JP2006515989A - Use of human ZVEN proteins and polynucleotides - Google Patents

Abstract

The present invention provides methods of using Zven1 and Zven2 polypeptides to enhance chemokine production. The invention also provides methods for treating bowel motility disorders and improving gastrointestinal function with Zven1 and Zven2 polypeptides.

Description

Background of the invention:
Optimal gastrointestinal function includes mixing and forward propulsion of contents in the stomach and intestine. Gastric emptying is sometimes abnormal in patients who have critical illness or recover from surgery. Recovery of gastrointestinal function and recovery of oral intake are important determinants in recovery from phenomena involving gastrointestinal function. Several phenomena occur in the gastrointestinal system, such as intestinal obstruction (post-operative and paralytic), chronic constipation, gastric paresis (including diabetic gastric paresis), intestinal pseudo-obstruction, dyspepsia, gastroesophageal reflux and vomiting Can lead to dysfunction.

  Disorders or disorders of gastrointestinal function that are impaired or unprotected include bowel obstruction and gastric insufficiency. Post-surgical bowel obstruction (POI) is a state of decreased intestinal motility, such as delayed gastric emptying, that occurs after surgery as a result of a disrupted muscular normal condition. It is especially a problem following abdominal surgery. Problems arise from the surgery itself, from the residual effects of anesthetics, and in particular from pain-reducing anesthesia and opiate derivatives used during and after surgery. Post-operative ileus can be classified as “no complications” lasting 2-3 days after surgery, or “paralytic” lasting 3 days or more after surgery. Thus, patients undergoing abdominal surgery with delayed recovery of gastrointestinal function have an extended hospital stay, which can lead to increased medical costs and substantially other complications. An estimated $ 750 million to $ 1 billion is spent every year on increased hospitalization due to postoperative ileus. Currently, there are no drugs approved for the treatment of this disease.

  In addition to the need for good therapeutics for postoperative ileus, there is a need for good therapeutics for diabetic gastric failure. Diabetic gastric failure paralysis is gastric paralysis caused by motility abnormalities in the stomach as a complication of type I and type II diabetes. Diabetic gastric palsy is characterized by delayed gastric emptying, postprandial dilatation, nausea and vomiting. In diabetes, it appears to be due to neuropathy, albeit also related to the loss of Cajal stromal cells (ICCs), which are the “pace marker cells” of the intestines.

  In the United States alone, there are at least 16 million diabetics, accounting for about 7% of the artificial. It continues to rise and is spreading all over the world. This issue is important because up to two thirds of diabetics have some degree of gastric paresis. Although long-term treatment is often required, the onset of symptoms is often acute. Furthermore, symptoms associated with diabetic gastric failure paralysis, such as delayed gastric emptying and vomiting, can cause water and electrolyte imbalances, poor glycemic control and subsequent complications. If severe enough, hospitalization for treatment of diabetes and treatment with intravenous fluids and nutrients is required.

The often acute nature of the onset of symptoms provides an opportunity for treatment by Prokinetic. Currently, there are very few drugs that effectively treat diabetic gastric paresis, those available have side effects and / or cannot be taken with other medications. Oral drugs are not resistant during severe onset and therefore require intravenous administration of prokinetic. In the United States, only two agents, erythromycin and metoclopramide, are available for treating gastric paresis.
Thus, there remains a need for therapeutic approaches for the treatment of gastric dysfunction.

Brief summary of the invention:
The present invention provides proteins useful for treatment in restoring gastrointestinal function and gastric emptying capacity. Other uses of Zven1 and Zven2 polypeptides are described in more detail below.

Description of the invention:
1. Overview:
The present invention is directed to the novel use of the previously described proteins Zven1 and Zven2. See US Patent Application Serial No. 09 / 712,529, currently issued as US Application No. 6,485,938. Zven1 and Zven2 are also industrially known as prokineticin 2 and prokineticin 1, respectively. As discussed herein, Zven1 and Zven2, and variants and fragments thereof can be used to modulate gastrointestinal function and gastric emptying. The receptors for Zven1 (prokineticin 2) and Zven2 (prokineticin 1) have been identified as G protein-coupled receptors, namely GPCR73a and GPCR73b. See Lin, D. et al., J. Biol. Cherra. 277: 19276-19280,2002. GPCR73a and BPCR73b receptor fluids are also known as PK-R1 and PK-R2.

  The present invention provides methods for using human Zven polypeptides and nucleic acid molecules encoding the same. An exemplary nucleic acid molecule comprising a sequence encoding a Zven1 polypeptide has the nucleotide sequence of SEQ ID NO: 1. The encoded polypeptide has the following amino acid sequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI TGACDKDSQC GGGMCCAVSI WVKSIRICTP MGKLGDSCHP LTRKVPFFGR RMHHTCPCLP GLACLRTSFN RFICLAQK (SEQ ID NO: 2). Thus, the Zven1 nucleotide sequence described herein encodes a polypeptide of 108 amino acids. A putative signal sequence for Zven1 polypeptide is present at amino acid residues 1-20, 1-21, and 1-22 of SEQ ID NO: 2. The mature form of the polypeptide comprises an amino acid sequence from amino acids 28-108, as shown in SEQ ID NO: 2.

  The long sequence as shown in SEQ ID NO: 2 is encompassed by the invention described herein. Its long form has the following amino acid sequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI TGACDKDSQC GGGMCCAVSI WVKSIRICTP MGKLGDSCHP LTRKNNFGNG RQERRKRKRS KRKKEVPFFG RRMHHTCPCL PGLACLRTSF NRFICLAQK (SEQ ID NO: 29). This long form of the putative signal sequence has a mature form comprising an amino acid sequence from amino acids 28-129 as shown in SEQ ID NO: 29.

  An exemplary nucleic acid molecule comprising a sequence encoding a Zven2 polypeptide has the nucleotide sequence of SEQ ID NO: 4. The encoded polypeptide has the following amino acid sequence: MRGATRVSIM LLLVTVSDCA VITGACERDV QCGAGTCCAI SLWLRGLRMC TPLGREGEEC HPGSHKVPFF RKRKHHTCPC LPNLLCSRFP DGRYRCSMDL KNINF (SEQ ID NO: 5). Thus, the Zven2 nucleotide sequence described herein encodes a polypeptide of 105 amino acids. The putative signal sequence of Zven2 polypeptide is present at amino acid residues 1-17 and 1-19 of SEQ ID NO: 5.

As described below, the present invention relates to amino acid residues 23 to 108 of SEQ ID NO: 2, to amino acid residues 28 to 108 of SEQ ID NO: 2, or from amino acid residues 28 to 108 of SEQ ID NO: 29, An isolated polypeptide comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to 129 is provided. Such an isolated polypeptide can specifically bind to an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Certain polypeptides can increase or decrease gastric contractility, gastric emptying and / or intestinal transit. An exemplary polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

  Similarly, the invention provides an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to amino acid residues 20 to 105 of SEQ ID NO: 5. Providing an isolated polypeptide comprising, wherein such isolated polypeptide specifically binds to an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 Can do. An exemplary polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

  The present invention also provides (1) amino acid residues 21 to 108 of SEQ ID NO: 2; (2) amino acid residues 22 to 108 of SEQ ID NO: 2; (3) amino acid residues 23 to 108 of SEQ ID NO: 2; Amino acid residues 82 to 108 of SEQ ID NO: 2; (5) amino acid residues 1 to 78 of SEQ ID NO: 2 (amide); (6) amino acid residues 1 to 79 of SEQ ID NO: 2; (7) amino acids of SEQ ID NO: 2 (8) amino acid residues 21 to 79 of SEQ ID NO: 2; (9) amino acid residues 22 to 78 (amide) of SEQ ID NO: 2; (10) amino acid residues of SEQ ID NO: 2 22-79;

  (11) amino acid residues 23 to 78 (amide) of SEQ ID NO: 2; (12) amino acid residues 23 to 79 of SEQ ID NO: 2; (13) amino acid residues 20 to 10 of SEQ ID NO: 2; (14) SEQ ID NO: (15) amino acid residues 20 to 79 of SEQ ID NO: 2; (16) amino acid residues 20 to 79 (amide) of SEQ ID NO: 2; (17) amino acid residues of SEQ ID NO: 2; (18) amino acid residues 21 to 79 (amide) of SEQ ID NO: 2; (19) amino acid residues 22 to 72 of SEQ ID NO: 2; (20) amino acid residues 22 to 79 of SEQ ID NO: 2 (amide) );

  (21) amino acid residues 23-72 of SEQ ID NO: 2; (22) amino acid residues 23-79 (amide) of SEQ ID NO: 2; (23) amino acid residues 28-108 of SEQ ID NO: 2; (24) SEQ ID NO: (25) amino acid residues 28-79 of SEQ ID NO: 2; (26) amino acid residues 28-79 (amide) of SEQ ID NO: 2; (27) amino acid residues of SEQ ID NO: 2 (28) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 75 to 79 of SEQ ID NO: 2; and (29) amino acid residues 75 to 78 (amide) of SEQ ID NO: 2. Also provide. The present invention also encompassed a polypeptide comprising an amino acid sequence comprising amino acid residues 28-129 as shown in SEQ ID NO: 29, and / or fragments thereof.

  Furthermore, the present invention provides (a) amino acid residues 20 to 105 of SEQ ID NO: 5, (b) amino acid residues 18 to 105 of SEQ ID NO: 5, (c) amino acid residues 1 to 70 of SEQ ID NO: 5, (d ) Amino acid residues 20 to 70 of SEQ ID NO: 5, (e) amino acid residues 18 to 70 of SEQ ID NO: 5, (f) amino acid residues 76 to 105 of SEQ ID NO: 5, (g) amino acid residues of SEQ ID NO: 5 And (h) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 82 to 105 of SEQ ID NO: 5. Exemplary polypeptides consist of amino acid sequences (a)-(h).

The invention further provides antibodies and antibody fragments that specifically bind to such polypeptides. Typical antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies. Exemplary antibody fragments include F (ab ′) ₂ , F (ab) ₂ , Fab ′, Fab, Fv, scFv, and minimal recognition isolation. The invention also encompasses anti-indiotype antibodies that specifically bind to such antibodies or antibody fragments. The invention further encompasses a composition comprising a carrier and a peptide, polypeptide, antibody or anti-idiotype antibody described herein.

