JPH04500904A - Polynucleotides encoding human proteoglycan peptide cores of effector cells of the immune response - 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] 17. Expression encoding the proteoglycan peptide core protein of claim 1 (ii) transforming a host with the molecule; (iii) Determine the sequence of the proteoglycan peptide core protein of the DNA molecule. expressed in a host, and (iv) Human proteoglycan peptide core protein produced by the expression Production of human proteoglycan peptide core protein by separating Law.

18. The human proteoglycan according to claim 17, wherein the host is a mammalian cell. Manufacturing method.

19, Formula: 5VQGYPTQRARYQWVR17) Pebuti)'.

20, peptide of formula: 5NKIPRLRTDLFPKTR.

21, peptide of formula: GSGSGSGSGSGSG.

22. Antibodies capable of binding human secreted proteoglycans or fragments thereof.

23. Claim 22 capable of binding to human secreted proteoglycan peptide core protein antibodies.

24, the antibody is attracted to the peptide of claim 19.20 or 21. 23. The antibody according to claim 22, which is a raised antibody.

25. The antibody of claim 22, wherein said antibody is a polyclonal antibody.

26. The antibody of claim 22, wherein said antibody is a monoclonal antibody.

Specification name of invention Encodes the human proteoglycan peptide core of effector cells of the immune response polynucleotide The present invention is disclosed in U.S. Patent Application No. 077224.035, filed July 13, 1988. This is a partial continuation application. Research based on this invention is supported by government funds. The Government has certain rights in this invention.

field of invention The present invention relates to the purification of human secretory granule proteoglycans and the assembly of their peptide cores. Concerning production by recombinant DNA technology. Additionally, the present invention provides human secretory granule proteins. Concerning the production of antibodies against oglycan.

A brief explanation of the aspects of the invention Secretory granule proteoglycans have at least one covalently attached to the peptide core. of sulfated glycosaminoglycan chains, all of which are highly acidic macromolecules. one of several families of mammalian proteoglycans [tantrapahi ( Proc of Tantravahi, R, V, et al.

Mat 1, Acad, Sci, U, S, A, +13:9207 (1986)]. Human proteoglycans were tested against a panel of monoclonal antibodies. differences in reactivity, differences in peptide mapping after proteolysis, and post-translation They are based on differences in size and, in some cases, differences in amino acid sequence. Various families of are presented. The proteoglycan family also final location (e.g., intracellular, extracellular and/or pericellular) ellular matrix), and carbohydrates attached to the peptide core. It is also classified according to the nature of the residue. For example, located in the extracellular matrix Proteoglycans are relatively sparse molecules that intercalate into the plasma membrane. It seems to belong to a group clearly different from the subfamily of highly aqueous proteoglycans. [Tantravahi, R.

V et al. (supra).

In humans, at least 12 molecules exist as the peptide core of proteoglycans. Currently, only a few of these proteins have complete primary structures and/or or chromosomal location is unknown, and no complete information is available for any proteoglycan. No gene encoding a unique peptide core has been isolated.

Derma present in the extracellular matrix surrounding fibroblasts The cDNA encoding the tan sulfate proteoglycan peptide core was found in the human embryonic line. Cloned from a fibroblast cell line [Krusius, T, ) and Proc of Ruoslhti (E,), Nat l, Ac1d, Sci, , U, S, A, 113 Near 6++3 (1 986)]. By its nucleotide sequence, three glycosaminoglycan starting points 40. with three sites for position and N-linked oligosaccharides. OOO A peptide core of Mr is predicted.

Additionally, cell surface glycoproteins may exist as proteoglycans. . For example, the invariant chains of transferrin-receptors and class II antigens are Proteoglycans on the plasma membrane of human skin fibroblasts and human lymphoid tissues, respectively. It is reported that [Giacoletto, K, S, ) et al. J, Exp, Med, 164:1422 (1986)].

cDs encoding those transferrin receptors and invariant proteins The deduced amino acid sequence of NA provides a serine-glycine glycosaminoglycan initiation The part has been identified. Human black containing chondroitin sulfate glycosaminoglycan Other proteoglycans on the cell surface of melanomas are loaded with ``Bumol''. +ol, T, F, ) and rice felt (R4cefeld, R, A , ) Proc, Natl, Acad, Sci, , U, S, A , 79:1245|1 249 (19+32). Part 240. Food with peptide core of OOOMr or more The gene responsible for this is estimated to be located on chromosome 15 [Retik 5science 231:1281 (1986 )].

A study of mast cells in rat hives revealed that proteog was stored within the cell granules. The existence of Likan was proven for the first time. However, it is involved in immune and inflammatory responses. mucosal mast cells, basophil leukocytes, eosinophilic leukocytes, eosinophilic leukocytes, macrophages. Many cells, such as cells, platelets, and natural killer cells, also contain their granules. It has become increasingly clear that they contain proteoglycans. protoplasm The proteoglycan family is present within the cell rather than in the membrane or extracellular matrix. These molecules are important for tumor surveillance in the cells mentioned above, and Important for host defense functions against bacterial, virus-1 fungal and parasitic pathogens This suggests that it is possible. Stevens, R. , L, ) Biology of Proteoglycans (1987) Intracellular Proteoglycans in Ce 1ls of theImIIlune SystemJ includes mast cells, halophilic Proteoglycans of basic leukocytes and natural killer cells are present in secretory granules There is evidence that these proteoglycans have unique structural features, and are implicated in the immune response. Their possible functions are outlined.

Boudon et al. [Proc, Natl, Aead, Sci, , U, S, A, 82:1322 (1985)] The cDNA of the itin sulfate proteoglycan peptide core was identified and sequenced.

Po1 of L2 rat yolk sac tumor (L2 rat yolk sac tumor) A) "cDNA clone p from a cDNA library prepared from mRNA" To select PG-1, the NH,-terminal protein sequence of L2 proteoglycan Oligonucleotides derived from two regions of the column were used. two different NH , - The use of oligonucleotides derived from the terminal amino acid sequence portions was essential for identifying cDNA clones. 17-m of one or the other It hybridizes with ar oligonucleotide, but does not hybridize with 111er. I've gotten some clones that don't. One of these clones can be partially was sequenced. The resulting sequence was completed with the appropriate 17-IIler probe described above. They do not have complete homology and are similar to the NH,-terminal peptide sequence of proteoglycans. There was no hood.

Deduced from the above peptide core cDNA clone of pβG-1 proteoglycan Depending on the amino acid sequence, the mature proteoglycan produced from this rat intestinal tumor cell The complete primary structure of the peptide core was determined. proteoglycan pepti Identified based on predicted amino acid sequence homology with the NH,-terminal amino acid sequence of Docore The hooded sequence of the proteoglycan peptide core has a calculated molecular weight of 10 It encodes a 104 amino acid core protein with a size of 190 Daltons.

The amino acid sequence of the rat L2 cell proteoglycan peptide core consists of three structural regions. It contains a 14-amino acid NH,-terminal region, followed by a 4-amino acid NH,-terminal region. 9 amino acid serine-glycine repeat region and 41 amino acid C0OH-terminal region The area continues. Although the functions of the NH,- and C00H-terminal regions are unknown, , which plays a role in determining the interactions of proteoglycans with both cell surface and extracellular molecules. This is thought to play a major role.

The function of the serine-glycine repeat region in the center of this molecule is also due to chondroitin sulfate. It is believed to serve as a recognition and receptor site for side chain attachment.

The binding of chondroitin sulfate and heparin chains to all proteoglycans is This is done through a 0-glycodyl bond with. Furthermore, glycine is a proteoglycan Glycine residues are also involved in glycosaminoglycan binding, as they are abundant in It is known that synthetic polymers containing alternating serine and glycine are peptides can serve as acceptors for glycosaminoglycan chain initiation. I can do it. The degree of serine O-glycosylation in L2 proteoglycans is approximately 60%, which means that at least 14 of the core proteins This suggests that the serine residue is responsible for the fundroitin sulfate chain. B Ourdon et al. found that the structure of the serine-glycine region was similar to that of rat heparin proteoglycerides. It is very similar to that predicted for the glycosaminoglycan binding region of Lycan. We noticed that the serine-glycine repeat region is present in at least proteoglycans. suggested that it may be a general feature of a subset of Heparin in rat mast cells The pronase-resistant glycosaminoglycan-binding region in proteoglycans is Contains only serine and glycine amino acids, so 15-20 serine residues and lysine residues alternate, and the heparin chain contains three serine residues. At least two of each of these were presented as being bound. rat mast cell hepari proteoglycans and rat egg yolk 1ml tumor proteoglycans. Both proteoglycans have identical or nearly identical glycosaminoglycan bonds. It seems to have an area. Serine and glycine codon usage ( codon usage) is extremely limited in the pPG-1 cDNA, 81% of serine residues are encoded by two of the six possible codons; 70% of glycines are encoded by one of four possible codons. There is.

Many other proteoglycans, in addition to chondroitin sulfate side chains, have It has N-linked oligosaccharides. These oligosaccharide chains are , threonine (serine) O-glycodyl or asparagine N-glycodylac It is linked to the proteoglycan peptide core via a receptor recognition sequence. Rat Even when the amino acid sequence of the L2 cell proteoglycan peptide phasing was investigated, the sequence :Asparagine oligosaccharide having X-asparagine-Y-serine No acceptor site found [Bourdon, M., A., et al. (ibid.) ), Prog of Hornis (Hugher;, R, C1), Biophys, Mo1. Biol, 26:189-268 (1973)].

Stevens et al., J. Biol. Chell, 260. : 14194-14200 (1985), the inventor and several other researchers Intracellular funding derived from interleukin-3-dependent mouse mast cells Structural analysis of the protein structure of Ichin E proteoglycan has been reported. This analysis The sum of glycine, serine, and glutamic acid/glutamine residues is It was found that it accounts for 70% of the total amino acids in the core protein of .

The authors reported that chondroitin sulfate E in mouse mast cells and hepatocellular carcinoma in rat serosa mast cells. Both phosphoproteoglycans are packaged within secretory granules and are Tease-resistant, highly sulfated glycosaminoglycans, high in serine and It is specified that they are similar in that they have a peptide core rich in glycine and glycine.

Furthermore, mouse fundroitin sulfate E proteoglycan is Bourdon Fundroitin sulfate broth isolated from rat egg yolk mass by et al. (ibid.) Slightly similar to G+7 Kan in terms of size and Ser, G]y, Glx content. It also clearly states that it has a peptide core.

Proc of T antravahi et al., Nat 1. Acad, Sci, , U, S, A, 83:9207-9210 (198U ), mast cell-derived mouse myeloma (BMMC) (intracellular fundolymphoma) (cells containing Itin sulfate E proteoglycan) and rat serosa mast cells (cells containing The same gene is expressed from cells containing intracellular heparin proteoglycans. In order to prove that II) PG described in Bourdon et al. (cited above) The use of the -1 probe and its subclones has been reported.

Summary of the invention The present invention provides secretory granule proteins present in the human promyelocytic leukemia cell line HL-60. cDNA and genomic fragment encoding the oglycan peptide protein Concerning the identification, characterization, and sequencing of analytes.

