JP2003521225A - Human phospholipase - Google Patents

Abstract

(57) Abstract The present invention provides human phospholipase (HPPL) and polynucleotides that identify and encode HPPL. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Furthermore, the present invention provides a method for diagnosing or treating or preventing a disease associated with the expression of HPPL.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a nucleic acid sequence and amino acid sequence of human phospholipase, and diagnosis, treatment and prevention of cancer, autoimmune disorder / inflammatory disease, and reproductive disorder using these sequences.

BACKGROUND OF THE INVENTION Triglyceride lipase is an enzyme that hydrolyzes ester bonds in triglycerides. Lipases are widely distributed in animals, plants and prokaryotes. In higher vertebrates, there are at least three tissue-specific isoenzymes, including pancreatic lipase, hepatic lipase, and gastric lipase. The structures of these three lipases are similar to each other and to lipoprotein lipase. Gastric lipase in the intestine aids in the digestion and absorption of dietary fat. Adipocytes contain lipases that break down stored triacylglycerols, which release fatty acids that are transported to other tissues that require them as fuel. Pancreatic lipase, hepatic lipase, and gastric lipase hydrolyze lipoprotein triglycerides and phospholipids. Pancreatic lipase,
The most conserved regions of hepatic lipase and gastric lipase are clustered around serine residues that are also present in prokaryotic lipases.

Phospholipases, a group of enzymes that catalyze the hydrolysis of membrane phospholipids, are classified into PLA1, PLA2, PLB, OLC, and PLD by cleavage of phospholipid bonds.
Phospholipases are responsible for many inflammatory reactions, producing arachidonic acid required for eicosanoid biosynthesis. Specifically, arachidonic acid is converted into inflammatory bioactive lipid mediators such as lysophospholipid-derived platelet activating factor and eicosanoids. Synthesis of arachidonic acid from membrane phospholipids is the rate-limiting step in the biosynthesis of four major classes of prostaglandins, prostacyclins, thromboxanes, and leukotrienes involved in pain, heat, and inflammation (Kaiser, E . other
(1990) Clin. Biochem. 23: 349-370.). Furthermore, leukotriene B4 is known to function in a feedback loop that further enhances PLA2 activity (Wi
jkander, J et al. (1995) J. Biol. Chem. 270: 26543-26549).

The secretory phospholipase A ₂ (PLA2) superfamily includes many heterologous enzymes with the common feature of hydrolyzing the fatty acid acyl ester bond at the sn-2 position of phosphoglycerides. Hydrolysis of glycerophospholipids releases free fatty acids and lysophospholipids. The activity of PLA2 produces biologically active lipids, hydroxy fatty acids, and precursors of platelet activating factor biosynthesis.

PLA2 was originally announced as a component of snake venom, but was later found in many species. PLA2 is divided into several major groups and subgroups by amino acid sequence, divalent cation requirement, and disulfide bond site. Type I, Type II, and Type III PLA2 consist of low molecular weight secretory Ca ²⁺ -dependent proteins. Type IV PLA2 is predominantly a 85 kilodalton Ca ²⁺ -dependent cytosolic phospholipase. Finally, the Ca ²⁺ -dependent PLA2 that constitutes V-form has been shown (Davidson, FF and D
ennis, EA, (1990) J. Mol. Evol. 31: 228-238; and Dennis, EF (1994
) J. Biol Chem. 269: 13057-13060).

The first well characterized PLA2 was type I, II found in snake venom and bee venom.
Type III and type III PLA2. These venom PLA2s share many properties with mammalian PLA2s, including a general catalytic mechanism, the same Ca ²⁺ requirements, and conserved primary and tertiary structure. In addition to its role in prey digestion, the venom PLA2 has actions that promote neurotoxicity, muscle toxicity, blood coagulation inhibition and inflammation in mammalian tissues. Such diverse pathophysiological effects occur due to the presence of specific and high affinity receptors for these enzymes in various cells and tissues (Lambeau, G. et al. (1995) J. Biol.
Chem. 270: 5534-5540).

There has been a description of type I, type IIA, type IIC and type PLA2 in mammalian and avian cells, initially characterized by tissue distribution, but the distinction is no longer absolute. Type I PLA2 was found in the pancreas, type IIA, type IIC were obtained from inflammation-related tissues (eg synovium), and type V was obtained from heart tissue. It is proposed that pancreatic PLA2 functions in the digestion of dietary lipids and plays a role in cell proliferation, smooth muscle contraction, and acute lung injury. Inflammatory PLA2 is a potent mediator of the inflammatory process and is highly expressed in synovial fluid and serum of patients with inflammatory diseases. These type II PLA2s are found in most human cell types examined and are expressed in a wide range of pathological processes such as septic shock, bowel cancer, rheumatoid arthritis, and epidermal hyperplasia. V type PLA2
Is cloned from brain tissue and is highly expressed in heart tissue. Human PLA2 has recently been cloned from fetal lung and is a mammalian PLA called type X due to its structural characteristics.
Is presumed to be the first member of a new group of. Other PLA2s have been cloned from various human tissues and cell lines, suggesting a wide diversity of PLA2s (
Chen, J. et al. (1994) J. Biol. Chem. 269: 2365-2368; Kennedy, BP, et al. (1995
) J. Biol. Chem. 270: 22378-22385; Komada, M., et al. (1990) Biochem. Biophy
s. Res. Commun. 168: 1059-1065; Cupillard, L. et al. (1997) J. Biol. Chem. 2
72: 15745-15752; and Nalefski, EA et al. (1994) J. Biol. Chem. 269: 18239-18.
249).

Upon stimulation, rat platelets secrete two groups of phospholipases, type II phospholipase A2 and phosphatidylserine phospholipase A1 (PS-PLA1). PS-PLA1, also called serine phospholipid-selective phospholipase A (PLA), was purified from rat platelets and was found to be a 55 kilodalton protein with active serine residues. The deduced amino acid 456 sequence contains a putative short N-terminal signal sequence and the GXSXG motif (SEQ IS NO: 10). PS-PLA1 is similar to mammalian lipases such as lipoprotein lipase, hepatic lipase, and pancreatic lipase in about 3
It has 0% homology. Residues surrounding the active serine of PS-PLA1 include histidine and aspartic acid. Serine, histidine, and aspartic acid residues,
Form a triad that is highly conserved in mammalian lipases. PS-PL
A1 hydrolyzes the fatty acid residue at the sn-1 position in lysophosphatidylserine and lysophosphatidylserine (Sato, T. et al. (1997) J. Biol. Chem. 272: 2192-2.
198).

PLA is involved in various disease processes. For example, PLA is found in pancreas, heart tissue, and inflammation-related tissues. It is proposed that pancreatic PLA functions in the digestion of dietary lipids and plays a role in cell proliferation, smooth muscle contraction, and acute lung injury.
Inflammatory PLA is a potent mediator of the inflammatory process and is highly expressed in synovial fluid and serum of patients with inflammatory diseases. In addition, inflammatory PLA is found in most human cell types examined and is expressed in a wide range of pathological processes such as septic shock, bowel cancer, rheumatoid arthritis, and epidermal hyperplasia.

The discovery of a novel human phospholipase and a polynucleotide encoding the same provides a novel composition useful for diagnosing, treating, and preventing cancer, autoimmune disorder / inflammatory disease, reproductive disorder and the like. Needs are met.

(Summary of the Invention) The present invention refers to "HPPL" as a general term, and individually "HPPL-1", "HPPL-2" and "HPPL-3".
Human phospholipase, which is a substantially purified polypeptide referred to as ". In one embodiment of the invention there is provided a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. The present invention also provides a polypeptide comprising an amino acid sequence in which one or more conserved amino acids of the amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 are substituted.

The present invention further provides a substantially purified variant having 90% or more amino acid identity with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. provide. The present invention also provides an isolated and substantially purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. The present invention also provides SEQ ID NO:
Provided is an isolated and purified polynucleotide variant sequence having 90% or more polynucleotide sequence identity with a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of 1-3 and fragments thereof. .

Furthermore, the present invention provides an isolated hybridisation under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. To provide a purified polynucleotide. The present invention also provides an isolated and purified polynucleotide containing a sequence complementary to a polynucleotide encoding a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. provide.

The present invention is also a method of detecting a polynucleotide in a sample containing nucleic acids, the method comprising: (a) hybridizing a complement of a polynucleotide sequence with a polynucleotide of at least one sample, and performing hybridization. A detection method comprising the step of forming a complex and (b) the step of detecting the hybridization complex, wherein the presence of the hybridization complex and the presence of the polynucleotide in the sample are correlated. . In one embodiment of the invention, the step of amplifying the polynucleotide prior to hybridization is included.

The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 4-6 and fragments thereof.
The present invention also provides an isolated and purified polynucleotide variant sequence having 90% or more polynucleotide sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 4-6 and fragments thereof. provide. Further, the present invention provides SEQ ID N
There is provided an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of O: 4-6 and fragments thereof.

The invention further provides an expression vector comprising at least one fragment of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. In another embodiment, the expression vector is contained within a host cell.

The present invention also provides a method for producing a polypeptide, which comprises (a) culturing a host cell containing an expression vector having the polynucleotide of the present invention under conditions suitable for expression of the polypeptide. , (B) recovering the polypeptide from the medium of the host cell.

The present invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof, together with a suitable pharmaceutical carrier. provide.

The present invention further provides a purified antibody that binds to a polypeptide selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. The invention also provides purified agonists and antagonists of the polypeptide.

The present invention also provides a method of treating or preventing a disease associated with reduced expression or activity of HPPL, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. There is provided a method of treatment or prophylaxis comprising administering to a patient an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide together with a suitable pharmaceutical carrier.

The present invention also relates to a method of treating or preventing a disease associated with increased expression or activity of HPPL, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3 and fragments thereof. Methods of treatment or prophylaxis are provided that include administering to the patient an effective amount of a polypeptide antagonist.

(Description of the Present Invention) Before explaining the protein and nucleic acid sequences and methods of the present invention, the present invention is not limited to the specific devices and materials and methods disclosed herein, and its embodiments are modified. Please understand what you can do. Also, the terminology used herein is for the purpose of describing particular embodiments only and is limited only by the scope of the following claims,
It should also be understood that it is not intended to limit the scope of the invention.

“A”, “the (these, etc.)” represent the singular in this specification and the claims.
Note that includes plural forms unless the context clearly indicates otherwise. Thus, for example, “a host cell” includes a plurality of host cells, “antibody” includes a plurality of antibodies, and equivalents well known to those skilled in the art.

All technical terms used herein, unless defined otherwise,
It has the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.
Although any apparatus and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, suitable apparatus and materials and methods are described herein.
All references cited herein are cited to describe and disclose the cell lines, protocols, reagents, vectors described in the references that may be used in connection with the present invention. The mere fact that the conventional invention is cited does not mean that the novelty of the present invention is impaired.

Definitions The term "HPPL" is substantially purified obtained from all species including natural, synthetic, semi-synthetic or recombinant (especially mammals including bovine, ovine, porcine, murine, equine and human). HPPL amino acid sequence.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of HPPL. This agonist either interacts directly with HPPL or interacts with components of biological pathways involving HPPL to regulate the activity of HPPL such as proteins, nucleic acids, carbohydrates, small molecules, or any other compound or The composition may be included.

The term “allelic variant” refers to another form of the gene encoding HPPL. An allelic variant sequence results from at least one mutation in a nucleic acid sequence, the variant mRN
A or a mutant polypeptide, and their structures and functions may or may not change. Some genes include those in which no naturally-occurring allelic variant is present, or one gene or many genes are present. Generally, mutations that give rise to allelic variant sequences are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations, either alone or concurrently with other mutations, occurs one or more times within a given sequence.

A “mutant” nucleic acid sequence that encodes HPPL refers to a polypeptide that has the same polypeptide as HPPL or at least one of the functional properties of HPPL, even though various nucleotide deletions, insertions, or substitutions have occurred. . This definition includes improper or unexpected hybridization of an HPPL-encoding polynucleotide sequence with an allelic variant at a position other than the normal chromosomal locus, as well as specific oligonucleotide probes of the HPPL-encoding polynucleotide. Includes polymorphisms that are easily or difficult to detect using. The encoded protein may also be mutated and may contain deletions, insertions, or substitutions of amino acid residues which result in a silent change and are functionally equivalent to HPPL. Intentional amino acid substitution is similar to the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of residues within a range that biologically or immunologically retains HPPL activity. Can be based on. For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and having polar uncharged side chains can include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and having uncharged side chains can include leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” refer to oligopeptides, peptides, polypeptides, protein sequences, or any fragment thereof, including naturally occurring and synthetic molecules. When the "amino acid sequence" is a naturally occurring protein molecule, "
Amino acid sequence "and like terms are not intended to limit the amino acid sequence to the complete, unaltered amino acid sequence associated with the protein molecules described.

The term “amplification” refers to making copies of nucleic acid sequences. Amplification is generally performed by the polymerase chain reaction (PCR) technique well known in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of HPPL. Antagonists are antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition that directly interacts with HPPL or interacts with components of biological pathways involving HPPL to regulate the activity of HPPL. May include proteins such as things.