  The present invention also provides an isolated nucleic acid molecule encoding a Zven polypeptide, wherein the nucleic acid molecule comprises (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, (b) SEQ ID NO: 2 (C) following stringent washing conditions, a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1, the nucleotide sequence of nucleotides 288-389 of SEQ ID NO: 1, or SEQ ID NO: A nucleic acid molecule that remains hybridized to the complement of the nucleotide sequence of any one of nucleotides 66-161 or nucleotides 288-389 of SEQ ID NO: 1, (d) comprising the nucleotide sequence of SEQ ID NO: 6 A nucleic acid molecule, (e) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 5, (f) following stringent washing conditions, followed by nucleotides 334-405 of SEQ ID NO: 4 Nucleic acid molecule consisting fault sequence, or is selected from the group consisting of nucleic acid molecules which are present remain hybridized to the complement of a nucleotide sequence of nucleotides 334 to 405 of SEQ ID NO: 4.

  Exemplary nucleic acid molecules include those molecules in which any difference between the amino acid sequence encoded by the nucleic acid molecule and its corresponding amino acid sequence of SEQ ID NO: 2 or 5 is due to a conservative amino acid substitution. . The present invention further contemplates an isolated nucleic acid molecule comprising the nucleotide sequence of nucleotides 132-389 of SEQ ID NO: 1, nucleotides 147-389 of SEQ ID NO: 1, and nucleotides 148-405 of SEQ ID NO: 4.

  The invention also includes vectors and expression vectors comprising such nucleic acid molecules. Such an expression vector comprises a transcription promoter and a transcription terminator, wherein the promoter is operably linked by a nucleic acid molecule, and the nucleic acid molecule is operably linked by the transcription terminator. The invention further encompasses recombinant host cells comprising these vectors and expression vectors. Exemplary host cells include bacterial, yeast, avian, fungal, insect, mammalian and plant cells. A recombinant host cell comprising such an expression vector comprises culturing such a recombinant host cell comprising said expression vector and producing a Zven protein, and optionally cultured recombinant It can be used to prepare Zven polypeptides by isolating Zven proteins from host cells. The invention further encompasses products produced by such methods.

  Furthermore, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any one of such an expression vector or a recombinant virus comprising such an expression vector.

  The invention also includes hybridizing (a) a Zven1 nucleic acid probe to either (i) a test RNA molecule isolated from a biological sample or (ii) a nucleic acid molecule synthesized from the isolated RNA molecule. Contacting under soy conditions (wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO: 1 or its complement), and (b) a nucleic acid probe and a test RNA molecule or synthesized To detect the presence of Zven1 RNA in a biological sample comprising detecting the formation of any hybrid of nucleic acid molecules, wherein the presence of the hybrid indicates the presence of Zven1 RNA in the biological sample We plan also the method. Similar methods can be used to detect the presence of Zven1 RNA in a biological sample, where the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of SEQ ID NO: 4, or its complement .

  The invention further comprises (a) contacting the biological hand sample with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or consisting of the amino acid sequence of SEQ ID NO: 5, wherein said contacting comprises biological In a biological sample that is performed under conditions that allow binding of the antibody or antibody fragment to the sample and comprises (b) detecting either the bound antibody or the bound antibody fragment Methods are provided for detecting the presence of a Zven polypeptide. Such antibodies or antibody fragments further comprise a detectable label selected from the group consisting of radioisotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels and colloidal gold.

  Exemplary biological samples include human tissues such as autopsy samples, biopsy samples, body fluids and digestive components, and the like.

  The present invention also provides kits for performing these detection methods. A kit for detection of Zven1 gene expression comprises a container comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1. (B) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO: 1, (c) a nucleic acid molecule comprising the complement of the nucleotide sequence of nucleic acid molecule (a) or (b), (d) A nucleic acid molecule which is a fragment of (a) consisting of at least 8 nucleotides, (e) a nucleic acid molecule which is a fragment of (b) consisting of at least 8 nucleotides, (f) consisting of at least 8 nucleotides (c) And (g) a fragment of a nucleic acid sequence as shown in SEQ ID NO: 12, 13, 15, 16, 17, 18, 19, 20, 23, or 24, or a nucleic acid sequence thereof It is selected from the formed group from RaNaru nucleic acid molecule.

  A kit for detection of Zven2 gene expression comprises a container comprising a nucleic acid molecule, wherein said nucleic acid molecule comprises (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 334 to 405 of SEQ ID NO: 4 (B) a nucleic acid molecule comprising the complement of the nucleotide sequence of (a), (c) a nucleic acid molecule that is a fragment of (a) consisting of at least 8 nucleotides, and (d) from at least 8 nucleotides Selected from the group consisting of nucleic acid molecules that are fragments of (b). Such a kit also comprises a second container comprising one or more reagents capable of indicating the presence of the nucleic acid molecule.

On the other hand, a kit for the detection of a Zven protein comprises a container comprising an antibody or antibody fragment that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or consisting of the amino acid sequence of SEQ ID NO: 5 Can comprise.
The present invention also provides an anti-idiotype antibody that specifically binds an antibody or antibody fragment that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or consisting of the amino acid sequence of SEQ ID NO: 5 Or an anti-idiotype antibody fragment.

  The present invention further includes SEQ ID NO: selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity 2. A variant Zven1 polypeptide comprising an amino sequence that shares identity with an amino acid sequence of 2, wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO: 2 is One or more conservative amino acid substitutions. The amino acid sequence of SEQ ID NO: 5 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or 95% identity or more; Provided is an exemplary variant Zven2 polypeptide comprising an amino sequence that shares identity, wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO: 5 is 1 or This is due to multiple conservative amino acid substitutions.

  The invention also provides a fusion protein comprising a Zven1 polypeptide component or a Zven2 polypeptide component. Such a fusion protein can further comprise an immunoglobulin component. A suitable immunoglobulin component is an immunoglobulin heavy chain constant region, such as a human Fc fragment. The present invention also encompasses isolated nucleic acid molecules that encode such fusion proteins.

  The present invention relates to defective intestinal obstruction in a mammalian subject in need of such treatment comprising administering to the mammalian subject a Zven1 polypeptide comprising the amino acid sequence of amino acid residues 23-108 of SEQ ID NO: 2. A method for treating contractile disease is provided. In one embodiment, the disease is diabetes. In another method, the disease is postoperative ileus. In another embodiment, the disease is sepsis-related gastrointestinal congestion or bowel obstruction.

  The present invention further includes the amino acid sequence of amino acid residues 23 to 108 of SEQ ID NO: 2, the amino acid sequence of amino acid residues 20 to 105 of SEQ ID NO: 5, or the amino acid sequence of amino acid residues 28 to 129 of SEQ ID NO: 29. A method of treating a defective ileal obstructive contractile disease in a mammalian subject in need of such treatment comprising administering to the mammalian subject a Zven1 polypeptide comprising: In one embodiment, the disease is diabetes. In another method, the disease is post-surgical ileus. In another embodiment, the disease is sepsis-related gastrointestinal congestion or bowel obstruction.

  The present invention further provides a method for regulating gastrointestinal contractility in a mammal comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2. . In one embodiment, the modulation is inhibition. In another embodiment, the polypeptide is performed in one or more administrations. In further embodiments, one or more administrations of the polypeptide stimulate gastrointestinal contractility and one or more administrations of the polypeptide inhibit gastrointestinal contractility. In another embodiment, the one or more administrations that stimulate gastrointestinal contractility are performed prior to the one or more administrations that inhibit gastrointestinal contractility. In another embodiment, the one or more administrations that inhibit gastrointestinal contractility are performed prior to the one or more administrations that stimulate gastrointestinal contractility. In a further aspect, therapeutic adjustment of gastric contractility is achieved. In another embodiment, the polypeptide is administered continuously for a period of time.

  The present invention also administers a polypeptide comprising the amino acid sequence of amino acid residues 20 to 105 shown in SEQ ID NO: 5 to a mammal in need thereof, followed by the amino acids of amino acid residues 28 to 108 of SEQ ID NO: 2. A method of stimulating gastrointestinal contractility comprising administering a polypeptide comprising a sequence is provided.

  The present invention also provides a method for regulating gastric emptying in a mammal comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2. . In one embodiment, the modulation is inhibition. In another embodiment, the polypeptide is performed in one or more administrations. In a further aspect, one or more administrations of the polypeptide stimulates gastric emptying and one or more administrations of the polypeptide inhibits gastric emptying. In another embodiment, the one or more administrations that stimulate gastric emptying are performed prior to the one or more administrations that inhibit gastric emptying. In another embodiment, the one or more administrations that inhibit gastric emptying are performed prior to the one or more administrations that stimulate gastric emptying. In a further aspect, therapeutic modulation of gastric emptying capacity is achieved. In another embodiment, the polypeptide is administered continuously for a period of time.

  The invention also provides a method of regulating gastric transit in a mammal comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2. In one embodiment, the modulation is inhibition. In another embodiment, the polypeptide is performed in one or more administrations. In a further embodiment, one or more administrations of the polypeptide stimulates gastric passage and one or more administrations of the polypeptide inhibits gastric passage. In another embodiment, the one or more doses that stimulate gastric transit are performed prior to the one or more doses that inhibit gastric transit. In another embodiment, the one or more doses that inhibit gastric transit are performed prior to the one or more doses that stimulate gastric transit. In a further aspect, therapeutic adjustment of gastric transit is achieved. In another embodiment, the polypeptide is administered continuously for a period of time.

  The invention also provides a method for treating gastric paresis in a mammal in need thereof, comprising administering the polypeptide to a mammal, wherein the polypeptide is an amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2. And provides a method wherein gastrointestinal contractility, gastric emptying or intestinal transit is improved. In one aspect, gastric paresis is associated with surgery. In another embodiment, the polypeptide is administered to the mammal before or after surgery. In another aspect, the polypeptide is administered to the mammal before or after the mammal provides a post-surgical meal. In another embodiment, the treatment is characterized by increased contractility in intestinal obstruction. In another embodiment, the gastric paresis is post-operative or paralytic ileus. In another aspect, gastric paresis is not related to surgery. In another aspect, gastric failure paralysis is associated with diabetes, false bowel obstruction, chronic constipation, reduced digestive function, gastroesophageal reflux disease, vomiting, paralytic gastric failure, sepsis, or drug consumption.

The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method of stimulating chemokine release in a mammal comprising:
The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method of stimulating chemokine release in a mammal comprising:

The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method of stimulating neutrophil infiltration in a mammal comprising:
The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method of inducing or increasing appetite or weight gain in a mammal is provided.

The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method for enhancing sensitization to thermal, mechanical or painful stimuli in a mammal is provided.
The invention also includes administering to a mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5. A method of reducing sensitization to thermal, mechanical or painful stimuli in a mammal. In one embodiment, the antagonist is an antibody that specifically binds to the polypeptide.

The present invention also provides a method of inducing angiogenesis in cardiac stem cells comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2. In one embodiment, the angiogenesis is induced ex vivo or in vitro.
The present invention also provides a method of inducing angiogenesis in cardiac stem cells, comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2. In one embodiment, the neoplasia is induced ex vivo or in vitro.

