MXPA97005366A - A novelty chemiocine expressed in fetal spleen, its production and a - Google Patents
A novelty chemiocine expressed in fetal spleen, its production and aInfo
- Publication number
- MXPA97005366A MXPA97005366A MXPA/A/1997/005366A MX9705366A MXPA97005366A MX PA97005366 A MXPA97005366 A MX PA97005366A MX 9705366 A MX9705366 A MX 9705366A MX PA97005366 A MXPA97005366 A MX PA97005366A
- Authority
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- Mexico
- Prior art keywords
- fsec
- nucleotide
- sequence
- seq
- nucleic acid
- Prior art date
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Abstract
The present invention provides nucleotide and amino acid sequences that identify and encode a novel chemokine (FSEC) that is initially found in human fetal spleen cells. The present invention also provides anti-sense molecules to the nucleotide sequences encoding FSEC, expression vectors for purified FSEC production, antibodies capable of binding to FSEC specifically, hybridization probes or oligonucleotides for the detection of nucleotide sequences encoding to FSEC, host cells genetically designed for the expression of FSEC, diagnostic tests for chemokine activation based on the nucleic acid molecules encoding the FSEC and antibodies capable of binding specifically to FS
Description
A NOVELESS OXYMYCINE EXPRESSED IN FETAL SPLEEN, ITS PRODUCTION AND USES
BACKGROUND TECHNIQUES Quinocines are a family of cytokines that are produced when the immune system responds to non-self antigens, such as invading microorganisms or antigens of an incompatible tissue type, and are associated with the trafficking of leukocytes under abnormal conditions. inflammatory, or diseased. Chemokines mediate the expression of particular adhesion molecules in endothelial cells, and generate gradients of chemoattractant factors, which activate specific cell types. In addition, chemokines stimulate the proliferation of epiploid types of rolulnr, and rpqul .... "I., activation of cells that carry specific receptors.I? SU a¡; activities demonstrate a high degree of target cell specificity Chemokines are small polypeptides, generally about 70-100 amino acids in length, 8-11 kD molecular weight and active over a concentration range of 1-100 nanograms / milliliter, initially isolated and purified from tissues inflamed and characterized in relation to its bioactivity More recently, chemokines have been discovered through molecular cloning techniques and have been characterized by structural as well as functional analysis Chemokines are related through a four-cysteine motif, which is based mainly on the spacing of the first two cysteine residues in the mature molecule. are assigned to one of two families, C-C chemokines. { a) and J chemokines C-X-C (ß). Although there are exceptions, C-X-C chemokines generally activate neutrophils and fibroblasts, whereas C-C chemokines act on a more diverse group of target cells that include monocytes / macrophages, basophils, eosinophils, T lymphocytes, and others. The known chemokines of both families are synthesized by many different types of cells, as reviewed in Thomson ?. (1 04) Tho Cytokinn llandbook,? Edition, Academic Press, New York City. The two groups of chemokines are described in turn. The C-C chemokines appear to have less N-terminal processing than the C-X-C chemokines. Known human and / or murine C-C chemokines include MIP-a and β; 1-309; RANTES and MCP-1. Inflammatory proteins of macrophage alpha and beta (MIP-IY and β) were first purified from the stimulated mouse macrophage cell line, and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allelic genes encode human MlP, and seven different genes encode MlP-lß. MlP-la and MlP-lß consist of 68-69 amino acids that are approximately 70 percent identical in their mature acid secreted forms. Both are expressed in T cells, B cells and monocytes stimulated in response to mitogens, anti-CD3 and endotoxin, and both polypeptides bind heparin. Although both molecules stimulate monocytes, MIP-lv chemoattracts the CD-8 subset of T lymphocytes and eosinophils, while MlP-lβ chemoattracts to the CD-4 subset of T lymphocytes. In the mouse, these proteins are known to stimulate myelopoiesis 1-309 was cloned from a human T cell line and, and shows 42% amino acid identity with the T 3 cell activation (TC? 3) gene cloned from the mouse. There is considerable nucleotide homology between the 5 'flanking regions of these two proteins, and they share an extra pair of cysteine residues not found in other chemokines. These similarities suggest that 1-309 and TCA3 are homologous species that have diverged over time in both sequence and function. RANTES is another C-C chemokine that is expressed in T cells (but not in B cells), in platelets, in some tumor cell lines, and stimulated rheumatoid synovial fibroblasts. In the latter, it is regulated by interleukins-1 and -4, transforming the nervous factor and the interferon-? . The cDNA cloned from the T cells encodes an 8 kD basic protein that lacks N-linkage glycosylation and can affect lymphocytes, monocytes, basophils, and eosinophils. The expression of RANTES mRNA is substantially reduced following the stimulation of T cells. The monocyte chemoattractant protein (MCP-1) is a protein of 76 amino acids that seems to be expressed in almost all cells and tissues when stimulated by a variety of agents. However, the objectives of MCP-1 are limited to monocytes and basophils, where it induces a flow of CP-1 receptor: calcium bound with protein G (Charo I, personal communication). Two other related proteins (MCP-2 and MCP-3) were purified from a human osteosarcoma cell line. CP-2 and MCP-3 have an amino acid identity of 62 percent and 73 percent, respectively, with MCP-1, and share their chemoattractant specificity for monocytes. The chemokine molecules have been reviewed in
Schall TJ (1994) Chemotactic Cytokines: Targets for Therapeutic Development. International Business Communications, Southborough, MA, pages 180-270; and in Paul WE (1993) Fundamental Immunology, Raven Press, New York City (NYC), pages 822-826.
In humans, the spleen releases the blood of microorganisms and antigens into particles, and / or generates antigens for foreign substances; sequesters and removes excess old and / or abnormal blood cells; regulates portal blood flow and is compromised in hematopoiesis during development or when the bone marrow alone can not produce enough blood cells. In the spleen, as well as in other tissues, leukocytes, including monocytes, macrophages, basophils, and eosinophils have important roles in the pathological mechanisms initiated by T and / or B lymphocytes. Macrophages in particular produce powerful oxidants and proteases that contribute to tissue destruction and secrete a range of cytokines that recruit and activate
Current techniques for the diagnosis of abnormalities in inflamed or diseased tissues depend mainly on the observation of clinical symptoms or serological analysis of tissues or body fluids for hormones, polypeptides or different metabolites. Frequently patients do not manifest clinical symptoms in the early stages of the development of the disease or tumor. On the other hand, serological analyzes do not always differentiate between invasive diseases and genetic syndromes that have overlapping or very similar ranges. In this way, the development of new diagnostic techniques that include the use of chemokines would provide immediate and accurate diagnosis of disease states and associated conditions, provide a better understanding of molecular pathogenesis, and provide the basis for the development of effective therapies.
DESCRIPTION OF THE INVENTION
The present invention provides a sequence of nucleotides encoding a novel chemokine initially found in the tissue of the human fetal spleen. The new gene, which is known as chemokine expressed in fetal spleen, or fsec (Incyte Clone No. 29592), encodes a polypeptide designated as FSEC, of the C-C chemokine family. The present invention relates to diagnostic tests for the physiological or pathological involvement of the spleen or other tissues wherein the FSEC is associated with a disease state, including steps to test a sample or an extract thereof with fsec DNA, fragments or oligomers thereof. Aspects of the invention include fsec anti-sense molecules; cloning or expression vectors containing nucleic acid encoding fsec; host cells or organisms transformed with expression vectors containing nucleic acid encoding fsec; FSEC purified and methods for the production and recovery of FSEC purified from host cells. The present invention also relates to the treatment of disease states and conditions associated with FSFC, such as inflammation.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide sequence for the chemokine expressed in fetal spleen (fsec) and the predicted amino acid sequence of the chemokine expressed in fetal spleen, FSBS (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) . Figure 2 shows the amino acid alignment of FSEC with other human chemokines of the C-C family. The alignments shown were produced using the PN? RT? R software multiple sequence alignment program (DNASTAR Inc, Madison Wl). Figure 3 shows a hydrophobicity analysis of FSEC based on the predicted amino acid sequence and composition. Figure 4 shows a related tree of human C-C chemokines. The phylogenetic tree was generated by the phylogenetic tree program of the DNASTAR software (DNASTAR Inc., Madison Wl) using the Clustal method with the PAM250 waste weight table.
