JPS62502939A - Identification of IgE binding proteins by molecular cloning - 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] Identification of IgE binding proteins by molecular cloning Field of the invention This invention belongs to the field of recombinant DNA technology, and more specifically, Concerning a method for producing a protein with specificity for IgE, which is approximately 31,000 daltons. do.

Background of the invention Both the production and action of immunoglobulin E (IgE) molecules are dependent on IgE-binding proteins. Much depends on quality. One type of IgE-binding protein is Contains cell surface receptors on basic cells, lymphocytes and other cells (1°2). Ma Receptors on skeletal and basophilic cells induce IgE-mediated (3,4) and on the other hand, receptors on lymphocytes. plays an important role in regulating IgE antibody responses (5,5). other Forms of IgE-binding proteins promote or suppress IgE antibody production Contains phosphorus, which acts like a lymph (, 5). In order to fully understand the IgE system, In order to explain the structural relationship of these proteins, the 11'4 structure of each of these proteins. It is important to establish the relationship of effects as well as the regulation of these gene expressions. So Therefore, the basic objective of this invention is to provide DNA for these related proteins. The goal is to invent molecular cloning.

Rat basophil leukemia cells (RBL) are found on basophil cells and mast cells. It has been widely used to study the high affinity IgE receptors that exist. cells from which IgE-binding proteins have already been isolated, and (reviewed in the literature 1, 4.7). one A well-studied IgE-binding protein is the M-155,000 glycoprotein. ri, r This has been studied in numerous laboratories (8.9.10). this protein is expressed on the surface of RBL cells, mask cells and basophil cells, and It is clear that these cells respond to IgE-binding properties with high affinity. Established.

The biochemical properties of the 1gE receptor with high affinity make it unique to RBL cells. Identification of other protein components that may be generated from the receptor's multiple subunits When expressing gender, it becomes somewhat complicated. Scientists from the group consisting of IgE- In the affinity generation of receptor complexes, M-33,000 (11,1 2) and M-10,000-To' (13) 2fili additional proteins were isolated. They also have bisensual By treating RBL cells or cell lysates with a cross-linking agent, ζM-approximately 55,0 00 glycoprotein and 1-" We show that these two proteins can be chemically cross-linked, as shown in Therefore, these proteins have high affinity for IgE receptors β and β, respectively. and the γ accessory unit, and the M-approximately 55,000 carbohydrate white matter is defined as the α accessory unit. It was defined as a cut.

Another group of scientists has conducted repeated affinity tests using IgE immunosorbents. In the generation of RBL IgE receptor by chromatography, M-55,00 0 glycoprotein r In addition to quality, M-30,000 to 33,000 proteins are separated. I let go. This protein has properties similar to a protein named β subunit. although the exact relationships between them remain firmly established. Ru. Not only that, the separation of either protein is based on its ability to independently bind IgE. It remains to be determined whether this is possible. Recently, β secondary M-30,000-33,00 which is different from knit but has IgE-binding properties 0 protein r have been identified within RBL cells (17).

The main object of this invention is to provide an IgE-specific The goal is to create and culture recombinant DNA molecules that produce sexual proteins. long For a range of purposes, using this in vitro obtained protein, Ig Mo+zeta-structural relationships of E-based proteins, structure-action relationships of each protein, and these The contents of the following references establishing the definition of gene expression for is incorporated herein by reference as set forth in the foregoing description.

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(Margin below) Summary of the invention A translatable IgH-specific protein with an apparent molecular weight of 31,000 Daltons A recombinant DNA molecule with a final product is produced. For molecular cloning, Double-stranded CDNA was synthesized from RBL mRNA fractionated on a sucrose gradient, and vector The molecule f hybridized to the mRNA encoding the IgE-binding protein of f131,000. We identified several genes that contain the same CDNAs. These cloned c Determine the DNA sequence of DNA 1 and find the amino acid sequence corresponding to the protein part in parentheses. The column was estimated. This cloned cDNA was identified in RBL cells. Child! It is thought that 31,000 IgE-binding proteins are commonly encoded; These cells may play an important role in basophil cells.