The present invention also provides a polypeptide comprising an amino acid sequence selected from amino acid residues 28-208 of SEQ ID NO: 2; amino acid residues 20-105 of SEQ ID NO: 5; and amino acid residues 28-129 of SEQ ID NO: 29 Is provided to a mammal in need thereof for a method of modulating gastrointestinal contractility, gastric emptying or intestinal passage in a mammal. In one embodiment, the polypeptide is administered orally, intraperitoneally, intravenously, intramuscularly or subcutaneously.
The present invention also provides an isolated nucleic acid comprising a nucleic acid sequence as shown in SEQ ID NO: 14.

  The invention also includes a step of growing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid as shown in SEQ ID NO: 14, a transcription promoter and a transcription terminator. A production method, wherein the promoter is operably linked by the nucleic acid, the nucleic acid is operably linked by the transcription terminator, and the protein encoded by the nucleic acid is produced by the recombinant cell. A featured method is provided. The present invention also provides a polypeptide produced by the above method.

  These and other aspects of the invention will become apparent upon reference to the following detailed description. In addition, various documents are identified below and are all incorporated by reference.

2. Definition:
In the following description, a number of terms are used extensively. The following definitions are provided to facilitate an understanding of the present invention.
As used herein, “nucleic acid” or “nucleic acid molecule” refers to a polynucleotide, eg, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), an oligonucleotide, a fragment produced by the polymerase chain reaction (PCR). And fragments generated by any of ligation, cleavage, endonuclease action and exonuclease action. Nucleic acid molecules are composed of monomers that are naturally occurring nucleotides (eg, DNA and RNA), or analogs of naturally occurring nucleotides (eg, α-enantiomer forms of naturally occurring nucleotides), or a combination of both. obtain.

  Modified nucleotides can have changes in the sugar moiety and / or in the pyrimidine or purine base moiety. Sugar modifications include substitution of one or more hydroxyl groups with halogens, alkyl groups, amines and azide groups, or sugars can function as ethers or esters. Furthermore, the total sugar component can even be replaced by sterically and electronically similar structures such as aza-sugars and carboxylic acid sugar analogs. Examples of modifications at the base component include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents.

  Nucleic acid monomers can even be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilide, phosphoramidate, and the like. The term “nucleic acid molecule” also encompasses so-called “peptide nucleic acids” comprising so-called naturally occurring or modified nucleobases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

The term “complement of a nucleic acid molecule” is a nucleic acid molecule having an opposite orientation relative to a complementary nucleotide sequence and a control nucleotide sequence. For example, the sequence 5 ′ ATGCACGGG 3 ′ is complementary to 5 ′ CCCGTGCAT 3 ′.
The term “contig” refers to a nucleic acid molecule having a series of consecutive identical or complementary sequences to other nucleic acid molecules. A contiguous sequence is said to “overlap” a length of nucleic acid molecule in whole or along part of the nucleic acid molecule.

The term “degenerate nucleotide sequence” refers to a sequence of nucleotides (as compared to a reference polynucleotide encoding a polypeptide) that includes one or more degenerate codons. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie, GAU and GAC triplets each encode Asp).
The term “structural gene” refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNC) that is translated into a sequence of amino acids characteristic of a particular polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that does not integrate into the genome of an organism. A nucleic acid molecule isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
A “nucleic acid molecule structure” is a single-stranded or double-stranded nucleic acid molecule that is combined in a non-naturally occurring configuration and modified through human intervention to include juxtaposed nucleic acid segments.

“Linear DNA” refers to non-circular DNA molecules having free 5 ′ and 3 ′ and ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
“Complementary DNA (cDNA)” is a single-stranded DNA molecule formed from an mRNA template by reverse transcriptase. Typically, a primer that is complementary to a portion of the mRNA is used to initiate reverse transcription. Those skilled in the art also use the term “cDNA” to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term “cDNA” also refers to a clone of a cDNA molecule generated from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription of a structural gene. Typically, the promoter is located in the 5 'non-coding region of the gene, closest to the transcription start site of the structural gene. Sequence elements within the promoter that function at the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSE: McGehee et al., Mol. Endocrinol. 7: 511 (1993)), cyclic AMP response elements (CRE) Binding sites for serum response elements (SRE: Treisman, Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements (GRE) and other transcription factors such as CRE / ATF (O'Reilly et al. , J. Biol. Chem. 267: 19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269: 25728 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3: 253 (1993)), and octamer factors (generally Watson et al., Eds., Molecular Biology of the Gene, 4 ^th ed. (The Benjamin Kummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau. Biochem. J. 303: 1 (1994)). If the promoter is an inducible promoter, the rate of transcription increases in response to the inducing agent. In contrast, the rate of transcription is not regulated by the inducing agent when the promoter is a constitutive promoter. Promoters that can be repressed are also known.

A “core promoter” contains essential nucleotide sequences for promoter function including TATA box and initiation of transcription. According to this definition, the core promoter may or may not have detectable activity in the absence of specific sequences that can enhance activity or confer tissue specific activity.
A “regulatory element” is a nucleotide sequence that regulates the activity of a core promoter. For example, a regulatory element can comprise a nucleotide sequence that binds to cellular factors that permit transcription exclusively or selectively in a particular cell, tissue or organelle. These types of regulatory elements are usually linked to genes that are expressed in a “cell-specific”, “tissue-specific” or “organelle-specific” manner.
An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

  “Heterologous DNA” refers to a DNA molecule or a population of DNA molecules that are not naturally present in a given host cell. A DNA molecule that is heterologous to a particular host cell can include DNA from a host cell species (ie, endogenous DNA) as long as the host DNA is combined with non-host DNA (ie, exogenous DNA). . For example, a DNA molecule comprising a non-host DNA segment that encodes a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule. Conversely, a heterologous DNA molecule can include an endogenous gene that is operably linked to a foreign promoter. As another example, a DNA molecule comprising a gene derived from a wild type cell is considered to be heterologous DNA when that DNA molecule is introduced into a mutant cell lacking the wild type gene.

A “polypeptide” is a polymer of amino acid residues, whether produced naturally or synthetically, linked by peptide bonds. Polypeptides of less than about 10 amino acid residues are usually referred to as “peptides”.
A “protein” is a macromolecule comprising one or more polypeptide chains. The protein can also include non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents are added by the cell in which the protein is produced and will vary with the cell type. Proteins are defined herein by their amino acid backbone; substituents, such as carbohydrate groups, are generally not specified, but can nevertheless be present.

A peptide or polypeptide encoded by a non-host DNA molecule is a “heterologous” peptide or polypeptide.
An “integrated genetic element” is a segment of DNA that is integrated into the chromosome of a host cell after the element has been introduced into the cell through human manipulation. In the present invention, the integrated genetic element is usually derived from a linearized plasmid that is introduced into the cell by electroporation or other techniques. The integrated genetic element is passed from the original host cell to its progeny.

  A “cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the ability to replicate autonomously in a host cell. Cloning vectors are typically transformed with one or a few restriction endonuclease recognition sites that allow the insertion of nucleic acid molecules in a determinable manner, without losing the essential biological function of the vector, and the cloning vector. A nucleotide sequence encoding a marker gene that is suitable for use in the identification and selection of cells. Marker genes typically include genes that confer tetracycline resistance or ampicillin resistance.

  An “expression vector” is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, an expression vector includes a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked” to the promoter. Similarly, a regulatory element and a core promoter are operably linked when the regulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector. As used herein, an example of a recombinant host is a cell that produces a Zven1 or Zven2 peptide or polypeptide from an expression vector. In contrast, such polypeptides are “natural sources” of Zven1 or Zven2, and can even be produced by cells lacking an expression vector.
An “integrated recombinant” is a recombinant host cell that allows heterologous DNA to be integrated into the cellular genomic DNA.

  A “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes. For example, the fusion protein comprises at least a portion of a Zven1 or Zven2 polypeptide fused by a polypeptide that binds an affinity matrix. Such fusion proteins provide a means for isolating large quantities of Zven1 or Zven2 using affinity chromatography.

  The term “receptor” refers to a cell-bound protein that binds to a bioactive molecule termed “ligand”. This interaction mediates the effect of the ligand on the cell. Receptors can be membrane-bound cytosole or nucleus; monomers (eg, thymus stimulating hormone receptor, β-adrenergic receptor) or multimers (eg, PDGF receptor, growth hormone receptor IL-3 receptor, GM -CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and typically an intracellular effector domain involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located on separate polypeptides that comprise a complete functional receptor.

  In general, binding of a ligand to a receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell leading to changes in the cell's metabolism. Metabolic phenomena often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased production of cyclic AMP, cellular calcium metabolism, membrane lipid metabolism, cell attachment, inositol lipid hydrolysis. Includes degradation and hydrolysis of phospholipids.

  The term “secretory signal sequence” refers to a DNA sequence that encodes a peptide that directs a larger polypeptide as a component of a larger polypeptide through the secretory pathway of the cell in which it is synthesized (“secreted peptide”). The larger polypeptide is usually degraded to remove the secretory peptide during movement through the secretory pathway.

  An “isolated polypeptide” is a polypeptide that is substantially free of contaminating cellular components, such as carbohydrates, lipids, or other proteinaceous impurities that are naturally associated with the polypeptide. Typically, an isolated polypeptide preparation is in highly purified form, ie, at least about 80% purity, at least about 90% purity, at least about 95% purity, greater than 95% purity. Or contain the polypeptide in a purity of 99% or higher. One means for indicating that a particular protein preparation contains an isolated polypeptide is a single band by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and Coomassie blue staining of the gel. Is due to the appearance of. However, the term “isolated” does not exclude the presence of the same polypeptide in other physical forms, such as dimers or other glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein to indicate a position within a polypeptide. Where the situation is possible, the terms are used with respect to a particular sequence or portion of the polypeptide to indicate accessibility or relative position. For example, a certain sequence located within the polypeptide at the carboxyl terminus of the subject sequence is located adjacent to the carboxyl terminus of the control sequence, but is not necessarily required at the carboxyl terminus of the complete polypeptide.
The term “expression” refers to the biosynthesis of a gene product. For example, in a structural gene, expression includes transcription of the structural gene into mRNA and translation of the mRNA into one or more polypeptides.

  The term “splice variant” is used herein to indicate an alternative form of RNA transcribed from a gene. Splice mutations occur in several transcribed RNA molecules, naturally occurring through the use of alternative splicing sites between, but usually low but separately transcribed RNA molecules, and transcribed from the same gene. Can bring about mRNA. A splice variant can encode a polypeptide having an altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors and synthetic analogs of those molecules.
The term “complement / anti-complement pair” refers to non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are basic members of a complement / anti-complement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. Where subsequent dissociation of a complement / anti-complement pair is desired, the complement / anti-complement pair preferably has a binding affinity of <10 ⁹ M ⁻¹ .