MODES OF CARRYING OUT THE INVENTION
Definitions As used herein, the term "chemokine expressed in fetal spleen" or "FSBS", refers to the polypeptide described in SEQ ID NO: 2, or a native fragment thereof, which is encoded by a mRNA transcribed to from the cDNA of SEQ ID N0: 1. FSEC can occur naturally or be chemically synthesized. As used herein, "fsec" in lowercase letters refers to a nucleic acid sequence, while "FSEC" in uppercase letters refers to a protein, a peptide, or an amino acid sequence. As used herein, the term "active" refers to those forms of FSEC that retain the biological and / or immunological activities of the FSEC that occurs naturally. As used herein, the term "FSEC that occurs naturally" refers to FSEC produced by human cells that have not been genetically engineered, and specifically contemplates different forms of FSEC arising from post-translational modifications of the polypeptide, including, but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As used herein, the term "derivative" refers to polypeptides derived from FSEC that occurs naturally, through chemical modifications such as ubiquitination, labeling (eg, with radionuclides, different enzymes, etc.), pegylation ( derivatization with polyethylene glycol), or by insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins. As used herein, the term "variant" or "recombinant variant" or "mutant" refers to any polypeptide that differs from FSEC that occurs naturally, through insertions, deletions, and amino acid substitutions, which are created using recombinant DNA techniques. Guidance can be found in determining which amino acid residues can be replaced, added or deleted without abrogating the activities of interest, such as cell adhesion and chemotaxis, by comparing the particular FSEC sequence with those of homologous cytokines, and minimizing the number of amino acid sequence changes made in regions of high homology or regions of known activity. Preferably, the "substitutions" of amino acids are the result of the replacement of an amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, that is, conservative replacements of amino acids. The "insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The allowed variation can be determined experimentally by making insertions, deletions, or amino acid substitutions in a systematic way, in the FSEC using recombinant DNA techniques and testing the resulting recombinant variants to see their activity. Wherever desired, the nucleic acid encoding
FSEC or an FSEC variant, can be genetically engineered to contain a "leader or signal sequence" that can direct the polypeptide through the membrane of a cell. As will be understood by an expert in this field, this sequence may be naturally present in 1 or. polypeptides of the present invention, or can be provided from heterologous protein sources by recombinant DNA techniques. As used herein, a "fragment," "portion," or "segment" of FSEC refers to a stretch of amino acid residues that is of sufficient length to display biological and / or immunogenic activity, and in the preferred embodiments will contain at least about 5 amino acids, at least about 7 amino acids, at least about 8 to 13, and in additional modalities approximately 17 or more amino acids. As used herein, an "oligonucleotide" or "fragment," "portion," or "segment" of polynucleotide refers to any stretch of nucleic acid encoding FSEC that is of sufficient length to be used as a primer in the polymerase chain reaction (PCR), or different hybridization methods known to those skilled in the art, for the purpose of identifying or amplifying identical or related nucleic acids. The present invention includes FSEC polypeptides purified from natural or recombinant sources, vectors and host cells transformed with recombinant nucleic acid molecules encoding FSEC. Different methods for the isolation of polypeptides and two FSECs can be performed by methods well known in the art. For example, such polypeptides can be purified by immunoaffinity chromatography, by using the antibodies provided by the present invention. Other different methods of protein purification well known in the art include those described in Deutscher M
(1990) Methods in Enzymology Volume 182, Academic Press, San
Diego; and Scopes R (1982) Protein Purification: Principies and
Practice Springer-Verlag, NYC, both incorporated herein by reference.
As used herein, the term
"recombinant" refers to a polynucleotide that encodes
FSEC, which is prepared using recombinant DNA techniques.
The polynucleotide encoding FSEC may also include allelic or recombinant variants and mutants thereof. As used herein, the term "probe" or "nucleic acid probe" or "oligonucleotide probe" refers to a portion, fragment, or segment of fsec that is capable of being hybridized to a desired target nucleotide sequence. . A probe can be used to detect, amplify or quantify the cDNA or endogenous nucleic acid encoding FSSE, by employing conventional techniques in molecular biology. A probe may be of variable length, preferably from about 10 nucleotides to many hundreds of nucleotides. As will be understood by those skilled in the art, the conditions of hybridization and the design of the probe will vary depending on the intended use. For example, a probe that is intended to be used in the polymerase chain reaction will be approximately 15 to 30 nucleotides in length, and may be part of a group of degenerate probes, ie, oligonucleotides that tolerate poor nucleotide couplings but that accommodate themselves by noting an unknown sequence; while a probe for use in Southern or Northern hybridizations may be a single, specific nucleotide sequence that is many hundreds of nucleotides in length. In accordance with the foregoing, a preferred probe for the specific detection of feec will comprise a polynucleotide or oligonucleotide fragment from a non-conserved nucleotide region of SEQ ID NO: 1. As used herein, "non-conserved nucleotide region" refers to a nucleotide region that is unique to SEQ ID NO: 1, and does not comprise a region that is conserved in the C-C chemokine family. The probes may be single-stranded or double-stranded, and may have specificity in hybridizations based on solution, cells, tissues or membranes, including in situ technologies and similar to the enzyme-linked immunosorbent assay. The present invention encompasses oligonucleotides, fragments or portions of the polynucleotides described herein, or their complementary strands used as probes. In the present invention, "oligonucleotides" or "oligonucleotide probes" are based on the nucleotide sequences described herein, which encode FSEC. Oligonucleotides comprise portions of the nucleotide sequence described herein, and contain at least about 15 nucleotides, and usually at least about 20 nucleotides and can include up to 60 nucleotides. Nucleic acid probes can comprise portions of the sequence having fewer nucleotides of about 6 kb, and usually less than about lkb. The oligonucleotide and nucleic acid probes of the present invention can be used to determine whether the nucleic acid encoding FSEC is present in a cell or a tissue, or to isolate identical or similar nucleic acid sequences from chromosomal DNA, as described Walsh PS et al. (1992 PCR Methods Appl. 1: 241-250). The nucleic acid probes of the present invention can be derived from naturally occurring or recombinant nucleic acids, single-stranded or double-stranded, or chemically synthesized. These may be labeled by nick translation, Klenow fill reaction, PCR or other methods well known in the art. In Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, NYC, both incorporated herein by reference, describe the probes of the present invention, their preparation and / or labeling. Alternatively, the recombinant variants encoding the polypeptides of the present invention or related polypeptides can be synthesized or identified through hybridization techniques known to those of skill in the art, by making use of "redundancy" in the genetic code. Different codon substitutions, such as silent changes that produce different restriction sites, can be introduced to optimize cloning within a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations can also be introduced to modify the properties of the polypeptide, to change ligand affinities to binding, interchain affinities, or polypeptide degradation or turnover index.
Detailed Description of the Invention The present invention provides a nucleotide sequence that uniquely identifies FSEC, a chemokine of the CC family, which was initially found expressed in the fetal spleen tissue, and was additionally found in inflamed adenoid tissue, thymus, elbow synovium, breast tissue, kidney tissue from a baby who died of anoxia, and tissue from the spine of an individual who died of respiratory failure. The use of FSEC and the nucleic acid sequences that encode it, is based on its characterization as a CC chemokine, and on the function of the known CC chemokines in the activation of monocytes, macrophages, basophils, eosinophils, T lymphocytes, and / or other cells that respond by producing abundant proteases and other molecules that can lead to tissue damage or destruction. The present invention is also based in part on the expression of FSEC in inflamed tissues, such as inflamed adenoid tissue, and possibly in expression in inflamed cells in other tissues that have immune involvement, such as in synovium, spleen and thymus, and in expression in stressed tissue cells. In accordance with the above, the nucleic acid sequences encoding FSEC and antibodies to FSEC provide the basis for diagnosing diseases or conditions related to the activation of monocytes, macrophages, basophils, eosinophils, T lymphocytes, and / or other cells that respond by the production of abundant proteases and other molecules that can lead to tissue damage or destruction. A specific diagnostic test for the expression of FSEC can accelerate the diagnosis of disease states associated with FSEC, thus providing an opportunity for timely treatment of those disease states before extensive tissue damage or destruction occurs. The present invention also relates to the use of nucleic acid sequences encoding FSEC and antibodies to FSEC to treat or ameliorate the symptoms of disease states or conditions associated with excessive FSEC expression, or associated with FSEC-related activation of monocytes. , macrophages, basophils, eosinoyiles, T lymphocytes, and / or other cells that respond by