This invention has a polypeptide that selectively binds IgE as a translation product. A method of producing a cDNA molecule is included, the method comprising the following steps. i.e. , has a molecular weight of approximately 31,000 daltons, and is isolated from the translation product of RBL mRNA. IgE-binding proteins that are precipitated by epilepsy and are not precipitated by normal rabbit serum. A step of isolating the genetic material encoding the repeptide, placing the genetic material in the vector. step of incorporating the vector, step of transferring the vector into the recipient organism, IgE-binding protein table the process of selecting and cloning host cells harboring the protein The process of collecting white matter.

The IgE-binding protein produced by the method described above has a specific interaction with anti-εBP antiserum. and binds to IgE-Sepharose 4B.

The IgE-binding protein produced by the method described above contains at least five methionyl atoms. As a molecule with a N-glycosylation residue, as a molecule with no potential N-glycosylation positions. And molecules! Separated independently from the 55,000 Dalton IgE-binding protein It is characterized as a molecule that can be

The present invention also provides a protein that specifically binds IgE of about 31,000 Daltons. Contains CDNAs characterized by white matter production.

is an IgE-binding protein with at least 5 methionine residues. molecule with no N-glycosylation positions and an additional 55,000 Daltons. It is a molecule that can be separated independently from the normal IgE protein.

This cDNA is a method for producing an IgE binding protein with a molecular weight of approximately 31,000 daltons. It is also characterized by the ability to react specifically with anti-epsilon BP antiserum. It is a molecule that specifically hybridizes with rnRNA from RBL cells.

Characterized by the production of an IgE binding protein with a molecular weight of approximately 31,000 daltons It is a cDNA, and contains the amino acid code of half of the carboxyl group terminal of the cDNA. cDNA whose code sequence is substantially as follows: 8 CA CTG ACA GTG CCC’T 8 CG entry ATCCCCCTTG CCT GCA GGA GTCACCCCT CGCATG CTG AT CACA ATCATA GGCACA GTG Eight G CCCACCGCA Eight people CAGT ATC, ACT CTG entering T TTC entering AG Eight people λ G GG Eight people CGACACCGCCTTCCACTTT, AACCCCCGCTT Cλ entering T GAG 8 people CAAC to G GA GTCATCGTG TGC 8 Person CACG AACCAG CAC Eight T Enter CTGG GGGACG GA A GAA 8GA CAG, TCA GCT TTCCCCTT GAG AGCGGCAA Enter Hachi CA To TTC °A ATA CAG GTCCTG GTT GAA GCCGACCACTTC eight GG'rT GCG GTC eight Person T GAT' GTT C to CTG TTG CAG TAT AACC ToT CGGAGC 8 people G 8 people CCTCAGG GAA ATC 8 GCC 8 people CTG GGG ATCATT GGTG entry CATA 8CCCTCACC, ACCGCT TCCCAC GCC entered TG entered TCT 8 people to GCC 8G to G G GGT GGG CCG CCA CCA GAA CTG CCCTGT GTG TTA TG 88 CG GCA 8 people CTTT GCA TTT C TCTCT CCT TAT ACT TCT TGT Eight G to CA TCC 8 TT 8 T 8 T 8 GT CTCGTG CTG AGA GAA 8 Enter person λ 8CCCCCCCCCCCC The characterized cDNAs described above encode the following proteins: THRLEtJ TIIRVAL PROTYR SP MET PlNo L EIJ PROGLY GLY VALILE ALA PIIE HIS P HE ASN PROARG PHE ASN GLU ASN ASN AR GARG VAL ILE VAL CYS ASN THRLYS GLN ASP ASN ASN’TRP GLYARG GLLI GLtJ AR G GLN SERIALA PIKE PROPHE GLU SERGLY LYSPrtOP) IE LYS ILE GLN VAL LEU VAL ' GLU ALA ASP) IIS PHE LYSVAL ALA VA L ASN ASP VAL HIS LEU LED GLN TYRASN HIS ARGMεT LYS ASN LEU ARG GLLl ILE SERGLN LEtJ GLY ILE ILE GLYASP ILE THRLED THRSERALA SEn lll5 ALA MET IL E ill This cDNA molecule contains an IgE binding protein with a molecular weight of approximately 31,000 daltons. The cDNA is also characterized by the annual production of white matter. Translated by ゛ It is a molecule that has 121 nucleic acids in a region without poly(A) addition. It is a molecule that typically has 15 to 21 bases in the AATAAA sequence, and has a membrane permeability. Does not have any oversequences (Trans II lembrane 5 sequences) A molecule of a sequence that does not have an N-linked carbohydrate junction. Furthermore, it is a molecule that exhibits hydrophilic properties.