The present invention encodes a human secretory granule proteoglycan peptide core protein. Provide the gene sequence of Furthermore, the present invention contains such gene sequences. Expression vectors, hosts transformed with the expression vectors, and genetic manipulation of recombinant secretory granule proteoglycan peptide core protein. A manufacturing method is provided.

In addition, the present invention provides an analysis of the human secretory granule proteoglycan peptide core protein. a gene sequence encoding a sense RNA, containing such a gene sequence; an expression vector, a host transformed with the expression vector, a genetically engineered , that is, a method for producing recombinant antisense RNA, and a method for producing human proteoglycans. These vectors can be used to inhibit expression of the peptide core protein gene. It provides a method for using

The present invention further provides antibodies that specifically recognize human secreted granule proteoglycans. do. This antibody is based on the consensus sequence of the cDNA encoding this protein. was generated for a common polypeptide with a sequence similar to the following.

Secretory granule proteoglycan cDNA, recombinant protein, antisense of the present invention RNA and antibodies play a role in the activation and activation of immune effector cells in health and disease. It is useful as a diagnostic probe for monitoring that association.

For example, this proteoglycan plays a role in killing tumor cells. - Since it is released from cells, the antibody of the invention can be used for cancer diagnosis and monitoring. Can be done. In other embodiments, the proteoglycan is also removed from mast cells during immune activation. The antibodies of the invention can also be used to monitor inflammatory and allergic diseases. can be done. In addition, human secretory granule proteoglycan peptide core protein quality is expressed during inflammatory responses associated with tissue damage and repair and during certain immune responses. Antibodies against human secretory granule proteoglycan peptide core protein tests to detect such reactions in humans and to track their progress. can be used regularly.

Different hematopoietic cells have different tags attached to this proteoglycan peptide core. It has a lot of carbohydrates. However, proteins of this family in various human hematopoietic cells Since the peptide core of rotoglycans is the same, the antibodies of the invention It recognizes all secretory granule proteoglycan peptide cores of vector cells. be.

Secretory granule proteoglycans interact with proteases. mast cell prote When an enzyme is combined with this proteoglycan, its degradation on large substrates Activity is significantly reduced. Therefore, the proteoglycan of the present invention further has protein May be useful in vitro and in vivo as protein degradation inhibitors Ru.

Drawing description 11'A: Restriction map and nucleotides of eDNA-H4 and its related cDNAs. Describes the direction of the de-sequencing method. cDNA-H4 originates from HL~60 cells Ru. rXJ and rAJ are sensitive to XnI and Accl, respectively. It means a site within each cDNA. Arrows indicate each sequenced cDNA. The orientation and length of subcloned fragments are shown.

! 2[il:HL-60 cells-consensus nucleotide sequence of derivatized cDNA, and Deduced amino acid sequence of translated proteoglycan peptide core. Arrow indicates signa The putative cleavage site of the peptide is shown. Stop codons are indicated by ***.

The numbers in the right and left columns represent the amino acid and nucleotide sequences, respectively. There is. X1nI and Aeel restriction sites are shown. c　D　　A　-H4 The 5° end of cDNA-Hl2 is 4 bp longer than that of cDNA-Hl9. The 5° end is 14 bp shorter. cDNA-H8 contains excess titanium at the 3° end of its cDNA. It differs from eDNA-H4 in that it has midine (shown in parentheses). .

Restriction map of the isolated human secretory granule proteoglycan peptide core gene.

! ±Flash: Gene encoding the peptide core of human secretory granule proteoglycans The nucleotide sequence of 5° flanking region, exon/intron joining The nucleotide sequences of the exons and the three exons are shown. in exon 1 The hydrophobic signal peptide and enzyme of the translated proteoglycan peptide core Square the serine-glycine-rich glycosaminoglycan binding region in exon 3. It is surrounded by

This is the nucleotide sequence of the mouse gene being coded. 5° Franchi Translated Pu The hydrophobic signal peptide of the rotoglycan peptide core is placed in a square within exon 1. It's surrounded. It has been suggested that it directs the addition of glycosaminoglycans to proteins. diacidic amino acid sequence and serine-glycine glycosaminoglycan binding region. The region is enclosed within exon 3 by a square. Below the polyadenylation site within exon 3 drawing a line.

Figure 6: ELI SA of rabbit anti-peptide 02 serum. Peptide 01 (0 ・・・・・・・・・○) and 02(・・・・・・・・・・・・) are combined to make a microphone. Separate rotator wells and add various dilutions of rabbit anti-peptide 02 serum to them. We investigated their reactivity to specific peptides. horseradish bello Add Xidase-Fundicogutated goat anti-rabbit antibody and then incubate for 2.2° After addition of di7-di[3-ethyl-benzthiazoline sulfonate, 49 Optical detection was performed on the spectroscopic side at 0 nm.

(B) 5DS-PAGE analysis of immunoprecipitates in cell lysates of HL-60 cells. be. (A) Cell lysates of [ssS]sulfate-labeled HL-60 cells were treated with anti-pep before immunoprecipitation with Peptide 021gG (lane 1) and in the presence of Peptide 02. Immunoprecipitated with anti-peptide 021gG (lane 2) and in the absence (lane 3). It was then analyzed. (B) Cell lysate of HL-60 cells was prepared using [368]methionine. After incubating the ink for 2 minutes (lane 1) or 10 minutes (lane 2) with the anti- Analyzed by immunoprecipitation with peptide 021gG. 10 minutes [ISS] Methio The Nin-labeled HL-60 cells were washed and the cell lysates were incubated with anti-peptide 02I. Methionine-containing enrichment (enriched medicament) before immunoprecipitation with gG um) culture medium for an additional 3 minutes (lane 3). 5 minute labeling HL Cell lysates of -60 cells were added to peptide 01 (lane 4) or peptide 02 (lane 4). Immunoprecipitation was carried out with anti-peptide 021gG in the presence of peptide 5). Immunization-pre-IgG psS']methionine-labeled protein nonspecifically immunoprecipitated with Shown below. The origin (ori) and Mr Mama are shown on the left and right sides of each panel. . Arrows indicate precursor peptide core and immunoprecipitated with anti-peptide 02 1gG. Mature proteoglycans are shown.

Brief description of preferred embodiments 1. Definition In the following description, we have expanded upon a number of terms used in recombinant DNA (rDNA) technology. used in the field. The specification and claims encompassing the scope given such words and phrases. The following definitions are provided so that the scope may be clearly and consistently understood.

1. “F” DNA sequence containing the template for RNA polymerase. Gene The RNA transcribed from may or may not encode a protein. protein The RNA that codes for quality is the so-called metsenger RNA (mRNA), In eukaryotic cells, it is transcribed by RNA polymerase ■. However, individual ■ An RNA sequence having a complementary sequence to the RNA sequence is transcribed, but it is not translated normally. RNA polymerase that is not translated ■It is also known that it constitutes the gene containing the template. It is being Such gene constructs are herein referred to as "antisense RNA gene". ” and such RNA transcripts are called “antisense RNA.” Because of the presence of a translation stop codon in the antisense RNA sequence, cannot always be translated.

“Complementary DNA” or “cDNA” genes are synthesized by reverse transcription of mRNA. The recombinant gene, in which intervening sequences (introns) have been removed, is encased in a smid or plasmid. DNA or other DNA sequences that contain the substantial biological function of the vehicle. Such NA sequences can be cleaved in a definable manner without loss of functionality, and their replication and One or a few endpoints into which the DNA can be spliced for cloning and cloning. Characterized by a donuclease recognition site. This cloning vehicle further describes its use in identifying cells transformed with this cloning vehicle. It can contain a marker suitable for. The marker is, for example, Tetracyc J Non-resistant or ambicisone-resistant. instead of “Re-Loaning Vehicle” Often the phrase "2 vectors" is used.

Expression Vehicle: A vehicle or vector similar to a cloning vehicle. However, after being transformed into a host, the gene cloned into it is expressed. It can appear. Cloned genes usually contain some type of promoter sequence. located below (i.e., operably coupled to) the control array's control array. ). Expression control sequences are operably linked to the vector in a prokaryotic host. engineered or operable in a eukaryotic host to express a gene It depends on whether it is designed to express the gene that is linked to the transcriptional elements such as enhancer elements, termination sequences, and tissue-specific elements. elements, and/or translation initiation or termination sites.

Proteoglycan. As used herein and in the claims, this term refers to proteoglycan. Contains glycosaminoglycan chains that are covalently linked to the core protein of oglycan. It refers to “human secreted proteoglycans,” which may or may not be processed. This phrase is also , the part of a proteoglycan that retains biological (functional or structural) activity If the peptide core protein contains fewer than its natural number of amino acids, such as fragments, peptide fragments of human secreted proteoglycans that have do. Functionally active examples include specific It is an activity that elicits a biological response. An example of structurally active is the natural valve recombinant protein. This is the activity of binding antibodies that also recognize proteins.

The term also refers to the natural human secretory granule proteoglycan peptide core protein or the sequence of amino acids flanking one or more of the following: The biological (functional) peptides contained in human secreted proteoglycans or structurally) is used to include peptides that exhibit activity.

The present invention relates to the expression of full-length human secretory granule proteoglycan peptide core protein, “Functional derivatives” of Mobaku, both with respect to Biology substantially similar to the biological activity of valve recombinant human secretory granule proteoglycans A protein that has functional activity (either functional or structural). human secretory condyle Functional derivatives of grain proteoglycan peptide core proteins achieve specific functions post-translational modifications, such as covalently attached carbohydrates, if necessary. It may or may not contain. The term “functional derivative” refers to the term “functional derivative” of a molecule. Also includes fragments, “variants,” “analogs,” or “chemical derivatives.” To be.

Fragment “Fragments” of molecules such as human secretory granule proteoglycans are , refers to a variant of the molecule, such as a peptide core or a variant of a peptide core. shall be.

Ryogurofu: “Mutants” of molecules such as human granule proteoglycans are substantially similar in structure or biological activity to any of the fragments thereof. shall mean similar molecules. Therefore, if two molecules have similar activities, The composition or secondary, tertiary or quaternary structure of one molecule is the same as that found in the other. The term is used herein even if the sequences of the amino acid residues are not identical or the sequences of the amino acid residues are not identical. These are considered variants when used in

! Placement “Analogues” of molecules such as human secretory granule proteoglycans refer to the entire molecule or its refers to a molecule that is substantially similar in function to any of the fragments shall be. As used herein, a chemical in which this is a non-nominal part of a molecule When a molecule contains a target moiety, it is said to be a "chemical derivative" of another molecule. this Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule.

This portion also reduces the toxicity of the molecule and eliminates unwanted side effects of the molecule. can be removed. The part that can mediate this effect is the Remington fiber. - Mechanical Sciences (Remington’s Phara+a Ceutical 5 Sciences) (1980) Methods of attaching such moieties to children are known to those skilled in the art.

■ Genetic engineering of human secretory granule proteoglycans The present invention Amino acid sequence of oglycan peptide core tandem, proteoglycan peptide Gene sequence encoding core tannoic mRNA or antisense mRNA a gene sequence, an expression vehicle containing the gene sequence, a host transformed therewith, and Recombinant human secretory granule proteins produced by expression in transformed hosts such as Includes theoglycan peptide core protein and antisense RNA. Main departure Ming also developed an antibody against human secretory granule proteoglycan peptide core protein. include.

