is that position 12 is a glutamic acid residue in S49.1. group, whereas in J774 it is aspartic acid. The first three amino acids and This difference in position 12

This is due to the heterogeneity between macrophages and T cells described below. Aminomi 30 The predicted signal sequences are numbered -30 to -1 and a line is drawn above the hydrophobic core. there was. The cleavage site of this predicted signal peptidase is the previously described Van) Ieijn. N-linked glycosylation sites, indicated by arrows between −1 and 1 according to e, are boxed and marked Stine residues are circled, the predicted protein sequence consists of two hydrophobic amino acids residue regions, and these regions are shown underlined in FIG. 1b. these territories Region ζ, putative signal sequence (nucleotides 64-153) and transmembrane encodes the link anchor sequence (nucleotides 709-769). 4 potential The N-linked glycosylation site (boxed area in Figure 1b) and the two interchain disulfides The mature protein contains four cysteine residues that can form a double bond, and the amino It is predicted that there are 185 extracellular domains. to serine and threonine The enriched region is located just before the transmembrane domain, with 30% of the residues located in these regions. It consists of amino M represented by two amino acids 155-185. This arrangement force) et al. A cytoplasmic domain of 26 amino acids is predicted. 1st array ↓ 30.040 Dal It is estimated to have a molecular weight of tons. This sequence is glycated with the four N-linked sites mentioned above. It is cosylated and, in some cases, also glycosylated at the 0-linkage site.

The extracellular domain consists of two internal repeats. Ami of predicted mature protein Noids 25-75 are homologous to amino acids 100-155, surrounding cysteine residues. These homologous clusters suggest structural repeat domains. These data numbers , the extracellular domain consists of repetitive domains defined by cysteine residues. It suggests that. Comparing this sequence with the Tanno sequence data bank reveals that Robulin molecule, MBC class I and class H proteins, β2 microglobulin and other genes of this supergene family Significant homology to , the homology between the extracellular domain and the rabbit V region is revealed by homologous clusters concentrated around cysteine residues in both proteins. Ta. This homology indicates that this Fe receptor contains two immunoglobulin-like domains. Each domain consists of 42 amino acids within a 70-80 amino acid domain. This suggests that it consists of a latent disulfide loop.

The most significant homology to this FcR is observed in the MHC class II protein Eβ, As shown in Figure 2a, 32% identity is observed over a 91 amino acid region. . Program rdf (Lipman et al., 5science 227:1435~ 14401985)) Random Sharaf ring of these two sequences using The optimal alignment shown in Figure 2a is extremely effective when It was found that the mean + 5 standard deviations. This homology to Eβ is in the β2 domain (Figure 2b), and the β2 domain itself is an immunoglobulin-like domain. Ru. This analysis also shows that the transmembrane proteins of these two proteins The homology of in is also clear.

In the -RN^ extracted from various cell lines, the J774 gene, designated as the α gene, The presence of a message corresponding to the eDN^ cloned from the cell line was analyzed.

Chitgwin et al., Biochem, 18:5294 (1979) and Le Hraeh et al., Biochem, 16:4743-4751 (1977). A general procedure was used. In summary, 111 ft of poly8RN^ was Fractionate on maldehyde gel, transfer to nitrocellulose, complete α probe (a) or is hybridized under stringent conditions with probe (b) constructed on the 5'α sequence. It was soybean. As is clear from Figure 3, macrophage system P388 Di, En Wide hybridization with Ell 3^, RAIII 2B4.7 and J774 band is detected. Reacts with FcγR monoclonal antibody 2.4G2 T cell line S49.1 contains higher molecular weight mRNA species, P2S5 has an equivalent The abundance (abundance) to two RN8 is shown, and WEHI 3^ is 1 A major band that migrates faster than 8S and a much smaller presence that migrates slower than 18S. 2.4 to 2-negative line CL, 7 (fibroblasts), and 2-cells with a number of bands. (fibroblasts) and L51789 (T cells) do not contain α transcripts and do not contain probes. The same macrophage and T cell RNA constructed in the 5° sequence of eDN^ (Figure 3b) When used in this study, the cell type specificity of α gene expression was evident. S4 No transcripts were detected with this probe in the 9.1 cell line, and P2S5 Only one species is detected in . Similarly, in WEHI 3^, 18 Low abundance species that migrate slower than S are not detected by this probe, α Similar results were obtained when constructing a 3゛ probe from cDN^ (Set-P st, data not shown).