This cDNA molecule reacts specifically with RNA species found in RBL cells. , molecular weight approximately 31. ooo is also characterized by the production of IgE binding proteins.

NaDodso, -PAGE of IgE-binding proteins isolated from translation products. It is an expression of analysis. A) RBL poly(A) + RNA was added to a 10-30% sucrose gradient. (See Reference 2 for RNA sedimentation coefficients in each fraction. 2). In addition, individual fractions were translated in vitro. Ta. Next, the IgE-binding protein in the translation product was isolated using IgE-Sepharose 4B. and separated. B) RNA fractionated on sucrose gradient (fractions 13-16 In vitro translation products from a pool of rabbit anti-εBP Immunoprecipitation with serum (lane A) or normal rabbit serum (lane b) did. The protein was analyzed on a 10% polyacrylamide gel.

Figure 2 shows the results from mRNA hybridized to cloned cDNA. Table of in vitro translation products of NaDodSO,-PAGE analysis of It is current.

hybridized with 136C9,13 cDNA immobilized on a nitrocellulose filter. The mRNA is in vitro.

and the translation product is a Sepha Rose 4B and mouse monoclonal 1gG+ (lane b) or mouse monoclonal IgE (lane C) or rabbit anti-εB For immunoprecipitation with P serum (lane d) or normal rabbit serum (lane e). was forced to Isolated white matter was analyzed on a 10% polyacrylamide gel. .

FIG. 3 shows the nucleic acid sequence of cloned cDNA136c9.13. this The amino acids predicted from the nucleic acid sequence are shown below this coding sequence.

Figure 4 shows the cloned 136C9,13 cDNA probe and various cell lines. Northern blot hybridization analysis for RNA from The results of lot bybridization analysis are shown. 5 types Cell lines, i.e. mouse IgE-secreting hybrid (26,82: lane a), mouse Mouse IgG, -secretory hybrid (109,3,L/-n b), RBL (lane c ), mouse mastocytoma P815 (lane d) and rat lymphoma IR9 Total poly(A)” RNA (5 μg each) from 83FC lane e) Glyoxylated and electrophoresed on a 2% agarose gel, nitrocellulose Pass through a filter and use a 136C9,,13 probe labeled with Kap. Interbreeded. Crossing conditions were 2×Denhardts/25mM P IPES, pH 6,8/25mM EDTAlo, 75M NaC store 150% hol NaD with maldehyde 10.2%.

d S O4/50 u g/m 11. Heat-denatured tRNA15 of DNA from heat-denatured salmon sperm at 0 μg/m Q: 42°C.

16 hours. Washing conditions were 15mM NaCQ/1.5mM sodium citrate. 10.1% NaDodSO4, 51°C.

Figure 5 shows NaDo'd of I-labeled IgE-binding protein from RBL cells. This is a representation of SO,-PAGE analysis.

(A) RBL (lane a), P1315 (lane b) or rat lymphoma Cell lysates from (Lane C) were purified with IgE as described in Example 1. - Purified by recurrent affinity on Sepharose 4B. By affinity At the end of the first cycle of purification, the protein was labeled with ■. (B) RBL cells The lysate is generated by affinity with IgE-Sepharose 4B and the binding protein was iodinated, soluted, and treated with lentil lectin-Sephas 4B. Conclusion The uncombined pellets are then 1) reacted with IgE-Sepharose 4B (reacted with lane a) or 2) rabbit anti-εBP (lane b) or normal rabbit Immunoprecipitation was performed with serum (lane C). The separated protein was washed with 10% polyacrylate. Analyzed on a Ruamide gel.

Detailed description of this invention This invention provides a method for producing IgE binding proteins, and a method for producing IgE binding proteins, as well as methods for use in recombinant DNA technology. This includes methods for producing cDNA molecules that can be produced. More specifically, the invention relates to the molecular Formation of a CDNA that produces a 31,000 Dalton IgE binding protein; It includes a method for producing a host/recipient organism capable of producing this protein.