The term “antibody” refers to intact molecules such as Fab and F (ab ′) ₂ capable of binding antigenic determinants, and fragments thereof, Fv fragments. Antibodies that bind HPPL polypeptides can be prepared using intact molecules, or fragments thereof, containing small peptides that immunize the antibody.
The polypeptide or oligopeptide used to immunize an animal (eg, mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically and is optionally conjugated with a carrier protein. It is also possible. Commonly used carriers that are chemically linked to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemonean (KLH). The conjugated peptide is then used to immunize the animal.

The term “antigenic determinant” refers to the region (ie, epitope) of a molecule that makes contact with a particular antibody.
Refers to. When a protein or a fragment of a protein is used to immunize a host animal, various regions of this protein are antibodies that specifically bind to an antigenic determinant (a specific region on the protein or a three-dimensional structure). Can induce the production of The antigenic determinant may compete with the intact antigen (ie, the immunogen used to elicit an immune response) to bind the antibody.

The term “antisense” refers to any composition containing a nucleic acid sequence complementary to the sense strand of a particular nucleic acid sequence. Antisense molecules can be produced by any method including synthesis or transcription. Once introduced into a cell, complementary nucleotides combine with the natural sequences made by the cell to form a duplex, which inhibits transcription or translation.
The expression "minus (-)" refers to the antisense strand, and the expression "plus (+)" refers to the sense strand.

The term “biological activity” refers to a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, the term “immunologically active” means that a natural or recombinant HPPL, synthetic HPPL or any oligopeptide thereof induces a specific immune response in a suitable animal or cell to induce a specific antibody. Refers to the ability to combine with.

The terms “complementary” and “complementarity” refer to polynucleotides that form base pairs and are naturally associated with each other. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". The complementarity between two single-stranded molecules may be partial, where only some of the nucleic acids bind, or there may be complete complementarity between the single strands resulting in perfect complementarity. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands, as well as in the design or use of peptide nucleic acid (PNA) molecules.

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” refers broadly to any composition comprising a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding HPPL or a fragment of HPPL can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be bound to a stabilizer such as sugar. In hybridization, the probe is developed in an aqueous solution containing salts (eg, NaCl) and detergents (eg, SDS: sodium dodecyl sulfate) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.

A “consensus sequence” is a nucleic acid sequence that has been sequenced to separate unwanted bases and is 5'and / or 3'using XL-PCR ^™ (Perkin Elmer, Norwalk, CT). Sequenced nucleic acid sequence or GELVI
EW fragment construction system (GCG, Madison, WI), etc. One or more Insight clones using computer programs for fragment construction,
And, in some cases, refers to nucleic acid sequences constructed by duplication of one or more public domain ESTs. Some consensus sequences are constructed by both extension and overlap.

The term “conservative amino acid substitution” refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, in particular its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile . Leu, Thr In general, in the case of conservative amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, eg β sheet or α helix conformation, b) the charge of the molecule at the substituted site or Hydrophobic and / or c) most of the side chains are retained.

The term “deletion” refers to a change in an amino acid sequence that lacks one or more amino acid residues, or a change in a nucleic acid sequence that lacks one or more nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of polynucleotide sequences include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide has at least one biological or immunological function of a natural molecule (unmodified molecule).
Encodes a polypeptide that maintains one. A derivative polypeptide is a polypeptide modified by glycosylation, polyethylene glycolation, or any similar process that maintains at least one of the biological or immunological functions of the original polypeptide. That is.

The term “fragment” refers to a unique portion of HPPL or a polynucleotide encoding HPPL that is identical to its parent sequence but shorter than that sequence. The maximum length of a "fragment" is the parent sequence minus one nucleotide / amino acid residue. For example, a fragment contains 5 to 1000 consecutive nucleotides or amino acid residues. Probe, primer, antigen,
The therapeutic molecule, or fragment used for other purposes, comprises at least 5, 10, 15,
It is the length of 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or 500 consecutive nucleotides or amino acid residues. Fragments may also be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may comprise a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids shown in the given sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the embodiments of the present invention.

Certain fragments of SEQ ID NO: 4-6 differ, for example, from other sequences in the same genome,
Includes a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 4-6.
Certain fragments of SEQ ID NO: 4-6 are useful, for example, in hybridization and amplification techniques, or similar methods of distinguishing SEQ ID NO: 4-6 from related polynucleotide sequences. The exact fragment length or region of SEQ ID NO: 4-6 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

Certain fragments of SEQ ID NO: 1-3 are encoded by certain fragments of SEQ ID NO: 4-6. Certain fragments of SEQ ID NO: 1-3 contain regions of unique amino acid sequence that uniquely identify SEQ ID NO: 1-3. For example, one fragment of SEQ ID NO: 1-3 is specifically
It is useful as an immunogenic peptide for producing an antibody that recognizes ID NO: 1-3. The exact fragment length or region of SEQ ID NO: 1-3 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. This includes partial similarity and complete similarity. The term “identity” can also be called “similarity”. Partially complementary sequences in which hybridization of the same sequence to the target nucleic acid is at least partially blocked are said to be "substantially similar." Inhibition of hybridization between the perfectly complementary sequence and the target sequence is tested using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under mildly stringent conditions. Substantially similar sequences or hybridization probes competitively suppress binding of the perfectly similar (identical) sequence to the target sequence under mildly stringent conditions. This does not mean that non-specific binding is allowed under mildly stringent conditions, but under mildly stringent conditions, the binding of two sequences to each other is specific (ie, selective). You have to interact. A second target sequence, which may not be said to be partially complementary (eg, less than 30% similarity or identity), can be used to test for the absence of non-specific binding. In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

The term “percent identity” or “% identity” with respect to a polynucleotide sequence refers to the residues of matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. It is a percentage. Such algorithms can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison W
I). This CLUSTAL V is for Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153, Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameter is Kt.
Set uple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. Percentage identity is reported by CLUSTAL V as the "percentage similarity" of aligned polynucleotide sequence pairs.

Alternatively, a set of commonly used and freely available sequence comparison algorithms is
NCBI, Bethesda, MD, and Internet (http://WWW.ncbi.nlm.nih.gov/BLAS
National Center for Biotechnology Information (NCB)
I) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J
Mol. Biol. 215: 403-410). The suite of BLAST software includes various sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available,
Used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequen
"ces" can be used interactively by accessing http://WWW.ncbi.nlm.nih.gov/gorf/b12.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program is commonly used with gaps and other parameters that set defaults. For example,
When comparing two nucleotide sequences, one may blast with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set to default parameters.
will use n. Such default parameters are, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on May be measured for the entire length of a given sequence, as determined by the sequence number of, or for shorter lengths, for example, fragments obtained from a given given sequence, for example at least contiguous , 20 or 30
, 40, 50, 70, 100, 200 nucleotide fragments. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

Nucleic acid sequences that do not show high identity may encode similar amino acid sequences due to the degeneracy of the genetic code. Degeneracy can be used to alter nucleic acid sequences to create multiple nucleic acid sequences each encoding substantially the same protein.

The term “percentage of identity” or “% of identity” as used for polypeptide sequences
"Is the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm." Methods of polypeptide sequence alignment are well known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, described in detail above, generally conserve charge and hydrophobicity at the site of substitution, and result in polypeptide structure (and thus function) as well.
Is saved.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program (supra). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignments, the percent identity of paired aligned polypeptide sequences is reported by CLUSTAL V as the "percentage of similarity."

Alternatively, the NCBI BLAST software suite is used. For example, when making a pairwise comparison of two polypeptide sequences, one would use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set with default parameters. . Such default parameters are, for example: Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on It may be measured over the entire length of the peptide sequence, or for shorter lengths, eg fragments derived from one large given polypeptide sequence, eg at least 15, 20, or 30, 40 contiguous. , 50, 70, 150 residues may be measured against the length of the fragment. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

A “human artificial chromosome (HAC)” can contain a DNA sequence of about 6 kb to 10 Mb in size, and is a linear, small-sized chain containing all the elements necessary for stable segregation and maintenance of mitotic chromosomes. It is a chromosome.

The term “humanized antibody” refers to antibody molecules in which the amino acid sequence of the non-antigen binding region has been altered in order to resemble a more human antibody while retaining its original binding ability.

“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms a base pair with a complementary single strand under defined hybridization conditions. Specific hybridization means that two nucleic acid sequences have high identity. Under conditions that allow annealing, specific hybridization complexes are formed and remain hybridized after the washing process. The washing process is particularly important in determining the stringency or stringency of the hybridization process, and under more stringent conditions, non-specific binding, ie binding of pairs of nucleic acid strands that are not perfectly matched. Is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those of skill in the art and are constant during hybridization, but the washing process involves changing the conditions during which to achieve the desired stringency. Is possible and hybridization specificity is obtained. Annealing conditions are, for example, at a temperature of 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100.
Includes μg / ml shear denatured salmon sperm DNA.

In general, hybridization stringency can be expressed in part by the temperature at which the wash process occurs. The wash temperature is usually selected to be about 5-20 ° C below the thermal melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is
It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The formula for calculating the Tm and the conditions for nucleic acid hybridization are well known, Sambrook, J. et al., 1989, Molecular Cloning : A Laboratory Manual , Volume 2 1-3, Cold Spring Harbor Press, Plainvi.
ew NY; Especially described in Chapter 2 of Volume 2.

High stringency hybridization between the polynucleotides of the present invention involves a wash step at about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is carried out at temperatures of 65 ° C, 60 ° C, 55 ° C, 42 ° C. The concentration of SSC is in the range of about 0.1-2xSSC in the presence of about 0.1% SDS. Normally,
Blocking agents are used to prevent non-specific hybridization. Such blocking agents include, for example, about 100-200 μg / ml denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v, for example RNA and
It can be used in specific cases such as hybridization of DNA. Useful modifications of these wash conditions are well known to those of skill in the art. Hybridization, particularly under highly stringent conditions, may suggest evolutionary similarities between the nucleotides. Such similarities strongly suggest that the nucleotides and encoded polypeptides play a similar role.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. Hybridization complexes may be formed in solution (eg, C ₀ t or R ₀ t analysis), or one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin). ,
Or a slide glass, or any suitable substrate for immobilizing cells and their nucleic acids)
Can be formed with another nucleic acid sequence fixed to.

The term “insertion” or “addition” refers to a change in an amino acid sequence or nucleic acid sequence in which one or more amino acid residues or nucleotides are added, respectively.

“Immune response” refers to conditions associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases and the like. These conditions are characterized by the expression of various factors such as cytokines and chemokines, other signaling molecules that affect the cell and systemic defense systems.

The term “microarray” refers to an array of different polynucleotides arranged on a substrate.

The term “element” or “array element” as used in the context of microarrays refers to hybridizable polynucleotides arranged on the surface of a substrate.

The term “modulation” refers to a change in the activity of HPPL. For example, modulation results in alteration of HPPL protein activity, or binding properties, or other biological, functional, or immunological properties.

The term “nucleic acid” or “nucleic acid sequence” refers to nucleotides, oligonucleotides, polynucleotides, or fragments thereof. In addition, DN of genomic or synthetic origin, which is a single-strand or double-strand and is a sense or antisense strand
A or RNA, peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.

“Operably linked” refers to the condition in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are very contiguous or contiguous if the regions encoding the two proteins must be joined in the same reading frame.

“Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent that comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. This terminal lysine renders the composition soluble. PNAs can bind preferentially to complementary single-stranded DNA or RNA to stop elongation of transcripts and polyethylene glycol to prolong life in cells.

The “probe” is a nucleic acid sequence encoding HPPL, a complementary sequence thereof, or a fragment thereof used for detecting the same or allelic nucleic acid sequence or a related nucleic acid sequence. A probe is an oligonucleotide or polynucleotide that has been isolated with a detectable label or reporter molecule attached. Typical labels include radioisotopes and ligands, chemiluminescent reagents, enzymes. A "primer" is capable of forming complementary base pairs and annealing to a target polynucleotide,
A short nucleic acid, usually a DNA oligonucleotide. After the primer anneals to the polynucleotide, it is extended by a DNA polymerase enzyme along the target DNA single strand. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.

The probes and primers used in the present invention include at least 15 of known sequences.
Of contiguous nucleotides. Longer probes and primers can be used to increase specificity. For example, at least 20 or 25, 30, 30, 40, 50, 60, 70, 80, 90, 100 contiguous of the disclosed nucleic acid sequences.
, 150 nucleotides. It should be understood that probes and primers that are considerably longer than the above examples can be used, and nucleotides of any length shown in the tables and drawings of the present specification and the sequence listing can also be used.

For the preparation and use of the probe and the primer, see, for example, Sambrook,
J. et al., 1989, Name "Molecular Cloning: A Laboratory Manual", 2nd
Editions 1-3 (Cold Spring Harbor Press, Plainview NY), or Ausubel, FM
In 1987, the name "Current Protocols in Molecular Biology" (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY), and Innis et al.,
In 1990, the name `` PCR Protocols, A Guide to Methods and Applications '' (Acade
mic Press, San Diego CA.). The set of primers for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research
, Cambridge MA), and the like, and can be derived from one known sequence using a computer program for such purpose.