According to the present invention, the sequence of the human secretory granule protein B1 thioglycan peptide core has been genetically engineered. The method for producing the peptide core involves cloning and gene sequences capable of encoding the peptide core. and expression of such gene sequences. As used herein, The phrase "gene sequence" shall mean a nucleic acid molecule (preferably DNA). Ru. The gene sequence that can encode the peptide core of human secretory granule proteoglycans is , derived from a variety of sources. These sources include genomic DNA, cDNA1 synthesis and combinations thereof. Preference for genomic DNA or mRNA A good source is the human promyelocytic leukemia cell line, HL-80.

The genomic DNA of the secretory granule proteoglycan peptide core protein of the present invention is derived from natural It may or may not contain a natural intron. In addition, such a genome D NA is the 5′ of the human secretory granule proteoglycan peptide core protein gene sequence. Promoter region and.

/or a 3' transcription termination region can be provided concomitantly. Furthermore, such a genome DNA is human secretory granule proteoglycan peptide core protein mRNA The gene sequence encoding the 5' untranslated region and/or the 3' untranslated region The coded gene sequence can be obtained concomitantly. The host cell produces mRNA and can recognize transcriptional and/or translational regulatory signals related to and protein expression. 5′ and/or 3′ of the native gene for regulation of transcription and W4 translation. Preserves untranscribed regions and/or 5′ and/or 3′ untranslated regions of mRNA. Can be held and used. Human secretory granule proteoglycan peptide coata Genomic DNA of the protein can be obtained by methods known to those skilled in the art (e.g., Guide to M o1ecular Cloning Techniques, S, L, Be rger et al6. eds, Academic Press (1 987)) from any cell containing human chromosome 10. can be extracted and purified.

Furthermore, human secretory granule proteoglycan peptide core protein mRNA can be obtained from those skilled in the art. Methods known to those skilled in the art (e.g. Guide to Molecular Cloni ng Techniques, S, L, Berger et al, e ds, Academic Press (1987)), human isolated from any cell that produces or expresses secreted granular groteoglycans, c. It can be used to produce DNA. Used RNAX1l products Naturally, Taisetsu protein is isolated from the cells that produce it, or in-house. In vitro, such as sucrose density gradient centrifugation, mRNA preparations can be analyzed for specific sequences. human secretory granules by methods commonly used to enrich them, or both. It is said that mRNA encoding proteoglycan peptide core protein is abundant. It is preferable that

For cloning into a vector, prepare such a suitable NA preparation (human genome DNA or cDNA), respectively, by cutting randomly or enzymatically. the recombinant gene (genomic DNA or cDNA) and ligate it into an appropriate vector. ) Manufacture a library.

human secretory granule proteoglycan peptide core protein or its functional derivatives. The encoding DNA sequence was ligated to plant ends for ligation and sticky ends, restriction enzyme digestion to obtain proper ends, and timely filling of sticky ends; alkaline phosphatase treatment to avoid undesired binding, and appropriate It can be inserted into a DNA vector according to conventional methods, including ligation with enzymes. I can do it. Methods for such operations have been described by Maniat, T., et al. and are known to those skilled in the art.

Contains human granule proteoglycan peptide core protein clone Screening libraries and human secretory granule proteoglycan peptide cores The protein clone was converted into human secretory granule proteoglycan peptide core protein DNA. A method for specifically selecting A, e.g. a) DN A-specific sequence of this protein. b) hybridize with a suitable nucleic acid probe containing the sequence; The natural mRNA that hybridizes to the clone is translated in vitro. Hybridization-selective translation analysis to further characterize translation products, or C) Contains a clone if the cloned gene sequence is capable of expressing mRNA. translated human secretory granule proteoglycan peptide coproduced by the host can be identified by immunoprecipitation of protein products.

A human secretory granule protein that can be used to identify clones for this protein. Oligonucleotide probes specific for the oglycan peptide core protein By knowing the amino acid sequence of the peptide core of a protein, it is possible to design it. Wear. Sequences of amino acid residues in peptides are herein referred to in their commonly used 3-letter notation or its 1-letter notation. these three letters For a list of 1 character display, please refer to Biochemistry, Lehninger , ^,, fourth Publishers, New York, NY (1970). Amino acid sequence is horizontal Where listed, the amine terminus is assumed to be on the left end, the carboxy terminus is assumed to exist at the right end. Amino acid residues in peptides are replaced by hyphens. It can be separated by twisting. These hyphens simply make it easier to understand the representation of the array. The intention is to

Genetic codes are synonymous, so they code for individual amino acids. One or more codons can be used for (Vatson, J. D., I. n: Mo1ecular Biology of the Gene, 3r d Ed, W, A, Benjamin, Inc, Menlo Par K, CA (1977), pp, 356-357). peptide fragment the oligonucleotide encoded by the oligonucleotide with the lowest degree of synonymy. Determine the amino acid sequence. This includes amino acids that are hooded by only one codon. Preferably, this is done by confirming the sequence that has.

Amino acid sequences can sometimes be encoded by only one oligonucleotide sequence. However, amino acid sequences are often determined by any of a set of similar oligonucleotides. can be coded as Importantly, all members of this set share the same peptide contains the oligonucleotide sequence encoding the fragment and thus the peptide fragment. may contain the same oligonucleotide sequence as the gene encoding the fragment, but Only one of the nucleotides in this set is identical to the sequence encoding the exon of this gene. Contains the otide sequence. This one exists in this set and other members of this set The gene encoding the peptide can hybridize to DNA even in the presence of To clone a single oligonucleotide, a set of unfractionated oligonucleotides is It can be used in the same way as oligonucleotides.

When using the genetic code (Watson, J, D, + In: Mo1ecu lar Biology of the Gene, 3rd Ed, T, A. Benjamin, Inc., Menlo Park, CA (197 7)), from the amino acid sequence, each of which hoods human secretory granule proteoglycans. identifying one or more different oligonucleotides that may Can be done. Individual oligonucleotides are virtually identical to actual human segmental granule proteoglytes. What is likely to constitute a Lycan coding sequence is the combination of abnormal base pairs and and the individual codons actually used in eukaryotic cells (coding for individual amino acids) ) can be evaluated by considering the frequency. This “codon usage rule” is L athe, R,, et al, J, Molec, Biol, 183:1- 12 (1985). Lathe's "codon usage rules" ”, the theoretical “ one oligonucleotide sequence containing the most likely nucleotide sequence, or a set of oligonucleotide sequences is identified.

Fragment of human secretory granule proteoglycan peptide core protein gene (or any such oligonucleotide or set of oligonucleotides) A suitable oligonucleotide, or set of oligonucleotides (complementary to Methods known to those skilled in the art (e.g. 5ynthesis and Applicati on of DNA and RIIA, S.A., Narang, ed.

, 1987. Academic Press, San Diego, C Human secretory granules synthesized by A) and cloned by methods known to those skilled in the art. Probes to identify and isolate grain proteoglycan peptide core protein genes It can be used as a block. For nucleic acid hybridization and clone identification Maniatis, T., et al. (in: Molecular CIoning, ALaboratory Manual, Co1d S pring t+arbor Laboratories, Co1d Spr ing Harbor+ NY (19g2no and Hames, B, D,... et al, (In: 1lucreic Acid Hybridizati on, A Practical Approach, IRL Press, Wa Shington, DC (1985)) and herein It constitutes part 1. Next, it was discovered that such hybridization was possible. analyzed these members of the gene library described above to find out what they contain. The extent and extent of human secretory granule proteoglycan peptide core protein coding sequences. and its characteristics.

of the desired human secretory granule proteoglycan peptide core protein DNA coding sequence. To facilitate detection, the DNA probe is labeled with a detectable group. child A detectable group such as is any group that has a detectable physical or chemical property. It can be a substance.

Such materials are well developed and commonly used in the field of nucleic acid hybridization. Any of the many labels useful in this manner may be applied to the present invention. Wear. Radioactive labels such as ``P, ``HSI,'' C%, ``S%,'' 1, etc. Especially useful. Any release that provides adequate signal and has sufficient half-life. Radioactive labels can be used. Oligonucleotides, e.g. Rig by, P, J, L.

et al, J, Mo1. Biol, 113:237 (1977). In the known manner described, by “nick translation” and e.g. Deen, C., et al., Anal, Biochem, 135: 456 (1983) by the T4 DNA polymerase replacement synthesis method described in , can be radioactively labeled.

Alternatively, when labeling with non-radioactive markers such as biotin, enzymes or fluorescent groups , polynucleotides are also useful as nucleic acid hybridization probes. Ru. For example, Leary, J. J., et al., Proc, Natl. Acad, Sci, UsA 80:4045 (1983); Renz, M. , et al, Nucl, Ac1ds, Res, 12:3435 (19 84): and Renz, M., EMBOJ, 6:817 (1983). Please refer.

So, in summary, we have not actually identified the human secretory condylar broteoglycan bebutide sequence. Then, the theoretical “most possible” DNA sequence that could hood such a peptide, and allows identification of a set of such sequences. Oligos complementary to this theoretical sequence by constructing the nucleotides (or the “most possible” oligonucleotide) human secretion (by constructing a set of oligonucleotides complementary to the set of for the identification and isolation of clones containing granule proteoglycan genes. A DNA molecule (or set of DNA molecules) that can function as a probe is obtained.

An alternative method for cloning human secretory granule proteoglycan genes involves cloning expression vectors. DN prepared from cells capable of expressing human secretory granule proteoglycans using A or more preferably by cloning the cDNA into an expression vector. Manufacture the library. This library is then applied to proteins, for example. human secretory granule proteins by screening libraries with antibodies that Screening for members expressing theoglycan peptide core protein do.

In another embodiment, the secretory granule proteoglycan encodes a peptide protein. In order to identify the human cDNA or genomic sequence of cDNA derived from the above-mentioned rat 2 cells is used for pPG-1. in a different species If it is necessary to consider mismatches between expressed sequences, e.g. The synthesized human genomic DNA was nicked under low stringency. Translated pPG-1 or pPGM (pPG-1 of 4 89bp 5spr-3' terminal fragment specific gene), Tantra vahi et al, Proc, Natl, Acad, Sci, US^8 3:9207 (1986).

Therefore, the above method is suitable for human secretory granule proteoglycan peptide core protein or or identify gene sequences that may encode fragments of this protein. Wear. In order to further characterize such gene sequences and produce recombinant proteins, It is desirable to express the proteins encoded by these sequences. This kind of manifestation These proteins express proteins with properties of human secretory granule proteoglycans. Identify clones. These properties include, among others, human secretory granule proteoglycans. specific binding ability to human secretory granules, ability to induce the production of antibodies that can bind to human secretory granules, and receptor cells. It has the ability to impart human division and granule proteoglycan-related functions to cells.