the production of abundant proteases. These diseases or conditions include, but are not limited to, diseases or conditions related to inflammation, such as viral or bacterial infection, including mononucleosis and malaria, diseases that affect immunoregulation, such as autoimmune diseases, rheumatoid arthritis or systemic lupus erythematosis.; inflammation associated with AIDS; inflammation associated with erythrocyte disease or blood flow, such as anemia of diseased cells and ß-thalassemia; and other physiological and pathological problems related to excessive production of proteases induced by FSEC, including mechanical injury associated with trauma. For example, an anti-fsec sense molecule can be administered to an individual subject to inflammation associated with FSSE, to inhibit the translation of endogenous fsec, thereby lessening the symptoms of inflammation. The present invention also relates to the use of FSEC or nucleic acid sequences encoding FSEC, to treat or lessen the symptoms of disease states or conditions where the immune system is compromised, such as in AIDS, where administration of FSEC or a nucleic acid encoding FSEC would replace or increase FSEC that occurs naturally, and would function to activate and attract other molecules of the immune system. The nucleotide sequences of the present invention that encode FSEC, or its complements, have numerous applications in techniques known to those skilled in the field of molecular biology. These techniques include the use of FSEC nucleotide sequences as hybridization probes to detect nucleotide sequences that encode FSSE in biological samples; the use as oligomers in the polymerase chain reaction to identify and / or amplify nucleotide sequences encoding FSEC; the use for mapping chromosomes and genes, the use in the recombinant production of FSEC, and the use in the generation of anti-sense DNA or RNA, which can inhibit the translation of FSEC. In accordance with the foregoing, the present invention provides a diagnostic test for the detection of nucleotide sequences encoding FSEC in a biological sample, which comprises the steps of combining the biological sample with a first nucleotide comprising the nyeleotide sequence. of SEQ ID NO: 1, or a fragment thereof, wherein said fragment is derived from a non-conserved region of this nucleotide, under conditions suitable for the formation of a nucleic acid hybridization complex; detecting the hybridization complex, wherein the presence of this complex correlates with the presence of a second nucleotide that encodes FSEC in the biological sample; and comparing the amount of the second nucleotide in the sample with a standard, thereby determining whether the amount of the second nucleotide varies from the standard, where the presence of an abnormal level of the second nucleotide correlates positively with a condition associated with the aberrant expression of FSEC. The first nucleotide can be labeled with a reporter molecule that allows the hybridization complex to be detected by measuring the reporter molecule. Additionally, the present invention also provides a diagnostic test for the detection of nucleotide sequences encoding FSEC in a biological molecule, which comprises the steps of combining the biological sample with polymorphism chain reaction primers under conditions suitable for amplification of the nucleic acid, wherein these primers comprise fragments of non-conserved regions of the nucleotide sequence of SEQ ID NO: 1, - detecting the amplified nucleotide sequences; and comparing the amount of nucleotide sequences amplified in the biological sample, with a standard, thereby determining whether the amount of the nucleotide sequence varies from the standard, where the presence of an abnormal level of this nucleotide sequence is positively correlated with a condition associated with the aberrant expression of FSEC. The present invention also provides methods for screening drugs or other agents that can specifically bind FSEC, thereby identifying potential therapy that can treat or ameliorate the symptoms of diseases or conditions associated with aberrant expression of FSEC. In accordance with the foregoing, the present invention provides a method for screening a plurality of compounds for the affinity of specific binding with FSSE or any portion thereof, which comprises the steps of providing a plurality of compounds; combining FSEC with each of a plurality of compounds for a sufficient time to allow fixation under suitable conditions; and detecting the binding of FSEC to each of the plurality of compound; -;, thereby identifying the compounds that specifically bind FSEC. The uses of the nucleotides encoding FSEC, described herein, are examples of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art. On the other hand, the nucleotide sequences described herein can be used in molecular biology techniques that have not yet been developed, with the proviso that the new techniques depend on the properties of the nucleotide sequences that are currently known, example, the genetic code of triplet, specific interactions of pairs of baees, etc. Those skilled in the art will note that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding FSEC can be produced, some with minimal nucleotide sequence homology to the nucleotide sequence of any known gene and which occurs naturally. The invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon selections. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of the naturally occurring FSEC, and may occur whenever the nucleotide sequence encodes the FSEC described in SEQ ID NO: 2, and all these variations will be considered as being specifically described. Although the nucleotide sequences encoding FSEC and / or FSEC variants are preferably capable of hybridizing to the nucleotide sequence of the FSEC gene that occurs naturally under stringent conditions, it may be desirable to produce nucleotide sequences encoding FSEC or to FSEC derivatives that possess a substantially different codon usage. The codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host, in accordance with the frequency with which the host uses the particular codons. Other reasons for substantially altering the nucleotide sequence encoding FSEC and / or FSEC derivatives without altering the encoded amino acid sequence include the production of RNA transcripts that have more desirable properties, such as a longer half-life, than the transcripts produced from the sequence that occurs naturally. Nucl.eotide sequences encoding FSEC can be linked to a variety of other nucleotide sequences by means of well-established recombinant? PN techniques (cf Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). The nucleotide sequences useful for binding to fsec include a variety of cloning vectors, for example, plasmids, cosmids, lambda phage derivatives, phagemids, and the like, which are well known in the art. The vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and the like. In general, the vectors of interest may contain a functional replication origin in at least one organism, convenient restriction sites sensitive to the endonuclease, and markers that may be selected for the host cell. Another aspect of the present invention is to provide fsec-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences that encode FSEC. Such probes for the detection of fsec-encoding sequences should preferably contain a nucleotide fragment of a non-conserved region of SEQ ID NO: 1. Such probes can also be used for the detection of sequences that encode the related chemokine and should preferably contain at least 50 percent of the nucleotides of a C-C coding sequence. The hybridization probes of the present invention can be derived from the nucleotide sequences of SEQ ID NO: 1, or from genomic sequences that include promoters, enhancer elements and feec introns that occur naturally. Hybridization probes can be labeled by a variety of reporter groups, including radionuclides such as P or J-S, or enzymatic labels such as alkaline phosphatase, coupled to the probe by avidin / biotin coupling systems, and the like, through of techniques known to those of skill in the art.
The polymerase chain reaction, as described in U.S. Patent Nos. 4,683,195; 4,800,195; and 4,965,188 provide additional uses for oligonucleotides based on the nucleotide sequences encoding FSEC. Such probes that are used in the polymerase chain reaction can be of recombinant origin, can be chemically synthesized, or a mixture of both, and comprise a nucleotide sequence from a non-conserved region of SEQ ID NO: 1 for use of diagnostic in the detection of fsec, or a degenerate group of possible sequences for the identification of closely related genomic sequences. Other means for producing fsec-specific hybridization probes include read cloning of nucleic acid sequences encoding FSEC and FSEC derivatives into vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available, and can be used to synthesize RNA probes in vitro. by the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleic acid encoding FSEC, portions thereof, or FSEC derivatives, can be produced entirely by synthetic chemistry, after which the gene can be inserted into any of the many available DNA vectors, using reagents, vectors and cells which are known in the art, at the time of filing this application. On the other hand, synthetic chemistry can be used to introduce mutations within the fsec sequence or any portion thereof. The nucleotide sequence of the nucleic acid encoding FSEC can be confirmed by DNA sequencing techniques. Methods for DNA sequencing are well known in the art. Conventional enzymatic methods employed the Klenow DNA polymerase fragment, SEQUENASE® (US Biochemical Corp, Cleveland, OH) or taq polymerase, to extend the DNA strands of a hardened oligonucleotide primer to the DNA template of interest. Methods have been developed for the use of both single-stranded double stranded chain templates and electrophoresed the chain termination reaction products in urea-acrylamide gels and were detected either by autoradiography (for precursors labeled by radionuclide) or by fluorescence (for precursors labeled by fluorescence). Recent improvements in the preparation, sequencing and mechanized analysis of the reaction, using the fluorescent detection method, allowed the expansion in the number of sequences that can be determined per day (using machines such as the Catalyst 800 and the Applied DNA sequencer Biosysteme 373).