Oligonucleotides used as primers are selected by computer programs well known in the art for primer selection. For example, OLIGO 4.06 software selects primer pairs for PCR, each up to 100 nucleotides,
And is useful for the analysis of large polynucleotides and oligonucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32,000 bases. Similar primer selection programs include additional features that expand capacity. For example, the PrimOU primer selection program (Genome Center at Uni
versity of Texas South West Medical Center, available from Dallas TX)
Since a specific primer can be selected from the megabase sequence, it is useful for designing a primer in a genome-wide scope. Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research,
(Available from Cambridge MA1) allows users to specify sequences that they would like to avoid as primer binding sites.
) ”Can be entered. In addition, Primer3 is especially useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from each source and modified to meet the needs of users). Can also). PrimerGen Program (UK Human Genome Mapping Pro
(available from the ject Resource Center, Cambridge UK), which designs primers based on multiple sequence alignments, so that they will hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Can be selected. Therefore, this program is useful for identifying unique and conserved oligonucleotide or polynucleotide fragments. The fragment of the oligonucleotide or polynucleotide identified by any of the selection methods described above is, for example, a PCR method or a sequencing primer, a microarray element, or a specific identifying a polynucleotide that is completely or partially complementary to a nucleic acid of a sample. It is useful for the hybridization technique such as the probe. The method for selecting the oligonucleotide is not limited to the above method.

As used herein, a “recombinant nucleic acid” is not a naturally occurring sequence but a sequence that is an artificial combination of two or more distant segments of a sequence. Often, this artificial combination is made by chemical synthesis, but it is more common to artificially manipulate distant segments of nucleic acid using genetic engineering techniques such as those described in Sambrook, supra. is there. The "recombinant nucleic acid" also includes a nucleic acid modified by simply adding or substituting a part of the nucleic acid. The recombinant nucleic acid may also include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such recombinant nucleic acids are vaccinia virus-based viruses that, when used to vaccinate a mammal expressing the recombinant nucleic acids, elicit a defensive immune response in the mammal. It can be part of the vector.

The term “sample” is used in its broadest sense. Samples suspected of containing HPPL-encoding nucleic acids or fragments thereof, HPPL itself include body fluids, extracts from cells, chromosomes or subcellular organelles isolated from cells, membranes, cells, and solutions. Genomic DNA, RNA, cDNA present in or immobilized on a substrate, tissues or tissue prints and the like may also be included.

The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. Point to. This interaction depends on the presence of a particular structure of the protein recognized by the binding molecule, eg, an antigenic determinant or epitope. For example, when the antibody is specific to the epitope "A", the reaction solution containing the unbound labeled "A" and the antibody is bound to the polypeptide containing the epitope A or the unbound unlabeled "A". Is present, the amount of labeled A bound to the antibody is reduced.

The term “substantially purified” refers to a nucleic acid sequence or amino acid sequence that has been removed from the natural environment and then isolated or separated so that the composition to which it is naturally associated has at least about 60 amino acids. % Or more, preferably about 75% or more, and most preferably about 90% or more.

“Substitution” is the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microspheres. Includes particles and capillaries. The substrate has various surface morphologies such as walls or trenches, pins, channels, pores, to which polynucleotides and polypeptides bind.

“Transformation” is the process by which exogenous DNA enters and changes the recipient cell. Transformation can occur under natural or artificial conditions by a variety of methods well known in the art and is performed by any well known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. be able to. This transformation method is
The choice depends on the type of host cell to be transformed. This method includes, but is not limited to, viral infection, electroporation, lipofection, and particulate irradiation. "Transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating autonomously, either as a plasmid or as part of a host chromosome. Further, a cell that transiently expresses the introduced DNA or the introduced RNA within a limited time is also included.

A “variant sequence” of a particular nucleic acid sequence refers to the default parameter setting “BLAST
2 Sequences "tool Version 2.0.9 (May-07-1999) shows that the specific nucleic acid sequence identity for a given length of a given nucleic acid sequence is at least 40%.
The nucleic acid sequence determined to be. Such nucleic acid pairs may, for example, be at least 50% or 60%, 70%, 80%, 85%, 90%, over a length.
It may exhibit 95%, 98%, or more identity. A variant sequence can be represented, for example, as an "allelic" variant sequence (described above) or a "splice" variant sequence, a "species" variant sequence, a "polymorphic" variant sequence. Splice variant sequences may have very high identities to the reference molecule, but alternative splicing of exons during mRNA processing may result in more or less polynucleotides. The corresponding polypeptide may have additional functional domains present in the reference molecule or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have a high degree of amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a given gene from a given gene in a given species. Polymorphic variant sequences may also include "single nucleotide polymorphisms" (SNPs) that differ by one nucleotide in the polynucleotide sequence. The presence of SNPs may be indicative of, for example, a population, a pathological condition, a characteristic of the pathological condition.

A “variant” of a particular polypeptide sequence refers to the default parameter setting “
Blast 2 Sequences "Tool Version 2.0.9 (May-07-1999) by blastp, the identity of the particular polypeptide sequence to a certain length of a certain nucleic acid sequence is determined to be at least 40% of the nucleic acid sequence That is. Such polypeptide pairs may, for example, be at least 50% or 60%, 70%, 8 in a length.
It may exhibit 0%, 85%, 90%, 95%, 98%, or more identity.

(Invention) The present invention is based on the discovery of a novel human phospholipase (HPPL) and a polynucleotide encoding HPPL in the diagnosis, treatment, and prevention of cancer, autoimmune disorders / inflammatory diseases, and reproductive disorders. It relates to the use of these compositions.

Table 1 shows the Incyte clones used to construct the full length nucleotide sequence encoding HPPL. Columns 1 and 2 show the SEQ ID NOs for the polypeptide and polynucleotide, respectively. Row 3 shows the Incyte in which the nucleic acid encoding each HPPL was identified.
The clone IDs of the clones are shown and column 4 shows the cDNA library in which those clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Insight clones for which the cDNA library is not shown were pooled
Derived from a cDNA library. The clone in column 5 was used to construct the consensus nucleotide sequence for each HPPL and is useful as a fragment in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the invention, column 1 SEQ ID N
O, column 2 is the number of amino acid residues in each polypeptide, column 3 is a potential phosphorylation site, column 4 is a potential glycosylation site, column 5 is an amino acid residue having a signature sequence and motif, column 6 is Homologous sequences identified by BLAST analysis, column 7 indicates the method of analysis and optionally the searchable database in which the method of analysis is available. The analysis method in column 7 can be used to characterize each polypeptide from sequence homology and protein motifs.

HPPL-1 has chemical and structural similarities to rat-derived type II phospholipase A-2 (GI 204319; SEQ ID NO: 7). Specifically, HPPL-1 and type II phospholipase A-2
Is 46%. HPPL-2 is a cytoplasmic phospholipase A2 derived from chicken.
(GI 508625; SEQ ID NO: 8) with chemical and structural similarity. For more information, see HPP
The identity of L-2 with cytoplasmic phospholipase A2 is 20%. HPPL-3 has chemical and structural homology with rat phosphatidylserine phospholipase A1 (PS-PLA1) (GI 1817556; SEQ ID NO: 9). In detail, the identity between HPPL-3 and PS-PLA1 is 8
It is 0%. HPPL-3 and PS-PLA1 each contain a short N-terminal signal sequence,
Conserved potential lipase serine active site at residue I160, and GXSXG (SEQ ID
NO: 10) Share the consensus pattern.

The columns of Table 3 show the tissue specificity and diseases or disorders associated with the HPPL-encoding nucleotide sequence. Column 1 of Table 3 shows the nucleotide sequence number (SEQ ID NO). Column 2 shows a fragment of the nucleotide sequence of column 1. For example, these fragments are useful in hybridization or amplification techniques that identify SEQ ID NO: 4-6 and distinguish SEQ ID NO: 4-6 from related polynucleotide sequences. Polynucleotides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the tissue category expressing HPPL and its percentage in total tissues expressing HPPL. Column 4 is HPPL
The diseases or abnormalities, symptoms associated with the tissues that express HPL, and their proportion in the total tissues that express HPPL are shown. Column 5 shows the vector used to subclon each cDNA library. HPPL-1 (SEQ ID NO: 1) is expressed in prostate, breast and testicular tumors, HPPL-2 (SEQ ID NO: 2) is expressed in ovarian, breast and brain tumors and HPPL-3 (SEQ ID NO: Note that: 3) is expressed in gastrointestinal and reproductive tissues.

The columns of Table 4 provide a description of the tissues used to create the cDNA library in which clones of the cDNA encoding HPPL were isolated. Column 1 shows the SEQ ID NO of the nucleotides, column 2 shows the cDNA library from which those clones were isolated, column 3 shows the cDNA of column 2
The origin and details of the tissue corresponding to the NA library are shown.

The present invention also includes variants of HPPL. Preferred variants of HPPL have at least about 80%, more preferably at least about 90%, most preferably at least about 95% amino acid sequence identity with the HPPL amino acid sequence, and are functional or structural of HPPL. Having at least one of the characteristics.

The present invention also includes polynucleotides encoding HPPL. In a particular embodiment, the invention comprises a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 4-6 encoding HPPL.

The present invention also includes variant sequences of the polynucleotide sequence encoding HPPL. In particular, such polynucleotide variants have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity with a polynucleotide sequence encoding HPPL. Have. A particular embodiment of the present invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4-6 and at least about 80%, more preferably at least about 90%, most preferably at least about 95% of a polynucleic acid sequence. A variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 4-6, having nucleotide sequence identity is included. Variant sequences of any of the above polynucleotides may encode an amino acid sequence having at least one functional or structural characteristic of HPPL.

Various polynucleotide sequences encoding HPPL that may be created by the degeneracy of the genetic code include those that have minimal similarity to polynucleotide sequences of any known naturally occurring gene. Those of ordinary skill in the art will understand. Thus, the present invention may include all of the possible polynucleotide sequence variations that may be created by the selection of combinations based on possible codon choices. These combinations are based on the standard triplet genetic code applied to the naturally occurring HPPL polynucleotide sequences, and are considered to be the clear disclosure of all mutations.

It is desirable that the nucleotide sequence encoding HPPL and its mutated sequences be capable of hybridizing to naturally occurring HPPL nucleotides under suitable selected stringent conditions, but with non-naturally occurring codons. It may be beneficial to create a nucleotide sequence that encodes HPPL or a derivative thereof with substantially different codon usage, such as inclusion. Codons can be selected based on the frequency with which a particular codon is utilized by the host to increase the rate at which peptide expression occurs in a particular eukaryotic or prokaryotic host. Without changing the encoded amino acid sequence,
Another reason to substantially alter the nucleotide sequences encoding HPPL and its derivatives is to produce RNA transcripts with favorable properties, such as longer half-life, than transcripts made from naturally occurring sequences.

The present invention also includes the production of DNA sequences encoding HPPL and its derivatives or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of the various available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to introduce mutations into the sequence encoding HPPL or any fragment thereof.

The present invention further provides polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, particularly SEQ ID NO: 4-6 and fragments thereof, under various stringent conditions. Included (e.g. Wahl, GM and SL Berger (
1987) Methods Enzymol. 152: 399-407; and Kimmel. AR (1987) Methods Enzy.
mol. 152: 507-511.). Hybridization conditions, including annealing and wash conditions, are described in the "Definitions".

Any of the embodiments of the present invention can be carried out using DNA sequencing methods well known in the art. This method includes, for example, the Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq Polymerase (Perkin Elmer)
, Thermostable T7 polymerase (Amersham, Pharmacia Biotech Piscataway NJ),
Alternatively, enzymes such as a combination of proofreading exonuclease and polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD) are used. Preferably, MICROLAB 2200 Liquid Transfer System (Hamilton, Reno, NV),
PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATALYST 80
Automate sequence preparation using a device such as 0 (Perkin-Elmer). Then ABI 373
Alternatively, sequencing is performed using the 377 DNA Sequencing System (Perkin-Elmer), MEGABACE 1000 DNA Sequencing System (Molecular Dynamics. Sunnyvale CA) or other methods known in the art. The resulting sequences are analyzed using various algorithms well known in the art (e.g., Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7;
Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New
York NY, pp. 856-853.).

A variety of PCR-based methods well known in the art can be used to extend the nucleic acid sequence encoding HPPL using partial nucleotide sequences to extract upstream sequences such as promoters and regulatory elements. To detect. For example, one method utilizing restriction site PCR is to amplify an unknown sequence from genomic DNA in a cloning vector using common and nested primers (eg, Sarkar, G. (1993) PCR Met.
See hods Applic 2: 318-322). An alternative method using the inverse PCR method uses primers that extend in a wide range of directions and amplify unknown sequences from a circularized template. This template is derived from a restriction fragment containing known genomic loci and sequences surrounding it (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). Capture PCR
A third method using the method involves PCR amplification of DNA fragments flanking known sequences of human and yeast artificial chromosome DNA (eg, Lagerstrom, M. et al. (1991) PCR Methods Ap.
See plic 1: 111-119). This method allows digestion and ligation with multiple restriction enzymes to insert the recombinant double-stranded sequence into a region of unknown sequence prior to PCR. It is also possible to obtain the unknown sequence using other methods well known in the art. (For example, Parker, JD et al. (1991) Nucleic Acids Res
. 19: 3055-3060). Furthermore, by using PCR, nested primers, and PROMOTER FINDER library (Clontech, Palo Alto CA), walking within genomic DNA is possible. This method does not require screening the library and is useful for looking for intron / exon junctions. In all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis software (National Bioscien
ces, Plymouth MN) or another suitable program etc.
It is designed to anneal to the template at a temperature of about 68 ° C to 72 ° C with 0 nucleotides, a GC content of 50% or more.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain large cDNAs. Furthermore, if the oligo d (T) library cannot produce a full-length cDNA, a randomly primed library, which often contains a sequence having the 5'region of the gene, is useful. Genomic libraries will be useful for extension of sequences into the 5'non-transcribed regulatory regions.