31KIgE-binding within the in vitro translation product of RBL mRNA Identification of synthetic proteins 98 Table BH62-502939 (7) Previously, we showed that on the surface of xenobu oocytes injected with RBL rnRNA. represents the expression of IgE-binding activity of (22) Expected IgE receptor 31 proteins on the NaDodSO,-PAGE gel compared to the alpha subunit. The abnormal intensity of the band may be due to the fact that this protein inherently has IgE7 binding activity. He suggested something. RBL mRNA fractionated with a Liy sugar gradient was transferred to rabbit reticulum In vitro in the lysate system of erythrocytes.

Translated by . This translation product (consisting of various translated proteins...data (not shown) was tested by affinity purification using an IgE immunosorbent. did. As is clear from Figure 1A, the 31Kffl white matter is The translation product of A was isolated substantially free of other white matter.

In addition to other high molecular weight proteins, M-30,000-3- 3.000 proteins were separated from RBL cell lysates using an IgE immunoadsorbent. (14-16). Polyclonal rabbits raised against these proteins It has been reported that the antiserum of G. elegans appears to react with protein components of both. (15). The protein at 31 detected above is Kosana M-30, OOO~3 3. 'OO0(7) Protein d4itondr The antiserum in parentheses is useful for research on 31K proteins. It was assumed that. Therefore, the reactivity of translation products with this serum was tested.

(For simplicity, this antiserum is defined as anti-IgE binding protein: anti-εBP. ). As shown in Figure 1B, the protein at 31 was expressed in the anti-εBP serum (lane a). immunoprecipitated from translation products, but not by normal rabbit serum (lane b). Not immunoprecipitated. 31 The K protein band is the main protein in the immunoprecipitate. 1+li- protein that is clearly immunoprecipitated and specifically immunoprecipitated. It should be noted that

Cloning of cDNA encoding 31KIbE-binding protein RBL mRNA from sucrose density gradient fractions 14 to 16 was combined with IgE-conjugated ry) was used to generate the protein-encoding gene. Approximately 1,00 One positive gene (136C9,13) was identified from 0 genes. (lane c), BSA C lane a) or IgG1 (lane b) hybridized to mRNA from RBL' cells that can be translated into protein to 31 that does not bind It will be done. Furthermore, the translated protein specifically reacts with anti-εBP antiserum. was shown (lane d vs. lane e). It was shown that it has an insert.

The nucleic acid sequence of 136C9,13 cDNA was determined and a single open reading frame was identified. As shown in Figure 3, this cDNA The coding sequence for 138 amino acids representing approximately half of the terminal end is shown. Ru. This cDNA sequence contains the 3' untranslated region as well as the poly(A) addition. 121 bases of 15 to 21 bases of a typical AATAAA sequence before the position Also includes nucleic acids. Homology searches for this nucleic acid database are based on the sequence mentioned above and which does not show any homology to other known proteins or DNA sequences. It was shown that This protein is shown on the hydropathic plot. Mushi 6 is hydrophilic, as shown by y pro, t) (29). This arrangement Inspection of the arrays for potential transmamble sequences or N It does not identify any attached carbohydrate junctions.

31KIgE-binding is uniquely expressed by RBL cells but not by cells lacking the receptor. expressed.

136C9, 13 ct), the IgE-binding properties of RBL cells with confidence that NA This cDNA detects the mRNA species present in RBL cells in order to associate it with However, it should be shown first that it is not detected in other cells lacking IgE receptors. It is. Northern plot crossbreeding experiments were conducted.

The results are shown in FIG. 136C9,13 cDNA is largely isolated from RBL cells. specifically hybridized with 1.11 cb and 1.6 kb mRNAs (ray C). Under more stringent conditions, only a 1.2 kb mRNA band was detected.

In addition, the 1.1 kb band, not the 1.6 kb band, is derived from the mouse mast cell tumor line P815. (lane d). These two mR All NAs are from rat lymphoma (lane e) or two mouse hives. Both IgE secreting ridoma (lane a) and 1gG+ (lane b) However, it was not found. Also, 1. lkb mRNA is expressed in obese mouse cells. Although it was detected in the cystoma cell line CXBG AGMCT-1, it was detected in the mouse macrophage cell line P. It was not detected in 388D1 (data not shown).