The nucleotide sequence of FSEC provides the basis for assays to detect inflammation or disease associated with abnormal levels of FSEC expression. The nucleotide sequence can be labeled by methods known in the art, and added to a sample of fluid or tissue from a patient, under hybridization conditions. After an incubation period, the sample is washed with a compatible fluid - foot optionally contains a dye if the nucleotide has been labeled with an enzyme or with another label or reporter molecule that requires a developer. After rinsing the compatible fluid, the dye is quantified and compared to a standard. If the amount of dye is significantly elevated, the nucleotide sequence has been hybridized with the sample, and the assay indicates the pre-eminence of inflammation and / or disease. The nucleotide sequence encoding FSEC can be used to construct hybridization probes to map that gene. The nucleotide sequence that is provided herein can be mapped to a chromosome and specific regions of a chromosome, using well-known techniques of genetic and / or chromosomal mapping. These techniques include in situ hybridization, analysis of binding against known chromosomal markers, screening by hybridization with libraries of chromosomal preparations selected by flow, specific for known chromosomes, and the like. Verma et al. (1988) Human Chromosomee: A Manual of Basic Techniques, Pergamon Press, NYC, among others, has described the fluorescent in situ hybridization technique of chromosome disseminations. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques can be correlated with additional genetic map data. Examples of genetic map data can be found in 1994 Genome Issue of Science (265: 1981f). The correlation between the location of fsec in a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help to narrow the region of DNA associated with that genetic disease. The nucleotide sequence of the present invention can be used to detect differences in the sequence of genes between normal, carrier and affected individuals, i.e., individuals subject to a disease or condition. The nucleotide sequences encoding FSEC can be used to produce purified FSEC, using well-known methods of recombinant DNA technology. Among the many publications that teach methods for the expression of genes after they have isolated these, is Goeddel (1900) Gene Expression Technology, Methods and Enzymology, Volume 185, Academic Press, San Diego. FSEC can be expressed in a variety of host cells, including cells of prokaryotic or eukaryotic origin. The host cells may be of the same species, in which the fsec nucleotide sequences are endogenous, or of a different species. The advantages of producing FSEC by recombinant DNA technology include obtaining adequate amounts of the protein for purification, and the availability of simplified purification procedures. FSEC can be expressed as a chimeric protein with one or more additional polypeptide domains, aggregated to facilitate the purification of proteins. Such purification facilitation domains include, but are not limited to, metal chelation peptides such as histidine-tryptophan modules, which allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a dissociating linker sequence, such as Factor XA or enterokinase (Invitrogen, San Diego CA), between the purification domain and the fsec coding sequence, may be useful in facilitating the expression of FSEC. The translation of the fsec cDNA can be carried out by subcloning the cDNA into an appropriate expression vector and transfecting this vector into an appropriate expression host. As described in Example VII, a preferred expression vector for the expression and purification of FSEC is one that allows expression of a fusion protein comprising FSEC and containing nucleic acid encoding 6 histidine residues followed by thioredoxin and a site Dissociation of enterokinase. The histidine residues facilitate purification in IMIAC (which in Spanish means affinity chromatography of immobilized metal ion, as described in Porath et al. (1992) Protein Expression and Purification 3: 263-281) while the dissociation site of Enteroquinaea provides a means to purify the chemokine of the fusion protein. The expression vector used for the generation of the cDNA libraries, which contains a promoter for the upstream of β-galactosidase of the cloning site, followed by a nucleotide sequence that contains the Met with amino terminal, and the 7 subsequent residues of β-galactosidase followed by a bacteriophage promoter useful for artificial priming and transcription, and a number of unique restriction sites, including Eco Rl, can also be used for the expression of FSEC. The induction of the bacterial chain - isolated with IPTG, using standard methods, will produce a fusion protein corresponding to the first seven residues of β -galactosidase, approximately 15 residues of "linker", and FSEC encoded within the cDNA. Since the cDNA clone inserts are generated by an essentially random process, there is a possibility in three that the included cDNA will be located in the correct frame for proper translation. If the cDNA is not in the proper reading frame, it can be obtained by deletion or insertion of the appropriate number of bases, by well-known methods including in vitro mutagenesis, exonuclease III digestion or bean nuclease, or inclusion of a linker. oligonucleotide. The FSEC will be expressed in the bacterial sevenma as described. The fsec cDNA can be released to other vectors known to be useful for the expression of proteins in specific hosts. Oligonucleotide amplimers containing cloning sites, as well as a DNA segment sufficient to hybridize to both sides of the target cDNA (25 bases) can be synthesized chemically by standard methods. These primers can then be used to amplify the desired segments of the gene by polymerase chain reaction. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene eegmentoe can be produced by digestion of the cDNA with appropriate restriction enzymes, and filling the missing gene segments with chemically synthesized oligonucleotides. The segments of the coding sequence of more than one gene can be ligated together, and cloned into appropriate vectors to optimize the expreration of the recombinant sequence. Suitable expression hosts for such chimeric molecules include, but are not limited to mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae , and bacteria cells such as E. coli. For each of these cellular systems, a useful expression vector may also include a replication origin to allow propagation in the bacteria and a selectable marker, such as the antibiotic-lactamase resistance gene, to allow selection in the bacterium. . In addition, the vectors may include a second selectable marker, such as the neomycin phosphotransferase gene, to allow selection in eukaryotic tranefected host cells. Vectors for eukaryotic host expiratory use may require RNA processing elements such as 3 'polyadenylation sequence, if such are not part of the cDNA of interest. Additionally, the vector may contain promoters or enhancers that increase the expreation of the gene. Such promoters are host specific and include MMTV, SV40, or metallothionin promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts, or alpha factor, alcohol oxidase or PGH promoters for yeast. Transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, ee can be used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of FSEC produced recombinantly from the conditioned medium can be recovered and analyzed using chromatographic methods known in the art. In addition to recombinant production, FSEC fragments can be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield R (1963) J Am C em Soc 85: 2149-2154). The protein synthesis in vitro can be performed using manual techniques, or by automation. Automated synthesis can be achieved, for example, using the Applied Biosystems 431A Peptide Synthesizer (Foster City, California), in accordance with the instructions provided by the manufacturer. Chemically diverse FSEC fragments can be synthesized per dose, and combined using chemical methods to produce full-length FSEC.
The FSEC for use in the induction of antibodies must be immunogenic. Peptides for use in the induction of FSEC-specific antibodies will comprise an amino acid sequence that comprises at least five amino acids and preferably at least 10 amino acids, such that the peptide retains the three-dimensional configuration of a portion of the FSEC that is presented naturally, and may contain the entire amino acid sequence of FSEC that occurs naturally. Short stretches of the amino acids of FSEC can be fused with those of another protein such as the limpet hemocyanin orifice and the chimeric molecule used for the production of anti- cuei by ... Those of skill in the art know different methods for the preparation of monoclonal and polyclonal antibodies for FSEC. In one approach, re obtains denatured FSEC from high-throughput reverse phase liquid chromatography separation, and is used to immunize mice or rabbits, using techniques known to those of skill in the art. Approximately 100 micrograms are suitable for immunization of a mouse, while up to 1 milligram can be used for the immunization of a rabbit. To identify mouse hybridomas, the denatured protein can be radioiodinated, and used to screen for potential murine B-cell hybridomas to find those that produce antibodies.
This procedure requires only small amounts of protein, such that 20 milligrams would be sufficient for labeling and tracing many thousands of clones. In another approach, the amino acid sequence of FSEC is analyzed, as deduced from the translation of the cDNA sequence, to determine regions of. high immunogenicity. For example, oligopeptides comprising hydrophilic regions, as shown in Figure 3, can be synthesized and used in suitable immunization protocols to generate antibodies. Ausubel FM et al. (1989, Current Protocols in Molecular Biology, John Wiley &Sons, NYC) describes the analysis to select the appropriate epitopes. The optimal amino acid sequences for immunization are usually in the C-terminus, the N-terminus and those intermediate polypeptide hydrophilic regions, which are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, the selected peptides, approximately 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431A, using fmoc chemistry, and are coupled to the hemocyanin 1-orifice mimetic (KLH, Sigma) by reaction with ether of M- maleimidobenzoyl-N-hydroxysuccinimide (MBS; cf Ausubel FM et al., supra). If necessary, a cysteine can be introduced at the N-terminus of the peptide to allow attachment to the orifice limpet hemocyanin, and the animals can be immunized with the peptide-hemocyanin complex limpede orifice in complete Freund's adjuvant. The resulting antisera can be tested for antipeptide activity by fixing the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with specific, purified goat anti-rabbit IgG. by affinity, labeled (radioactive or fluorescent). Hybridomas can also be prepared and screened using standard technique. Hybridomae of interest can be detected by screening with labeled FSEC to identify those fusions that produce the monoclonal antibodies with the desired specificity. In a typical protocol, re-coat plate wells (FAST, Becton-Dickinson, Palo Alio, CA) with rabbit-anti-mouse specific antibodies (or suitable Ig antispecies) at approximately 10 milligrams / milliliter. The coated wells are blocked with 1 percent bovine serum albumin, they are washed and exposed to supernatants of the hybridomas. After incubation, the wells are exposed to labeled FSEC at 1 milli gram / milliliter. The clones that produce antibodies will be fixed in a labeled amount of FSEC, which can be detected on the background. Such clones are expanded and subjected to 2 cloning cycles in limiting dilution (1 cell / 3 wells). Cloned hybridomas are injected into mice treated with pristane to produce ascites, and the monoclonal antibody can be purified from mouse ascitic fluid by affinity chromatography, using Protein A. Monoclonal antibodies with affinities of at least 10 M ", preferably from 109 to 10 or stronger, will typically be done by standard procedures as described by Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and in Goding (1986) Monoclonal Antibodies: Principies and Practice, Academic Press New York City, both incorporated herein by reference, Antibodies specific for particular FSEC can be produced by inoculating an api or i.ule animal with the polypeptide or an antigenic fragment.An antibody is specific for FSEC if produces against all or part of the FSEC, and is fixed to all or part of the FSEC.The induction of antibodies includes not only stimulation of an immune response by injection into animals, but also analogous paeoe in the production of synthetic antibodies or other specific binding molecules, such as the screening of recombinant immunoglobulin libraries (Orlandi R. et al. (1989) PNAS 86: 3833 -3837, or Huse WD et al. (1989) Science 256: 1275-1281) or in vitro stimulation of lymphocyte populations. Current technology (Winter G. and Milstein C. (1991) Nature 349: 293-299) allows a number of highly specific binding reagents based on the principles of antibody formation. These techniques can be adapted to produce molecules that specifically bind to FSEC. The present invention also relates to the use of nucleic acid sequences encoding FSEC and antibodies to FSEC to treat or ameliorate the symptoms of disease states or conditions associated with excessive expression of FSEC, such as inflammation associated with FSEC, such as viral or bacterial infection; diseases that affect immunoregulation such as autoimmune diseases, - inflammation associated with AIDS; e 'inflammation associated with trauma. For example, a fsec anti-sense molecule can be administered to an individual subject to inflammation associated with FSEC to inhibit the translation of the endogenous fsec, thereby lessening the symptoms of inflammation. The present invention also relates to the use of FSEC or nucleic acid sequences encoding FSEC to treat or ameliorate the symptoms of disease states or conditions where the immune system is compromised, such as in AIDS. Under these conditions, the administration of FSEC or the nucleic acid encoding FSEC would replace or increase FSEC that occurs naturally, and function to activate and attract other molecules of the immune system. Antibodies, inhibitors, receptors or antagonists of FSEC (or other treatments for excessive production of FSEC, hereinafter abbreviated TEC), can provide different effects when administered therapeutically. The TECs will be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium, preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary in accordance with the characteristics of the antibody, inhibitor , receptor or antagonist being formulated and the condition to be treated. The characteristics of TEC include molecule solubility, half-life and antigenicity / immunogenicity; These and other characteristics can help define the effective carrier. Native human proteins such as TECs are preferred, but organic or synthetic molecules that re-trace drug traces can be equally effective in particular situations. The TECs can be sent by known administration routes, including but not limited to local creams and gels; spray and transmucosal spray, patch and transdermal bandage; injectable, intravenous and washed fondulations; and liquids and pills administered orally, particularly formulated to resist the acid and enzymes of the stomach. The particular formulation, exact dose, and route of administration will be determined by the attending physician and will vary according to each specific situation. Such determinations are made by considering multiple variables such as the condition to be treated, the TEC to be administered, and the particular pharmacokinetic profile of the TEC. Additional factors that may be taken into account include the patient's disease status (eg, severity), age, weight, sex, diet, time of administration, combination of drugs, reaction sensitivities , and tolerance / response to therapy. Long-acting ECT formulations can be administered every 3 or 1 day, every week, or once every two weeks, depending on the half-life and evacuation rate of the ECT. The amounts of normal doses may vary. from 0.1 to 100,000 micrograms, up to a total dose of approximately 1 gram, depending on the route of administration. Guidance is given in the literature regarding particular doses and methods of application; see United States Patents Number 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different TECs, and that the administration targeting the spleen may need to be shipped in a different manner from that for the shipment directed to another organ or tissue.