Sequencing or PCR using a commercially available capillary electrophoresis system
It is possible to analyze or confirm the size of the nucleotide sequence of the product. For more information,
Capillary sequencing uses a flowable polymer for electrophoretic separation, and a laser-activated fluorochrome specific for four different nucleotides and a CCD camera used to detect emitted wavelengths. It is possible.
The output / light intensity is determined by the appropriate software (eg GENOTYPER and SEQUENCE NAVIGA
TOR, Perkin-Elmer) is used to convert to an electric signal, and the process from sample loading to computer analysis and electronic data display can be controlled by computer. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA, which may be present in small amounts in a particular sample.

In another embodiment of the invention, the polynucleotide sequence encoding HPPL or a fragment thereof is used in a recombinant DNA molecule to express HPPL, a fragment thereof or a functional equivalent in a suitable host cell. Is possible. Due to the inherent duplication of the genetic code, alternative DNA sequences may be produced which encode substantially the same or functionally equivalent amino acid sequences, which sequences are available for cloning and expression of HPPL.

The nucleotide sequences of the invention can be recombined using methods generally known in the art to alter the HPPL coding sequence for a variety of purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Nucleotide sequences can be recombined using DNA mixing with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, and the like.

According to another embodiment, the sequence encoding HPPL can be wholly or partially synthesized using chemical methods well known in the art (eg, Caruthers. MH et al. (1980)).
) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acid
s Res. Symp. Ser. 225-232). Alternatively, chemical methods can be used to synthesize HPPL itself or fragments thereof. For example, peptide synthesis can be performed using a variety of solid phase techniques (eg, Roberge, JY et al. (1995) Science 269:
See 202-204). Alternatively, automation of synthesis may be achieved using, for example, the ABI 431A Peptide Synthesizer (Perkin Elmer). In addition, the amino acid sequence of HPPL, or any portion thereof, may be altered by direct synthetic alterations and / or combinations with sequences from other proteins or any portion thereof using chemical methods. It is possible to make a polypeptide.

This peptide is used for preparative high performance liquid chromatography (eg, Chiez, RM
And FZ Regnier (1990) Methods Enzymol. 182: 392-421)). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton. T. (1983) Proteins , Structures and Molecular Properties , WH Freeman, New York, NY).
.

To express the biologically active HPPL, the nucleotide sequence encoding HPPL or a derivative thereof is inserted into a suitable expression vector. This expression vector contains the elements necessary for the regulation of transcription and translation of the coding sequence inserted into a suitable host. These elements include regulatory sequences such as enhancers, constitutive and expression inducible promoters, 5'and 3'untranslated regions in the vector and polynucleotide sequences encoding HPPL. Such elements vary in length and specificity. With particular initiation signals, it is possible to achieve more efficient translation of sequences encoding HPPL. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. If the sequence encoding HPPL and its initiation codon, upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However,
In-frame AT if only the coding sequence or a fragment thereof is inserted
Exogenous translational control signals, including the G start codon, must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of natural and synthetic sources. The efficiency of expression can be increased by the inclusion of enhancers suitable for the particular host cell system used. (For example, Scharf, D. et al. (19
94) Results Probl. Cell Differ. 201-18-162.).

Methods well known to those skilled in the art can be used to produce expression vectors containing sequences encoding HPPL and suitable transcriptional and translational regulatory elements. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination technology. (
For example, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual ,
Cold Spring Harbor Press, Plainview NY, chapters 4 & 8, and 16-17; and
Ausubel, FM et al. (1995) Current Protocols in Molecular Biology , John Wi
ley & Sons, New York NY, ch. 9 and 13-1-4).

A variety of expression vector / host systems may be utilized to retain and express sequences encoding HPPL. These include, but are not limited to, microorganisms such as recombinant bacteriophage, plasmids, or bacteria transformed with cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors. (Eg baculovirus) infected insect cell lines, viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg Ti or pBR322 plasmids).
Plant cell lines transformed with, and animal cell lines. The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for the polynucleotide sequence encoding HPPL. For example, routine cloning, subcloning of a polynucleotide sequence encoding HPPL,
PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (G
A multifunctional E. coli vector such as IBCO BRL) can be used. Ligation of sequences encoding HPPL into multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method for identification of transformed bacteria containing recombinant molecules. In addition, these vectors can be used to generate in vitro transcription of cloned sequences, dideoxin screening, single chain rescue by helper phage, and nested deletions. (See, for example, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.). For example, when a large amount of HPPL is required for antibody production, a vector that induces HPPL expression at a high level can be used. For example, a vector containing a strongly inducible T5 or T7 bacteriophage promoter can be used.

Yeast expression systems can be used to express HPPL. Various vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH promoter can be used for yeast Saccharomyces cerevisiae or Pichia pastoris . Furthermore, such vectors induce either the secretion of the expressed protein or its retention within the cell, integrating foreign sequences into the host genome for stable growth. (For example, Ausubel .; and Bitter, GA et al. (1987) Method above.
s Enzymol. 153: 51-794: Scorer. CA et al. (1994) Bio / Technology 121-181-18
Plant systems can also be used to express HPPL. Transcription of the sequence encoding HPPL, for example, viral promoters such as 35S and 19S promoter derived from CaMV alone,
Alternatively, it is promoted in combination with an omega leader sequence derived from TMV (Takamatsu, N. et al. (1987) EMBO J 6: 307-311). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992) Mc
See Graw Hill NY, pp.191-196).

In mammalian cells, a variety of virus-based expression systems may be utilized. When adenovirus is used as an expression vector, the HPPL coding sequence can be linked to an adenovirus transcript / translation complex consisting of a late promoter and a triple leader sequence. Insertion into the nonessential E1 or E3 region of the viral genome makes it possible to obtain live virus expressing HPPL in infected host cells (Logan, J.
And Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells. For high level expression of proteins, SV40 or EBV based vectors can be used.

Human artificial chromosomes (HACs) can be used to supply fragments of DNA that are larger than those expressed in and contained in a plasmid. HACs of about 6kb-10Mb for treatment
Are prepared and delivered by conventional delivery methods (liposomes, polycationic amino polymers, or vesicles). (For example, Harrington.JJ et al. (1997) Nat Genet. 15: 3.
45-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of HPPL in cell lines is desirable. For example, an expression vector can be used to transform a cell line with a sequence encoding HPPL. Such expression vectors contain replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are grown in enriched medium for about 1-2 days before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selective mediators, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques suitable for that cell type.

Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or apr ^- used in the cell. (For example, Wigler, M. et al. (1
977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, and als or pat confers resistance to chlorsulfuron (cHPPLsulfuron) and phosphinotricin acetyltransferase (eg, phosphinotricin acetyltransferase). , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14). Further genes available for selection have been described in the literature, eg trpB and hisD which alter what the cell needs for metabolism (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad.
. Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (GFP; Clon
tech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin and other visible markers are used. Green fluorescent protein (GFP) (Clon
tech, Palo Alto, CA) can also be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to a particular vector system (eg, Rhodes, CA et al. (19
95) Methods Mol. Biol. 55: 121-791).

Even if the presence / absence of the expression of the marker gene indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of the gene. For example, if the HPPL coding sequence is inserted within a marker gene sequence, transformed cells containing the HPPL coding sequence can be identified by the lack of marker gene function.
Alternatively, the marker gene can be placed in tandem with the sequence encoding HPPL under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, a host cell that contains a nucleic acid sequence encoding HPPL and expresses HPPL can be identified using a variety of methods well known to those of skill in the art. These methods include
Protein biological tests or immunological methods including DNA-DNA or DNA-RNA hybridization, PCR methods, membrane-based, solution-based, or chip-based techniques for detection and / or quantification of nucleic acids or proteins. Assay included,
It is not limited to these.

HP using either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring PL expression are well known in the art. Such techniques include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence activated cell sorters (FACS) and the like. Two-site, monoclonal-based immunoassay using a monoclonal antibody that reacts with two non-interfering epitopes on HPPL
Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton. R
Et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Paul
.MN, Sect. IV; Coligan, JE et al Current Protocols in Immunology , Greene
Pub. Associates and Wiley-Interscience, New York. NY; and Pound, JD (
1990) Immunochemical Protocols , Humans Press, Totowa NJ).

Various labeling and conjugation methods are well known to those of skill in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to HPPL-encoding polynucleotides include oligo-labeling, nick-translation, end-labeling, or labeled nucleotides. Includes the PCR amplification used. Alternatively, the HPPL coding sequence, or any fragment thereof, can be cloned into a vector to generate an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be used for the synthesis of RNA probes in vitro by the addition of a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. . These methods are described, for example, in Amersham Pharmac
ia Biotech and Promega (Madison WI), US Biochemical Corp (Cleveland OH
) Can be used with various kits commercially available. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding HPPL are cultured under conditions suitable for the expression and recovery of this protein in cell culture. Whether the protein produced by a transformed cell is secreted or remains intracellular depends on the sequence and / or the vector used. It will be understood by those skilled in the art that the expression vector containing the polynucleotide encoding HPPL can be designed to contain a signal sequence that induces the secretion of HPPL that penetrates the prokaryotic cell membrane and the eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (
lipidation), and acylation, but is not limited thereto.
Post-translational processing which cleaves a "prepro" or "pro" form of the protein can be used to identify target proteins, folds and / or activities. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular machinery and characteristic mechanisms for post-translational activity are available in the American Type Culture C
Ollection (ATCC; Bethesda, MD) and is selected to ensure correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural or modified or recombinant nucleic acid sequence encoding HPPL is linked to a heterologous sequence which results in translation of the fusion protein of any of the host systems described above. For example, a chimeric HP containing a heterologous moiety that can be recognized by a commercially available antibody
PL proteins may facilitate the screening of peptide libraries for inhibitors of HPPL activity. In addition, the heterologous protein portion and the heterologous peptide portion are
Commercially available affinity substrates can be used to facilitate purification of the fusion protein. Such parts include glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-Hi.
s, FLAG, c-mc, hemagglutinin (HA), but are not limited thereto. GST and MBP, Trx, CBP, 6-His immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
Each of the metal chelate resins allows purification of the cognate fusion protein. FLAG
, C-mc, and hemagglutinin (HA) allow purification of the immunoaffinity of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, HPPL can be cleaved from the heterologous moiety after purification by genetically engineering the fusion protein to include a proteolytic cleavage site between the HPPL coding sequence and the heterologous protein sequence. Methods for expressing and purifying fusion proteins are described in Ausubel. (1995, supra ch 10). A variety of commercially available kits can be used to facilitate expression and purification of the fusion protein.

In another embodiment of the invention, it is possible to synthesize radiolabeled HPPL in vitro using the TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega). These systems connect transcription and translation of sequences encoding proteins that are operably linked to the T7 or T3, SP6 promoters. Translation occurs in the presence of a radiolabeled amino acid precursor, which is, for example, ³⁵ S methionine.

Fragments of HPPL can be produced by direct peptide synthesis using solid phase techniques as well as recombinant products (see, eg, Creighton, pp. 55-60. Supra). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed using, for example, a 431A Peptide Synthesizer (Perkin Elmer). Various fragments of HPPL are synthesized separately and then combined to produce the full length molecule.

Treatment There are chemical and structural similarities between certain regions of HPPL and human phospholipases, for example in the context of sequences and motifs. Furthermore, HPPL expression is closely associated with cancer, inflammation, and immune responses, and gastrointestinal and reproductive tissues. Therefore, HPPL is believed to play a role in cancer, autoimmune disorders / inflammatory diseases, and reproductive disorders. In the treatment of diseases associated with increased HPPL expression or activity, it is desirable to reduce HPPL expression or activity. In addition, in the treatment of diseases associated with decreased expression or activity of HPPL, it is desirable to increase expression or activity of HPPL.

Therefore, in one embodiment, HPPL or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of diseases associated with decreased expression or activity of HPPL. Examples of such diseases include, but are not limited to, cancer, including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and the like,
Specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen , Testicular, thymic, thyroid, and uterine cancer, and also autoimmune disorders / inflammatory diseases, including acquired immune deficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, Allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candid ectodermal dystrophy (APECED), bronchi Inflammation, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema,
Lymphotoxin-induced transient lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, hypersensitivity Colorectal syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Scheegren's syndrome, Systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation and viral and bacterial infections, fungal infections, parasites Infectious diseases, protozoal infections, helminthic infections, trauma, and reproductive disorders, including abnormal prolactin production, fallopian tube disease, ovulation abnormality, and endometriosis, estrus Normal, infertility, including menstrual cycle abnormalities, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune disorders, ectopic pregnancy, and malformations, and breast cancer, fibrocystic Mastopathy and mastosis, including spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male breast cancer and gynecomastia .