An “anti-idiotype antibody” is an antibody that binds to the variable region domain of an immunoglobulin. As used herein, an anti-idiotype antibody binds to the variable region of an anti-Zven1 or anti-Zven2 antibody, and thus the anti-idiotype antibody mimics an epitope of Zven1 or Zven2.
“Antibody fragments” are a portion of an antibody, eg, F (ab ′) ₂ , F (ab) ₂ , Fab ′, Aab and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-Zven1 monoclonal antibody fragment binds to an epitope of Zven1.

  The term “antibody fragment” also refers to a synthetic or genetically constructed polypeptide that binds to a specific antigen, eg, a polypeptide comprising light chain variable regions, an “Fv” fragment comprising heavy and light chain variable regions. A recombinant single chain polypeptide molecule ("svFv protein") in which the L and H chain variable regions are linked by a peptide ringer, and a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein comprising various domains derived from rodent antibodies and complementary determining regions, whereas the remainder of the antibody molecule is derived from a human antibody.
A “humanized antibody” is a recombinant protein in which the murine complementarity-determining regions of a monoclonal antibody are transferred from murine immunoglobulin H and L variable chains to a human variable domain.

A “detectable label” is a molecule or atom that can be conjugated to an antibody component to produce a molecule useful for diagnosis. Examples of labels that can be detected include chelating agents, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions or other marker components.
The term “affinity label” refers to a polypeptide that can be bound to a second polypeptide to provide purification or detection of the second polypeptide or to provide a site for binding of the second polypeptide to a substrate. Used herein to indicate a peptide segment. In principle, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity label. Affinity labels are poly-histidine series, ie protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991), glutathione S transferase (Smits and Johnson, Gene 67; 31, 1988), Glu-Glu affinity labeling (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82: 7952-4, 1985), Substance P, ie Flag ^TM peptide (Hopp et al., Biotechnology 6 : 1204-1210, 1988), streptavidin binding peptides, or other antigenic epitopes or binding domains. See generally Ford et al., Protein Expression and Purification 2: 95-107, 1991. Nucleic acid molecules encoding affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ; Eastman Kodak, New Heven, CT; New England Biolabs, Beverly, Mass.).

A “naked antibody” is an intact antibody that is not conjugated by a therapeutic agent as opposed to an antibody fragment. Naked antibodies include polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
As used herein, the term “antibody component” encompasses both intact antibodies and antibody fragments.

  A “target polypeptide” or “target peptide” is an amino acid sequence that contains at least one epitope and is expressed on a target cell, eg, a tumor cell, or a cell bearing an infectious agent antigen. T cells recognize the peptide epitope provided by the major histocompatibility complex molecule to the target polypeptide or target peptide, and typically lyse the target cell or other immunity to the target cell example. Replenish cells and thereby kill target cells.

  An “antigenic peptide” binds a major histocompatibility complex molecule to form an MHC-peptide complex that is recognized by T cells, thereby causing a cytotoxic lymphocyte response based on provision to the T cell. Is a peptide that induces Thus, the antigenic peptide binds to the appropriate major histocompatibility complex molecule and induces a cytotoxic T cell response, such as cytolysis or specific cytokine release to target cells that bind or express the antigen. Can do. Antigenic peptides can be conjugated with respect to class I or class II major histocompatibility complex molecules on antigen-donor cells or target cells.

  In eukaryotes, RNA polymerase II catalyzes the transcription of structural genes to produce mRNA. The nucleic acid molecule is designed to include an RNA polymerase II template, where the RNA transcript has a sequence that is complementary to the sequence of a particular mRNA. RNA transcripts are referred to as “antisense RNA” and nucleic acid molecules that encode antisense RNA are referred to as “antisense genes”. Antisense RNA molecules can bind to the mRNA molecule and result in inhibition of mRNA translation.

  "An antisense oligonucleotide specific for Zven1" or "Zven1 antisense oligonucleotide" can form a stable trimer with (a) a part of the Zven1 gene, or (b) mRNA of the Zven1 gene An oligonucleotide having a sequence capable of forming a stable dimer with a portion of a transcript. Similarly, an “antisense oligonucleotide specific for Zven2” or “Zven2 antisense oligonucleotide” can form a stable trimer with (a) a portion of the Zven2 gene, or ( b) An oligonucleotide having a sequence capable of forming a stable dimer with a part of the mRNA transcript of the Zven2 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalytic center. The term encompasses RNA enzymes, self-splicing RNAs, self-degrading RNAs and nucleic acid molecules that perform their catalytic function.
An “external guide sequence” is a nucleic acid molecule that directs an endogenous ribozyme, ie, RNase P, to a specific species of intracellular mRNA resulting in degradation of the mRNA by RNase P. A nucleic acid molecule that encodes an external guide sequence is referred to as an “external guide sequence gene”.

  The term “variant Zven1 gene” refers to a nucleic acid molecule that encodes a polypeptide having an amino acid sequence that is a modification of SEQ ID NO: 2. Such variants include naturally occurring polymorphic Zven1 genes and synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 2. Additional variant forms of the Zven1 gene are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein. A mutant Zven1 gene can be identified by determining whether the gene hybridizes under stringent conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or its complement. Similarly, mutant Zven2 genes and mutant Zven2 polypeptides can be identified for SEQ ID NOs: 4 and 5, respectively.

  On the other hand, mutant Zven genes can even be identified by sequence-comparison. Two amino acid sequences have “100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned by maximal response. Sequence comparison can be performed using standard software programs such as those included in the LASERGENE bioinformation computer site manufactured by DNASTAR (Madison, Wisconsin).

Other methods for comparing two nucleotide or amino acid sequences by determining optimal alignment are well known to those skilled in the art (eg, Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.), “Information Superhighway and Computer Databases of Nucleic Acids and Proteins.” In Methods in Gene Biotechnology, Pages 123-151 (CRC Press. Inc. 1997 ), and Bishop (ed.), Guide to Human Genome Computing, 2 nd Edition (Academic Press, Inc. 1998) see). Specific methods for determining sequence identity are described below.

  Regardless of the particular method used to identify the mutant Zven1 gene or mutant Zven1 polypeptide, the mutant gene or polypeptide encoded by the mutant gene specifically binds to the anti-Zven1 antibody. It can be characterized by its ability to Similarly, a mutant Zven2 gene product or mutant Zven2 peptide is characterized by the ability to bind specifically to an anti-Zven2 antibody.

  The term “allelic variant” is used herein to indicate any of a plurality of gene alternatives of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation and can result in phenotypic polymorphism within a population. Genetic mutations can be silent (no change in the encoded polypeptide) or encode a polypeptide having an altered amino acid sequence. The term allelic variant is also used herein to indicate a protein encoded by an allelic variant of a gene.

The term “orthology” refers to a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences between orthologs are the result of specification.
“Paralogs” are different but structurally related proteins produced by living organisms. The para body appears to arise through gene duplication. For example, α-globin, β-globin and myoglobin are para bodies of each other.

The present invention encompasses functional fragments of the Zven1 and Zven2 genes. In the present invention, a “functional fragment” of a Zven1 (or Zven2) gene is a nucleic acid molecule that encodes a portion of a Zven1 (or Zven2) polypeptide that specifically binds to an anti-Zven1 (anti-Zven2) antibody. To mention.
It is understood that the molecular weight and length of the polymer are approximate values due to inaccuracies in standard analytical methods. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ± 10%.

3. Generation of human Zven1 and Zven2 genes:
A nucleic acid molecule encoding the human Zven1 gene is obtained by screening a human cDNA or genomic library using a polynucleotide probe based on SEQ ID NO: 1. Similarly, a nucleic acid molecule encoding the human Zven2 gene can be obtained by screening a human cDNA or genomic library using a polynucleotide probe based on SEQ ID NO: 4. Those techniques are standard and well established.

  As illustrated, a nucleic acid molecule encoding a human Zven1 gene can be isolated from a human cDNA library. In this case, the first step is to prepare a cDNA library using methods well known to those skilled in the art, for example, by isolating RNA from tissues such as testis or peripheral blood lymphocytes. In general, RNA isolation techniques provide a method for degrading cells, a means for inhibiting RNase-directed degradation of RNA, and a method for separating RNA from DNA, protein and polysaccharide contaminants Should.

For example, total RNA can be obtained by freezing tissue in liquid nitrogen, grinding the frozen tissue with a nipple and pestle, lysing cells, extracting the ground tissue with a phenol / chloroform solution, except, and can be isolated by separating the impurities remain the RNA by selective precipitation with lithium chloride (see, for example, Ausubel like., (eds.), Short Protocols in Molecular Biology, 3 rd Edition pages4-1~4- 6 (John Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [See “Wu (1997)”]). On the other hand, total RNA can be isolated by extracting the ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating the RNA from contaminants by differential centrifugation (eg, Chirgwin et al. Biochemistry 18:52 (1979); Ausubel (1995) P.4-1-4-6; Wu (1997) P.33-41).

In order to construct a cDNA library, poly (A) ⁺ RNA should be isolated from the total RNA preparation. Poly (A) ⁺ RNA can be isolated from total RNA using standard techniques of oligo (dT) -cellulose chromatography (eg, Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408 (1972 ); Ausubel (1995) P. 4-11-4-12).
Double stranded cDNA molecules are synthesized from poly (A) ⁺ RNA using techniques well known to those skilled in the art (see, eg, Wu (1997) P. 41-46). In addition, commercially available kits can be used to synthesize double-stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, Wis.), And STRATAGENE (La Jolla, CA).

A variety of cloning vectors are suitable for construction of the cDNA library. For example, a cDNA library can be prepared in a vector derived from a bacteriophage, such as λgt10 Bacterium. For example, Huynh et al., “Construction and Screening cDNA Libraries in λgt10 and λ11” in DNA Cloning: A Practica Approach Vol. I, Glovar (ed.), Page 49 (IRL Press, 1985); Wu (1997) at pages 47 See -52.
Alternatively, the double-stranded cDNA molecule can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), LAMDAGEM-4 (Promega Corp.), or other commercially available vectors. Suitable cloning vectors are also available from the American Type Culture Collection (Manassas. VA).