These results demonstrate that the T-cell line S49.1 and macrophage-like lines P388 and WEH I3^ is a 5° cross-hybridized when probed with a complete probe. and 3° ends contain RNA species that are not homologous to macrophage α transcripts. , analysis of two other T cell lines, EL-4 and NI-36, detected in S49.1. A transcript of a similar size to that of the sample was detected (data not shown).

These transcripts and the genetic basis of their expression are the same. To determine the size of the eDN^DNA library, size-fractionated S49. was constructed and probed with the complete cDNA^α DNA block. Positive clone frequency 0.1 % and characterized by restriction mapping and DNA sequence analysis.

Figure 4a shows the physical map and sequencing method of a T cell transcript designated β1. Figure 4b shows the nucleotide sequence and predicted amino acid sequence of this protein. The procedure used was used to obtain a physical map and sequence of macrophage DNA. The procedure was the same as that for 1st stroke 1. Starting with 8TG at nucleotide 340 One open reading frame terminating at leotide 1326 was detected. This protein before denaturation The expected molecular weight of the compound is 36.750 Daltons. Nucleotide 42 (position +1 ), which are the determining amino acids of this protein. Matches the mino-terminal sequence. The sequence preceding this N-terminus has a characteristic hydrophobic core (upper This signal sequence encodes a signal sequence with a The extracellular domain, which is not homologous to the signal sequence of the product (see Figure 1b), is shown in Figure 5. As shown, the α sequence starts at amino acid 4 of the β1 sequence and continues to amino acid 174, and the 5% match. The transmembrane and cytoplasmic domains of the β1 sequence are similar to the sequence Not expected to be homologous to similar domains. 5° and 3° of these two transcripts. No sequence homology is observed in the translated domains.

S49. I. Sequences of four additional tryptic peptides for FeγR were determined. . Is the 10 amino acid sequence 5QVQASYTFK the β1 sequence? 1F50~5 Confirmed on 9th. The sequence of 8 amino acids l5FFHNEK is located at positions 120-127. was confirmed. ETLPEEVGEY is at position r112 in the 13 amino acid sequence EM 22 F235 t, lt&4:l:, 13 amino acid sequence TEAEN TITYSLLK was confirmed at positions 271-283.

Since both the α and β1 sequences were obtained from a cell line derived from B&lb eight mice, , 5% of sequence variation in the highly conserved m extradomain arises from allelic variation. It was long. To confirm that the β1 transcript was derived from a second gene, we tested α and β genes were determined using Southern blot analysis of DNA obtained from various isogenic strains. We created a map of the nom sequence. As shown in Fig. 6 (cut with aql and completely DNA probed with a probe detects polymorphisms associated with this gene and Three restriction fragments are generated. 5° and 3° This polymorphism, as determined by reprobing with a probe, is located in the 3′ of the α gene (data not shown), these same DNA preparations were transformed into complete β1 cDNA. When probed with a DNA probe (Fig. 6b), the 3° polymorphism flag associated with the α gene was detected. The fragment has been replaced with a 2.4 kb non-polymorphic fragment. These de The α and β transcripts contain highly conserved sequences encoding extracellular domains. This suggests that it is derived from a different gene containing . Low stringency conditions (25% formamide, 10% KM dextran, 5X SSC 40°C; final wash Clean solution = 2x SSC1, 1% SDS at 40°C) with both α and β probes. Hybridization can result in additional limiting flora that cross-hybridize. component was detected, which is the third gene of this gene family. (not shown).