The discovery of RBL mRNA encoding the 31KIgE-binding protein indicates that this protein is actually translated in vivo in a seed cell system that binds IgE. He urged us to prove that. The lysate of RBL cells is IgE-Sepharose. The bound proteins are solubilized and eluted, and the IgE-Sepharose When re-adsorbed, the proteins were separated in addition to. Binds non-specifically to IgH A two-cycle affinity purification procedure is required to remove the proteins It was found that (14.15). In combination with this method, affinity At the end of the first cycle of purification, the white matter is labeled with a radioactive element. It is advantageous to obtain a protein exhibiting highly specific radioactivity.

Mouse mast cell JHriP815 cells (lane b) and rat B lymphoma No protein was isolated from either cell (lane C) and both proteins were present in RBL cells. It was shown that it is unique to the cell.

Next, as described above, the protein 31 derived from RBL cells was detected as IgE- It can be argued that the protein is isolated to prove that it is a binding protein. It is necessary to show that white matter can be separated independently from higher molecular weight components.

the latter protein (alpha accessory unit), which is very heavily glycosylated However, is known to bind to various lectins (30); on the other hand, Non-glycosylated (or lightly glycosylated) IKK protein The above proof is due to the fact that it binds very weakly or not at all to lectins. , was carried out by using a lectin adsorbent. Therefore, the first Proteins bound to IgE-Sepharose 4B and then eluted from it It is believed that white matter can react with lentil lectin-Sepharose 4B. The free and unbound material was treated with IgE immunoadsorbent.

As shown in Figure 5B (lane a), the protein at 31 is actually IgE-Sepha It was the main protein isolated by Rose 4B. This result shows that in RBL cells This is the same as the case for the 31K protein translated in vitro, which was observed in As such, it alone reacts with anti-εBP antiserum, leading to immunoprecipitation with εBP serum. It was found that the 31K protein was specifically immunoprecipitated (see Figure 5B, relationships for proteins identified in vitro translated products. Strengthen (support) the connection.

Example 1 Embodiments of this invention are divided into the following stages: 1. Preparation of RNA; RNA in vitro.

translation in Il, cDNA cloning and screening ■, immunoprecipitation (A) Separation of IgE-bound normal white matter by IgE-Sepharose 4B: inv One time method used for translation products in itro.

Separation of IgE-binding proteins by purification using .

(C) Immunoprecipitation with rabbit anti-εBP serum.

CD) Electrophoresis, Northern blot hybridization and DNA sequence analysis.

Cell lines and reagents: The cell lines used were RBL [18° National Institute Kindly provided by Dr. Metzger, Institute of Health ], mouse mast cell 11ilIiP815 (19), rat phosphorus Poma IRQ83F [20, Originally Bazin t'! Originated from 7 samurai Zanetti of the Medical Biology Institute (provided by Dr. Zanetti) and anti-dinitrophenic Mouse hybridomas secreting 1gG (DNP) IgE or 1gG (21) It is. Mouse monoclonal IgE specific for DNP (Hl-DN P-ε-26,82) and IgG, (II-DNP-α1-109.3) (2 1) Goat anti-rabbit IgG (GARG) antibody (21) and IgE immunization was extracted from RBL cells by repeated chromatography on adsorbent (anti-εBP:15). Rabbit antiserum adjusted against the protein purified by Finity was previously explained. The immunoadsorbent was prepared by combining 10 ulg of protein with 1 g (dry mQ) of CN. Prepared by bonding to Sepharose 4B (from Pharmaceutical Paper) activated with Br. did.

Preparation of RNA and in vitro translation of RNA Total cytoplasmic RN from various cellular sources by standard phenol/chloroform method Extraction of A, oligo(dT)-cellulose affinity, chromatography separation of poly(A)1 and RBL poly(A)'' RNA by sucrose density gradient All images have been reported (22). In vitro translation of mRNA was performed using a rabbit reticulocyte lysis chamber system [New York] in the presence of [S]methionine. New England Nuclear] It was done using

cDNA cloning and screening. Transfer double-stranded cDNA to a sucrose gradient. Prepared from RBL mRNA fractionated with and used to transform lCo11C600 (23).

Cross-selection/in vitro translation methods are screened for positive genes. used for leaning (24).