To amplify the cloned cDNA molecule, the cDNA library is inserted into a prokaryotic host using standard techniques. For example, a cDNA library can be introduced into competent E. coli DH5 cells obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).
Human genomic libraries can be prepared by means well known in the art (see, eg, Ausubel (1995) p. 5-1-5-6; Wu (1997) p. 307-327). Genomic DNA lyses the tissue with the detergent Sarkosyl, digests the lysate with proteinase K, cleans the insoluble debris from the lysate by centrifugation, precipitates the nucleic acid from the lysate using isopropanol, and chlorides it. It can be isolated by purifying the resuspended DNA on a cesium density gradient.

  DNA fragments that are suitable for the generation of genomic libraries are obtained by random shearing of genomic DNA or by partial digestion of genomic DNA with restriction endonucleases. Genomic DNA fragments can be transformed into vectors according to conventional techniques, such as the use of restriction enzyme digestion to provide the appropriate termini, the use of alkaline phosphatase treatment to avoid undesired ligation of DNA molecules, and ligation with ligase as appropriate. For example, it can be inserted into a bacteriophage or cosmid vector. Techniques for such manipulation are well known in the art (see, for example, Ausubel (1995) p. 5-1-5-6; Wu (1997) p. 307-327).

  Nucleic acid molecules encoding human Zven1 or Zven2 genes are also obtained using polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences based on the nucleotide sequences described herein. General methods for screening libraries by PCR are, for example, Yu et al., “Use of The Polymerase Chain Reaction to Screen Phage Libraries”. In Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Application, White (ed.), p. 211-215 (Humana Press, Inc. 1993).

Furthermore, techniques for using PCR to isolate related genes are described in, for example, Preston, “Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members,” in Methods in Molecular Biology, Vol. 15: PCR Protocols; Current Metods and Applications. White (ed.), Pages 317-337 (Humana Press, Inc. 1993).
On the other hand, genomic libraries are obtained from commercial sources such as Research Genetics (Huntsville, AL) and ATCC (Marassas, VA).

Libraries containing cDNA or genomic clones can be screened with one or more polynucleotide probes based on SEQ ID NO: 1 using standard methods (see, eg, Ausubel (1995) p. 6-1-6-11). See
The anti-Zven antibody produced as described below can also be used to isolate a DNA sequence encoding the Zven gene from a cDNA library. For example, antibodies can be used to screen a λgt11 expression library, or antibodies can be used for immunoscreening following hybrid selection and translation (eg, Ausubel (1995) p. 6-12-6-16; . such as Margolis, "Screening λ expression libraries with untibody and Protein Probes" in DNA Cloning 2: (. eds). Expression Systems, 2 nd Edition, such as Glover p 1-14 see (Oxford University Press 1995)).

  Alternatively, the Zven gene can be obtained by synthesizing nucleic acid molecules using long oligonucleotides that prime each other and the nucleotide sequences described herein (see, for example, Ausubel (1995) p. 8-8). (See 8-9). Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least 2 kb in length (Adang et al., Plant Molec. Biol. 21: 1131 (1993). Bambot et al., PCR Methods and Applications 2: 266 (1993), Dilfon et al., “Use of the Polymerase Chain Reaction for the Rapid Constrauction of Synthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications. White ( ed.), Pages 263-268. (Humana Press. Inc. 1993), and Holowachuk et al., PCR Methods Appl. 4: 299 (1995)).

  The nucleic acid molecules of the invention can also be synthesized by “gene opportunity” using protocols such as the phosphoramidite method. When chemically synthesized double stranded DNA is required for applications such as gene or gene fragment synthesis, the individual complementary strands are produced separately. Generation of short genes (60-80 base pairs) is technically simple and can be achieved by synthesizing complementary strands and then annealing them. However, for the production of long genes (300 base pairs or more), special measures are required since the coupling efficiency of individual cycles is rarely 100% during chemical DNA synthesis.

  To overcome this problem, synthetic genes (double strands) are assembled in a modular fashion from single stranded fragments that are 20-100 nucleotides in length. For reconsideration of polynucleotide synthesis, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA (ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53: 323 (1984), etc. Et al., Proc. Nat'l Acad. Sci. USA 87: 633 (1990).

  The sequence of the Zven cDNA or Zven genomic fragment can be determined using standard methods. The Zven polynucleotide sequences disclosed herein can also be used as probes or primers to clone the 5 'non-coding region of the Zven gene. Promoter elements from the Zven gene can be used to direct the expression of heterologous genes in, for example, transgenic animals, or tissue of patients undergoing gene therapy. Identification of genomic fragments containing the Zven promoter or regulatory elements can be accomplished using well-established techniques such as deletion analysis (see generally Ausubel (1995)).

  Cloning of the 5 'flanged sequence also facilitates the production of Zven protein by "gene activation" according to the method disclosed in US Patent No. 5,641,670. Briefly, the expression of an endogenous Zven gene in a cell introduces a DNA construct comprising at least a target sequence, a regulatory sequence, an exon, and an unpaired splice donor site into the Zven locus. It is changed by doing. The target sequence is a Zven 5 'non-coding sequence that allows homologous recombination of a structure having an endogenous Zven locus, whereby the sequence within the structure is operatively linked by an endogenous Zven coding sequence Will come to be. In this case, the endogenous Zven promoter is replaced or supplemented by a row of other regulation, providing enhanced tissue specific or, on the other hand, regulated expression.

4). Mutant generation:
The present invention provides various nucleic acid molecules, such as DNA and RNA molecules, that encode the Zven polypeptides disclosed herein. One skilled in the art will readily recognize that considerable sequence variation is possible between these polynucleotide molecules in view of the degeneracy of the genetic code. SEQ ID NOs: 3 and 6 are degenerate nucleotide sequences that encompass all nucleic acid molecules that encode the Zven polypeptides of SEQ ID NOs: 2 and 5, respectively.

  One skilled in the art will provide all RNA sequences that each encode SEQ ID NO: 2 by replacing the degenerate sequence of SEQ ID NO: 3 with U instead of T, and the degenerate sequence of SEQ ID NO: 6 is also It will be appreciated that substituting U for T replaces all RNA sequences encoding SEQ ID NO: 5, respectively. Accordingly, the present invention provides a Zven polypeptide-encoding nucleic acid molecule comprising nucleotides 66-389 of SEQ ID NO: 1, and RNA equivalents thereof, and nucleotides 91-405 of SEQ ID NO: 4, and RNA equivalents thereof. A Zven polypeptide-encoding nucleic acid molecule comprising is designed.

  Table 1 shows the single letter codes used within SEQ ID NOs: 3 and 6 to indicate degenerate nucleotide positions. “Solution” is the nucleotide indicated by the code letter. “Complement” refers to the code for the complementary nucleotide. For example, the code Y indicates either C or T, and its complement R indicates A or G, A is complementary to T, and G is complementary to C.

  Degenerate codons used in SEQ ID NOs: 3 and 6, including all possible codons for a given amino acid, are shown in Table 2.

  One skilled in the art will understand that some ambiguity is introduced in the determination of degenerate codons that are representative of all possible codons encoding individual amino acids. For example, a degenerate codon for serine (WSN) can encode arginine (AGR) under certain circumstances, and a degenerate codon for arginine (MGN) under certain circumstances can be serine ( AGY) can be coded. A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by a degenerate sequence can encode a variant amino acid sequence, but one skilled in the art will recognize such variants by reference to the amino acid sequences of SEQ ID NOs: 2 and 5. Sequences can be easily identified. Variant sequences can be readily tested for functionality as described herein.

  Different species can exhibit “selective codon usage”. Generally, Grantham, et al., Nuc. Acids Res. 8: 1893-912, 1980; Haas, et al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiera, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. et al. Mol. Biol. 158: 573-97, 1982, Sharyp and Matassi, Curr. Opin. Genet. Dev. 4: 851 (1994), Kane, Curr. Opin. Biotechnol. 6: 494 (1995), and Makrides, Microbiol. Rev. 60 : See 512 (1996).

  As used herein, the terms “selective codon usage” or “selective codon” are used most frequently in certain cell types and thus refer to possible codons encoding individual amino acids. Technical term referring to protein translation codons that prefer one or a few representatives (see Table 2). For example, the amino acid threonine (Thr) is encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon; in other species, for example, insect cells In yeast, viruses or bacteria, different Thr codons are preferred.

  Selective codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. For example, the introduction of selective codon sequences into recombinant DNA enhances the production of the protein by making it more efficient by translation of the protein within a particular cell type or species. Thus, the degenerate codon sequence disclosed in SEQ ID NO: 3 is routinely used in the art and serves as a template for optimizing polypeptide expression in the various cell types and species disclosed herein. To do. Sequences containing selected codons can be tested for expression in various species and tested for functionality disclosed herein.

  The invention further provides polypeptides and nucleic acid molecules that represent counterparts from other species (orthos). Such species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species. Of particular interest are Zven polypeptides from other mammalian species such as pigs, sheep, cows, dogs, cats, horses and other primate ligands. The orthoform of human Zven can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zven. Appropriate sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. Next, a library is prepared from positive tissue or cell line mRNA.

  Zven-encoded cDNAs can be isolated in various ways, for example by probing with complete or partial cDNA or with one or more degenerate probes based on the disclosed sequences. cDNA can also be cloned by the polymerase chain reaction using primers designed from the representative human Zven sequences disclosed herein. In a further method, a cDNA library is used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody against the Zven polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

  One skilled in the art will recognize that the sequences disclosed in SEQ ID NOs: 1 and 4 represent a single allele of human Zven1 and Zven2, respectively, and that allelic variation and alternate splicing are expected to occur. Let's go. Allelic variants of the nucleotide sequences disclosed herein can be cloned by probing cDNA or genomic libraries from different individuals according to standard methods. Allelic variants of the nucleotide sequences shown in SEQ ID NOs: 1 and 4, eg, those variants including silent mutations and those variants in which the mutation results in an amino acid sequence change are the allelic variations of SEQ ID NOs: 2 and 5 Like the body protein, it is within the scope of the present invention.

  A cDNA molecule produced from another spliced mRNA that retains the properties of a Zven polypeptide is included within the scope of the present invention, as is a polypeptide encoded by such a cDNA and mRNA. . Allelic and splice variants of those sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard methods known in the art.

  In a preferred embodiment of the invention, an isolated nucleic acid molecule will hybridize under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence disclosed herein. For example, such a nucleic acid molecule comprises, under stringent conditions, a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1, nucleotide 288 of SEQ ID NO: 1 A nucleic acid molecule consisting of the nucleotide sequence of 389, a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 4, a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 334 to 405 of SEQ ID NO: 4, or a nucleotide sequence which is the complement of such a sequence It can hybridize to a nucleic acid molecule. In general, stringent conditions are selected to be about 5 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence will hybridize to a suitably matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide sequences have some degree of complementarity. Mismatch base pairs in the hybrid double helix can be tolerated, but the stability of the hybrid is affected by the degree of mismatch. The Tm of the mismatch hybrid decreases by 1 ° C. for every 1-1.5% base pair mismatch. Changing the stringency of the hybridization conditions allows control over the degree of mismatch that may be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Stringent hybridization conditions include a hybridization buffer having a temperature about 5-25 ° C. below the thermal melting point (T _m ) of the hybrid and Na ^{+ up} to 1M.