! Figure 7a of C in JfflllJ and macrophage 7 shows β1 cDN^D Results of RN^ plot analysis of macrophages and Ti follicle systems probed with NAb. This probe detects the S49.1 transcript and is highly effective in the macrophage system. It is expected to cross-hybridize to the α transcript. However, this plot Comparing the results in Fig. 4a and Fig. 4a, we can see that the transcripts of different patterns are in different locations. It is clear that it can be detected in abundance. In particular, -EHI 3^ and P2S5 are the same. , and one of the transcripts is the same as the S49.1 transcript. Appearance has mobility. As shown in Figure 7b, this means that the homology to the α gene is Using a 5'βl probe constructed in the 5' untranslated region of this transcript, which is not observed. It is confirmed by In the macrophage system, this probe The transcript is detected. As a result, the β gene is expressed in macrophages. and that the transcripts detected in J774, RAM and P388D1 are S 49. l It turns out that the transcript has a different size from that detected in the T cell line. . To study these macrophage β transcripts, J774 cDN^DN The A library was screened by common unique sequences derived from the β gene. profit Restriction enzyme maps of the clones identified are those of these clones and the β1 cell line S49.1. It was proved that the difference from the transcript lies in the deletion of the Xmn site. The matrix designated as β2 The physical map and sequencing strategy of clophage β transcripts are shown in Figure 8a and The octide sequence and deduced amino acid sequence used to obtain these are shown in Figure 8b. The procedure followed is the same as that for roasting.

1 2: T for domain.

Suboosu The β1 and β2 sequences contain identical coding and non-coding sequences over their entire length. However, in the β1 sequence, there is 1 immediately after nucleotide 783 of the β2 sequence. 38 nucleotides have been inserted (nucleotides 1066 to !204, 4blJ), this sequence allows the cytoplasmic domain of the β1 transcript to 46 amino acids will be inserted. As a result of this insertion, S49. 1 and T cell lines EL-4 and NI-36 (data (not shown), β1 gene-specific radioactivity generated from 77 RN^ polymerase RNase protection using Olabel RNA probe (X+*n-Pst, Section 4a) (see figure), 500 nulls were obtained for S49.1 and Ni-367 cell lines. A β1 gene-specific protected fragment consisting of cleotide was detected, but the It was not detected in the lophage line P388D1. S49.1 and - In 36, in addition to the protected fragment of 500b, P2S5 Di 382 to eomigrate with clophage protected fragments A bp fragment was detected. This fragment is protected by β2 transcripts. matches the expected fragment size. These results indicate that the β gene is Suggests that it is transcribed into cells and macrophages. In addition, in T cells , a transcript with a 138 bp insertion is detected. This is added to T cell FeγRβ. This can be quite simply explained by an alternative splice pathway that gives rise to additional exons. . The predicted molecular weight of β2 protein before any post-translational deception is 31,886. be.

Functional domains of FcγR: expression in transfected cell lines current Evaluate the functional role of the structural heterogeneity described for the FC receptor and To determine whether one or more polypeptide chains are required for Gund binding, the FC receptor Pter-negative cells were transfected with these CDNA clones. For transcription Expression vector (pcEXV-3) utilizing the SV40 early promoter (Mil J. Immunol, 134:4212-4217 (1985 Generated by cloning the sequence encoding β1 or β2 cDNA into I did the actual thing. pGCc confers resistance to 0418.

s3neo (Southern, et al., J, Mol. Appl. Ge net, 1 □ 327-341 (1982)) and the above plasmid disaster was used to co-transfect 878H1 mouse melanoma follicles.

After 10 days of culture in G418-containing medium, monoclonal antibody 2.4G2 binding Screen each clone by rosetting it with human red blood cells <Albino, other 1M01 & Ce1l Biol, 5: 69 2-697 (1985) @ Teru). After O-ning positive cells, their Fc γ receptor activity was tested.