■ Expression of human secretory granule proteoglycans and their functional derivatives in 11 human granules In order to express the grain proteoglycan peptide core protein, a suitable host requires transcription and translation signals that can be recognized by the obtained by the above method a cloned human secretory granule proteoglycan peptide, preferably in double-stranded form, The sequence encoding the core protein is placed in an expression vector to control transcriptional expression. operably linked to a sequence that functions in a host cell, either prokaryotic or eukaryotic. recombinant human secretory granule proteoglycan peptide core protein or or functional derivatives thereof can be produced. Human secretory granule proteoglycan Which strand of the peptide core protein-encoding sequence controls transcriptional expression? Human secretory granule proteoglycans, depending on whether they are functionally linked to Generating antisense RNA of can peptide core protein or a functional derivative thereof. can be made to appear.

By expressing the proteoglycan peptide core protein in different hosts, Post-translational modifications that can change the properties of theoglycans may result in different results. be. The present invention preferably provides human secretory granule proteoglycan peptide core protein or its functional induction It encompasses the expression of the body. Particularly preferred eukaryotic hosts are in vivo or in tissue culture. It is a mammalian cell. Mammalian cells contain recombinant proteoglycan peptide coretan It imparts post-translational modifications to proteins, including those found in natural proteoglycans. Glycosylation and/or folding at the same or similar sites is included. Most preferably, the mammalian host cells include rat-1 fibroblasts, Mast cells derived from mouse bone marrow, mouse obese cells made permanent by bovine Rusten's sarcoma virus normal murine obese cells co-cultured with murine fibroblasts, or mouse fibroblasts. lds et al, “Fibroblasts and lungs producing Kirsten sarcoma virus” Persistence of murine connective tissue-type mast cells at multiple stages of differentiation by co-culturing cells. Hisaka”, J, Biol, Chem, 263: 12783-1279 1 (1988) (see Example 5 below).

Nucleic acid molecules such as DNA contain expression control sequences that contain transcriptional regulatory information. A polypeptide is said to be “capable of being expressed” when it has a sequence that is "operably linked" to a nucleotide sequence encoding a polypeptide. ing.

Functional linkage is such that expression of the sequence is under the influence or control of the regulatory sequence. A linkage that joins a sequence to a regulatory sequence (or sequences). two DNA sequences sequence (e.g., human secretory granule proteoglycan peptide core protein encoding sequence and the sequence of the promoter region linked to the 5” end of this coding sequence) are: Human secretory granule proteoglycan peptide core protein by induction of promoter function When lllRNA of a protein-coding sequence results in being transcribed, and the nature of the bond between two DNA sequences (1) to introduce a frameshift mutation. (2) Human secretory granule proteoglycan mRNA, antisene does not interfere with the ability of the expression control sequences to express the sequence RNA or protein, or (3) Human secretory granule proteoglycans transcribed by promoter region sequences They are said to be functionally linked when they do not interfere with the ability of the template to bind. That is, professional The motor region is used when the promoter is able to transcribe the DNA sequence. operably linked to a DNA sequence.

The exact nature of the regulatory regions required for gene expression depends on the species or cell type at which translation begins. 5° non-transcribed and 5° untranslated (non-coding) sequences involved, respectively, e.g. TATA Contains boxes, cap sequences, CAAT sequences, etc. In particular, 5 °Non-transcriptional control sequences contain transcriptional control sequences for functionally linked genes. This will include a region containing a promoter for.

Expression of the human secretory granule proteoglycant protein in eukaryotic hosts suggests that such control regions, preferably eukaryotic and translational control signals that function in a normal host. Viruses that infect nuclear cells, such as adenovirus, lie papillomavirus, and monkeys It can also be derived from the genome sequences of viruses, herpesviruses, etc. these Regulatory signals bind to specific genes that allow for high level expression in host cells It is preferable to do so.

In eukaryotes, where transcription is not linked to translation, such control regions depends on whether the cloned sequence contains methionine or not. may or may not provide the tar methionine (AUG) codon. It can be anything. Typically, such regions contain RNA in the host cell. A promoter region sufficient to initiate synthesis will be included. Translation possible Promoters derived from heterologous complement mammalian genes encoding the mRNA product are preferred. especially under the condition that it functions as a promoter in the host cell. Strong promoters like those of cutin, collagen, mion, etc. can be used. Preferred eukaryotic promoters include mouse metallothione In I gene promoter rHa+++er, D,, et al,, J, Mo1. Appl, Gen, L: 273-288 (1X 82)]; herpesvirus TK promoter [McKnight, S. , Ce1l 31: 355-365 (1982)l]; S V 40 first phase promoter [Benoist, C,, et al, +Nature (L ondon) 290.3o4-31o(x9g1); in yeast, yeast ga14 Gene promoter [Johnston, S. A., et al. Proc, Nat], Acad, Sci, USA 7X : 6971-6975 (1982); 5ilver, P, A,, et al, , Proc, Natl, Acad, rci, USA 81:5951-5955 (1984)], and the genetics of glucose degradation. Child promoters can also be used.

As is widely known, eukaryotic RNA translation encodes the first methionine. starts at the codon that is Therefore, human secretory granule proteoglycans a DNA sequence encoding a peptide core protein or a functional derivative thereof; The binding between eukaryotic promoters eliminates any intervening codons encoding methionine. It is preferable to ensure that it does not contain. If such a codon is present, fusion Protein production (AUG fudon encodes human secretory granule proteoglycans) in the same reading frame as a DNA sequence) or in the same reading frame as a DNA sequence Futo mutation (sequence in which the AUG codon codes for human secretory granule proteoglycan) is not in the same reading frame).

If desired, a fusion product of the human secretory granule proteoglycan bebutide core protein You can also create one. For example, human secretory granule proteoglycan bebutidophata When a protein-encoding sequence is combined with a sogenic sequence, it can be isolated from a specific host. secretion of a protein, or the ability to fractionate a protein in a particular host. cormorant. such signal sequences with or without specific protease sites. It may be designed to facilitate subsequent removal of the signal peptide sequence.

Alternatively, the protein's natural signal sequence can be used.

repression or activation so that expression of functionally linked genes can be regulated Transcription initiation regulatory signals can be selected that are capable of inducing transcription. to change the temperature Regulatory signals that are temperature sensitive and can therefore inhibit or initiate expression are important and subject to chemical regulation, for example intermediate metabolites. Also, secretory granule broteoglycan peptide protein mRNA and antiserum Both transcribed RNAs are in transcribed form but different promoters or other transcribed It is important that the constructs provided contain regulatory elements and that human secretory granule proteoglycerols are Antisense RNA expression induces mRNA expression of lycanpeptide protein. and/or by inhibition of the human secretory granule proteoglycan bebutidof. Suppression of mRNA expression of protein is carried out by inducing antisense RNA expression. cormorant.

Translation signals are derived from human secretory granule proteoglycan peptide core proteins. This is not necessary when it is desired to express a sense RNA sequence.

If desired, encodes human secretory granule proteoglycan peptide core protein. The 3° untranscribed and/or untranslated region of the sequence is subjected to the cloning method described above. Therefore, it can be obtained. This 3° non-transcribed region is used for its transcription termination regulatory sequence element. It is also possible to retain the 3rd non-red translation region in its translation termination control sequence element. Elements that can be retained for and also polyadenylated in eukaryotic cells It can also be kept for. Naturally expressed Funtrol sequence signal is used by the host When the sequence does not function satisfactorily in this host cell, it is possible to replace it with a sequence that is functional in the host cell. I can do it.

Vectors of the invention may include other operably linked regulatory elements, such as enhancer sequences. , or confer tissue- or cell-type-specific expression on operably linked genes. DNA elements can also be included.

A number of vector systems are available for transforming mammalian cells with DNA constructs of the invention. human secretory granule proteoglycan peptide core protein. Is it desirable to insert the protein DNA construct into the chromosomal DNA of the host cell? , or whether it is desired to exist in extrachromosomal form.

Human secretory granule proteoglycan DNA encoding sequence and functional linkage The promoter is a linear molecule or more preferably capable of autonomous replication. A non-replicating DNA (or RNA) component that is any closed covalent circular molecule that cannot be Human secretory granule proteoglycans when introduced into recipient eukaryotic cells as offspring Expression of the protein will occur by transient expression of the introduced sequences.

In a preferred embodiment, genetically stable transformants are transferred to a vector system or a transformation system. human secretory granule proteoglycan peptide core protein The protein DNA is integrated into the host chromosome.

Such integration can occur naturally within the cell, and is most preferred. In some cases, vectors that functionally insert themselves into the host chromosome are used, e.g. virus vectors, transposons or other DNA elements (DNA into chromosomes) This can also be aided by transformation with A sequences that promote integration of the A sequence. place A vector capable of integrating the desired gene sequence into the mammalian host cell chromosome is used. There is.

Cells that have stably integrated the introduced DNA into their chromosomes carry the expression vector in their chromosomes. also introduce one or more markers that allow selection of host cells containing the Select by. This marker may confer lethal resistance, for example. (resistance to antibiotics, heavy metals like copper, etc.) selection marker remains Genes can be directly linked to the DNA gene sequence to be expressed. , and can also be introduced into the same cell by co-transformation.

In another embodiment, the introduced sequence is a protein capable of replicating autonomously in the recipient host. into a lasmid or viral vector. A wide variety of Any vector like this can be used for this purpose.

Important factors in selecting a particular plasmid or viral vector include: i.e. recognizes recipient cells containing the vector and Ease of selection from non-containing recipient cells: desirable for certain hosts the number of copies of the vector; and the ability to “shuttle” the vector between host cells of different species. The question is whether it is desirable to do so.

Preferred eukaryotic plasmids include bovine papilloma virus, Watanya virus, S those derived from V40 and, in yeast, plasmids containing a 2 micron ring. etc., or derivatives thereof. Such plasmids are not known in the art. It is well known [Botstein, D.

et al, , Miami Wntr, Symp, 19: 265- 274 (1982); Broach, J.R., “she Utan Yo〕L μ standing 1 ogy of pressure rainbow noodle Adan ha Sane Goen 11J 匡匡对Badan” Stripping In heritance'', Co1d Spring Harbor Labor atory, Co1d Spring BarbOrl NY+ p, 445 -470 (1981); Broach, J, R, Ce1l 28: 203-204 (1982)@；B ollon, D, P, et al, J, CI in, He matol, 0nco1.10: 39-48 (198O); Man can be obtained. For example, MSV to express a cloned gene. - using LTR promoter and co-transfected with Hell/f-virus. to amplify the plasmid copy number and insert this plasmid into the chromosome of the host cell. A vector or DNA sequence containing a mammalian construct(s) that can be integrated into a Once prepared for use, this DNA construct(s) can be transferred by a variety of suitable means. The host cell is then introduced into a suitable host cell by any of the following methods:

After introducing the vector, the recipient cells are grown in selective media and then transformed into vector-containing cells. Select for cell growth. human by expressing the cloned gene sequence(s). Secretory granule proteoglycan peptide core protein or fragments of this protein. This results in the production of components. This expression is continuous in transformed cells. or under control, e.g. induction of differentiation of transformed cells (e.g. neuroblastoma cells) (by administration of promothiochloride/uracil) followed by expression of can.

The expressed protein is subjected to conventional conditions such as extraction, precipitation, chromatography, Isolate and purify by affinity chromatography, electrophoresis, etc.