In another embodiment, HPPL or a fragment or derivative thereof is expressed for the treatment or prevention of diseases associated with decreased expression or activity of HPPL, including but not limited to the diseases listed above. It is also possible to administer the resulting vector to a patient.

In yet another embodiment, substantially purified HPPL is used for the treatment or prevention of diseases associated with decreased expression or activity of HPPL, including but not limited to the diseases listed above. It is also possible to administer the pharmaceutical composition containing it to a patient together with a suitable pharmaceutical carrier.

In yet another embodiment, an agonist that regulates the activity of HPPL for the treatment or prevention of diseases associated with decreased expression or activity of HPPL, including but not limited to the diseases listed above. It is also possible to administer to a patient.

In a further embodiment, a patient may be administered an antagonist of HPPL for the treatment or prevention of disorders associated with increased expression or activity of HPPL. Examples of such disorders include, but are not limited to, cancer, autoimmune disorders / inflammatory disorders, and reproductive disorders. In one embodiment, antibodies that specifically bind HPPL can be used directly as an antagonist or indirectly as a targeting or delivery mechanism to deliver the drug to cells or tissues expressing HPPL.

In another example, the complement of a polynucleotide encoding HPPL for the treatment or prevention of diseases associated with increased expression or activity of HPPL, including but not limited to the diseases listed above. It is also possible to administer a vector that expresses

In another example, any protein, antagonist, antibody, agonist, complementary sequence, vector of the invention can be administered in combination with another suitable therapeutic agent. One of ordinary skill in the art can select suitable therapeutic agents for use in the combination therapy according to conventional pharmaceutical principles. The combination with therapeutic agents may bring about a synergistic effect in the treatment or prevention of the various diseases listed above. Using this method, it is possible to obtain a medicinal effect with a small amount of each drug, and it is possible to reduce the possibility of a wide range of side effects.

HPPL antagonists can be prepared using methods common in the art. More specifically, purified HPPL can be used to make antibodies, or a library of therapeutic agents can be screened to identify those that specifically bind to HPPL. HPPL
Antibodies can also be produced using a method generally used in this field. Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chains,
Fab fragments and fragments produced by Fab expression libraries are included. However, it is not limited to these. For treatment, neutralizing antibodies (ie,
Those which inhibit the formation of dimers) are particularly preferred.

For the production of antibodies, various hosts, including goat, rabbit, rat, mouse, human and others, are injected with HPPL or any fragment or oligopeptide thereof with immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and surfactants such as dinitrophenol. However, it is not limited thereto. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly preferable.

Oligopeptides, peptides, or fragments used to elicit antibodies to HPPL are preferably composed of at least about 5 amino acids, generally about 10 or more amino acids. These oligopeptides or peptides, or fragments thereof, are preferably identical to a portion of the amino acid sequence of the native protein and also include the entire amino acid sequence of small naturally occurring molecules. Short stretches of HPPL amino acids can be fused with sequences of another protein, such as KLH, to produce antibodies to the chimeric molecule.

Monoclonal antibodies against HPPL can be generated by any cell line in culture, using any technique which produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler,
G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. Met.
hods 81-8-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030.
Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing the mouse antibody gene onto the human antibody gene developed for the production of “chimeric antibodies” are used to obtain molecules with suitable antigen specificity and biological activity. (For example, Morrison, SL et al. (1984) Proc.
Natl. Acad. Sci. 81-4851-4855; Neuberger, MS et al. (1984) Nature 312: 6.
04-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, the techniques described for the production of single chain antibodies are applied using methods well known in the art to produce HPPL-specific single chain antibodies. Antibodies with related specificities but of different idiotype composition can also be generated by chain mixing from random combinatorial immunoglobulin libraries (e.g., Burton DR (1991) Proc. Natl.
Acad. Sci. 88: 11120-3).

Antibodies can be induced by in vivo production in a population of lymphocytes, or by screening immunoglobulin libraries or a panel of highly specific binding reagents, as shown in the literature. (See, for example, Orlando, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833.
-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibodies containing specific binding sites for HPPL can also be produced. For example,
Such fragments include F (ab ') fragments produced by pepsin digestion of antibody molecules and Fabs produced by reducing the disulfide bridges of the fragments to F (ab').
Fragments are included, but are not limited to. Alternatively, generating a Fab expression library allows for rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 12.
See 75-1281).

A variety of immunoassays are used to screen to identify antibodies with the desired specificity. Numerous protocols for competitive binding, or immunoradioactivity, using either polyclonal or monoclonal antibodies with isolated specificity are well known in the art. HPPL is commonly used for such immunoassays.
Included is the measurement of complex regulation between the antibody and its specific antibody. Two-site monoclonal-based immunoassays are generally used with monoclonal antibodies reactive to two non-interfering HPPL epitopes, but competitive binding assays can also be used (Pound, supra).

Using various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques, H 2
Assess the affinity of the PPL antibody. Affinity is represented by the binding constant Ka, which is the molar concentration of HPPL antibody complex divided by the molar concentration of free antibody and free antigen under equilibrium conditions. The measured value Ka of the polyclonal antibody reagent having a heterogeneous affinity for many HPPL epitopes represents the average affinity or binding activity of the HPPL antibody. Specific HPPL
The Ka of the monoclonal antibody reagent monospecific for the epitope represents a true measure of affinity. High affinity antibody reagents with Ka values of 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays where the HPPL antibody complex must withstand vigorous manipulation. The low affinity antibody reagent having a Ka value of 10 ⁶ to 10 ⁷ L / mol is preferably used for immunopurification and similar treatments in which HPPL must be finally dissociated from the antibody in an activated state. (Catty, D. (1988) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, DC; Liddell, JE and Cryer,
A. (1991) A Practical Guide to Monoclonal Antibodies , John Wiley & Sons
, New York NY).

The antibody titer and binding activity of polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, at least 1
Polyclonal antibody reagents containing 2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, are commonly used in processes in which the HPPL antibody complex must be precipitated. Specificity of antibodies and antibody titer, binding activity, quality of antibodies and guidelines for usage in various applications are publicly available. (Eg Catty, supra
, And Coligan et al., Supra).

In another embodiment of the invention, the polynucleotide encoding HPPL, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In certain embodiments, if the complementary sequence of the polynucleotide encoding HPPL is suitable for blocking the transcription of mRNA, it can be used. In particular, cells can also be transformed with sequences complementary to the polynucleotide encoding HPPL. Thus, the complementary molecule or fragment can be used to regulate the activity of HPPL, or the regulation of gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the sequence encoding HPPL, or from various positions along the coding region.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia, or various bacterial plasmids can also be used to deliver the nucleotide sequence to the target organ, tissue or cell population. Methods which are well known to those of skill in the art can be used to produce vectors expressing nucleic acid sequences complementary to HPPL-encoding polynucleotides (see, eg, Sambrook et al., Supra, and Ausubel et al., Supra). .

The gene encoding HPPL can be stopped by transforming cells or tissues with an expression vector that expresses high levels of a polynucleotide encoding HPPL or a fragment thereof. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Even when not integrated into DNA, such vectors continue to transcribe mRNA molecules until impaired by endogenous nucleases. Non-replicating vectors also have transient expression that lasts for a month or more, and may last even longer if suitable replication elements are part of the vector system.

As described above, gene expression can be regulated by designing a sequence complementary to the regulatory 5 ′ or regulatory region of the gene encoding HPPL or an antisense molecule (DNA or RNA, PNA). . For example, it is possible to use an oligonucleotide derived from the transcription start site from about -10 to about +10 from the start site. Similarly, it can be blocked using "triple helix" and base pairing methods.
The triple helix structure is useful because it prevents the double helix from expanding sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances with triplex DNA have been described in the literature (eg Gee, JE et al. (1994) In: Hub.
er, BE and BI Carr, Molecular and Immunologic Approaches , Futura Pu
See blishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes, enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand scission.
For example, recombinant hammerhead ribozyme molecules that specifically and effectively catalyze the nucleotide strand scission of the sequence encoding HPPL are included.

A specific ribozyme cleavage site in any potential RNA target is identified by the subsequent sequence GU.
Initially identified by scanning the target molecule against ribozyme cleavage sites including A, GUU, and GUC. Once identified, a short RNA sequence of 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is evaluated for secondary structural features that impair the function of the oligonucleotide. Is possible. The suitability of candidate targets can also be assessed by testing their ease of hybridization with complementary oligonucleotides using a ribonuclease protection assay.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made for the synthesis of nucleic acid molecules using methods well known in the art. These methods include methods of chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, RNA molecules may be generated in vitro and in vivo by transcription of a DNA sequence encoding HPPL, such DNA sequences using various RNA vector promoters, such as T7 or SP6, of various vectors. Can be incorporated into. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

Modification of RNA molecules can increase intracellular stability and prolong half-life. Possible modifications include the addition of flanking sequences at the 5'and / or 3'ends of the molecule, or the use of phosphorothionate or 2'O methyl rather than phosphodiesterase linkages within the backbone of the molecule. , Are not limited to these. Unique to PNA production, this concept is that adenine, cytidine, guanine, thymine, which are not readily recognized by endogenous endonucleases,
And the acetyl-, methyl-, thio-, and similar modified forms of uridine, as well as the inclusion of non-conventional bases such as inosine, queosine, and wybutosine, in whole of these molecules. Can be expanded to.

[0146] vectors available are a number of ways of introducing into a cell or tissue, in vivo, are equally suitable for use in in vitr o, and ex vivo. For ex vivo treatment, the vector is introduced into hepatocytes taken from a patient and cloned and propagated for autologous transplant back into the same patient. Transfection, ribosome injection or delivery by polycationic aminopolymers can be performed using methods well known in the art (eg Goldman, CK et al. (1997) Nature Biotechnology 15: 462.
-66 :).

Any of the above methods of treatment can be applied to any subject in need of treatment including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits and monkeys.

Another embodiment of the present invention relates to the administration of a pharmaceutical or sterile composition together with a pharmaceutically acceptable carrier for all of the above mentioned therapeutic effects. Such a pharmaceutical composition comprises HPPL, an antibody against HPPL, a mimetic, an agonist, an antagonist, an inhibitor of HPPL, or the like. The composition can be administered alone or with one or more additional agents such as stabilizers to a sterile biocompatible pharmaceutical carrier.
Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water and the like. This composition can be administered alone or with another agent such as a drug or hormone.

The pharmaceutical composition used in the present invention can also be administered using any number of routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, ectopic, sublingual, It also includes, but is not limited to, the rectum.

In addition to active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable excipients and adjuvants that facilitate the formulation of the active compounds into pharmaceutically usable agents. The carrier may be included. Detailed techniques for formulation and administration are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers allow tablets, pills, dragees, capsules, and pharmaceutical compositions to be ingested by patients.
It is formulated as a liquid, gel, syrup, mud, or suspension.

Medicaments for oral use include tablets or dragee cores (after grinding if desired) by mixing the active compound with solid drug additives and treating the resulting granule mixture. cores). Suitable excipients include lactose, sucrose, mannitol, or sugars including sorbitol, starch from corn, wheat, rice, potato, or other plants, methylcellulose, hydroxypropylmethylcellulose, or cellulose such as sodium carboxymethylcellulose, Gum, including gum arabic and gum tragacanth, carbohydrates or protein excipients such as proteins such as gelatin and collagen.
If desired, a disintegrant or solubilizer such as cross-linked polyvinylpyrrolidone, kanten, alginic acid, or its salt sodium alginate is added.

Dragee cores are used with a suitable coating such as a concentrated sugar solvent. Such concentrated sugar solvents may include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solvents, and suitable organic or mixed solvents. Dyestuffs or pigments are added to the tablets or dragee coatings for identification of the product or to indicate the quantity of active compound, ie the dose.

Pharmaceutical formulations for oral use include push-fit capsules made of gelatin, encapsulated capsules made of gelatin with a coating such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in admixture with excipients or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the active compound, with or without stabilizer,
It may be dissolved or suspended in a suitable solution such as a fatty oil, solution, or polyethylene glycol solution.

Pharmaceutical agents suitable for parenteral administration are formulated in aqueous solution, but physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological buffered saline are preferred. Aqueous suspension injections may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable hydrophilic solutions or vehicles include fatty oils such as sesame oil, ethyl oleate, triglycerides or synthetic fatty acids such as ribosomes. Non-lipid polycationic amino polymers are used for delivery purposes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for concentrated solutions.

For local or nasal administration, suitable penetrants that penetrate certain barriers are used in the formulation. Such penetrants are well known to those of ordinary skill in the art.

The pharmaceutical compositions of the present invention may be prepared using methods well known in the art, such as conventional mixing, dissolving, granulating, sugar coating, elevating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Can be manufactured.

Pharmaceutical compositions are formulated as salts and can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid. Salts are more soluble in water or other protic solvents than are the corresponding free base systems. Another drug form comprises some or all of 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, with a pH range of 4.5-5.
5, a lyophilized powder that is buffered prior to use can be used.

After the pharmaceutical compositions have been prepared, they are packaged in suitable boxes and labeled as a drug for the indicated symptoms. For administration of HPPL, such labels will include amount, frequency and mode of administration.

Suitable pharmaceutical compositions for use in the present invention include compositions that contain an effective amount of the active ingredients to achieve the objectives. Those of ordinary skill in the art can determine a dose that is sufficiently effective based on their ability.