It is contemplated that conditions or diseases of the spleen or other tissues that activate monocytes, macrophages, basophils, eoeinophils, or other leukocytes, may precipitate damage that may be treatable with ECTs. These conditions or diseases can be diagnosed specifically by the diagnostic tests discussed above, and these tests should be performed in cases of suspected inflammation or viral or bacterial infections, including mononucleosis and malaria; inflammation associated with AIDS; mechanical injury associated with trauma; diseases that affect immunoregulation such as rheumatoid arthritis or systemic lupus erythematosis; and diseases of erythrocytes or blood flow, including anemia of diseased cells and ß-thalassemia. The following examples are provided to illustrate the present invention. These examples are provided for illustration and are not included for the purpose of limiting the invention.
EXAMPLES
I Isolation of the mRNA and Construction of cDNA libraries The fsec cDNA sequence was initially identified among the sequences comprising the human fetal spleen library. This library was constructed from pooled human fetal spleen tissues from Stratagene Inc. (Cat. # 937205; 11011 N Torrey Pines Rd, La Jolla, CA 92037). The synthesis of the cDNA was primed using oligo dT hexamers, and the synthetic adapter oligonucleotides were ligated over the ends of the cDNA allowing their insertion into the UNI-ZAP M vector system (Stratagene, Inc). This allows a construction of the lambda library of greater unidirectional efficiency (sense orientation) and the convenience of a plasmid system with blue / white color selection to detect clones with cDNA inserts. The quality of each cDNA library was screened using either DNA probes or antibody probes, and then phagemid pBluescppt (Stratagene Inc) was rapidly removed in living cells. The phagemid allows the use of a plasmid system for the characterization sene i 1 l. of the insert, sequencing, site-directed mutagenesis, the creation of unidirectional deletions and the expression of fusion proteins. The phage particles of each library were infected within the host strain of E. coli XL1-BLUE (Stratagene Inc). The high transformation efficiency of the XLI-BLUE increases the probability of obtaining rare clones, sub-represented from the cDNA library.
II Isolation of cDNA Clones The phagemid forms of individual cDNA clones were obtained by the in vivo excision process, in which the XLI-BLUE was simultaneously infected with an auxiliary phage fl. Proteins derived from both lambda phage and auxiliary phage fl initiated a new DNA synthesis from the sequences defined on the target lambda DNA and created a smaller molecule of single-stranded circular phagemid DNA, which included the sequences of DNA of the plasmid Pbluescript and the insert of the cDNA. The phagemid was released from the cells and purified, then used to re-infect fresh bacterial host cells (SOLR, Stratagene Inc), where the double-stranded phagemid DNA was produced. Because the phagemid carries the gene for β-lactamase, newly transformed bacteria were selected on a medium containing ampicillin. Phagemid DNA was purified using the QIAWELL-8 Plasmid Purification System from the QIAGEN DNA Purification System (QIAGEN Inc., 9259 Eton Ave.
Chatsworth, CA 91311). This technique provides a fast and reliable high-throughput method for dissolving bacterial cells and for isolating highly purified phagemid DNA. The DNA levigado of the purification resin was suitable for the secuta Sarniento of DNA and other analytical manipulations.
III Sequencing of the cDNA Clones The cDNA inserts were sequenced in part from randomized isolates of the human fetal spleen library. The cDNAs were sequenced by the method of Sanger F. and AR Coulson (1975; J. Mol. Biol. 94: 441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with four Peltier Thermal Cyclers (PTC200). of MJ Research, Watertown MA) and the DNA Sequencing Systems 377 or 377 of Applied Biosystems (Perkin Elmer) and the determined reading frame.
IV Homology Search of the Clones of the cDNA and Deduced Protein Each cDNA was compared to the sequences in the Gene Bank using a search algorithm developed by Applied
Biosystems and were incorporated into the Analysis System of
INHERIT sequence. In this algorithm, the Language of
Pattern Specification (developed by TRW Inc) to determine regions of homology. The three parameters that determined how the sequence comparisons were run were the window size, the window offset and the error tolerance. Using a combination of these three parameters, the DNA database was examined to look for sequences containing regions of homology to the sequence in question, and the appropriate sequences were labeled with an initial value. Subsequently, these regions of homology were examined using dot matrix homology diagrams to distinguish regions of homology from casual couplings. Smith-Waterman alignments were used to visually display the results of the homology search. Peptide and protein sequence homologies were determined using the INHERIT 670 Sequence Analysis System in a manner similar to that used in DNA sequence homologies. The Pattern Specification Language and the parameter windows were used to search the protein databases for the sequences containing regions of homology to which they were marked with an initial value. Matrix dot homology diagrams were examined to distinguish regions of significant homology from casual couplings. BLAST was used, which in Spanish means Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36: 290-300; /.ltschul, SF et al. (1990) J Mol Biol 215: 403-10) , to look for local sequence alignments. The Basic Local Alignment Search Tool produces alignments of both nucleotide and amino acid sequences to determine the similarity of the sequence. Due to the local nature of the alignments, the Basic Local Alignment Search Tool is especially useful in determining accurate couplings or identifying identification homologs. The Basic Local Alignment Search Tool is useful for links that do not contain gaps. The fundamental unit of the output information of the Basic Local Alignment Search Tool algorithm is. the High Marking Segment Pair (HSP). A High-Dial Segment Pair consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximum and for which the alignment mark approximates or exceeds a threshold or limitation marking set by the user. The focus of the Basic Local Alignment Search Tool is to look for Elevated Marking Segment Pairs between a sequence in question and a database sequence, to evaluate the statistical significance of any found couplings, and to report only those couplings that satisfy the threshold of meaning selected by the user. parameter E establishes the statistically significant threshold for reporting the couplings of; the sequence of the database. E is interpreted as the upper limit of the expected frequency of the casual presentation of a High Marking Segment Pair (or set of High Marked Segment Pairs) within the context of the complete search of the database. Any database sequence whose coupling satisfies E in the output information of the program is reported. The nucleotide and amino acid sequences for the chemokine expressed in fetal spleen, FSEC, are shown in Figure 1 (SEQ ID N0: 1 and SEQ ID N0: 2), respectively. Inheritance analysis revealed that clone Incyte 29592. db (see Figure 2), contained a significant portion of the nucleotide sequence illustrated in SEQ ID N0: 1 encoding FSEC. The 5 'nucleotide sequence encoding FSEC was obtained using polymerase chain reaction technology. In the first round of amplification, a specific oligomer was used for a portion of the chemokine 29592. db, TCC TTC CTT CTG GTC CTC GGT TCC, and another specific one for the vector containing the cDNA inserts of fetal spleen, GGA AAC AGC TAT GAC CAT G, to identify and amplify the 5 'fsec nucleotide sequence. In the second round of amplification, a set of nested oligomers was used, ie an internal one for the previous 5 'bligomer, consisting of the nucleotides CTT GGA ATT CAC TCC GGG CTC CCT CTG CAC G, and the primer specific for the sequence of the vector (shown above), to further amplify the cDNA. the missing 5 'sequence was isolated using ECO Rl (which digests the DNA within the restriction site underlined in the previous internal oligomer), was subjected to electrophoresis and ligated to the 3' portion of the chemokine sequence from 29592 db. The full-length molecule designated 29592 was sequenced again.
V Identification v Full-length Sequencing of the Gene The nucleotide sequence of fsec is homologous to, but distinctly different from, any known C-C chemokine molecule. The complete nucleotide sequence for fsec is shown as the clone Incyte 29592. When looking for the three predicted poorerings of the frequency against the protein data baees, such as SwissProt and PIR, no exact couplings were found with the possible translations of fsec. Figure 2 shows the comparison of FSEC with other C-C chemokines. The regions of homology between the chemokines, including the definitive C-C motif, are shaded. The phylogenetic analysis (Figure 4) shows how closely the FSEC is related to other well characterized human C-C chemokines. The most related of these molecules are grouped together on the right side of the figure.