Figure 9 shows the results obtained by O-ning β2 CDNA in the expression vector. No results shown. 2. A stable line expressing the 432 epitope is rabbit anti-5R. It strongly bound to BC-opsonized sheep red blood cells (SRBC). This fact is F cγ receptor function v1t! This is what is shown. Furthermore, this bond is monochrome This binding was inhibited by the null antibody 2.4G2 in a concentration-dependent manner. is shown to be specific for the receptor. Cloning in an expression vector by the β1 insert Transfected cells also showed a similar binding pattern.

hmm Untransfected 878H1 cells, FcγRβ sequence is SV40 Transfection with one inserted in the opposite direction with respect to the early promoter Negative results were obtained using 5RBC cells and non-antibody coated cells. (data not shown). From the above experiments, it was determined that the cDNA clone was expressed. proteins are displayed on the surface of the 111 cells and bind the antigen-antibody complex in a specific manner. It has been shown that it can mediate binding. Mouse L cells or monkey CO8 cells, etc. Experiments with other cell lines confirm that these sequences identify FCC retarders. It was suggested that the ability is not cell type specific.

Two genes encoding Fcγ receptors have been identified. One of them is named α It is expressed in macrophage cell lines and peritoneal macrophages. Another one The other is called β and is expressed in macrophages and T cell lines. These genetics The offspring consists of two repeating N-terminal domains, each with two glycosylation sites. and an intramolecular disulfide loop-forming moiety consisting of 42-45 amino acids. Il! with It encodes a transmission protein. This structural prediction is based on N-linked glycosylation. obtained by an in vivo labeling method that showed the existence of four silage sites. This is consistent with the results obtained. 95% homology for extracellular regions of γ and β genes While there is sex, that ji! The parts that cover the permeable and cytoplasmic regions are completely different. This is seen between the FCγ receptors of lymphocytes and macrophages. The differences in function suggested that some of them are derived from different signals 'IRa. It is something to do.

However, cell-specific splicing machinery may result in different protein production. Considering this, there is yet another level of complexity in the genetic system. It is thought that there is a bell.

The β gene is transcribed in both T cells and Mac[1)7-di, but TIB cells Analysis of the specific transcript (β1) revealed that 138 nucleotides were added. Therefore, there are 46 Fcγ receptors in the cytoplasmic region of the Ti cell-based Fcγ receptor. of amino acids will be inserted. According to RNase protection experiments, β1 transcript is found in T cell line, but J774, P388D1 or RAW manager Not found in clophage cell lines. This is probably another β gene. may have arisen from different splicing routes. Early macrophage cell The cell line P388 and WEHI3A have the same appearance as the T cell line. Since β1 splicing is present in macrophages, β1 splicing is It is suggested that this is regulated in a developmental stage during post-maturation. Above amino Although the effect of acid insertion is not yet understood, the intracytoplasmic region of βIFcγR Because it is longer, it is difficult to interact with cytoplasmic or membrane proteins involved in signal transduction. It is possible that the effects will be different. Three translocations derived from α and β genes The structure of the photograph is outlined in Figure 10.

The sequences obtained for these receptors suggest that FCγ receptors are isolated from epithelial cells. The polylQA receptor (Mostov, et al., Nature 308:37-43 (1984)). , was found to belong to the immunoglobulin sequence family. overall imnog In addition to homology with Robulin, several other important points of homology have been identified for FcγRs. It was.

The IB extravesicular region of α and β genes is very similar to the β2 region of MHC class ■ protein Eβ. is homologous to In contrast, polyIQ receptors are similar to FCγ receptors as described above. There is no significant homology, suggesting that this is more relevant to Fcγ receptors than to class II antigenic determinants. This suggests that there is a distant relationship between the two.

Another significant homology detected in the membrane-inducing region of the α-chain but not in the β-chain is that It is found between one of the membrane permeable regions of the cetylcholine receptor chain. Y oung et al. (Proc, N atl, △cad, Sci, ao: 1636-1640 (1983)) and Nelson et al. (l4Cl in, T nycst, 76: 500-507 (1985)) is an FC7 receiver. have reported on the ion channel activity corresponding to the binding of ligands to Ru. The roles played by various structural regions of the Fc receptor protein are currently under investigation. Currently being investigated.