For any composition, the therapeutically effective dose can be estimated initially either in tumor cell assays, eg, of tumor cells, or in animal models. Usually, a mouse, a rabbit, a dog, a pig, or the like is used as the animal model. Animal models can also be used to determine the appropriate concentration range and route of administration. Based on such treatment, a beneficial dose and administration route for human can be determined.

A medically effective dose is related to the amount of active ingredients, such as HPPL or fragments thereof, HPPL antibodies, HPPL agonists or antagonists, inhibitors, etc. that ameliorates symptoms or conditions. Medicinal efficacy and toxicity can be calculated, for example, by calculating the ED ₅₀ (50% of the population has a medicinal effect on dose) or LD ₅₀ (50% of the population is fatal on dose) statistics. , Can be determined by standard drug techniques in cell culture or animal studies. Dosage ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD _{50 /} ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human application. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the patient sensitivity, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Dosage factors considered include disease severity, general health of the patient, age, weight and sex of the patient, time and frequency of administration, concomitant medications, response susceptibility, and treatment. Contains the response. Long-acting pharmaceutical compositions may be administered every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.

The usual dosage varies depending on the route of administration, but is about 0.1-100,000 μg.
Up to about 1 gram. Guidance on specific dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art will utilize formulations of nucleotides that are different than the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide is
It will be specific to a particular cell, condition, location, etc.

Diagnosis In another example, a patient who is diagnosed with a disease in which an antibody that specifically binds to HPPL is characterized by the expression of HPPL, or is treated with HPPL or an agonist, antagonist, or inhibitor of HPPL. Used in an assay to monitor Antibodies useful for diagnosis are formulated in the same manner as described in treatment.
Diagnostic assays for HPPL include methods that use antibodies and labels to detect HPPL from human body fluids or from cells and tissues. These antibodies, used with or without modification, can be labeled by covalent or non-covalent attachment of reporter molecules. Various reporter molecules known in the art are used, some of which have been mentioned above.

Various protocols for measuring HPPL, including ELISA, RIA, and FACS, are well known in the art and provide the basis for diagnosing altered or abnormal levels of HPPL expression. The value of normal or standard HPPL expression is determined by binding a body fluid or cell collected from a subject such as a normal mammal, for example, a human, with an antibody against HPPL under conditions suitable for complex formation. To do. The amount of canonical complex formation can be quantified by various methods such as photometric. Amounts of HPPL expression, control and disease in the subject, samples from biopsy tissue are compared to standard values. The deviation between the standard value and the subject is the parameter for diagnosing the disease.

According to another embodiment, the polynucleotide encoding HPPL can also be used for diagnosis. The polynucleotide used includes an oligonucleotide sequence,
Included are complementary RNA and DNA molecules, and PNAs. This polynucleotide is used to detect and quantify the expression of genes in biopsied tissues that express HPPL that can be correlated with disease. Use this diagnostic assay to check for the absence, presence, and overexpression of HPPL,
Monitor the control of HPPL levels during treatment.

In certain embodiments, it is possible to identify a nucleic acid sequence encoding HPPL by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising a gene sequence encoding HPPL or a closely related molecule. is there. The specificity of the probe, whether it is made from a highly specific region such as a 5'regulatory region or a region with a slightly low specificity of a conserved motif, and the stringency of hybridization or amplification are Will identify only the natural sequence that encodes HPPL, or only the natural sequence that encodes the allele or related sequence.

Probes may also be utilized in the detection of related sequences and may have at least 50% sequence identity with any sequence encoding HPPL. The hybridization probe of the present invention of interest can be DNA or RNA, and has the sequence of SEQ ID NO: 4-6,
Alternatively, it may be derived from a genomic sequence containing the HPPL gene promoter, enhancer and intron.

As a method of producing a hybridization probe specific to HPPL-encoding DNA, there is a method of cloning a polynucleotide sequence encoding HPPL and an HPPL derivative into a vector for producing an mRNA probe. Such vectors are commercially available, well known to those skilled in the art, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. Hybridization probes can be labeled with a population of different reporters such as radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase linked to the probe by an avidin / biotin binding system.

[0171] HPPL-encoding polynucleotide sequences can be used to diagnose disorders associated with HPPL expression. Non-limiting examples of such diseases include cancer, including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
There are teratocarcinomas, and more specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate. , Cancer of the salivary gland, skin, spleen, testis, thymus, thyroid, and uterus, as well as autoimmune disorders / inflammatory diseases, including acquired immunodeficiency syndrome (AIDS).
) And adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular Endocrine candid ectodermal dystrophy (APECED
), Bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis , Glomerulonephritis, Goodpasture syndrome, gout, Graves' disease,
Hashimoto thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, Rheumatoid arthritis, scleroderma, Scheegren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, and viruses Infectious and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminthic infections, trauma, and reproductive disorders, including prolactin production abnormalities and fallopian tubes. Disease, ovulation disorders, and endometriosis, estrus disorders, menstrual cycle disorders, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial cancer and ovarian cancer, uterine fibroids, autoimmune disorders, ectopic pregnancy, and Malformation Including infertility, breast cancer, fibrocystic mastopathy, and milk leakage, spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, Includes male breast cancer and gynecomastia. The polynucleotide sequence encoding HPPL can be obtained by the Southern method, the Northern method, the dot blot method, other membrane-based techniques, the PCR method, the dipstick (dip stick
It can be used in microarrays that utilize bodily fluids or tissues collected from patients to detect the expression of stick, pin, ELISA based assays, and mutant HPPL. Such qualitative or quantitative methods are well known in the art.

In a particular form, the nucleotide sequence encoding HPPL is associated with a disease associated with
It will be particularly useful in assays to detect the above mentioned diseases. The nucleotide sequence encoding HPPL could be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the samples are washed and the signal is quantified and compared to standard values. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the nucleotide sequence encoding HPPL in the sample reveals the presence of the associated disease. Using such assays, in animal studies, clinical trials, or in monitoring individual patient care,
It is possible to estimate a particular therapeutic effect.

Normal or canonical expression profiles are established to provide a basis for diagnosis of diseases associated with HPPL expression. This can be achieved by ligating the body fluid or cells extracted from a normal subject, either animal or human, with the sequence encoding HPPL or a fragment thereof under conditions suitable for hybridization or amplification. . Standard hybridizations can be quantitated by comparing the values obtained from normal subjects to those from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared with those obtained from subjects who exhibit symptoms of the disease. Deviation between standard and subject values is used to establish the presence of disease.

Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay can be repeated on a regular basis to estimate whether the level of expression in the subject has begun to approach the values shown in normal patients. It is possible. The results of repeated assays can be used to see the effect of treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual predispose to the development of the disease and can provide a method of detecting the disease before actual clinical symptoms develop. is there. This type of clearer diagnosis allows medical professionals to use preventative or aggressive therapies early to prevent the development or progression of cancer.

Further diagnostic uses of oligonucleotides designed from the sequence encoding HPPL can include the use of PCR. Such oligomers are chemically synthesized,
It can be produced enzymatically or in vitro . The oligomer is preferably
It includes a fragment of a polynucleotide encoding HPPL or a fragment of a polynucleotide complementary to a polynucleotide encoding HPPL, and is used for identifying a specific gene or condition under optimal conditions. Further, the oligomer can be used for detecting and / or quantifying a closely related DNA or RNA sequence under slightly mild stringent conditions.

Methods that can be used to quantify the expression of HPPL include radiolabelling or biotin-labeling of nucleotides, coamplification of regulatory nucleic acids, and standard curves with the results added. (For example, Melby, PC et al. (1993) J. Imm
unol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The quantitation rate of a large number of samples was accelerated by using a high throughput assay in which the oligomer of interest was included in different dilutions and quantified by spectrophotometry or non-color response.

In another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein are used as targets in microarrays. Microarrays are used to monitor the expression levels of a large number of genes at the same time and to identify gene mutations, mutations and polymorphisms. This information is useful in determining gene function, interpreting the genetic basis of disease, diagnosing disease, and monitoring and developing the activity of therapeutic agents.

Microarrays are prepared, used, and analyzed by methods well known in the art. (For example, Brennan, TM et al. (1995) US Pat. No. 5,474,796; Schena, M. et al. (1996) Pro.
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT application number W
O95 / 251116; Shalon, D. et al. (1995) PCT application number WO95 / 35505; Heller, RA et al. (1
997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Pat.No. 5,605,662) Another embodiment of the invention also uses a nucleic acid sequence encoding HPPL. Thus, it is possible to generate hybridization probes useful for mapping naturally occurring genomic sequences. This array maps to:
Specific chromosomes, specific regions of chromosomes or artificially generated chromosomes, for example, human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 products or single chromosome cDNA libraries. (For example, Harrington, 1.3. Other (1
997) Nat Genet. 15: 345-355; Price, CM (1993) Blood Rev. 5-87-134, and Trask, BJ (1991) Trends Genet. 7: 149-154) in sit fluorescence hybridization ( FISH) will correlate with other physical chromosome mapping techniques and genetic map data (see, eg, Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.). Examples of genetic map data can be found in various scientific journals or the World Wide Web site of the Online Mendelian Inheritance in Man (OMIM). On a physical chromosome map
The correlation between the location of the gene encoding HPPL and a particular disease, or the predisposition to a particular disease, helps determine the region of DNA associated with such a disease. The nucleotide sequences of the present invention may also be used to detect differences in gene sequences between normal, carrier and infected individuals.

The genetic map can also be extended using physical mapping techniques such as in sit hybridization of chromosome preparations and binding analysis using established chromosomal markers. By placing the gene on the chromosome of another mammal, such as a mouse,
Relevant markers are often revealed, even if the number or arm of a particular human chromosome is not known. The new array by physical mapping
It can also be assigned to a chromosome arm. This is valuable information for researchers studying genetic disorders using positional cloning or other gene discovery techniques. The location of the disease or syndrome is, for example, 11q22-
Once roughly determined by gene binding to a particular gene region such as 23, any sequence mapping for that region represents a relevant gene or regulatory gene for further investigation (eg, Gatti, RA et al. (1988). Nature 336: 577-580
See). Further, by using the nucleotide sequence of the present invention of interest, a normal person, a holder,
That is, a difference in chromosomal position due to translocation, inversion, etc. between infected persons may be detected.

In another embodiment of the invention, HPPL, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen a library of compounds in any of a variety of drug screening techniques. Fragments used for such screening may be free in solution, immobilized on a solid support, retained on the surface of cells, or present intracellularly. Formation by binding the complex between HPPL and the agent being tested may be measured.

Another method used for drug screening is used to increase the screening throughput of compounds with suitable binding affinity for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is washed with HPPL or a fragment thereof. Bound HPPL is then detected by methods well known in the art. Purified HPPL can also be coated directly on plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding HPPL specifically compete with the test compound for binding HPPL.
In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with HPPL.

In another example, nucleotide sequences encoding HPPL are used in developing molecular biology techniques, including, but not limited to, currently known nucleotides, such as the triplet code and specific base pair interactions. New technologies can be provided that depend on the characteristics of the sequence.

Those skilled in the art will be able to make full use of the present invention without further explanation given the preceding description. Therefore, the examples described below are for illustrative purposes and do not limit the invention.

All patent applications, patents and publications mentioned above and below, especially US applications [1998]
Attorney docket number PF-0625P filed on October 27, 2010 and US application [attorney docket number PF-0663P filed January 21, 1999] are hereby incorporated by reference. .

Example 1 Preparation of cDNA Library RNA was purchased from Clontech, or isolated from the tissues listed in Table 4. A tissue was homogenized and dissolved in a guanidinium isothiocyanate solution. On the one hand, another tissue is homogenized and dissolved in phenol, or TRIZOL (
Life Technologies), guanidinium isothiocyanate and a suitable modifier such as a single phase solution of phenol. The lysate was centrifuged in cesium chloride or extracted with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

Extraction and precipitation of RNA with phenol was repeated as many times as necessary to increase the purity of RNA. In some cases, RNA is treated with a DNA degrading enzyme. Most libraries have oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN).
Valencia CA), OLIGOTEX mRNA purification kit (QIAGEN) was used to isolate poly (A +) RNA. Alternatively, another RNA isolation kit such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX) was used to isolate directly from tissue lysates.

[0189] Alternatively, frozen tissue was placed on a Brinkmann Homogenizer Polytron PT-3000 (Brinkman).
n Instruments, Westbury NY) and homogenized and dissolved in guanidinium isothiocyanate solution. RNA from Stratagene's RNA isolation protocol (Stratagene
gene, La Jolla CA). RNA was extracted twice with acidic phenol, precipitated with sodium acetate and ethanol, resuspended in RNAase-free water and treated with DNAase. Poly (A +) RNA was added to the OLIGOTEX kit (QIAGEN, Inc, Chatsworth
, CA).

Using the poly (A +) RNA, the SUPERSCRIPT Plasmid System (Life Technologies
CDNA synthesis and preparation of each cDNA library were performed according to the recommended protocol in (1). The cDNA was fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia Biotech), and the cDNA of over 400 bp was labeled with pINCY (Incyte Pharmaceuticals, PalocAlto CA).
). Recombinant plasmid is DH5α competent cells or ELECTROMA
X cells (Life Technologies) were transformed.