VI Anti-sense analysis Knowledge of the correct cDNA sequences, complete with the expressed chemokine nbvedoeoe genes, will allow their use in anti-sense technology in the investigation of the functioning of the gene. Either oligonucleotides, genomic fragments or cDNAs comprising fsec anti-sense strands can be used, both in vitro and in vivo to inhibit the expression of the specific protein. Such a technology is currently well known in the art, and probes can be designated at several places along the nucleotide sequence. The gene of interest can be effectively diverted by treating cells or whole test animals with said anti-sense sequences. Frequently, the function of the gene can be ascertained by observing the behavior at the cellular, tissue or organism level (for example, mortality, loss of differentiated function, changes in morphology, etc.). In addition to using the sequences constructed to interrupt transcription of the open reading frame, modifications of gene expression can be obtained by means of deeigning anti-sense sequences to intron regions, promoter / enhancer elements, and even to transaction regulatory genes. . Similarly, inhibition can be achieved using the Hogeboom base pairing methodology, also known as "triple helix" base pairing.
VII FSEC Expression The nucleotide sequences encoding FSEC were cloned into an expression vector that includes a T7 promoter followed by a starter methionine (ATG) codon, followed by seie histidine codons, followed by an E. coli TrxA gene. (which encodes thioredoxin), followed by sequence coding for a dissociation site of enterokinase and the nucleotide sequences encoding FSEC. Empirical studies associated with the dissociation of signal sequences indicate that dissociation occurs at or near the C-terminus of a predicted hydrophobic region located at the N-terminus of the full-length protein. The hydrophobicity profile of FSEC is shown in Figure 3, and based on this profile, residue 16 (proline) of SEQ ID NO: 2 was selected as the N-terminal amino acid residue for the expression of mature FSEC. To determine the naturally processed N-terminal amino acid residue of mature FSEC, the nucleotide sequence described in SEQ ID NO: 1 is ligated into an expression vector appropriate for eukaryotic expression. The expression vector comprising SEQ ID NO: 1 is transfected into an appropriate host cell, and the transfected host cell is subjected to conditions suitable for expression of the protein. The amino acid sequence of the processed mature protein is confirmed by N-terminal protein sequence analysis techniques known to those skilled in the art. The expression vectors described above containing the 6 codons of histidine were used to transform a host cell, culture of the host cell was induced with IPTG and the expressed protein was subjected to SDS denaturing polyacrylamide gel electrophoresis, the nucleic acid was partially purified from the expression vector using the miniprep procedure of Sambrook supra which produced the DNA super rolled Approximately 100 nanograms of DNA were used to transform the host bacterial cell, W3110 / DE3. The W3110 / DE3 was constructed using the ATCC W3110 and the DE3 lambda lysogenization kit commercially available with Novagen. DE3 lysogens are usually less suitable than their origin, W3110, and are adapted to use super-coiled DNA for efficient transformation. A single transformant of the FSEC transformation was selected and used to inoculate a 5 milliliter culture of L broth containing ampicillin. Each 5 milliliter culture was grown overnight (12-15 hours) at 37 ° C with shaking. The next day, 1 milliliter of the previous night's culture was used to inoculate a 100 milliliter culture of L broth with ampicillin in a 500 milliliter flask and allowed to grow at 37 ° C with shaking until the OD600 of the culture reached 0.4-0.6. If the inoculated cells are allowed to grow beyond an OD600 of 0.6, they will begin to reach a stationary phase and induction levels will be reduced. At the time of inoculation, a 5 milliliter sample was removed, placed on ice and used as a previous induction sample (or 0 hour). When the cell culture reached an OD600 of 0.6, 400 microliters of an IPTG extract solution of 100mM was added for a final concentration of 0.4mM. They were allowed to grow for 3 hours at 37 ° C with shaking. The induction analyzes were determined by means of the 5 milliliter aliquot sample of the culture at intervals of 1 hour to 6 hours and analyzing them on denaturing SDS polyacrylamide gel electrophoresis. It appears that the fusion protein accumulated in both the soluble and insoluble fraction of the cells. The maximum induction of FSEC was presented at 2 hours. Growth for more than 4 hours resulted in dissolution in the crop and reduced total yields of the desired protein due to proteolysis. Five milliliter aliquots were obtained from cell cultures at 0 and 2 hours and centrifuged for 5 minutes at 3000 RPM at 4 ° C. The supernatant was aspirated and the granules were subjected to a freeze-thaw pause to help dissolve the cells. The granule was again suspended in TE (10 mM Tris-HCl pH 8.0, lmM EDTA pH 8.0) at 4 ° C at a volume calculated as: vol TE (μl) = (OD600) (250), and added an equivalent volume of Sample Load pH Regulator (Novex) of 2X SDS to each sample. The samples were boiled for 5 minutes and 10 microliters of each sample were loaded per row. The expected molecular weight of the fusion protein comprising FSEC is 23,145 Daltons. The FSEC analysis expressed on a 14 percent SDS-polyacrylic amide gel shows an apparent molecular weight of approximately 32 kDa. N-terminal protein sequence analysis of this band produced a histidine residue chain that is consistent with the predicted sequence. A second funtion protein was constructed and expressed. lacked the six histidine residues. This fusion protein also had an apparent molecular weight greater than its predicted value. The sequence analysis of the N-terminal protein of this product was coupled with the thioredoxin sequence, which is consistent with the expected results.
VIII Isolation of FSEC Recombinant FSEC was expressed as a chimeric protein having six histidines followed by the thioredoxin gene (TrxA from E. coli) with a dissociation site of the enterokinase between the TrxA protein and FSEC. Histidines were added to facilitate purification of the protein. The presence of histidines allows purification on IMIAC chromatography (Porath supra).
IX Diagnostic Test Using FSEC Specific Antibodies
Particular FSEC antibodies are useful for the diagnosis of prepatological conditions, and chronic or acute diseases that are characterized by differences in the amount or distribution of FSEC. It is possible that FSEC is specific for tissue abnormalities or pathologies where they are found. Diagnostic tests for FSEC include methods that use an FSEC antibody and a label to detect FSSE in body fluids, tissues or extracts from said human tissues. The polypeptides and antibodies of the present invention can be used with or without modification. Frequently, polypeptides and antibodies will be labeled by binding them, either covalently or non-covalently, with a substance that provides a signal that can be detected. A wide variety of labels and conjugation techniques are known and have been widely reported in the scientific and patent literature. Appropriate labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. The patents that instruct on the use of said labels include the Patents of the United States of North America Numbers 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in U.S. Patent No. 4,816,567, incorporated herein by reference. A variety of protocols for measuring soluble or membrane-bound FSEC are known in the art, using either polyclonal or monoclonal antibodies specific for that FSEC. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell (FACS) screening. It preferred; a two-site monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on FSEC, but a competitive binding assay may be employed. These assays are described, inter alia, in Maddox, DE et al. (1983, J Exp Med 158: 1211).
X Purification of FSEC Native Using Specific Antibodies Native or recombinant FSEC is purified by immunoaffinity chromatography using antibodies specific for FSSE. An immunoaffinity column is constructed by covalently coupling the an? I-FSEC antibody to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune sera, either by precipitation with ammonium sulfate, or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Alternatively, monoclonal antibodies are prepared from mouse ascites fluid by precipitation of ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently bound to a chromatographic resin such as activated Sepharose of CnBr (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derived resin is washed in accordance with the manufacturer's instructions. Such an immunoaffinity column is used in the FSEC purification by preparing a fraction from the cells containing FSEC in a soluble form. This preparation is derived by means of the solubilization of the whole cell or a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble FSEC can be secreted which contains a signal sequence in a useful amount within the medium in which the cells are growing. A preparation containing soluble FSEC is passed over an immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of FSEC (for example, the ionic strength is regulated in the presence of a detergent). The column is levigated under conditions that break the antibody / FSEC binding (eg, a pH regulator of 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and FSEC is harvested.
XI Determination of Chemotaxis Induced by FSEC or Cellular Activation The chemotactic activity of FSEC is ured in a 48-well microchemotaxis chamber (Falk WR et al. (1980) J Immunol Methods 33: 239). In each well, the compartment is separated and separated by a filter that allows the passage of the cells in response to the chemical gradient. Cell culture medium such as RPMT 1640
(Sigma, St. Louis MO) containing FSEC is placed on the side of a filter, usually polycarbonate, and cells suspended in the same medium are placed on the opposite side of the filter. Sufficient incubation time is allowed for the cells to travel through the filter in response to the concentration gradient across the filter. The filters of each well are recovered, and they determine and quantify the cells that are attached on the side of the filter that is in front of the FSEC. The specificity of chemoattraction is determined by performing the chemotaxis assay on specific cell populations. First, the blood cells obtained are fractionated by venipuncture by centrifugation of the density gradient and the chemotactic activity of FSBS is tested on enriched populations of neutrophils, peripheral blood mononuclear cells, monocytes and lymphocytes. Optionally, said populations of enriched cells are further fractionated using CD8 + and CD4 specific antibodies for the negative selection of CD4 + and CD8 + enriched T cell populations, respectively. Another trial elucidated the chemotactic effect of FSEC on activated T cells. There, subsets of unfractionated T cells or fractionated T cells are cultured for 6 to 8 hours in tissue culture vessels coated with the CD-3 antibody. After this activation of CD-3, the chemotactic activity of FSEC is tested as described above. Many other methods for obtaining enriched cell populations are known in the art.