The various embodiments of the invention described above can be utilized and applied by those skilled in the art. You will get it. For example, for patients with immune disorders, the necessary Fc receptor – does not express proteins and therefore has a sufficient or complete response to infection It is possible to trace the cause to the cells and organs that never occur. Appropriate traits Fc receptor protein produced in vitro by culture of transformed cells a prosthetic organ having the protein in it, or a transformed cell that produces the protein (physiological Organs etc. that are inoculated with (acceptably acceptable) are considered.

As a second use/application example, human DNA corresponding to the DNA disclosed herein is used. The use of the DNA of the present invention for the purpose of obtaining. Established using a mouse probe Human l"c receptor protein was synthesized according to the DNA hybridization technology developed. It is possible to identify the location of the DNA that expresses the protein and hybridize with it. Ru. These techniques are well known to those skilled in the art.

Claims (25)

[Claims] 1. Substantially pure nucleotide sequences expressing Fc receptor proteins. 2. Claim 1, wherein the receptor protein is an Fcr receptor protein. Nucleotide sequences as described in Section. 3. Claim 1, wherein the receptor protein is an Fcra receptor protein. The nucleotide sequence according to item 2. 4. Claim 1, wherein the receptor protein is an Fcrb receptor protein. The nucleotide sequence according to item 2. 5. Claim that the Fcrb receptor protein is an Fcrb receptor protein. The nucleotide sequence according to range item 4. 6. The claimed Fc receptor protein is Fcrb2 receptor protein. The nucleotide sequence according to range item 4. 7. The nucleotide sequence according to claim 3 having the following sequence: [There is an array] 8. The nucleotide sequence according to claim 5 having the following sequence: [There is an array] 9. The nucleotide sequence according to claim 6 having the following sequence: [There is an array] 10. Substantially pure Fcr receptor protein. 11. The method according to claim 10, wherein the protein is Fcra protein. pure Fcr receptor protein. 12. The substantial substance according to claim 10, wherein the protein is an Fcrb protein. pure Fcr receptor protein. 13. The substance according to claim 12, wherein the protein is Fcrbl protein. Essentially pure Fc receptor protein. 14. 13. The substantially pure protein of claim 12, wherein the protein is Fcrb2. Fcrb receptor protein. 15. The substantially pure protein of claim 11 having the following amino acid sequence: Park: [There is an array] 16. The substantially pure protein of claim 13 having the following amino acid sequence: Npaku: [There is an array] 17. The substantially pure protein of claim 14 having the following amino acid sequence: Npaku: [There is an array] 18. Eukaryotic cells were isolated from Fca. Consists of Fcbl and Fcb2 receptor proteins Substantially pure nucleic acids expressing Fc receptor proteins selected from the group The transformed cells are cultured under conditions suitable for expression, and the protein sequence is transformed with the protein sequence. and purifying the expressed protein. How to get protein. 19. Fc. linked within a portion of carrier DNA. Composed of Fcb and Fcb2 Substantially pure nucleic acids expressing an FC receptor protein selected from the group consisting of: An Fc receptor protein expression vector containing a tide sequence. 20. 20. The vector according to claim 19, which is pcEXV-3. 21. FC receptor selected from the group consisting of Fca, Fcb and Fcb2 A eukaryotic cell line transformed with a foreign nucleotide sequence that expresses a protein. 22. An infusion containing a physiologically acceptable cell line according to claim 21. Munogloalin receptor organ analog. 23. Samples containing substantially pure human DNA were transfected with mouse Fc receptor receptors. a mouse DNA sample expressing the protein, and the mouse DNA and complementary human ON The hybrid DNA is contacted under conditions suitable for hybridization between A and A. processing the sample to separate the hybrid DNA from non-hybrid DNA; human Fc receptor protein, which consists of removing the mouse DNA from the human Fc receptor protein. A method of obtaining substantially pure human DNA for expression. 24. The DNA is Fcr. Fcra. A group consisting of Fcrb1 and FCrb2. 24. The method of claim 23, wherein the selected receptor protein is expressed. 25. Substantially pure human DNA obtained by the method according to claim 23.