In some cases, RNA was provided to Stratagene and a cDNA library corresponding to Stratagene was created. Otherwise, UNIZAP vector system (Stratagene)
Alternatively, a cDNA library was prepared by using the SUPERSCRIPT plasmid system (Life Technologies) to synthesize cDNA by a recommended method known in the art or a similar method.
(See, eg, Ausubel, 1997, supra, Units 5.1-6.6). Reverse transcription is oligo d (T)
Or started with random primers. A synthetic oligonucleotide adapter was attached to the double-stranded cDNA and the cDNA was digested with a suitable restriction enzyme or restriction enzymes. For most libraries, SEPHACRYL S 1000 or SEPHAROSE
CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech
), And the size of the cDNA (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT1 plasmid (Life Technolo
gies), plNCY (Incyte Pharmaceuticals, Palo Alto CA), etc., and the cDNA was ligated to the compatible restriction enzyme site of the polylinker of a suitable plasmid. Use this recombinant plasmid with Stratagene XL1-Blue, XL1-BIueMRF, SOLR, or Life Technologi.
The cells were transformed into competent E. coli cells containing es DH5α or DH10B and ELECTROMAX DH10B.

2 Isolation of cDNA clones Plasmids were recovered from host cells by in vivo excision utilizing the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Minipreps
DNA purification system (Promega) and AGTC Miniprep purification kit (Edge Biosystems, G
aithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QI
Plasmids were purified using at least one of the AWELL 8 Ultra Plasmid Purification System, REAL Prep 96 Plasmid Kit. After precipitation, it was resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without freeze-drying.

Alternatively, plasmid DNA was amplified from host cell lysates by a high throughput direct ligation PCR method. (Rao, VB (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and transferred to 384-well plates for storage and the concentration of amplified plasmid DNA is quantified using PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Measured.

3 Sequencing and Analysis cDNA sequencing reactions can be performed using standard methods or ABI CATALYST 800 (
Perkin-Elmer) thermal cycler or PTC-200 thermal cycler (MJ Research)
HYDRA Microdispenser (Robbins Scientific) or MICROLAB 2200 (Ham
ilton) High throughput equipment such as combination with liquid transfer system. c
For the preparation of a DNA sequencing reaction, a reagent from Amersham Pharmacia Biotech or a reagent contained in an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer) was used. c
For electrophoretic separation of DNA sequencing reactions and detection of labeled polynucleotides, the MEGABACE 1000 DNA Sequencing System (Molecular Dynamics
), ABI PRISM 373 using standard ABI protocol and base pair calling software
Alternatively, the 377 Sequencing System (Perkin-Elmer), other sequence analysis systems well known in the art, was used. The open reading frame of the cDNA sequence is standard (Ausubel, 1997
, Unit 7.7), supra. Some of the cDNA sequences were selected and extended by the method described in 5 of this example.

Construction and analysis of the polynucleotide sequences obtained from the sequencing of the cDNA was performed in combination with software utilizing algorithms well known to those skilled in the art.
Table 5 outlines the tools, software, algorithms utilized, their description, references, and threshold parameters used. Column 1 of Table 5 is the tools and programs used, algorithms, column 2 is a brief description thereof, column 3 is a reference cited as part of this specification by reference, column 4 is a sequence of two sequences. The score, probability value, and other parameters used in the evaluation of the degree of coincidence are shown (the higher the probability value, the higher the homology between sequences). MACDNASIS PRO software (Hitachi Softwa
re Engineering, S. San Francisco CA) and LASER GENE software (DNASTAR)
Was used.

Confirmation of polynucleotide sequences is accomplished by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vectors and linkers, poly A sequences and mask ambiguous base pairs. I went there. next,
Using databases based on BLAST, FASTA and BLIMPS, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases and BLOCKS, PRINTS, DOMO, PRODOM and PFAM databases. Annotations were obtained by querying these sequences against sequences selected from. These sequences were constructed into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed and screened for open reading frames with programs based on BLAST and FASTA, BLIMPS. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and the GenBank database (above) and Swi
These full-length sequences were analyzed by querying databases such as ssProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite, or Hidden Markov Model (HMM) based protein family databases such as PFAM. HMMs use probabilities to analyze the consensus primary structure of gene families (eg, Eddy
, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365).

The programs described above for the construction and analysis of full-length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NO: 4-6. Fragments of about 20 to 4000 nucleotides are useful for hybridization and amplification and have been described in the invention above.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of transcripts of a gene, labeled nucleotides on the membrane to which RNA from a particular cell type or tissue is bound. With sequence hybridization (see, for example, Sambrook, supra,
Chapter 7; and Ausubel. FM et al., Supra, Chapters 4 and 16).

Using similar computer technology for BLAST, GenBank or LIFESEQ (Inc
Search for identical or related molecules in nucleotide databases such as yte Pharmaceuticals). This assay is much faster than many membrane-based hybridizations. You can also change the sensitivity of the computer search so that any particular match
It can be established whether it is classified as an exact match or a homologous match. The search criterion is the product score defined as (% sequence identity x% maximum BLAST score) / 100. The product score takes into account both the similarity between the two sequences and the length of the sequence match. For example, if the product score is 40, the match is 1 to 2
Exact within% error, at 70 the match would be exact. Similar molecules are usually identified by selecting those molecules that show a product score in the range of 15-40, although lower scores may identify related molecules.

The results of the Northern analysis are reported as the percent distribution of the library in which transcripts encoding HPPL occurred. The analysis also includes classification of the cDNA library by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, development, endocrine,
Includes gastrointestinal, hematopoiesis / immunity, musculoskeletal, neural, reproductive, urinary. Disease categories include cancer, inflammation / trauma, cell proliferation, nerves, pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percentage) of specific expression in each tissue and the ratio of expression in each disease.

5 Extension of Polynucleotide Encoding HPPL The full-length nucleic acid sequence of SEQ ID NO: 4-6 was prepared using oligonucleotide primers designed from suitable fragments of the full-length molecule. The fragment was extended and produced. One primer was synthesized to initiate the 5'extension of the known fragment and the other primer was synthesized to initiate the 3'extension of the known fragment. Starting primers were approximately 22 to 30 nucleotides in length and approximately 5 nucleotides in length using OLIGO 4.06 software (National Biosciences) or other suitable program.
It had a GC content of 0% or higher and was designed to anneal to the target sequence at a temperature of about 68-72 ° C. The nucleotides were extended so that hairpin structures and primer-primer dimers did not occur.

This sequence was extended with selected human cDNA libraries. If more than one extension is required or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by the PCR method using a method known in the art. PCR is PTC-200
A 96-well block plate was performed using a thermal cycler (MJ Research, Inc.). The reaction mixture was prepared by using template DNA and 200 nmol of each primer, a buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, and Taq DNA polymerase (Amers).
ham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (Stratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1 3 minutes at 94 ° C Step 2 15 seconds at 94 ° C Step 3 1 minute at 60 ° C Step 4 2 minutes at 68 ° C Step 5 Repeat steps 2 and 16-30 20 times Step 6 5 minutes at 68 ° C Step 7 4 Storage at ℃ Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 57 ° C for 1 minute Step 4 68 ° C for 2 minutes Step 5 Repeat Steps 2 and 16-30 20 times Step 6 68 ° C for 5 minutes Step 7 4 Store at ℃.

The DNA concentration in each well was 100 μl of PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes) dissolved in 1 × TE and 0.5 μl of undiluted PCR product.
s, Eugene OR) is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent and then measured. Flu this plate
Scan with oroskan II (Labsystems Oy, Helsinki, Finland) and measure the fluorescence of the sample to quantify the concentration of DNA. A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence.

The extended nucleotides were desalted and concentrated before being transferred to a 384 well plate,
CviJI Cholera virus endonuclease (Molecular Biology Research, Madiso
n WI) and sonicated or sheared before religation to pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments and the agar was digested with Agar ACE (Promega). T4 Ligase (New Engla
clones extended with nd Biolabs, Beverly MA) to pUC 18 vector (Amersham
Religation to Pharmacia Biotech) and treatment of restriction site extensions with Pfu DNA polymerase (Stratagene) to transfect competent E. coli cells. Transfected cells are selected and transferred to media containing antibiotics, individual colonies are cut and LB / 2
It was cultured overnight at 37 ° C. in a 384-well plate of X carbenicillin culture solution.

Cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was PCR amplified using DNA polymerase (Stratagene) according to the following procedure. Step 1 3 minutes at 94 ° C Step 2 15 seconds at 94 ° C Step 3 1 minute at 60 ° C Step 4 2 minutes at 72 ° C Step 5 Repeat steps 2 and 16-30 29 times Step 6 5 minutes at 72 ° C Step 7 4 Store at ℃. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the above conditions. Dilute the sample with 20% Dimethysulphoxide (1-15, v / v) and use DYENAMIC DIREC
T-Kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cyc
Sequencing was performed using the le sequencing ready reaction kit (Perkin-Elmer).

Similarly, following the procedure described above, the nucleotide sequence of SEQ ID NO: 4-6 was utilized to obtain a 5'regulatory sequence using an oligonucleotide designed for this extension and a suitable gene library.

6 Labeling and Use of Individual Hybridization Probes With hybridization probes derived from SEQ ID NO: 4-6,
Screen for cDNA, mRNA, or genomic DNA. Special mention is made of labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software such as OLIGO 4.06 software (National Bioscience), 50 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham, Chicago, IL). And T4 polynucleotide kinase (DuPont NEN
, Boston MA). The labeled oligonucleotide was loaded onto a SEPHADEX G-25 ultrafine exclusion dextran bead column (Amersha
m Pharmacia Biotech). An aliquot containing 10 ⁷ counts of labeled probe per minute was treated with the following endonucleases, Ase I, Bgl II.
, Eco RI, Pst I, Xba1 or Pvu II (DuPont NEN) for use in a typical membrane-based hybridization analysis of human genomic DNA.

DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are sequentially washed at room temperature, eg, under conditions of max 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. The hybridization patterns are visualized and compared by autoradiography or another imaging means.

7 It is possible to synthesize array elements on the surface of a substrate using a microarray chemical bonding method and an inkjet device (see eg Baldeschweiler, supra). Using an array similar to dot blotting or slot blotting, heat, UV,
It is placed and bonded to the surface of the substrate using mechanical or chemical bonding methods. A typical array can be made manually or using available methods and machines and can include any suitable number of elements. After hybridization, unhybridized probe is removed and a scanner is used to determine the level and pattern of fluorescence. Scanned images can be analyzed to determine the degree of complementarity and the relative amount / expression level of each probe that hybridizes to the element on the microarray.

Full-length cDNAs, expressed gene sequence fragments (ESTs), or fragments thereof, can form the elements of a microarray. A fragment suitable for hybridization is LASERG
Selection can be made using software known in the art such as ENE software (DNASTAR). A full-length cDNA corresponding to one of the nucleic acid sequences of the invention, EST,
Alternatively, those fragments, or cDNA arbitrarily selected from the cDNA library related to the present invention, are aligned on a suitable substrate such as a glass slide. cDNA is, for example, UV
It is immobilized on slides using UV cross-linking, then heat and chemical treated and finally dried (eg Schena, M. et al. (1995) Science.
270: 467-470; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used to hybridize to the element on the substrate. This substrate is analyzed as described above.

8 Complementary Polynucleotides A sequence complementary to the sequence encoding HPPL or any portion thereof is used to reduce or inhibit the expression of naturally occurring HPPL. Although the use of oligonucleotides containing from about 15 to about 30 base pairs is described, essentially the same method can be used with smaller or larger sequence fragments. Appropriate oligonucleotides are designed using Oligo 4.06 software (National Biosciences) and HPPL coding sequences. The most unique 5'to inhibit transcription
A complementary oligonucleotide is designed from the sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to prevent the ribosome from binding to the HPPL-encoding transcript.

9 HPPL Expression HPPL expression and purification can be performed using bacterial or virus based expression systems. For the expression of HPPL in bacteria, the cDNA is subcloned into a suitable vector containing an antibiotic resistance and an inducible promoter that enhances the transcription level of the cDNA. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. BL21 recombinant vector
Transform into a suitable bacterial host such as (DE3). Bacteria with antibiotic resistance express HPPL when induced with isopropyl β-D thiogalactopyranoside (IPTG).
Expression of HPPL in eukaryotic cells is expressed in insect or mammalian cell lines by the Autographica californica nuclear pleiotropic virus (AcM), commonly known as baculovirus.
NPV). The nonessential polyhedrin gene of baculovirus is replaced with the HPPL-encoding cDNA either by homologous recombination or by bacterial-mediated gene transfer involving transfer plasmid mediated. The infectivity of the virus is maintained,
The strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is often used to infect Spodoptera frugiperda (Sf9) insect cells, but can also be used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (For example, Engel
hard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V.
(1996) Hum. Gene Ther. 7: 1937-1945.).

In most expression systems HPPL is used, for example, glutathione S transferase (GST).
, Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, the affinity-based purification of the recombinant fusion protein from crude cell lysate can be performed rapidly in one step. The 26-kilodalton enzyme GST from Schistosoma japonicum allows purification of fusion proteins with immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharma
cia Biotech). After purification, the GST moiety can be proteolytically cleaved from HPPL at specific manipulation sites. FLAG, a peptide of 8 amino acids, allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His in which 6 histidine residues are continuously extended enables purification with a metal chelate resin (QIAGEN). The method of protein expression and purification is described by Ausu
bel (1995, supra, ch 10, 16). The following assay can be performed directly using HPPL purified by these methods.