Some chemokines also produce a non-chemotactic cell activation of neutrophils and monocytes. This is tested via standard urements of neutrophil activation such as actin polymerization, increased activity of respiratory burst, degranulation of asurophilic granule and mobilization of Ca ++ as part of the path of signal transduction. The assay for the mobilization of Ca ++ involves previously loading the neutrophils with a fluorescent probe whose emission characteristics have been altered by the Ca ++ fixation. When the cells are exposed to an activation stimulus, the flow of Ca ++ is determined by observing the cells in a fluorometer. The urement of Ca ++ mobilization has been described in Grynkievicz G et al. (1985) J Biol Chem 260: 3440, and in McColl S et al (1993) J Immunol 150: 4550-4555, incorporated herein by reference. The responses of degranulation and respiratory burst in monocytes are also ured (Zachariae COC et al. (1990) J Exp Med 171: 2177-82). Additional ures of 1 to monocyte activation are the requirement for adhesion molecule expiation and cytokine production (Jiang Y et al. (1992) J Immunol 148: 2423-8). The expression of the adhesion molecules also varies with the activation of the lymphocyte (Taub D et al (lacH) Science 260: 355-358).
XII Drug Tracing FSEC, or biological or immunological fragments thereof, are used to screen compounds in any of the varieties of drug tracing techniques. The FSEC or a fragment thereof, which is employed in said test, is either free in the solution, adhered to a solid support, supported on a cellular surface or located intracellularly. A method for drug screening utilizes eukaryotic or prokaryotic host cells which have been stably transformed with recombinant nucleic acids that express FSEC or a fragment thereof. The drugs are screened compared to said cells transformed into competitive binding sites. These cells, either in a viable or fixed form, are used for standard fixation tests. One can measure, for example, the formation of a complex between the FSEC or a fragment thereof, and the agent that is being tested. In an alternative way, the decrease in complex formation between the FSEC and its target cell, monocyte, etc., caused by the agent being tested is monitored. In this manner, the present invention provides methods of screening drugs or other agents which can cause inflammation and disease. These agents comprise contacting a drug or agent to be tested with an FSEC polypeptide or fragment thereof and making tests to (i) search for the presence of a complex between the agent and the FSEC polypeptide or the fragment, or ( ii) looking for the presence of a complex between the FSEC 'polypeptide or the fragment and the cell, by methods well known in the art. In such competitive binding assays, the FSEC polypeptide or fragment is labeled. After a suitable incubation, the FSEC polypeptide or fragment is separated from that which is present in the fixed form, and the amount of the free or un-complexed label is a measure of the ability of the particular agent to bind to the FSEC or to interfere with the FSEC compliant L / cell. Another technique for screen tracking provides a high throughput screening for compounds that have an adequate binding affinity to Ion pol i pected "FSEC and is described in detail in European Patent Application 84/03564, published on SEPTEMBER 13, 1984, incorporated herein by reference Briefly stated, a plurality of different peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Testing with the FS C polypeptide is washed. Then the FSEC polypeptide is detected by methods well known in the art. The purified FSEC can also be directly coated on the plates for use in the drug screening techniques mentioned above. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug scrambling assays in which the neutralizing antibodies capable of binding to FSEC compete specifically with a test compound to bind to the FSEC polypeptide or fragments thereof. In this form, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with the FSSE.
XIII Rational Drug Design The objective of the rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, for example, agonists, antagonists, or inhibitors. Any of these examples can be used to form drugs that are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (see Hodgson J (1991) Bio / Technology 9: 19-21, incorporated herein by reference). In one approach, the three-dimensional structure of a protein of interest, or of a protein inhibitor complex, is determined by X-ray crystallography, by computer modeling or, more typically, by a combination of two approaches. Both the manner and charges of the polypeptide must be ascertained to elucidate the structure and to determine the active site (s) of the molecule. Useful information can be obtained with respect to the structure of the polypeptide by modeling based on the structure of the homologous proteins. In both cases, the relevant structural information is used to designate chemokine-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown in Braxton S and Wells JA (1992 Biochemistry ?: 77or, 7801) or which act as inhibitors, agonists, or antagonists of peptides that occur naturally, as shown in Athauda SB et al. (1993 J Biochem 113: 742-746), incorporated herein by reference. It is also possible to isolate a specific target antibody, selected by functional assay, as described above, and then dissolve its crystal structure. This approach, in principle, yields a farmanucleus on which the design of the drug can be based. It is possible to deviate the protein crystallography in its entirety by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. Like the mirror image of a mirror image, the anti-idiotypic binding site would be expected to be an analogue of the original receptor. The anti-idiotics can then be used to identify any peptides isolated from chemically or biologically produced peptide libraries. The isolated peptides would then act as the farmanucleus. Using the methods described below, a sufficient amount of FSEC can be made available for performing analytical studies such as X-ray crystallography. In addition, knowledge of the FSEC amino acid sequence described here will give guidance to those they employ computer modeling techniques in l.uqar of or in addition to X-ray crystallography.
XIV Identification of FSEC Receptors The purified FSEC, or a fragment thereof, can be used to characterize and purify specific cell surface receptors and other binding molecules. It is very likely that cells that respond to FSEC by chemotaxis or other specific responses express a receptor for FSEC. The radioactive tags are incorporated within the FSEC, or a fragment thereof, by various methods known in the art. A preferred embodiment is the labeling of primary amino groups in FSEC with Bolton-Hunter I reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529), which has been used to label other chemokines without concomitant loss of biological activity (Hebert CA et al. (1991) J Biol Chem 266: 18989; McColl S et al. (1993) J Immunol 150: 4550-4555). The carrier cells of the receptor are incubated with labeled FSEC. The cells are then washed to remove unfixed FSEC, and the FSEC bound to the receptor is quantified. The data obtained using different concentrations of FSEC is used to calculate the values for the number and affinity of receptors. The FSEC labeling is useful as a reactive pai.i \ purification of your rece; specific tor. In an affinity purification mode, the FSEC is covalently coupled to a chromatography column. The carrier cells are removed from the receptor, and the extract is passed over the column. The receptor is fixed to the column by virtue of its biological affinity for the FSEC. The receptor is recovered from the column and subjected to sequencing of the N-terminal protein. This amino acid sequence is then used to designate degenerate Leotide oligonucleotide probes for the cloning of the receptor gene.
In an alternative method, the mRNA is obtained from the carrier cells of the receptor and converted into a cDNA library. The library is transfected into a population of cells, and those cells expressing the receptor are selected using FSEC fluorescently labeled. The receptor is identified by recovering and sequencing the recombinant DNA from highly labeled cells. In another alternative method, antibodies, preferably monoclonal, are placed against the surface of the cells carrying the receptor. The monoclonal antibodies are screened to identify those that inhibit the binding of labeled FSEC. These monoclonal antibodies are then used in the affinity purification or cloning of receptor expression. Soluble receptors or other soluble binding molecules are similarly identified. The labeled FSEC is incubated with extracts or other appropriate materials derived from the spleen. After incubation, the FSEC complexes larger than the size of the purified FSEC are identified by a sorting technique is < ~ Pin the size, such as, for example, size exclusion chromatography or density gradient centrifugation and purify by methods known in the art. The soluble receptors or the binding protein (s) are subjected to an N-terminal sequencing to obtain sufficient information for the identification of the database, if the soluble protein is known, or for cloning, if The soluble protein is unknown. All publications and patents mentioned in the above specification are incorporated herein by reference. It is considered that the above written specification is sufficient to enable one skilled in the art to practice the invention. In fact, various modifications of the manners described above for carrying out the invention are intended to be within the scope of the following claims which are obvious to those skilled in the field of molecular biology or related fields.
LIST OF SEQUENCES
(1) GENERAL INFORMATION: (i) APPLICANT: Incyte Pharmaceuticale, Inc.
(ii) TITLE OF THE INVENTION: A NOVELTY CHEMIOCINE EXPRESSED IN FETAL SPLEEN, ITS PRODUCTION AND USES (iii) NUMBER OF SEQUENCES: 9
(iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Incyte Pharmaceuticals, Inc. (B) STREET: 3174 Porter Drive (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: UNITED STATES OF NORTH AMERICA (F) CODE POSTCARD: 94304
(v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: Compatible with IBM (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ Version 1.5
(vi) CURRENT APPLICATION DATA: (A) TCP REQUEST NUMBER: To Be Assigned
(B) DATE OF SUBMISSION: January 19, 1996 (C) CLASSIFICATION:
(vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 08 / 375,346 (B) DATE OF SUBMISSION: January 19, 1995
(viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Luther, Barbara J. (B) REGISTRATION NUMBER: 33,954 (C) REFERENCE NUMBER / CASE: PF-0026 PCT
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-852-0195
(2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 719 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) GENOTECA: HUMAN FETAL SPLEEN (B) CLON: 29592
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
GCAGGGCTCA CATTCCCAGC CTCACATCAC TCACACCTTG CATTTCACCC CTGCATCCCA 60
GTCGCCCTGC AGCCTCACAC AGATCCTGCA CACACCCAGA CAGCTGGCGC TCACACATTC 120
ACCGTTGGCC TGCCTCTG T CACCCTCCAT GGCCCTGCTA CUJUC TCA GCCTGCTGGT? A?