Human secretory granule proteoglycan DNA obtained by the above method was fed. By definition, the sequence is a human granule proteoglycan peptide core protein. sequence encoding the protein and then the human secretory granule proteoglycan peptide. Can be used to obtain core protein antisense RNA gene sequences (This antisense RNA sequence will give the peptide core 1RN (as it is the sequence found on the mirror opposite the strand that transcribes A). Also, this a This is achieved by operably linking the antisense DNA strand to a promoter in an expression vector. Human secretory granule proteoglycerides are produced in transformed cells by transformation with this vector. It is also possible to obtain a host capable of expressing lycan antisense RNA. antisene secreted granule proteoglycans in a highly specific manner using SRNA and its expression. endogenous humans to inhibit or repress transcription or translation of the peptide core gene. secretory granule proteoglycan peptide to interact with core DNA or RNA be able to. Antisense RNA probes to block gene expression The use of In the following description, reference is made to various methods well known to those skilled in the field of immunology. do. Standard references describing the general principles of immunology include Catty, D. [Antibodies, A. Practical Approach , Vol. 1゜IRL Press, Washington, DC (198 -8) Ko; K1ein, J, [Immunology: The 5 science of Ce1l-Noncell Discriminatio n, John Wiley & 5ons, New York (1982)] : K ennett, R et al. [Monoclonal Antibodies , Hybridoma: A New Dimension in Biolo gica solo nalies, Plenum Press, New Y.

1, 13. Burdon, R, et al, eds), El Sevier, Amsterdam (1984); and E. Sen, H, N, [@icrobiology, 3rd Ed, (Davis, B, D, et al, ), @Harp er & Rovi, Ph1ladelphia (1980)]. It will be done.

An antibody is a compound that reacts specifically with a molecule, thereby causing the molecule to bind to the antibody. It is said that molecules can be [combined] when they can be combined. “Epito The term "p" refers to the part of Habten that can be recognized and bound by an antibody. Ru. An antigen can have one or more epitopes. What is “antigen”? , inducing an animal to produce antibodies capable of binding to an epitope of this antigen. It is something that can be done. The above-mentioned specific reaction means that the antigen is expressed in a highly selective manner. Reacts with the corresponding antibody but with many other antibodies induced by other antigens This indicates that the

As used herein, "antibody (Ab) j" or "monoclonal antibody (Mab) J" The term refers to the intact molecule as well as its fragments capable of binding antigen [ For example, Fab and F(ab')2 fragments] . Fab and F (ab') = fragments are Fc fragments of intact antibodies. The non-specific tissue binding of intact antibodies is eliminated from the circulation more rapidly. [Wahlet al., J. Nucl. Med, 24: 316-325 (1983).

Antibodies of the invention are prepared by any of a variety of methods. For example, human secretion Expressing granule proteoglycan peptide coretan 7 protein or its fragments Administering these cells to animals and binding them to human secreted m-grain proteoglycans. The production of serum containing polyclonal antibodies can be induced. good In a preferred method, human secretory granule proteoglycan peptide core protein fragments are chemically synthesized and purified by HPLC to be substantially free of impurities. I'll do my best. Such preparations are then introduced into animals to obtain polypeptides with high specific activity. Produce clonal antiserum.

Monoclonal antibodies can be produced using the hybridoma method. immunization of an animal with a secreted granule proteoglycan antigen. like this Lung cells from a different animal are removed and fused with the appropriate myeloma cell line. any suitable Any suitable myeloma cell line can be used in accordance with the present invention. However, Amer ican Type Culture Co11ection (Rockville Permanent myeloma cell lines (S, P, O) available from Le, Maryland) It is preferable to use After fusion, W a nd s.

J, R, et al. [Gastroenterology 80 2225-232 (1 The obtained hybridoma cells were selected in HAT medium as described in [981)]. selectively maintained and then cloned by limiting dilution. Then, such a selection The hybridoma cells obtained were assayed for human secretory granule proteoglycan anti- Clones that secrete antibodies capable of binding the antigen are identified.

By applying the above method, human secretory granule proteoglycan protein It is possible to obtain another cell line that can produce antibodies that recognize the epitope. can.

for both highly and poorly conserved regions of the human proteoglycan peptide core. Antibodies are involved in the biosynthesis and dysbiosis of human secretory granule proteoglycans in normal and pathological states. This is useful for research on the control of chemical reactions. Furthermore, these antibodies can be used clinically to A pathological condition in which the expression of secretory granule proteoglycan peptide core protein is abnormal. The progress of the condition can be monitored.

The following examples are intended to illustrate the invention and do not limit the scope of the invention. It's not something you do.

Example 1 Dan 1. YO L is allowed N included Gohijin 1 U J2 12'iR:ift & U ball! two-legged two-three 235-promyeloid leukemia cell line HL-60 is a chondroitin sulfate broteog IJ A transformed human that synthesizes can and stores these proteoglycans in one secretory granule. It is a cell. Under certain in vivo conditions, this cell can be transformed into neutrophils, mononuclear cells, and macrophages. phages, eosinophils and basophils can be induced to differentiate into two similar cells. Wear. HL-60 cells (line CCL240: American I Sociny Thai Collection, Rockville, Maryland) (BRL, Gaithersburg, MD) to remove total RNA. C5 of Chevewin et al., Biochemistry 18: 5294 (1979) Purified by CQ density gradient centrifugation technique. Oligo(dT)-Cellulose (Cola Volatile Research, Wiltham, Massachusetts) Chromatography ( Avive, Ecchi, Heilator, He, Blok Natl 2 Acad Suki Ni Poly(A)'' RNA obtained by Nisei 69:1408 (1972)) was DNA (Okakajima, Ecchi, Hiherc, P, Mol Cell Paiol 2:1 61-170 (1982). The obtained cDNA is a T4 DNA polymer. (Biolaps, Beverley, MA), internal EcoR1 site with blunt ends with methylation and cDNA linked to EcoRI poly-linker. there were. 7 by Sepharose CL-4B (Pharmacia) chromatography 500bp cDNA to A option L cD N A to dephosphorylated λgtlQDNA Connected. Ethelicia fu1ba C600Hf1 strain), complexity >lXl0' infected with the resulting recombinant phage obtained in a library containing H.L. −,60 cell-induced cDNA library was incubated at 37°CT[a-”P]dCTP (3 000Ci/+nmol; New England Nuclear, Boston, Masachi hybridization buffer with nick-translated pPG-1 (50% formamide, 5XSSC (0,15M N5cQ715mM citric acid) Sodium), 2x Denhardt's buffer, 0 , 1% F'te'//l/W acid (SDS), 1mM EDTA, 100μ g/ml salmon sperm DNA carrier and 10 mM sodium phosphate).

Filter at 37°C, 1.0Xssc, 0.1% SDS, 1mM EDTA, and 10 mM sodium phosphate, pH 7.0, under low stringency conditions. Rai In Braly, Habo 500. Apply the OOO recombinant to the plate and designate the clone (d esignated) cDNA-84 was isolated (Figure 1). HL-60 thin cD using a cell-induced cDNA library (-50o, ooo recombinant) as a probe. Screened again using NA-H4. Highly strict conditions (55℃ + 0.2xSS C, 1mM EDTA, 0.1% SDS and ], OmM sodium phosphate, pH 7,0) Below, 30 clones hybridized with cDNA-H4 were assembled into a library. Separated from secondary screening.

Individual HL-60 cell-derived cDNAs and their subclonal fragments were converted into M13++ +p18 and M13mp19 (Ammarjam, Arlington Heights, Illinois) ) and aligned both mirrors of Sanger et al., Proceedings A-H4. S -The fencing method is shown in Figure 1.

HL-60i secretory granule proteoglycan peptide core cDNA, common nucleus of A The leotide sequence is shown in Figure 2.

A 249bp EcoRI-)ECORI fragment is the EcRI fragment of cDNA-Hl9. isolated from the oR■ digest. The nucleotide sequence of this fragment (Table 1) is Denylation site (underlined) and poly(A)″ tail (underlined) contain the expected sequence . This fragment contains the genome that harbors this proteoglycan peptide core gene. hybridizes to the fragment and thus probably represents the last 249 bp of the transcript.

cDNA encoding the peptide core of HL-60 secreted granule proteoglycan Common nucleotide sequence at the 3' end (Avraham et al., Proceedings National Le Academy Sub-Science Usconii, 86: 3763-3 767 (1989no.

GAATTCTTM-AGGATrATGC-TTTAATGCTG-TTA TCTATCT-TATTGTTCTT-GAAAATACbT- GCATrTITTG-GTATCATGTT-CAACCAACAT-C ATTATGAM -TTAATTAGAT-TCCCATfGCC- ATAAMTGGC-TTTAAAGMT-ATATATATAT-TTTTA AAGTA-GCTTGAGAAG-CAAATTGGC`- GGTAATATT-CATACCTAAA-TTAAGACTCT-GAC TrGGATT -GTGMTTATA-ATGATATGbC- CCTTTrCTTA-TAAAAAACAAA-AAAAAAAATAA-T implementation Example 2 Five different human/mouse (lines 13C2, 24Bs) , 1711.462TG and 175) and 12 different human/hamster (line 35A2.35A4.35B5.35C1, 35D3.35D5.35E4.3 5F1.35F3.89E5 and 95A4) Somatic cell hybrids with BamHI Disassembled. The resulting fragments were resolved by agarose gel electrophoresis and DNA plotted. The samples were analyzed under high stringency conditions using cDNA-84 as a probe. CD The percent mismatch of the NA-84 probe to each human chromosome is listed in Table H. It was decided that. ○, OO discordant fractions are in HL-60 cells; The secretory granule proteoglycan peptide core gene is located on chromosome 10. and

Table■ Isolation of cDNA-H4 using DNA from human/li dental somatic cell hybrids formula Different human/hamster and human/mouse somatic cells\Eve1 runod cell lines and DNA from the control was analyzed for its hybridization to cDNA-H4, 2-fold formation. did. Column names are +/+, hybrid throat type for cDNA/eDNA-H4. hybridization and staining to -/-1 cDNA-H4. No color bodies, 10/-, hybridization but no chromosomes, and -/ +, No Eve 1 le.

There is no cell formation, but there are chromosomes. For calculation of the unmatched fraction of each chromosome, Divide the sum of the +/- and -/ten columns by the sum of the +/ten, -/-1 tens/- and -/- columns. 1 The 9q+ division may be lysed by leukocytes from two different X/19 translocation carriers. This figure shows the der19 transposed chromosome of the clone derived from No. X and X The q section shows unprocessed X and derX translocated chromosomes. Blance et al. Okem Jiefu) To-17 River 03 1-1059 (1979).

Example 3 Human genome encoding secreted granule proteoglycans and related proteins Identification of the nucleotide sequence of rat 2 cell derived cDNA, pPG-1 (Bo urdon et al.

Proc, Natl, Acad, Sci, USA 82:1322 (1 secretory granules from Bam old digests of human genomic DNA using A genomic fragment encoding the proteoglycan peptide protein was identified.

DNA plots were probed with ppc-1 or pPG-M under high stringency conditions. when probed or probed with pPG-M under low stringency conditions. did not result in any hybridization, but pPG-1 caused low stringency. At least 10 DNA fragments were observed when probed under normal conditions. Ta. The large number of DNA fragments detected were derived from L2 cell proteoglycan peptides. A multilayer protein with a repeat sequence similar to the repeat sequence encoding the glycine-glycine repeat region. - Suggests that the gene family exists in humans.