10 Demonstration of HPPL activity HPPL activity can be demonstrated in an in vitro hydrolysis assay using 1-palmitoyl-2- [1-14C] oleoyl phosphatidylcholine containing vesicles (Sigma-Aldrich). HPPL
The activity of phospholipase A ₂ can be demonstrated by analysis of cleavage products isolated from the hydrolysis reaction mixture.

Vesicles containing 1-palmitoyl-2- [1- ¹⁴ C] oleoyl phosphatidylcholine (Sigma-Aldric
h) is 12.5 mg unlabeled 1-palmitoyl-2- [1-1 of 2.0 μCi radiolabeled phospholipid.
4C] oleoyl phosphatidylcholine mixed, dried under nitrogen and 150 mM Tris-
Add 2.5 ml of HCL (pH 7.5) and sonicate and centrifuge the mixture to prepare. Store this supernatant at 4 ° C. The final reaction mixture was 2.0 mM taurochenodeoxycho.
0.25 ml Hank's buffered saline supplemented with late, 1.0% bovine serum albumin, 1.0
mM CaCl ₂ (pH 7.4), 150 μg 1-palmitoyl-2- [1- ¹⁴ C] oleoyl phosphatidylch
Oline vesicles containing various amounts of HPPL diluted in PBS. After incubating for 30 minutes at 37 ° C., 20 μg each of lysophosphatidylcholine and oleic acid are added respectively as carriers and each sample is extracted to obtain total lipid. The lipids are separated by thin layer chromatography using two solvents: chloroform: methanol: acetic acid: water (63: 35: 8: 4) until the surface of the solvent is halfway up the plate. This process was repeated until hexane: ether: acetic acid (86:
Continue with 16: 1). The region containing lipid is visualized with I ₂ vapor, the region is scraped off, and radioactivity is measured with a scintillation counter. The addition of high amounts of HPPL to the assay mixture increases the amount of radioactivity released as fatty acids, but the amount of radioactivity released as lysophosphatidylcholines remains low. This demonstrates that HPPL cleavage products at the sn-2 position, but not at the sn-1 position, are characteristic of the activity of phospholipase A ₂ .

Alternatively, HPPL activity is measured by hydrolysis of the fatty acid acyl residue at the sn-1 position of phosphatidylserine. HPPL in stoichiometric amount in a suitable buffer
And the tritium [H3] -labeled substrate phosphatidylserine. After incubation for a suitable time, the hydrolyzed reactants are separated from the substrate by chromatographic methods. The amount of acylglyerophosphoserine produced is measured by counting tritiated products with a scintillation counter. Set various control groups to compensate for background noise and unincorporated substrate. The final count of the tritiated enzyme product [H3] -acylglyerophosphoserine is directly proportional to the activity of HPPL in the biological sample.

11 Functional Assay The function of HPPL is at physiologically elevated levels in mammalian cell culture systems.
Evaluation is by expression of the HPPL coding sequence. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels.
Such vectors include pCMV SPORT ^™ (Life Technologies.) And pCR 3.1 (In
Vitrogen, Carlsbad, CA), both containing the cytomegalovirus promoter. For example, 5 to 10 μg of the recombinant vector is transiently transfected into a human cell line derived from endothelium or hematopoiesis by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows to distinguish between transfected and non-transfected cells. In addition, by expressing the labeled protein, the cDNA
The expression from the recombinant vector can be accurately predicted. Such labeled proteins include green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion protein. Automatic flow cytometry using a technology based on laser optics (F
CM) is used to identify transfected cells expressing GFP or CD64-GFP and to assess their apoptotic status and other cellular characteristics. In addition, FCM detects and weighs uptake of fluorescent molecules that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include nuclear DNA measured by staining DNA with propidium iodide.
Changes in contents, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and changes in protein expression on cell surface and intracellular as measured by reactivity with specific antibodies. , Changes in plasma membrane composition measured by binding of fluorescent-complexed annexin V protein to the cell surface. Flow cytometry is by Ormerod, MG (1994) Flow Cytometry Oxford, New York,
It is listed in NY.

The effect of HPPL on gene expression depends on the sequence encoding HPPL and CD64 or CD64-G.
Highly purified cell populations transfected with either FP can be evaluated. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods known in the art. The expression of mRNA encoding HPPL and other genes of interest can be analyzed by Northern analysis or microarray technology.

12 Production of Antibodies Specific for HPPL Polyacrylamide gel electrophoresis (PAGE; eg, Harrington, MG (1990)
Methods Enzymol. 1816-3088-495) or HPPL substantially purified by other purification techniques are used to immunize rabbits according to standard protocols to generate antibodies.

Alternatively, the HPPL amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions, and the corresponding oligopeptides synthesized and used to determine well known methods to those of skill in the art. To produce antibodies. Selection of suitable epitopes, such as those near the C-terminus or within adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).

Usually, an oligopeptide having a length of about 15 residues was synthesized by fmoc method chemistry using an Applied Biosystems peptide synthesizer ABI 431A peptide synthesizer (Perkin-Elmer), and N-maleimidobenzoyl-N-hydroxyl was synthesized. KLH (Sigma-Aldrich, St. Louis M by reaction with succinimide ester (MBS)
O) to enhance immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity and anti-HPPL activity of the obtained antiserum, the peptide or HPPL was bound to a substrate, blocked with 1% BSA, reacted with rabbit antiserum and washed, and then radioactive iodine was added. Labeled goat anti-rabbit IgG
React with.

13 Purification of Spontaneous HPPL Using Specific Antibodies Spontaneous HPPL or recombinant HPPL are substantially purified by immunoaffinity chromatography using antibodies specific for HPPL. The immunoaffinity column is constructed by covalently linking an anti-HPPL antibody with a resin for activation chromatography such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

The culture medium containing HPPL is passed through an immunoaffinity column, and the column is washed under conditions capable of preferentially adsorbing HPPL (for example, with a buffer having a high ionic strength in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and HPPL (for example, with a buffer of pH 2-3 or with a high concentration of chaotropic ion such as urea or thiocyanate ion), and HPPL is recovered.

Identification of 14 HPPL-Interacting Molecules HPPL or a biologically active fragment thereof was labeled with ¹²⁵ I Bolton-Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Label with. The candidate molecules pre-arranged in the multiwell plate were labeled with HPPL.
Incubate with, wash and assay all wells with labeled HPPL complex. Using the data obtained at various HPPL concentrations, the candidate molecule and HPPL
Calculate numerical values for relationships, affinities, and numbers.

Those skilled in the art will be able to make various modifications of the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the present invention has been described with reference to particular preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such particular embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to generate HPPL-encoding full length sequences (SEQ ID NO), clone identification number, cDNA.
A library and a cDNA fragment are shown.

Table 2 lists the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and
The method, algorithm, and searchable database used for the analysis of HPPL are shown.

Table 3 shows selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders and conditions associated with these tissues, and each DNA. Shows the cloned vector.

Table 4 shows the tissues used to create the cDNA library in which the cDNA clone encoding HPPL was isolated.

Table 5 shows the tools, programs, and algorithms used in the analysis of HPPL, their description, references, and threshold parameters.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Sequence list]

[Brief description of drawings]

Figure 1: LASERGENE software multi-sequence alignment program (DNASTE
R, Madison WI), HPPL-l (Incyte clone number 2641779
SEQ ID NO: 1) and type II phospholipase A-2 (GI 204319; SEQ ID NO: 7) are shown.

FIG. 2A shows an amino acid sequence alignment between HPPL-2 (Incyte clone number 1430683; SEQ ID NO: 2) and cytoplasmic phospholipase A2 (GI 508625; SEQ ID NO: 8).

FIG. 2B shows an amino acid sequence alignment between HPPL-2 (Incyte clone number 1430683; SEQ ID NO: 2) and cytoplasmic phospholipase A2 (GI 508625; SEQ ID NO: 8).

FIG. 2C shows an amino acid sequence alignment between HPPL-2 (Incyte clone number 1430683; SEQ ID NO: 2) and cytoplasmic phospholipase A2 (GI 508625; SEQ ID NO: 8).

FIG. 3A shows an amino acid sequence alignment between HPPL-3 (Incyte clone number 1316804; SEQ ID NO: 3) and rat phosphatidylserine phospholipase A1 (PS-PLA1) (GI 1817556; SEQ ID NO: 9). Show.

FIG. 3B shows an amino acid sequence alignment between HPPL-3 (Incyte clone number 1316804; SEQ ID NO: 3) and rat phosphatidylserine phospholipase A1 (PS-PLA1) (GI 1817556; SEQ ID NO: 9). Show.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/10 A61P 7/00 4H045 5/14 7/06 7/00 9/10 101 7/06 11 / 00 9/10 101 11/06 11/00 13/08 11/06 13/12 13/08 15/00 13/12 15/08 15/00 15/10 15/08 17/00 15/10 17/02 17/00 17/06 17/02 17/12 17/06 19/02 17/12 19/08 19/02 19/10 19/08 21/04 19/10 25/28 21/04 29/00 25 / 28 101 29/00 31/00 101 31/04 31/00 31/10 31/04 31/12 31/10 35/00 31/12 37/00 35/00 37/02 37/00 37/08 37 / 02 43/00 111 37/08 C07K 16/40 43/00 111 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/16 D 1/21 C12Q 1/68 A 5 / 10 C12N 15/00 ZNAA 9/16 5/00 A C12Q 1/68 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, S, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Bandman, Olga California, USA 94043 Mountainview Anna Avenue 366 (72) Inventor Gegler, Karl Jay United States California 94025 Menlo Park Oakland Avenue 1048 (72) Inventor Coley, Neil Sea United States California 94040 Mountain View # 30 Dale Avenue 1240 (72) Inventor Bogun, Mariah Earl United States California 94577 San Leandro Santiago Road 14244 (72) Inventor Azimzai, Yarda United States California 94545 Hayward Rock Springs Drive 2045 (72) Inventor Lal, Pretty United Carifo Nya State 95054 Santa Clara Russ Drive 2382 (72) Inventor Ryu, Dung Aina Em USA California 95136 San Jose Park Belmont Park 55 F Term (reference) 4B024 AA01 AA11 BA11 CA04 CA07 CA09 CA20 DA02 DA06 EA02 EA04 GA11 HA13 HA14 4B050 CC01 CC03 DD11 FF09E FF14E FF15E FF16E LL01 LL03 LL05 4B063 QA01 QQ43 QQ44 QQ53 QR08 QR32 QR40 QR42 QR56 QR62 QS16 QS25 QS34 QX02 4B065 AA26X AA58X AA72X AA87X AA90X AA93Y AB01 AC14 BA02 BD14 CA31 CA44 CA46 4C084 AA02 AA06 AA17 BA02 CA18 CA34 CA36 CA53 DC01 NA14 ZA152 ZA162 ZA362 ZA452 ZA512 ZA532 ZA552 ZA592 ZA662 ZA682 ZA812 ZA892 ZA942 ZA962 ZB052 ZB072 ZB112 ZB132 ZB152 ZB262 ZB272 ZB332 ZB352 ZB352 ZB352 ZB352 ZA352

Claims (20)

[Claims] 1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3, and fragments thereof. 2. A substantially purified variant having at least 90% amino acid sequence identity with the amino acid sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. An isolated and purified polynucleotide variant sequence having at least 90% polynucleotide sequence identity with the polynucleotide of claim 3. 5. An isolated and purified polynucleotide which hybridizes with the polynucleotide of claim 3 under stringent conditions. 6. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 3. 7. A method of detecting a polynucleotide comprising: (a) hybridizing the polynucleotide of claim 6 with at least one nucleic acid in a sample, thereby forming a hybridization complex; b) a step of detecting the hybridization complex, the step of detecting the presence of the hybridization complex correlating with the presence of the polynucleotide in the sample. Law. 8. The detection method according to claim 7, further comprising a step of amplifying the polynucleotide before the hybridization. 9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5, and fragments thereof. 10. An isolated and purified polynucleotide variant sequence having at least 90% polynucleotide sequence identity with the polynucleotide of claim 9. 11. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 9. 12. An expression vector comprising at least one fragment of the polynucleotide of claim 3. 13. A host cell containing the expression vector of claim 12. 14. A method for producing a polypeptide, comprising: (a) culturing the host cell according to claim 13 under conditions suitable for expression of the polypeptide; and (b) a medium for the host cell. And a step of recovering the polypeptide from the same. 15. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable medical carrier. 16. A purified antibody that specifically binds to the polypeptide of claim 1. 17. A purified agonist of the polypeptide of claim 1. 18. A purified antagonist of the polypeptide of claim 1. 19. A method of administering HPPL expression or activity, which comprises the step of administering an effective amount of the pharmaceutical composition of claim 15 to a patient in need of treatment or prevention of a disease associated with reduction of HPPL expression or activity. A method of treating or preventing a disorder associated with depression. 20. An increase in the expression or activity of HPPL, comprising the step of administering an effective amount of the antagonist of claim 18 to a patient in need of treatment or prevention of a disease associated with the increase in expression or activity of HPPL. A method of treating or preventing a related disorder.