  Higher temperature stringency at lower temperatures can be achieved by the addition of formamide to lower the Tm of the hybrid at about 1 ° C. for each individual 1% formamide in the buffer solution. Generally, such stringent conditions include a temperature of 20-70 ° C. and a hybrid buffer containing 6 × SSC and 0-50% formamide. A high degree of stringency can be achieved at a temperature of 40-70 ° C. and with a hybridization buffer having 4 × SSC and 0-50% formamide. High stringency conditions typically include a hybridization buffer having a temperature of 42-70 ° C. and 1 × SSC and 0-50% formamide. Different degrees of stringency can be used during hybridization and washing to achieve maximal specific binding to the target sequence. Typically, the wash following hybridization is performed with an increased degree of stringency to remove unhybridized polynucleotide probes from the hybridized complex.

The above conditions are meant to act as a guide, and it is well within the ability of one skilled in the art to adapt those conditions for use with a particular polypeptide hybrid. The T _m for a particular target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a fully matched probe sequence. Those conditions that affect T _m include the size and base pair content of the polynucleotide probe, the ion temperature of the hybridization solution, and the presence of destabilizing agents in the hybridization solution.

  Many equations for calculating Tm are known in the art and are specific for various lengths of DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences (eg, Sambrook et al. , Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1988); Ausubel et al., (Eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds. ), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227 (1990)).

  Sequence analysis software, such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, Calif.), As well as sites on the Internet, analyze a given sequence and are defined by the user Means are available for calculating Tm based on other criteria. Such a program can also analyze a given configuration under defined conditions and identify appropriate probe sequences. Typically, hybridization of long polynucleotide sequences of 50 or more base pairs is performed at a temperature about 20-25 ° C. below the calculated Tm. For small probes of 50 base pairs or less, hybridization typically occurs at Tm or 5-10 ° C. or lower. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.

  The length of the nucleotide sequence affects the rate and stability of hybridization. Small probe sequences, ie 50 base pairs or less, quickly reach equilibration with complementary sequences, but do not form stable hybrids. Incubation times (approximately minutes to hours) can be used to achieve hybridization. Longer probe sequences equilibrate more slowly, but even at lower temperatures, they form more stable complexes. Incubation is allowed to proceed overnight or longer. In general, the incubation can be performed for a period equal to three times the calculated cot time. The cot time, ie, the time it takes for a polynucleotide sequence to reassociate, can be calculated for a particular sequence by methods known in the art.

  The base pair composition of the polynucleotide sequence results in the thermal stability of the hybrid complex, thereby affecting the choice of hybridization temperature and the ionic strength of the hybridization buffer. The AT pair is less stable than the GC pair in aqueous solutions containing sodium chloride. Therefore, the higher the GC content, the more stable the hybrid. An equal distribution of G and C residues within the sequence also contributes positively to hybrid stability. Furthermore, the base pair composition can be manipulated to change the Tm of a given sequence. For example, 5-methyldeoxycydine can be substituted with deoxycytidine, and 5-bromodeoxyuridine can be substituted with deoxycytidine, and 5-bromodeoxyuridine can be substituted with thymidine to increase Tm, and 7-deaz-2'-deoxyguanosine can be replaced by guanosine to reduce its dependence on Tm.

The ionic concentration of the hybridization buffer also affects the stability of the hybrid. Hybridization buffers are generally blocking agents such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, milk powder (BLOTTO), heparin or SDS, and Na ⁺ sources such as Contains SSC (1 × SSC: 0.15: N NaCl, 15 mM sodium citrate) or SSPE (1 × SSPE: 1.8 M NaCl, 10 mM NaH ₂ PO ₄ , 1 mM EDTA, pH 7.7). Typically, the hybridization buffer contains 10 mM to ¹ M Na ⁺ .

Addition of destabilizing or denaturing agents such as formamide, tetraalkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the _Tm of the hybrid. Typically, formamide is used at a concentration of up to 50% to allow more convenient and low temperature incubations to be performed. Formamide also acts to reduce non-specific background when using RNA probes.

  As illustrated, a nucleic acid molecule encoding a mutant Zven1 polypeptide is 50% formamide, 5 × SSC (1 × SSC: 0.15 M) by means of a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement). Sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution (100 × Denhardt's solution: 2% (w / v) Ficoll 400, 2% (w / v) Polyvinylpyrrolidone and 2% (w / v bovine serum albumin), hybridized overnight at 42 ° C. in a solution comprising 10% dextran sulfate and 20 μg / ml denatured and sheared salmon sperm DNA. obtain.

  Those skilled in the art can take into account the variability of their hybridization conditions. For example, the hybridization mixture can be incubated at a high temperature, for example at about 65 ° C., in a solution that does not contain formamide. In addition, premixed hybridization solutions are available (eg, EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.

  Following hybridization, the nucleic acid molecules can be washed to remove unhybridized nucleic acid molecules under stringent conditions or under high stringency conditions. Typical stringent wash conditions include washing at 55-65% with a 0.5 × -2 × SSC solution containing 0.1% sodium dodecyl sulfate (SDS). For example, a nucleic acid molecule encoding a mutant Zven1 polypeptide is a nucleic acid molecule consisting of nucleotide sequences of nucleotides 66-161 of SEQ ID NO: 1, nucleotides 288-389 of SEQ ID NO: 1, or their complements under stringent wash conditions Wherein the wash stringency is 0.5 × -2 × SSC solution containing 0.1% SDS at 55-65 ° C., for example 0.5 × SSC solution containing 0.1% SDS at 55 ° C., or Equivalent to 2 × SSC solution containing 0.1% SDS at 65 ° C.

  In a similar manner, a nucleic acid molecule encoding a mutant Zven2 variant hybridizes under stringent wash conditions with a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 334-405 of SEQ ID NO: 4, or its complement, wherein The cleaning stringency is 0.5 x to 2 x SSC solution containing 0.1% SDS at 55 to 65 ° C, for example, 0.5 x SSC solution containing 0.1% SDS at 55 ° C, or 0.1% SDS at 65 ° C. Equal to 2 × SSC solution containing One skilled in the art can easily produce equivalent conditions, for example, by replacing SSC in the wash solution with SSPE.

  Typical high stringency wash conditions include washing with a solution of 0.1 × -0.2 × SSC containing 0.1% sodium dodecyl sulfate (SDS) at 50-65 ° C. As illustrated, a nucleic acid molecule encoding a mutant Zven1 polypeptide can have a nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1, a nucleotide sequence of nucleotides 288-389 of SEQ ID NO: 1 under high stringency wash conditions, or Hybridizes with nucleic acid molecules consisting of their complements, wherein the wash stringency is between 0.1 × to 0.2 × SSC solution containing 0.1% SDS at 50 to 65 ° C., for example at 50 ° C. , 0.1 × SSC solution containing 0.1% SDS, or 0.2 × SSC solution containing 0.1% SDS at 65 ° C.

  Similarly, a nucleic acid molecule encoding a mutant Zven2 variant hybridizes under high stringency washing conditions to a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 334-405 of SEQ ID NO: 4, or its complement, wherein The wash stringency is from 0.1x to 0.2x SSC solution containing 0.1% SDS at 50-65 ° C, eg, 0.1x SSC solution containing 0.1% SDS at 50 ° C, or at 65 ° C. Equivalent to 0.2 × SSC solution containing 0.1% SDS.

  The invention also provides an isolated Zven1 polypeptide having substantially similar sequence identity to the polypeptide of SEQ ID NO: 2 or their ortho form. The term “substantially similar sequence identity” means a polypeptide having at least 85%, 90%, 95% or 95% sequence identity to the sequence shown in SEQ ID NO: 2 or their ortho form Is used herein to indicate. Similarly, the present invention also provides an isolated Zven2 polypeptide having a sequence identity of 85%, 90%, 95%, or 95% or more with respect to the sequence shown in SEQ ID NO: 5 or an ortho form thereof. .

  The present invention also contemplates Zven variant nucleic acid molecules that can be identified using the following two criteria: determination of similarity between the amino acid sequence of SEQ ID NO: 2 or 5 and the encoded polypeptide; Hybridization assay. For example, a Zven1 gene variant has (1) the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1 under stringent wash conditions equivalent to a 0.5 × -2 × SSC solution containing 0.1% SDS at 55-65 ° C. Hybridizes with a nucleotide sequence of nucleotides 288-389 of SEQ ID NO: 1 or a complement thereof, and (2) 85%, 90%, 95% or 95% to the amino acid sequence of SEQ ID NO: 2 Nucleic acid molecules encoding polypeptides having the above sequence identity are included.

  On the other hand, the Zven1 variant is (1) the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1 under high stringent wash conditions, equivalent to a 0.1 × -0.2 × SSC solution containing 0.1% SDS at 50-65 ° C. Hybridizing with a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 288-389 of SEQ ID NO: 1, or the complement thereof, and (2) 85%, 90%, 95% or 95 with respect to the amino acid sequence of SEQ ID NO: 2 It can be characterized as a nucleic acid molecule that encodes a polypeptide having a sequence identity greater than or equal to%.

  In addition, certain Zven2 gene variants are (1) the nucleotide sequence of nucleotides 334-405 of SEQ ID NO: 4 under stringent wash conditions equivalent to a 0.5 × -2 × SSC solution containing 0.1% SDS at 55-65 ° C. Or a nucleic acid that hybridizes with a nucleic acid molecule comprising its complement and (2) encodes a polypeptide having a sequence identity of 85%, 90%, 95%, or 95% or more to the amino acid sequence of SEQ ID NO: 5 Includes molecules. On the other hand, the Zven2 mutant gene is (1) nucleotides 334-405 of SEQ ID NO: 4 under high stringent wash conditions, equivalent to a 0.1 × -0.2 × SSC solution containing 0.1% SDS at 50-65 ° C. Hybridizes with a nucleic acid molecule comprising the sequence, or its complement, and (2) encodes a polypeptide having 85%, 90%, 95% or more than 95% sequence identity to the amino acid sequence of SEQ ID NO: 5 It can be characterized as a nucleic acid molecule.

  % Sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and henikoff and Henikoff, Pruc.Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, the two amino acid sequences consist of 10 gap initiation penalties, 1 gap extension penalty, and Henikoff and Henikoff as shown in Table 3 (amino acids are indicated by the standard single letter code) ) Using the “BLOSUM62” rating matrix to match that score to optimize it. The% identity is then calculated as follows: ([total number of identical matches] / [longer sequence length + in the longer sequence to match the two sequences Number of gaps introduced]) (100).