Example 4 Isolation and characterization of the human secretory granule proteoglycan bebutidofa gene HL-60 cell-induced proteoglycan cDNA, cDNA-H4 subgroup The loan fragment was converted into [α-”P]dCTP (3G (10C4/mmol; DuP ont-New England Nuclear) and nicktra (Maniatis, T., et al., Mo1ecula r Cloning: A Laboratory Manual, Co1d Spring Harbor Laboratory, Co1d Spri ng Hsrbor, New York, pp109-114 (1985) ) or random briming (Feinberg, A.P. et al. , 1na1. Bioche+a 165:224-229 (1983)) specific activity >10”cpm/l19, then EMBL3 human genome library In Braley (Klickstein, L, B, +et al,, J, Exp, Med, 165:1095-1112 (1987)), Plaque/ ~106 recombinants were screened by X hybridization.

Nitrocellulose filter (Millipore, Bedford, Muri at 42°C in 50% formamide, 0.75M NaC6, 75mM citric acid. acid sodium, 5X Denhardt buffer, 0.1% SDS, 1mM EDTA, 100μ9/l salmon sperm DNA carrier and 10IIIM Probed in sodium phosphate. Bring the nitrocellulose filter to 55°C 30mM Nac4.3mM sodium citrate, 0.1% SDS, 1 Washed with mMEDTA and 10mM sodium phosphate (pH 7.0).

Using full-length 650 base pair (bp) HL-60 cell-derived cDNA, cDNA-H4. Several independent clones were obtained. However, the 5″ flanking region of the gene has a good To obtain a representative sample, the 136bp 5° → Kpn I fragment of cDNA-H4 was The human genome library was screened again using the A clone was isolated. A clone's restriction map maps each sample of its DNA to A ccl, BamHI, EcoRI, HindI! I, Kpnl or 5 alI (New England Biolabs, Beverly, MA ). The digested material was electrolyzed in a 1% agarose gel. The separated DNA fragments were separated using a nitran membrane (Schleicher and Schleicher). 5chuell.

Keene, NH) (Southern, E, M,, J, Mo1.Bio 98:503-517 (1975)). The obtained DNAP cDNA-84 specificity 5' (5' → Kpn I and 5° → Xm nT) and 3′ (λmnl→3′ and AceI→3′) fragments. .

Nucleotide sequence analysis of human secretory granule proteoglycan genes Bluescript human genome fragments using dual-enzyme polarized Sigitgan ligation reactions (Stratagene, La Jolla, CA) into a plasmid vector. subcloning, improving recombination efficiency and maintaining subclone orientation (Kurt z, D, T, et al, Gene 13:145-152 (198 0)). Recombinant transformants were identified by colony hybridization and Restriction mapping was performed by a method similar to that used above for the phage clones. Ta. Determination of double-stranded DNA sequences (Sanger, F. et al., Pro c, Natl, Acad, Sci, LISA 74:5463-5467 ( 1977)): Zhang, H, et al, Nuc, Ac1d, R eS, 15:1220-1227ted, 5tates Biochemi eal, C1eveland, OH) and [α-55S]d ATP (1000Ci/mmol: Amersham) was used to prepare the plasmid sample. I went directly to Book Clone. Universal oligonucleotide bly? −( SK, KS, T3. T7 and M13 rev: Stratagen e) to determine the first and last 300 nucleotide sequences in each subclone fragment. Decided on the row. The sense strand (sense 5 strand) and the antisense strand of the genome fragment Based on the nucleotide sequence of the antisense 5 strand, Two oligonucleotides each having a length of 18 nucleotides were Microchemistry Facility, Cambridge.

Applied Biosystems 380 oligonucleotide in MA Synthesized on a synthesizer. Use these oligonucleotides as primers of the next 200-250 nucleotides in each direction of the duplex DNA using The continuous nucleotide sequence was determined. Additional oligonucleotides complementary to different regions of the cDNA Determination of sequences from exons in both directions using oligonucleotides as primers The line was extended. Nucleotide sequence data are The software was installed and edited on an IBM-PC using the Cage software. De database search and mouse secretory granule proteoglycan peptide core gene Comparison of homology with and other genes was performed using the Dana-Farber Cancer In5titude, Boston.

Mo1ecular Biology Computer Re5e located in MA This was done using a computer available at Arch, Re5source.

A human genome library was scanned using a full-length 650 bp cDNA-H4 probe. When cleaned, six independent clones were isolated (λHG-PGI (numbered λ) IG-PC6). There are 6 restrictions on this rack loan map. all clones #) <5' 12 kb EcoRI - + lacking EcoRI fragment , did not hybridize to the 136 bp 5'→Kpn■ fragment of cDNA-H4. Ta. The genomic library was rescreened with the 5' fragment of CDNA H4. At the same time, two additions named λHG-PG7 and λHG-PG8, respectively. A target clone was isolated. Analysis by restriction mapping revealed that clone λHG-PG6 , λHG-PG7 and λHG-PG8 were found to contain overlapping genomic sequences. However, when taken together, the human secretory granule proteoglycan peptide core Contains the full-length gene encoding.

Detailed restriction mapping of these subclones revealed that this gene It was revealed that it was 5 kb and had 3 exons. 1st and 2nd exercise There is an intron of about 8 kb between the exons 2 and 3, and about 6 kb between the exons 2 and 3. kb intron is present. Both introns have the nucleotide sequence “GTAAG” It begins and ends with the array "CAG". Analysis of the nucleotide sequence of this gene According to encodes a hydrophobic signal peptide. Exon 2 is 49 of the peptide core The amino acid portion (amino acid residues 28 to 76) is covered by a hydrophobic signal. It is believed that the peptide becomes the N-terminus of the molecule after being removed in the endoplasmic reticulum. workman Exon 3 (634 bp) is the largest exon and the remaining 82 exons of the translated molecule. It encodes amino acids and the full-length 3' untranslated region of the mRNA transcript. The above 82 amino acids are , a 17 amino acid sequence (residues 77-93), which is serine-glycine rich glycosaminoglycan addition region, 18 amino acid serine-glycine enriched region (remaining groups 94-111) and the C-terminus of the translated molecule (residues 112-158).

Determination of the transcription initiation site of the human secretory granule proteoglycan peptide core gene S1 nuclease mapping analysis revealed secreted granule proteoglycerides in HL-60 cells. We identified the transcription initiation site of the human gene encoding the peptide core of Lycan. . The 4kb SalT→old ndIIl fragment of genomic clone λHG-PC7 is blue. Script (named pB5sH3). oligonucleotide (5°→ACTGCATTTGAGTAGCTT→3゛) is the cDNA-H4 The antisense strand corresponding to residues 39-59 was synthesized. This oligonuc Ion g of leotide was hybridized to 4 μ2 of alkaline denatured pB5sH3. , similar to the above except that the complementary strand of DNA was labeled with [α-”P]dATP. Synthesized under the following conditions. A 400bp antisense DNA probe was added to a synthetic 8M tube. Separated by electrophoresis on a Rare/8% polyacrylamide gel. single strand radiolabel DNA fragments were identified by autoradiography, electroeluted, and ethanol precipitated. I let it happen. 50,000 epm of a standard radiolabeled DNA fragment was introduced into HL-60 cells. Total RNA (Chirgin, J. M. et al., Biochemis try18:5294-5299 (1979)) ~15μ9 or HL-60 se Le-induced poly(A)’RNA (Aviv, H, et al, Proc, N atl, Acad, Sci, USA 69:1408-1412 (1972) ) 1 H9, 80% formamide, 400mM NaCl2, ImMEDT A and 40mM Pipes (pH 6,4) at 48°C for 16 hours/ ＼Ibridize. 3” P-DNA/RNA hybrid with Sl nuclease (Pharmacia) and incubated for 60 minutes with 100 units. After the reaction is complete, remove the standard sample. It was extracted with phenol and precipitated with ethanol at -80°C. ImMEDTA and 1 0mM Dingris-H (J! (pl’18.o) 3uQ and formamide draw 4 uQ of ding buffer was added to the precipitated preparation.

Boil the preparation and place it on an 8M urea/8% polyacrylamide sequencing gel. , 3ffp labeled p B R322 (New England BiolabS) of pBSH3 primed with the same oligonucleotide as the MS p I digest of Loaded with a sequencing ladder. against two negative controls. , S1 nuclease reaction is tRNA (Bethesda Re5earch Labs) or MBBC (Razin, E. et at., J. Biol. Chet 257:7229-7236 (19g2)) All RN A L5u9 were done in parallel.

HL-60 cell-derived total RNA A poly(A)” RNA contains the ~82 nucleotide of the probe. Otide was protected from degradation by Sl nuclease.

Therefore, the putative transcription initiation site of HL-60 cells for this gene is It was concluded that it was located 53 bp upstream of the site. 1P sign 5 “Anti” The sense 400 bp DNA fragment was tR It is not protected when cultured with NA or mouse mast cell RNA. HL-60 This putative transcription initiation site in cells is associated with 8MMC-induced mast cells and basophilic cells. Ukemia Cell (Bourdon, M, A, et al, Mo1.Ce1 Biol, 7:33-40 (1987)), but is expressed in two egg yolks in rats. Bursal malignant cells (Bourdon, M, ^, et al, Mo1. Ce1l Biol, 7:33-40 (1987)). Corresponds to the putative transcription start site.

Figure 3 is a restriction map of human genes. Figure 4 shows flanking and intron sequences Gene encoding human secretory granule proteoglycan peptide core protein containing The nucleotide sequence of

Cloning of mouse genes Contains the gene encoding the mouse secretory granule proteoglycan peptide core protein. A 15 kb mouse genome fragment containing mouse secretory granule proteolytic proteins was isolated using the method described above. BMMC cDNA (cDNA-M6) encoding the glycan peptide core [α, sap] dCTP-labeled 450bp Ace I → 3′ gene-specific fragment By using 5au3AI digest of BAL B/C7 mouse liver DNA (A vraham, S.

et al, Proc, Natl, Acad, Sci, USA 86:3 763-3767 (1989)). It was cloned by screening. Nucleotide of this gene The code sequence and deduced amino acid sequence are shown in FIG.

Either human or mouse genes have ~30 bp amplification at their transcription start site. Classic TATA box (Breathnach, R, e tal, Ann, Rev. Biochem, 50:349-383 (1 981)) or GC-enriched elements (Sehgal, et al. Mo1. Ce11. Biol, 8:3160-3187 (1988)). I haven't done it. A secreted granule proteoglycan peptide expressed in hematopoietic cells. The docore gene appears to have an abnormal promoter. other human pros Regarding the 5° flanking region for the theoglycan peptide core gene, Genes encoding other proteoglycan peptide cores have not been described so far. No comparison could be made with the child. However, some known ci for other genes Human and mouse secretory granule proteoglycans matching s-acting restriction elements. There are some nucleotide sequences within these 5' flanking regions of the petidocore gene. exist. Importantly, the human (residues -1 to -119) gene and mouse (residues -119) 119 bp nucleotide sequence of the transcription start site of the base-1 to -125) gene The discovery is that 96% of the nucleotides present upstream are identical. Ru.