TCTCTGGACT TCCCCAGCCC CAACTCTGAG TGGCACCAAT GATGCTGAAG ACTGCTGCCT 240
GTCTGTGACC CAGAAACCCA TCCCTGGGTA CATCGTGAGG AACTTCCACT ACCTTCTCAT 300
CAAGGATGGC TGCAGGGTGC CTGCTGTAGT GTTCACCACA CTGAGGGGCC GCCAGCTCTG 360
TGCACCCCCA GACCAGCCCT GGGTAGAACG CATCATCCAG AGACTGCAGA GGACCTCAGC 420
CAAGATGAAG CGCCGCAGCA GTTAACCTAT GACCGTGCAG AGGGAGCCCG GAGTCCGAGT 480
CAAGCATTGT GAATTATTAC CTAACCTGGG GAACCGAGGA CCAGAAGGAA GGACCAGGCT 0
TCCAGCTCCT CTGCACCAGA CCTGACCAGC CAGGACAGGG CCTGGGGTGT GTGTGAGTGT 600
GAGTGTGAGC GAGAGGGTGA GTGTGGTCAG AGTAAAGCTG CTCCACCCCC AGATTGCAAT 660
GCTACCAATA AAGCCGCCTG GTGTTTACAA CTAAAAAAAA AAAAAAAAAA AAAAAAAAA 719
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 98 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(vii) IMMEDIATE SOURCE: (A) GENOTECA: HUMAN FETAL SPLEEN (B) CLON: 29592
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser Pro
1 5 10 15 - Wing Pro Thr Leu Ser Gly Thr Asn Asp Wing Glu Asp Cys Cys Leu Ser
25 30 Val Thr Gln Lys Pro He Pro Gly Tyr He Val Arg Asn Phe His Tyr
40 45 Leu Leu He Lys Asp Gly Cys Arg Val Pro Wing Val Val Phe Thr Thr
50 55 60 Leu Arg Gly Arg Gln Leu Cys Wing Pro Pro Asp Gln Pro Trp Val Glu 65 70 75 80
Arg Lie Lie Gln Arg Leu Gln Arg Thr Be Ala Lys Met Lys Arg Arg 85 90 95 ~
Being
(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide gone (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Me t Ala
1 5 10 15
Leu Cys Asn Gln Phe Ser Wing Ser Leu Wing Asp Thr Pro Thr Wing 20 25 30 Cys Cys Phe Ser Tyr Thr Ser Arg G ln He Pro Gl n Asn Phe H e A
40 45 Asp Tyr Phe G l u Thr Ser Ser G ln Cys S er Lys Pro Gly Va l H e Phe 50 55 60 Leu Thr Lys Arg Ser Arg Gln Val Cys Wing Asp Pro Ser Glu Glu Trp
65 70 75 80
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Wing 85 90
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
Met Lys Leu Cys Val Thr Val Leu Ser Leu Leu Met Leu Val Wing Ala
1 5 10 15
Phe Cys Ser Pro Wing Leu Ser Wing Pro Met Gly Ser Asp Pro Pro Thr
25 30 Wing Cys Cys Phe Ser Tyr Thr Wing Arg Lys Leu Pro Arg Asn Phe Val
40 45 Val Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Ala Val Val
50 55 60 Phe Gln Thr Lys Arg Ser Lys Gln Val Cys Wing Asp Pro Ser Glu Ser 65 70 75 80
Trp Val Gln Glu Tyr Val Tyr Asp Leu Glu Leu Asn 85 90
(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 91 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(xi) 'SEQUENCE DESCRIPTION: SEQ ID NO: 5
Met Lys Val Ser Ala Ala Arg Leu Al a Val He Leu He Ala Thr Ala
1 5 10 15
Leu Cys Wing Pro Wing Being Wing Being Pro Tyr Being Ser Asp Thr Thr Pro
25 30 Cys Cys Phe Wing Tyr He Wing Arg Pro Leu Pro Arg Wing His He Lys
40 45 Glu Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Wing Val Va l Phe
50 55 60 Val Thr Arg Lys Asn Arg Gln Val Cys Wing Asn Pro Glu Lys Lys Trp 65 70 75 80
Va l Arg Glu Tyr He Asn Ser Leu Glu Met Ser 85 90
) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 78 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
Gly Leu Wing Wing Wing Leu Leu Val Leu Val Cys Thr Met Wing Leu Cys 1 5 10 15 Ser Cys Wing Gln Val Gly Thr Asn Lys Glu Leu Cys Cys Leu Val Tyr 20 25 30 Thr Ser Trp Gln He Pro Gln Lys Phe He Val Asp Tyr Ser Glu Thr 35 40 45 Pro Pro Gln Cys Pro Lys Pro Gly Val He Leu Leu Thr Lys Arg Gly 50 55 60 Arg Gln He Cys Wing Asp Pro Asn Lys Lys Trp Val Gln Lys 65 70 75
(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TCCTTCCTTC TGGTCCTCGG TTCC 24
(2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGAAACAGCT ATGACCATG 19
(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH '31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTTGGAATTC ACTCCGGGCT CCCTCTGCAC G 31
Claims (15)
1. A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence as recited in SEQ ID NO: 2 or its complement.
2. The polynucleotide of Claim 1, wherein the nucleic acid sequence is set forth in SEQ ID NO: 1.
3. An expression vector comprising the polynucleotide of Claim 2.
4. A host cell comprising the expression vector of Claim 3.
5. A nucleic acid probe comprising an unconserved fragment of the polynucleotide of Claim 2
6. an anti-sense molecule comprising a polynucleotide sequence complementary to at least a portion of the polynucleotide of Claim 2.
7. A method for producing a polypeptide comprising the sequence as depicted in SEQ ID NO: 2, the method comprising: a) culturing the host cells of Claim 4 under conditions suitable for expression of the polypeptide, and b) recovering said polypeptide from the cell culture.
8. A chemokine expressed in purified fet .-- 1 spleen having the amino acid sequence as depicted in SEQ ID NO: 2.
9. A chemokine expressed in purified fetal spleen having the N-terminal amino acid residue of the residue 16, Proline, of SEQ ID NO: 2.
10. An antibody specific for the purified polypeptide of claim 9.
11. A diagnostic composition for the detection of nucleic acid sequences encoding chemokine expressed in fetal spleen (FSEC ), which comprises the nucleic acid probe of Claim 5.
12. A diagnostic test for the detection of nucleotide sequences encoding chemokine expressed in fetal spleen (FSBS) in a biological sample, comprising the steps of: a) combining the biological sample with a first nucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof, wherein said fragment is derived from a non-C region; stored from the nucleotide, under conditions suitable for the formation of a dp complex nucleic acid hybridization; b) detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a second nucleotide encoding FSEC in the biological sample; and c) compare the amount of the second nucleotide in? said sample with a standard, determining by the same whether the amount of the second nucleotide varies from said standard, wherein the presence of an abnormal level of the second nucleotide correlates positively with a condition associated with the aberrant expression of FSEC. The diagnostic test of Claim 12, wherein the first nucleotide is labeled with a reporter molecule, and the hybridization complex is detected by measurement of said reporter molecule. 14. A diagnostic test for the detection of nucleotide sequences encoding chemokine expressed in fetal spleen (FSBS) in a biological sample, comprising the steps of: a) combining the biological sample with polymerase chain reaction primers under conditions suitable for nucleic acid amplification, wherein the primers comprise fragments from non-conserved regions of the nucleotide sequence of SEQ ID NO: 1, b) detecting amplified nucleotide sequences; and c) comparing the amount of amplified nucleotide sequences in said biological sample to a standard, thereby determining whether the amount of the nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said nucleotide sequence is correlated positively with a concentration associated with the aberrant expression of FSEC. 15. A method for screening a plurality of compounds for their specific binding affinity with the polypeptide of Claim 8, or any portion thereof, which comprises the steps of: a) providing a plurality of compounds; b) combining the chemokine expressed in fetal spleen (FSBS) with each of a plurality of compounds, for a sufficient time! to allow fixation under suitable conditions; and c) detecting FSEC binding to each of the plurality of compounds, thereby identifying compounds that specifically bind FSEC.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/375,346 US5605817A (en) | 1995-01-19 | 1995-01-19 | DNA encoding chemokine expressed in fetal spleen |
US08375346 | 1995-01-19 | ||
PCT/US1996/000920 WO1996022374A1 (en) | 1995-01-19 | 1996-01-19 | A new chemokine expressed in fetal spleen, its production and uses |
Publications (2)
Publication Number | Publication Date |
---|---|
MX9705366A MX9705366A (en) | 1997-10-31 |
MXPA97005366A true MXPA97005366A (en) | 1998-07-03 |
Family
ID=
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