In summary, a group of 12 chimeric (chimeric) plasmids to a nitrocellulose filter and convert the filter into a complete RBL Incubate with RNA, elute the hybridized RNA and in vitro Translated in trO. Translation products are immunoprecipitated (see below) with IgE =Bound protein detected. Treat each gene in the positive group in the same way. This cell line, which has been rapidly developed by American type culture Collection (American Type Culture Co11ec tion) as No. 53107 and 1. Under the terms of the Budapest Treaty It will be available in

, an equal amount of 2XTENT (IXTENT- 20mM) Lis-HCu, pH 7,5/10mM EDTA/150mM Na CQ, diluted with 1% Trasylol) and 1% at 4°C. Rabbit gamma globulin (RγG)-Sepha Mixed with Rose 4B. Next, the supernatant was mixed with 50μ of IgE-Sepharose 4B. Mixed for 3 hours at 4°C. The beads were added to 4 mL of TENT-1% Triton X- Wash 4 times with 100% NaDodSO and an additional 4 mQ of TENT-0°05% NaDodSO. Washed once with 4. The bound protein was prepared by boiling for 3 minutes. 2.5mM Tris-HC, p, H6, 8/2% NaDodSO,/10% glyc Elution was performed with 2-mercaptoethanol containing 15% Serol.

(B) IgE-Ig by repeated affinity purification using Sepharose 4B Separation of E-binding proteins: used on cell lysates. This method first It is essentially the same as the reported method (14, 15) and described in the literature (22). The contents of these documents may not be cited. is incorporated herein by. In summary, cell lysate (typically 5X107 cells) were purified by the first affinity as described in (A), and 200!1 portion of 0.5N acetic acid/1% Triton-X, respectively. Elute three times with 75μα of 2M Tris-H (4, pH 8°8/ 1. Neutralize with Triton X-100 and perform a second affinity purification. I did it. In this study, at the end of the first affinity purification, Still bound to IgE-Sepharose 4B by the Lamin-T method (25), zr While preparing the protein, the protein was treated with 1 mCl of NaI and 10 μg of chloramine-T. using a solution of 30μ 0.1M sodium phosphate solution, pH 7.5, Labeling with radioactive elements was performed at 4°C for 60 seconds. Add 4 more beads Wash the TENT-1% Triton three times and remove the bound proteins as described above. It was eluted.

(C) Immunoprecipitation using rabbit anti-εBP serum. ! gE-Sepharose, 4 In vitro isolated from RBL cells using B (200 μQ.) Normal rabbit serum (NRS ) was added. This mixture was incubated for 1 hour, then GA for 1 hour. It was reacted with RG-Sephazer 4B (50 μl). 1μ of this supernatant for 1 hour. React with rabbit anti-εBP serum containing m3μ, then GARG-Sepha for 1 hour. It was mixed with 0's 4B (50μ included). Wash the beads and remove the bound proteins. It was eluted as described above. All methods described above were performed at 4°C.

IgE-bound to Cepharose 4B, iodinated, and The eluted proteins as described in method (B) were transferred to lentil lecithin-Sepharo 4B (pharmaceutical grade. 10mMI-Lis-HCQ., pH 7.410.15M NaC 1/1 1:1 suspension suspended in Triton X-100/f: 100 μL of J solution” ) and mixed for 1 hour at 4°C.

(E) Electrophoresis, Northern blot hybridization and DNA sequence analysis. protein N aDodSO*/polyacrylamide gel electrophoresis analysis, by citing The Laemmli apparatus (26) incorporated herein is r It was done using Gel containing normal white matter labeled with S, radioactive photographic engraving Hansa (EN”) IANCE, 221 l29 1 Fluorescence photography was taken: The gel containing the protein labeled with I was dried and released. I took radiation photos. RNA was glyoxylated for Northern blot analysis. electrophoresed on a 2% agarose gel and passed through a nitrocellulose filter. in Nick's Translation Method (27), which is incorporated by reference herein. Hybridization was performed using a cDNA probe labeled with P. This arrangement of DNA Dideoxy Primer Extension Method ( 28).

industrial applications The availability was discussed earlier in this specification. Specifically, (1) The IgE-binding protein of 31 is transferred from RBL mRNA in vitro. (2) A protein encoding this protein has been identified. The cDNA encoding the protein was found to be novel: and (3) this protein. specific in cell types of species known to have IgE-specific receptors. It was discovered that it was shown to be expressed in Corresponds to a specific part of the expected array Further research using antibodies and cDNA probes against synthetic peptides In addition, the specific expression of the described gene in IgE-receptor positive cells, localization of this protein within these cells and IgE-mediated basophilia. The role of this protein in cellular and mast cell activation should be established. be. Therefore, this protein is useful in treating allergic reactions as IgE5. J preparation or used to absorb IgE, that is, as a "gE absorber" It can be done.