This degree of identity is similar to that of other 119 bp regions within the exons of the genes of these two species. It is thicker (exceeding) the degree obtained when comparing the Suggestions that a limiting element exists in this conserved nucleotide region It is.

Example 5 by the mouse secreted proteoglycan peptide core gene in rat-1 fibroblasts. Transfect Fisher rat-1 fibroblasts with 10% fetal bovine blood. liquid, 2ml L-glutamine, penicillin 100U/x (i and strept Dulbecco's modified basal medium (DMEM) supplemented with 100 μg/xQ of mycin The cells were grown at 37°C in a 5% CO, a-air atmosphere.

DNA transfections were performed essentially as previously described. (Southern, P, J, et al, J, Mo1. Appln, G en1:327-341 (1982)). Simply put, a 3-4X10' rack To-1 fibroblasts and mouse genome clone λMG-PG 1 and selectivity for 12-24 hours before transfection with car psv2 neo. Placed in 10crA plastic culture dishes containing DMEM.

In the presence of air bubbles, containing λMG-PGI DNA5u9 and psv2netO,5u9 250mM solution of calcium phosphate 0. SzQ 280+nM NaCQ, 10mM KCf2.12 + nM dextrose, 1.5mM sodium phosphate and and 0.5xl of 5h + M HE P E S (pH 7,1)! drip onto and a calcium phosphate/DNA precipitate is generated. After incubation for 30 minutes The resulting precipitate was added to a fibroblast culture dish, and after 10 to 18 hours, DNA precipitation was performed. The precipitate was removed. Wash the transfected cells twice with growth medium and After 24 hours of collection, the cells were treated with trypsin and split at a ratio of 1:6.

The resulting fibroblasts were placed in a new loam plastic dish and treated with gentamicin ( Gibco) 500 μ9/xQ in DMEM for 2 to 3 weeks. Culture medium Pick up gentamicin-resistant colonies of cells individually in a cloning cylinder. and grown as a cell line in a medium containing 100 μg/l1lI2 gentamicin. I let it grow.

Rat-1 fibroblasts transfected with mouse proteoglycan genes Cellular RNA and DNA plot analysis Total RNA was analyzed using guanidinium thiocyanate. (Chirgin, J.

M, et al., Biochemistry 18:5294-5299 ( 1979); G11sin, V, etal, Biochemistr y13:2633-2637 (1974)), Mouse BMMC, Rat from To1 fibroblasts and transfected rat-1 fibroblasts. Prepared. RNA (5 μ9/lane) was analyzed on a 1% formaldehyde-agarose gel. Electrophoresis was carried out using zetabind (Tho+nas, P, S, Proc, Na tl, ^cad, sci, USA 77:5201-5205 (1980)) was moved to. The resulting RNA plot was plotted with radiolabeled ACcI of cDNA-M6 → 3' fragment in a hybridization bath at 42°C for 24 hours. It was cultured in Professional.

The plates were washed under high stringency conditions and autoradiography was performed.

Remove the proteoglycan peptide core probe from the plot by high temperature washing and remove the proteoglycan peptide core probe from the plot. Reprobe the cells with actin cDNA and quantify the mRNA loaded in each lane. did.

Transfection of DNA into mouse liver, rat liver, rat)-1 fibroblast cells and B11n, N, et al. isolated from isolated rat-1 fibroblasts. hcleic Ac1ds Res, 3:2303-230111 (1976) ), the standard specimens are respectively Xmn I SBamHI SBgl II, Ssp I 5Sau3A　I, HindI[I or Ecor　I for 4 hours at 37°C (10 μg/digestion). The fragments were analyzed by agarose gel electrophoresis. under degrading conditions using the AceI→3' fragment of mouse cDNA-M6 as a probe. Hybridization analysis was performed.

Mouse secretory granule proteins in transfected rat-1 fibroblasts. Expression of mouse genes encoding the peptide core of rotoglycans λMG-PCI contains its promoter region and contains all mouse secretory granule proteoglycans. Contains the can peptide core gene and that this mouse genome clone To demonstrate that it is expressed in other mammalian cells, rat-1 fibroblasts were λMG-, PGl and the dominant neo encoded by plasmid psv2 neo co-transfected with a resistance-selective marker. neo resistance transformer Seventeen independent clones of transfected rat-1 fibroblasts were isolated and Both were extended.

Total RNA was extracted from BMMC, neotransfected rat-1 fibroblasts and and co-transfected rat-1 fibroblast cell line. Ta.

Gene-specific secretory granule proteoglycan peptide core probes are non-trans hybridize to transcripts in RNA plots of infected fibroblasts. I couldn't do it. However, in mouse BMMC and co-transfection 1.0 kb RNA in two of the treated rat-1 fibroblast cell lines. We were able to hybridize. Transfected fibroblasts? We performed brimer extension analysis using RNA from The decision was made. RNA from transfected cells was used as an RNA template. When used as -100 nucleotides in length A DNA product with a high quality was obtained.

Another primer corresponding to residues 39-59 of cDNA-M6 was added.

When used in Sei, a -60 nucleotide DNA product was obtained.

The above two types of rat-1 fibroblasts transfected with genomic DNA Created from a clone and digested with BglIr, Xmn1.5alI or BamHI did. The DNA plot of the digested material was These transfected rat-1 fibroblasts were probed with isomeric fragments. Proof that the cell line is included in the mouse proteoglycan genome sequence. I made it clear. Mouse proteoglycan probe was obtained by BglII digestion of murine DNA. 2.7 kb fragments present in the liver DNA and bar 1 fibroblasts hybridized to a 7.5kb fragment present in the BglI digest of both cell DNAs. I lost it. Transfected fibroblasts have 2.7kb and 7.5k bD N There is a lot going on with cells. Mouse liver DNA and fiber with equal amounts of gene-specific probes BglI of blast cell DNA [hybridized to the 2.7 kb fragment present in the digested material] Based on the relative intensity of cell lines, each fibroblast cell line has a population of 10 to 20 cells. and two to three mouse secretory granule proteoglycan peptide core genes. It can be incorporated into Nomu.

Transfection with defrin in Chinese hamster ovary cells A peptide copolymer of fibroblast-induced dermatan sulfate broteogsocan called (Yamaguchi, Y, e tal, Nature 336:244-246 (198g)). Also , in CO5-7 cells, T cell induction of invariant chain proteoglycans associated with Ia. carried out by a cDNA encoding the peptide core (Miller, J. et al, Proc, Natl.

^cad, sci, USA 85:1359-1363 (1988)). deer However, using a genomic clone containing the entire proteoglycan peptide core gene, Transfection has not been reported to date.

Example 6 An amino acid that recognizes the natural HL-60 secreted granule proteoglycan peptide core protein Production of antibodies against peptides with amino acid consensus sequences 16 amino acid peptide 02 [Ser-Asn-Lys-11e-Pro-Ar g-Leu-Arg-Thr-Asp-Leu-Phe-Pro-Lys-Th r-Arg was chemically synthesized and bonded to hemocyanin, and New Zealand Injected into White Rabbit. This peptide corresponds to residues 64-79 of the translated molecule and the core region that precedes the serine-glycine-rich gutsosaminoglycan addition region. It is.

Antibody that specifically recognizes the peptide core protein of human secretory granule proteoglycans The induction of was tested as follows. Peptide (3jI9) in 0.25% glue Presence of curlaldehyde in calf-whole limpet hemocyanin (Sigma) Combine polyclonal antibodies with 5xg in Zealand White Rabbit. Bound peptides were raised using standard immunization techniques. Antibody titer in whole serum is enzyme It was measured using a linked immunosorbent assay (ELISA).

Each microtiter well contains 1 μl of synthetic peptide in phosphate-buffered saline. 9 and cultured overnight at 4°C. Remaining protein binding sites in the wells were reduced to 1% (V /V) Float by incubation with bovine serum albumin (Sigma) for 1 hour. After cooling, the wells were diluted with phosphate buffer containing 1% (w/v) Tveen20. - Washed with saline solution. Rabbit serially diluted in phosphate buffered saline Add serum and add horseradish peroxidase-conjugated goat anti-rabbit IgG ( Bio-rad, RicbIIlond, CA) was added. then the well 2,2'-azinodi[3-ethylbenzthiazine sulfonate co-dye (B oehringer-Mannheim, 1ndianaPolis, IN) In addition, it was analyzed using a colorimetric method. For predicted amino acid residues 24-39 of cDNA-84 The corresponding peptide 01 (Ser-Val-Gin-Gly-Tyr-Pro-Th r-GIn-Arg-Ala-Arg-Tyr-G1n-Trp-1al-Ar g) was synthesized and used in ELISA to confirm the specificity of rabbit serum. anti - Peptide IgG was analyzed by ammonium sulfate precipitation and ion exchange chromatography. Partially purified by

Anti-peptide IgG (-30 μg) was added to protein A-Sepharose beads ( Sigma) with 100 μa of a 15% (w/v) suspension in RIPA buffer. The cells were incubated for 1 hour at room temperature. Obtained protein A-Sepharose-1gG? j[combination body with protein A-Sepharose for 24 hours, then protein A-Sepharose. [''S Comethionine-labeled or sulfate-labeled HL-50 cells were added per 5 x 10” cells. RIPA cell decomposition product containing 1xQ amount was added.

Protein A-Sepharose/Antipeptide-18-24 hours at 4°C with 1gG After incubation, the beads were incubated with 10 mM unlabeled methionine or unlabeled sodium sulfate. Contains 0.1% bovine serum albumin, 0.5% Tween 20 and 10mM Phosphate. 3 times by centrifugation in phase buffered saline (pH 7.2). Washed thoroughly. Suspend the beads in 60 μl of Lae+u+1 i buffer, Bound radiolabeled antigen was eluted by incubation for 5 minutes at 5°C. eluate Electrophoresed in 15% 5DS-PAC, E-gel, Fumassy Brilliant Blue autoradiographed using Kodak XAR-5 film. turned into

In ELISA J5, the antiserum gave half-maximal binding at approximately 500-fold dilution. (Figure 6). Antipeptide 02 serum contains predicted amino acid residue 2 of the same cDNA. Peptide 01 corresponding to peptides 4 to 39 could not be recognized (Fig. 6). Before Immune serum was also unable to react with bound peptide 02. Antiserum 1μ g was preincubated with 1μ9 of peptide 02 for 60 min at 25°C. At that time, no immunoreactivity was observed in ELISA.

Using the IgG-enriched fraction of Antipeptide 02 serum, Protein A-Seph Wallose-binding antibodies recognize early translational secretory granule peptide cores and mature proteoglycans. I decided whether or not I should know. Standout 20. OOOMr protein [SSS Comechi] It was specifically immunoprecipitated from a 2-minute lysate of onin-labeled HL-60 cells, and a slight amount of gel was detected. specific immunoprecipitation. After 10 min pulse and 5 min chase, 20 ．． 000 Mr[”S]methionine-labeled proteins were less obvious, but macromolecular has increased somewhat. [ISs]Methionine-labeled macromolecules induce cells to p[sl sulfate. After overnight radiolabeling with acid salts, precipitated [”s]sulfate-labeled proteoglycans were It was the correct size. Shen Yi to protein - Sepharose immune 1gG was inhibited by pre-incubation with 1 μ9 of synthetic peptide 01. Anti-Peptide 02 antibody is a precursor to mature HL-60 cellbroteoglycans. It was concluded that the peptide core recognizes the peptide core.