  One skilled in the art understands that there are many established algorithms for aligning two amino acid sequences. The Pearson and Lipman “FASTA” similarity search algorithm is suitable for testing the level of identity shared by one amino acid sequence disclosed herein and the amino acid sequence of a putative Zven1 or Zven2 variant. It is a simple protein alignment method. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85: 2444 (1988), and Pearson, Meth. Enzymol. 183: 63 (1990).

  Briefly, FASTA will first consider the highest density of identity (if the ktup variable is 1) or paired, without considering the sequence in question (eg, SEQ ID NO: 2) and conservative amino acid substitutions, insertions or deletions. Characterize the sequence by identifying regions shared by test sequences that have any of the identities (if ktup = 2). Next, the 10 regions with the highest density of identity are reevaluated by comparing the similarity of all paired amino acids using an amino acid substitution matrix and the end of the region is the highest “Arranged” to include only those residues that contribute to the score. If there are several regions with a score higher than the “cut-off” value (calculated by the expected formula based on sequence length and ktup value), the trimmed initial region is the region Are tested to determine if they can be combined to form an approximate in-line array with the gap.

  Finally, the Needleman-Wunsch algorithm (Needleman and winsch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J), where the highest score region of the two amino acid sequences allows for amino acid insertions and deletions. Appl. Math. 26: 787, 1974). Preferred parameters for FASTA analysis are: ktup = 1, gap opening penalty = 10, gap extension penalty = 1 and substitution matrix = BLOSUM62. These parameters can be introduced into the FASTA program by adjusting the score matrix file ("SMATRIX"), as described in Appendix 2 of Pearson, Meth. Eizzymol. 183: 63 (1990).

  FASTA can also be used to determine the sequence identity of nucleic acid molecules using ratios as disclosed above. For nucleotide sequence comparison, the ktup value can be 1-6, preferably 3-6, most preferably 3, with other parameters set above. Other parameters may be set as follows: gap opening penalty = 10 and gap extension penalty = 1.

  The invention encompasses nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes compared to the amino acid sequences disclosed herein. For example, a variant comprising one or more amino acid substitutions of SEQ ID NO: 2 or 5 is obtained, wherein an alkyl amino acid is substituted for an alkyl amino acid in the Zven1 or Zven2 amino acid sequence, and an aromatic amino acid is in the Zven1 or Zven2 amino acid Substituted for aromatic amino acids, sulfur containing amino acids substituted for sulfur containing amino acids in Zven1 or Zven2 amino acid sequences, substituted for hydroxy containing amino acids in hydroxy containing amino acids Zven1 or Zven2 amino acid sequences, acidic amino acids Zven1 Or substituted for an acidic amino acid in the Zven2 amino acid sequence, a basic amino acid substituted for a basic amino acid in the Zven1 or Zven2 amino acid sequence, or a dibasic monocarboxylic acid amino acid in the Zven1 or Zven2 amino acid sequence Monocarboxylic acid Substituted for mino acid.

  Among common amino acids, “conservative amino acid substitutions” are indicated by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and Tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

  The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments representing highly conserved regions of more than 500 groups of related proteins [Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89: 10915 (1992)]. Thus, the BLOSUM62 substitution frequency can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the invention. Although it is possible to design amino acid substitutions based solely on chemical properties (as discussed above), the term “conservative amino acid substitution” is represented by a BLOSUM62 value greater than −1. Mention substitution. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), more preferably conservative substitutions are at least 2 (eg, 2 or 3). Characterized by the BLOSUM62 value of

  A particular variant of Zven1 or Zven2 has at least 70%, at least 80%, at least 90%, at least 95% or more than 95% sequence identity to its corresponding amino acid sequence (ie, SEQ ID NO: 2 or 5) Wherein the variation in amino acid sequence is due to one or more amino acid substitutions.

  Conservative amino acid changes in the Zven1 and Zven2 genes can be introduced by substituting nucleotides for the nucleotides listed in any one of SEQ ID NOs: 1 and 4, respectively. Such “conservative amino acid” variants are obtained, for example, by oligonucleotide directed mutagenesis, linker scanning mutagenesis, mutagenesis using the polymerase chain reaction and similar methods (Ausubel (1995)). pages 8-10-8-22; and McPhrson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).

  The proteins of the present invention also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include trans-3-methylproline, 2,4-metaproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, Hydroxyethyl homocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-aza Includes phenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for introducing non-naturally occurring amino acid residues into proteins.

  For example, in vitro systems can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. For example, Rovertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Meth. Enzymol. 202: 301,1991; Chung et al., Science 259: 806-09, 1993; and Chung et al. ., Proc. Natl. Acad. Sci. USA 90: 10145-49, 1993.

  In the second method, translation is performed in Xenopus oocytes by microinjection of mutagenized mRNA and chemically aminoamylated suppressor-tRNA (Turcatti et al., J. Biol. Chem. 271: 1991-98, 1996). In a third method, E. coli cells are obtained in the absence of the natural amino acid (eg, phenylalanine) that is to be replaced and the desired non-naturally occurring amino acid (eg, 2-azaphenylalanine, 3-aza In the presence of phenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). Non-naturally occurring amino acids are introduced into proteins in place of their natural counterparts. See Koide et al., Biochem. 33: 7470-46, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be replaced by Zven amino acids.
Amino acid sequence analysis shows that Zven1 and Zven2 share several motifs. For example, one motif is “AVITGAC [DE] [KR] D” (SEQ ID NO: 8), where acceptable amino acids for a given position are shown in square brackets. This motif is present in Zven1 at amino acid residues 28-37 of SEQ ID NO: 2 and Zven2 at amino acid residues 20-29 of SEQ ID NO: 5. Another motif is “CHP [GL] [ST] [HR] KVPFFX [KR] RXHHTCPCLP” (SEQ ID NO: 9), where the acceptable amino acids for a given position are shown in square brackets, And “X” can be any amino acid residue. This motif is present in Zven1 at amino acid residues 68-90 of SEQ ID NO: 2 and Zven2 at amino acid residues 68-82 of SEQ ID NO: 5. The present invention includes peptides and polypeptides comprising these motifs.

  Sequence analysis also shows that Zven1 and Zven2 include various conservative amino acid substitutions with respect to each other. Thus, a particular Zven1 variant can be designed by modifying that sequence to include one or more amino acid substitutions that match the Zven2 sequence, and a particular Zven1 variant can be one or more that matches the Zven1 sequence. It can be designed by modifying the sequence to include multiple amino acid substitutions. Such variants can be constructed using Table 4 which provides typical conservative amino acid substitutions found in Zven1 and Zven2. Although Zven1 and Zven2 variants can be designed with any number of amino acid substitutions, some variants have at least about X amino acid substitutions (X is 2, 5, 7, 10, 12, 14, 16, 18 and Selected from the group consisting of 20).

  Essential amino acids in the polypeptides of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989). Bass et al., Proc. Natl. Scad. Sci. USA 88: 4498-502, 1991), Coombs and Corey, “Site-Directed Mutagenesis and Protein Engineering”, Proteins: Analysis and Design, Angeletti (ed.) Ppages 259-311 (Academic Press, Inc. 1998)). In the latter technique, a single alanine mutation is introduced at every residue in the molecule, and the resulting mutant molecule is used to identify amino acid residues that are critical to the activity of the molecule. Are tested for biological activity, eg, the ability to bind an antibody, as disclosed below. See also Hilton et al., J. Biol. Chem. 271: 4699-5708, 1996.

  The location of the Zven1 or Zven2 receptor binding domain is also determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, along with putative contact site amino acid mutations. It can also be determined by physical analysis. See, for example, de Ves, Science 255: 306 (1992), Smith, J. Mol. Biol. 224: 899 (1992), and Wlodaver, FEBS Lett. 309: 59 (1992). . Furthermore, Zven1 or Zven2 labeled with biotin or FITC can be used for expression cloning of Zven1 or Zven2.

  Multiple amino acid substitutions are known in mutagenesis and screening methods such as Reidhaar-Olson and Sauer (science 241: 53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989). And is tested using the method disclosed in In short, their authors suddenly randomize multiple positions in a polypeptide, screen functional polypeptides, and then determine the range of possible substitutions at each position. Disclosed are methods for sequencing mutagenized polypeptides. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 30: 10832-10837,1991; Ladner et al., US Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204), and regions Includes directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

  Variants of the disclosed Zven1 or Zven2 nucleotide and polypeptide sequences are described in Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51, 1994 and WIPO published WI97 / Can be generated through DNA shuffling as disclosed by 20078. Briefly, mutant DNA molecules are generated by in vitro homologous recombination by random fragmentation of the parent DNA, followed by assembly using PCR, resulting in randomly introduced point mutations. This technique can be improved with parental DNA families, such as allelic variants or DNA from different species, to introduce additional variability during the process. Further interaction of the desired activity selection or screening, mutagenesis and assay provides rapid “evolution” of the sequence by selecting for the desired mutation while simultaneously selecting for deleterious changes. To do.

  Mutagenesis methods as disclosed herein can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides in host cells. Mutagenized DNA molecules encoding biologically active polypeptides or polypeptides that bind to anti-Zven1 or anti-Zven2 antibodies are recovered from the host cells and immediately using modern equipment. Can be arranged. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide of interest and can be applied to polypeptides of unknown structure.

  The present invention also encompasses “functional fragments” of Zven1 or Zven2 polypeptides and molecules encoding such functional fragments. Normal deletion analysis of nucleic acid molecules can be performed to obtain functional fragments of nucleic acid molecules encoding Zven1 or Zven2 polypeptides. As illustrated, a DNA molecule having the nucleotide sequence of SEQ ID NO: 1 can be digested with Bal3 T nuclease to obtain a series of deletions. The fragment is then inserted into an expression vector with the correct reading frame aligned, and the expressed polypeptide is isolated and tested for cell-cell interactions or the ability to bind Zven antibodies. One method for exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions, or to use stop codons to identify the production of the desired fragment . On the other hand, specific fragments of the Zven gene can be synthesized using the polymerase chain reaction.

  Methods for identifying functional domains are well known to those skilled in the art. For example, studies based on cleavage at either or both ends of interferon are illustrated by being summarized by Horisberger and Di Marco, pharmac. Ther. 66: 507 (1995). In addition, standard techniques for functional analysis of proteins are e.g. Treluter et al., Molec. Gen. Genet. 240: 113 (1993), Content et al., “Expression and preliminary deletion analysisi of the 42 kDa 2-5A synthetase. induced by human interferon ”, in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), Pages 65-72 (Nijhoff 1987), Herschman,“ The EGF Enzyme ”, in Cortrol of Animal Cell Proliferation , Vol. 1, Boynton et al., (Eds.) Pages 169-199 (Academic Press 1985), Counailleau et al., J. Biol. Chem. 270: 29270 (1995); Fukunaga et al., J. Biol. Chem. 270: 25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50: 1295 (1995); and Meisel et al., Plant Molec. Biol. 30: 1 (1996).