Modifications that are obvious to those skilled in the art may be made without departing from the scope of the claimed invention. Wear. For example, genetic material used to form cDNA may be IgE-specific. by introducing other human cell types containing isomeric binding receptors or RBL It may also be introduced using other cellular materials other than mRNA.

Although this invention has been described with respect to rat IgE-binding proteins, the method includes: 1. Related human equivalent proteins or siderophones It is equally applicable to producing cDNA useful for detection.

FIGLJIIE l b -93 to -69 to -46 to 1314151617 m, BP NR8 Fraction Amber FIGUnEl: 2 Kuse - 屓handling boar t; translation 4 le drop bcde To 31------ BSA, 19G11cIE castle εBP NRS exemption, a anti-inflammatory agent 1” IGUI (E 4 2.12- 1.57- ! 21C; UIIE 5 procedural aids October 21, 1986

Claims (10)

[Claims] 1. cDN having a polypeptide that selectively binds IgE as a translation product A method for producing molecule A, which has a molecular weight of approximately 31,000 Daltons and has an RBL immunoprecipitated from mRNA translation products and precipitated by normal rabbit serum. isolating genetic material encoding a non-stallable IgE-binding polypeptide; Introducing the above genetic material into a vector, transferring the vector to a recipient organism; selecting and cloning a host cell bearing expression of said 1gE-binding protein; produce genes, A method comprising steps of recovering the obtained protein. 2. Reacts specifically with anti-εBP antiserum and binds to 1gE-Sepharose 4B An IgE-binding protein produced by the method according to claim 1. 3. As a molecule with at least 5 methionine residues, as a molecule substantially free of potential N-glycosylation positions, and Characterized as a molecule that can be isolated independently from the 55,000 Dalton IgE protein. 2. An IgE-binding protein produced by the method according to claim 1. 4. Characterized by the method for producing an IgE-specific binding protein of approximately 31,000 daltons cDNA that can be attached. 5. A cDNA characterized by a method for producing a translation product, said translation product teeth, IgE-binding protein of approximately 31,000 Daltons with at least 5 methio A molecule having a nin residue and no N-glycosylation position, as well as cD, a molecule that can be isolated independently from the 55,000 dalton IgE protein N.A. 6. Characterized by a method for producing an IgE binding protein with a molecular weight of approximately 31,000 Daltons. cDNA with at least 570 base pairs; A molecule that specifically reacts with Chiron BP antiserum, and A cD characterized by being a molecule that specifically hybridizes mRNA from RBL cells. N.A. 7. The IgE-binding protein with a molecular weight of approximately 31,000 Daltons was characterized by the production method. the amino acid sequence of the carboxy terminal half of the cDNA is substantially [There is an array] A cDNA characterized by: 8. Characterized by a method for producing an IgE binding protein with a molecular weight of approximately 31,000 Daltons. A protein that is translated from a cDNA containing Half of the amino acid sequence is essentially [There is an array] A protein characterized by: 9. Characterized by the preparation of an IgE binding protein with a molecular weight of approximately 31,000 Daltons. It is a cDNA molecule that contains 121 nucleic acids within 3 untranslated regions. A molecule with The poly(A) addition site is typically preceded by 15 to 21 bases of the AATAAA sequence. A molecule with a sequence that does not have a transmembrane sequence. , a molecule having a sequence that does not have an N-linked carbohydrate attachment position, and A cDNA molecule characterized by being a molecule exhibiting hydrophilicity. 10. Characterized by the production method of an IgE binding protein with a molecular weight of approximately 31,000 Daltons cDNA characterized by specifically reacting with RNA species from RBL cells. A molecule.