As shown in Figure 7, the thickness of the immunoprecipitated peptide core protein is approximately 13,000 mm. Dalton, and the peptide core has lost its 27 amino acid signal peptide. 5tevens et al, J, Biol, Chem, 263 Near 27g (1988).

Example 7 Separation of proteoglycan proteins chromatography, gel electrophoresis, affinity chromatography, or Isolation using standard protein separation techniques in this field, such as in vitro immunoextraction techniques. can be released. For example, this proteoglycan can be extracted by the following procedure. (Stevens, R.L., et al., Journal Biological Research) Mistry, 260:14194-14200 (1985)).

The BMMC pellet was treated with 1% Zvitzurgent 3-12 containing protease inhibitors. CsC] (density 1 ．． Add 2.35 ml of 4M guanidine HCl (GnHCl) containing 4g/ml) do. These detergent-GnHCl proteoglycan extracts were then In a typical experiment, 48 ml of extract is obtained from approximately 2X 10” BMMC. , among which 3X10' are radioactively labeled. The collected extracts were kept at 17°C. Centrifuged at 195,000x for 48 hours, the gradient eluate was It was divided into two equal fractions, designated D, (lower part) and R, (upper part). Cs C] Gradient fractionation or subsequent ion exchange or gel filtration chromatography The fundroitin sulfate E proteoglycan moiety in the fraction from Fluor (Hydrofluor) 12. Test each fraction during 5m I. Increasing the amount of liquid scintillation, TRACOL ANALYTIC MARK III liquid scintillation Establishment of 358 and 3H radioactivity in radiolabel-proteoglycans using a radiolabel counter It was measured by For protein, bovine serum albumin was used as a standard substance. or by optical density at 28 Qnm.

Nucleic acids were measured at a wavelength of 260 nm. The bottom fraction of each CsC1 gradient is 5 0000 Mr-blocked dialysis tube system and 24 hours at 4°C against 1M NacI. Dialysis for an additional 24 hours followed by 1M urea containing Tris-HCl, pH 7.3. Dialyze against. The dialysate was adjusted to 4M in urea by adding solid urea. DE equilibrated in 4M urea, 0.05M Tris-HCl, pH 7,8 Applied to a 0.8×29 Cm column of AE-52. Ion exchange column is 4M urea , washed with 35 ml of 0.05 M Tris-HCl, pH 7,8, and washed with fundroitin. E sulfate proteoglycan was incubated with NaC+ (0- Elution was carried out using a linear gradient of 80ml (0.1M). Collect 2 ml of fractions, Proteoglycan-rich fractions were treated with either 5S or sH radioactivity. Observe a portion of the fraction and search for pre-labeled cells. Collected, dialyzed against 0°IMNH, HCO, at 4°C for 48 hours, and lyophilized. Ta. This material was mixed with 0.4M GnHCIlo, 1M sodium sulfate, 10.1m Redissolve the Sepharofluoride in the same buffer in 100 μm of S-HCl, pH 7.0. CL-4B 0.6X100cmO column, 1.5ml/hour from the column. It was eluted at a flow rate between. Collect 5 ml of fraction Q, radioactivity and 280 Absorption at nm was measured. Collect the proteoglycan-containing fractions, IMNH.

Dialyzed against HCO3 and lyophilized.

Example 8 40, American Type Culture Collection, Bethesda, Maryland) enriched medium [fetal bovine serum 10 (V/V)%, jmML-glutamine, Q, 1 m M non-essential amino acids, penicillin 100 U/ml and streptomycin 100 Medium RPMI-1640 medium (Gibco, Grand Island) supplemented with μg/ml The cells were cultured at 37°C in a humidified atmosphere with 0.5% CO. .

For [”S]methionine labeling, HL-60 cells were prepared from methionine-free, dialyzed cows. Breincubate for 10 minutes at a concentration of 107 cells/ml in fetal serum-enriched medium. did. Then [18S]methionine (129Ci/mmol; Amersham, Approximately 500 μCi/ml (Arlington, Illinois) was added. HL-6 0 cells were further incubated at 37°C for 2-10 minutes and incubated with cooling for 10 minutes. Centrifuged at 2°Xg and washed in enriched medium at 4°C. pulse-chase In experiments, HL-5Q cells were labeled with [”S]methionine for 10 minutes and Wash as above and resuspend in normal enrichment medium for an additional 5 minutes at 37°C. Ta. [”S]] 5xlQ’ part of methionine labeled HL-60se was buffered with RIPA. liquid [0,15M NaC], 1% dioxycholate, 1% Nonident P-40, 1% SDS, 10mM N-ethylmaleimide, 2mM phenylethylbutylene Ruphonyl, 10mM NaF and O, IM) Lis-MCI, pH'7゜2] The immunoprecipitate of the lysate was dissolved in 5DS-polyacrylamide as described below. Analysis was performed by dog gel electrophoresis (SDS-PAGE).

[35S HL-60 cell for Sl sulfur travel] (-4000Ci/mmol; DuPont-New England Nuclear, Boss IX 10' in enriched medium containing 50 μCi/ml Incubate for 1 hour at a density of cells/ml or 18 hours at a density of 2X10" cells/ml. Cubated. Centrifuge the release-resistant labeled cells at 120Xg for 10 minutes at 4°C. chondroitin sulfate A (Miles Scientific, Neighborville, Inc.) Linoi) 100 μg and heparin 100 μg (Sigma, St. Louis, Mizzou) li) 1% Zvittagendt 3-12 (containing glycosaminoglycan carrier) Add 150 μl of ``Lupiochem, San Diego, California'') to each cell pellet. and then 1.35 ml of 4MGnHCl, O, 1M sodium sulfate and O, 1M Tris-MCI was added. Sephadec samples of each lysate and supernatant. G-25/PD-10 column (Pharmacia, Piscataway, Newja - chromatography) to convert ["Sl sulfate] into a polymeric substance. Uptake was quantified.

[“For the isolation of Sl sulfa-travel HL-60 cell proteoglycans, Solid CsCI was added to the remainder of the cell lysate to a final density of 100 g/ml.

- After centrifugation at 100,000Xg for 48 hours, the bottom 33% of each CsCl gradient was dialyzed against 0.5M sodium acetate for 24 hours, and further diluted with O, 1M hydrogen carbonate. Dialyzed against ammonium for 24 hours. The dialysate was lyophilized and reconstituted in 0.4 ml of water. It was completely dissolved. Some parts were cut off [! 118] Sulfur label-proteoglycan sample for 30 minutes in the presence or absence of 10 ug of Pronese (Calbiochem). The digestate was incubated with 4M GnHC+, 0.1M sodium sulfate. Sepharose equilibrated with Lithium, Q, IM) Lis-HCl, pH 7.2. It was applied to a 0.8×85 cm column of CL-6B (Pharmacia). as a control ['a%S]Sulfur-labeled chondrosarcoma proteoglycans The samples were simultaneously analyzed for sensitivity to Bronese.

Pronase-sensitive fundrosarcoma proteoglycans are extracellular matrix proteins. , distinguished from secretory granule proteoglycans.

After pronase treatment, H160 cells [sSS]] Sulfur Travel Proteo No substantial changes in the hydrodynamic magnitude of the Glyca hawk were observed, whereas in the rat [ ``Sl sulfa-travel chondrosarcoma proteoglycans are susceptible to degradation. won. These results indicate that proteoglycans resist pronase digestion. ing.

Although the present invention has been described in detail and in connection with specific embodiments, the gist of the invention and It is understood that various changes and modifications may be made to the invention without departing from its scope. It is obvious to those skilled in the art.

FIG. FIG.2 ~to #C) IL (490nm) FIG, 1A FJG, “lB international search report ++11#rRIlt61111^a@hleasy”””orT/niQ/n1OR +PCT10S89103051

Claims (26)

[Claims] 1. Substantially pure human secretory granule proteoglycan peptide core protein or derivatives or fragments thereof. 2. The peptide core protein is linked to a gene present on chromosome 10 of HL-60 cells. The substantially pure human secretory granule proteoglycan of claim 1 encoded by Peptide core protein. 3. Claim that human proteoglycan peptide core protein is synthesized in a biological system Substantially pure human proteoglycan peptide core protein of item 1. 4. 4. The substantially pure human protein of claim 3, wherein said biological system is a cultured mammalian cell. Theoglycan peptide core protein. 5. The peptide core protein is transcribed from recombinant DNA. 5. The substantially pure human protein of claim 4, which is translated from mRNA of P. a. Oglycan peptide core protein. 6. The protein (i) 18 amino acid glycosaminoids consisting essentially of alternating serine and glycine Contains Noglycan attachment area (ii) is present in its natural form in secretory granules in effect cells of the immune system; and (iii) the substantially pure substance of claim 1 or 2 which is resistant to proteolytic degradation. human proteoglycan peptide core protein. 7. The fruit of claim 6, wherein the gene sequence encoding said protein consists of recombinant DNA. Qualitatively pure human proteoglycan peptide core protein. 8. Substantially pure human secreted proteoglycan peptide core protein DNA or is its fragment. 9. The sequence of the DNA is [There is an array] The substantially pure human secreted proteoglycan peptide core protein of claim 8, which is DNA or its fragments. 10. Coating the peptide core protein of the human secretory granule proteoglycan of claim 1. A recombinant DNA molecule consisting of a nucleic acid sequence containing a code. 11. 11. The recombinant DNA of claim 10, wherein said molecule further comprises a cloning vector. molecule. 12. 12. The recombinant DNA molecule of claim 11, wherein said vector is capable of expressing said DNA molecule. 13. Claim that said vector is capable of expressing antisense RNA to said DNA molecule Recombinant DNA molecule or fragment thereof according to item 11. 14. A host cell transformed with the engineered DNA molecule of claim 10. 15. 15. The host cell of claim 14, wherein said cell is a mammalian cell. 16. 16. The host cell of claim 15, wherein said mammalian cell is a mouse mast cell. 17. (i) Encoding the proteoglycan peptide core protein of claim 1; (ii) transforming a host with the molecule; exchange, (iii) converting the sequence of the proteoglycan peptide core protein of the DNA molecule into the host protein; manifest in the Lord, and (iv) human proteoglycan peptide core protein produced by the expression A method for producing human proteoglycan peptide core protein, which comprises separating . 18. The human proteoglycan according to claim 17, wherein the host is a mammalian cell. Manufacturing method. 19. Peptide with formula: [has sequence]. 20. Peptide with formula: [has sequence]. 21. Peptide with formula: [has sequence]. 22. An antibody capable of binding to a human secreted proteoglycan or a fragment thereof. 23. Claim 22 capable of binding to human secreted proteoglycan peptide core protein. antibodies. 24. The antibody is attracted to the peptide of claim 19, 20 or 21. 23. The antibody according to claim 22, which is a raised antibody. 25. 23. The antibody of claim 22, wherein said antibody is a polyclonal antibody. 26. 23. The antibody of claim 22, wherein said antibody is a monoclonal antibody.