  The present invention also contemplates functional fragments of the Zven1 or Zven2 gene having an amino sequence change compared to the amino sequence of SEQ ID NO: 2 or 5. Mutant Zven genes can be identified on the basis of the structure by determining the level of identity with the disclosed nucleotide and amino acid sequences, as described above. Another approach to identify mutant genes based on the structure is that the nucleic acid molecule encoding the potential mutant Zven1 or Zven2 gene is, as discussed above, SEQ ID NO: 1 or Determining whether it can hybridize to a nucleic acid molecule having a nucleotide sequence of 4.

  The invention also provides a polypeptide fragment or peptide comprising an epitope-bearing portion of a Zven1 or Zven2 polypeptide described herein. Such a fragment or peptide comprises an "immunogenic epitope that is part of a protein that elicits an antibody response when the complete protein is used as an immunogen. An immunogenic epitope-bearing peptide is Can be identified using standard methods (see, eg, Geysen et al., Proc. Natl. Acad Sci. USA 81: 3988, 1983).

  In contrast, a polypeptide fragment or peptide comprises an “antigenic epitope”, a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous range of amino acids, and the antigenicity of such epitopes is not destroyed by denaturing agents. It is known in the art that relatively short synthetic peptides that mimic protein epitopes can be used to stimulate the production of antibodies to proteins (see, eg, Sutcliffe et al., Science 219: 660, 1983). ) Accordingly, the antigenic epitope-bearing peptides and polypeptides of the present invention are useful for raising antibodies that bind to the polypeptides described herein.

  Antigenic epitope-bearing peptides and polypeptides preferably comprise at least 4 to 10 amino acids, at least 10 to 15 amino acids, or about 15 to about 30 amino acids of the amino acid sequence of SEQ ID NO: 2 or 5. Such epitope-bearing peptides and polypeptides can be generated by fragmentation of Zven1 or Zven2 polypeptides or chemical peptide synthesis, as described herein. In addition, epitopes can be selected by phage display of random peptide libraries (eg, Lane and Stephen, Curr. Opin. Immunol. 5: 268, 1993, and Cortese et al., Curr. Opin. Biotechnol. 7: 616, (See 1996).

  Standard methods for identifying epitopes and generating antibodies from small peptides comprising the epitope are, for example, Mole, “Epitope Mapping,” in Methods in Molecular Biology, Vol. 10, Manson (ed.), Pages 105-16 (The Humana Press, Inc., 1992), Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), Page 60 -84 (Cambridge University Press 1995), and Coligan et al. (Eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons, 1997) Is done.

  Regardless of the specific nucleotide sequence of the mutant Zven1 or Zven2 gene, the gene encodes a polypeptide that can be characterized by its ability to specifically bind to anti-Zven1 or anti-Zven2 antibodies.

  In addition to the above uses, the polynucleotides and polypeptides of the invention are useful as educational tools in laboratory implementation kits for processes related to genetics and molecular biology, protein chemistry and antibody production and analysis. . Because of its unique polynucleotide and polypeptide sequences, the Zven1 or Zven2 molecule can be used as a standard for testing purposes or as “unknown”.

  For example, Zven1 or Zven2 polynucleotides can be used to prepare students for expression constructs for bacterial, viral or mammalian expression, including fusion constructs, where Zven1 or Zven2 is the gene to be expressed. For teaching; for determining restriction endonuclease degradation sites of polynucleotides; for determining mRNA and DNA localization of Zven1 or Zven2 polynucleotides in tissues (ie, Northern and Southern blots, and polymerase chain reaction) And) as an aid for the identification of related polynucleotides and polypeptides by nucleic acid hybridization. By way of example, a student provides two fragments of about 123 base pairs and 201 base pairs of PstII digestion of a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 66-389 of SEQ ID NO: 1 and about 46 base pairs of HaeIII digestion. And will generate 278 base pair fragments.

  Zven1 or Zven2 polypeptide is expressed for teaching of antibody preparation; for protein identification by Western blot; for protein purification; expressed Zven1 or Zven2 polypeptide as a ratio to total protein expressed For determining the weight of peptides; for identifying peptide degradation sites; for coupling amino and carboxyl terminal labels; for amino acid sequence analysis; and biological activity of both native and labeled proteins It can be used as an aid for in vitro and in vivo monitoring of (ie protease inhibition). For example, a student may generate five fragments with an approximate molecular weight of 148, 4337, 1909, 2402, and 2939 by digestion of unglycosylated Zven1 with the endopeptidase Lys C, and glycosylation with BNPS or NCS / urea It will be found that digestion of unvenated Zven1 produces fragments with approximate molecular weights of 5231 and 6444.

  Zven1 or Zven2 polypeptides can also be used for analytical skills, eg, mass spectrometry, circular dichroism to determine the conformation of the four alpha helices, and atomically detailed conformation. X-ray crystallography can also be used to teach nuclear magnetic resonance spectroscopy to represent the structure of proteins in solution. For example, a kit containing Zven1 or Zven2 can be given to the student to analyze. Since the amino acid sequence is known by the professor, ie the protein is given to the student as a test to determine or develop the student's skill, the professor You will know if you have analyzed or not. Since every polypeptide is unique, the educational availability of Zven1 or Zven2 will be unique in itself.

  An antibody that specifically binds to Zven1 or Zven2 encodes the antibody as a teaching aid to teach students how to prepare an affinity chromatography column to purify Zven1 or Zven2. Can be used as teaching aids for cloning and sequencing polynucleotides, and therefore as an exercise in teaching students how to design humanized antibodies. The Zven1 or Zven2 gene, polypeptide or antibody is packaged by a reagent company and marketed to educational institutions for students to gain molecular biology skills. Because individual genes and proteins are unique, individual genes and proteins create a unique challenge and learning experience for students in the experimental training section. Such educational kits comprising a Zven1 or Zven2 gene polypeptide or antibody are considered to be within the scope of the present invention.

  For any Zven polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily use the information shown in Tables 1 and 2 above to generate a sufficient degenerate polynucleotide sequence encoding the variant. Can be generated. In addition, one skilled in the art can use standard software for various Zven1 or Zven2 variants based on the nucleotide and amino acid sequences described herein. Accordingly, the present invention encompasses a computer readable medium encoded by a data structure providing at least one of the following sequences: SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

  Suitable forms of computer readable media include magnetic media and optically readable media. Examples of magnetic media include hard or fixed drives, random access storage (RAM) chips, floppy disks, digital linear tape (DLT), disk curses and ZIP disks. Optically readable media include compact discs (eg, CD—read only (ROM), CD—rewritable (RW) and CD—rerecordable), and digitally rollable / video discs (DVDs) ( For example, it is exemplified by DVD-ROM, DVD-RAM, and DVD + RW.

5. Generation of Zven fusion proteins:
The Zven fusion protein can be used to express a Zven polypeptide or peptide in a recombinant host and to isolate the expressed Zven polypeptide or peptide. One type of fusion protein comprises a peptide that guides a Zven polypeptide from a recombinant host cell. In order to direct a Zven polypeptide into the eukaryotic secretory pathway, a secretory signal sequence (also known as a signal peptide, leader sequence, prepro sequence or presequence) is provided in a Zven expression vector. The The secretory signal sequence is derived from Zven1 or Zven2, but a suitable signal sequence can also be derived from another secreted protein or synthesized de novo.

  The secretory signal sequence is operably linked to the Zven1 or Zven2-coding sequence, ie the two sequences are correctly aligned in reading frame and direct the newly synthesized polynucleotide into the secretory pathway of the host cell. Placed in. The secretory signal sequence is usually located 5 ′ to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences can be located elsewhere in the nucleotide sequence of interest (eg, Welch Etc., see US Pat. No. 5,037,743; Holland et al., US Pat. No. 5,143,830).

Zven1 or Zven2, or another protein produced by a mammalian cell (eg, a tissue type plasminogen activator signal sequence as described in US Pat. No. 5,641,655) can be expressed in a recombinant mammalian host as Zven1 or Although useful for expression of Zven2, the yeast signal sequence is useful for expression in yeast cells. Examples of suitable yeast signal sequences are those derived from yeast pairing events α-factor (encoded by MFα1 gene), invertase (encoded by SUC2 gene) or acid phosphatase (encoded by PHO gene) belongs to. For example, Romanos such as, "Expression of Cloned Genes in Yeast " in DNA Cloning 2:. (. Eds) A Practical Approach, 2 nd Edition, Glover and Hames, see p.123-167 (Oxford University Press 1995) .

  In bacterial cells, it is often desirable to express heterologous proteins as fusion proteins in order to reduce toxicity, increase stability, and increase the recoverability of the expressed protein. For example, Zven1 or Zven2 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide. Glutathione S-transferase fusion proteins are typically soluble and can be easily purified from E. coli lysates on immobilized glutathione columns. In a similar approach, a Zven1 or Zven2 fusion protein comprising a maltose binding protein polypeptide can be isolated by an amylose resin column, and a fusion protein comprising the C-terminus of the cleaved protein A gene is: It can be purified using IgG-Sepharose.

Established techniques for expressing heterologous polypeptides as fusion proteins in bacterial cells are Williams et al., “Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies”, in DNA Cloning 2: ^{A Practical Approach, 2 nd Edition,} Glover and Hames (Eds.), described by p.15-58 (Oxford University Press 1995). In addition, commercially available expression systems can be used. For example, the PINPOINT Xa protein purification system (Promega Corporation, Madison, Wis.) Provides a method for isolating a fusion protein comprising a polypeptide that is biotinylated during expression by a resin comprising avidin. To do.

  Peptide labels that are useful for isolating heterologous polypeptides expressed by prokaryotic or eukaryotic cells include polyhistidine labels (with affinity for nickel-chelating resins), c-myc labels, calmodulin binding proteins ( (Isolated by calmodulin affinity chromatography), substance P, RYIRS label (binding to anti-RYIRS antibody), Gln-Gln label and FLAG label (binding to anti-FLG antibody). See, for example, Luo et al., Srch. Biochem. Biophys. 329: 215 (1996), Morganti et al., Biotechnol. Appl. Biochem. 23: 67 (1996), and Zheng et al., Gene 186: 55 (1997). That. Nucleic acid molecules encoding such peptide labels are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO).

  Another form of fusion protein is a Zven1 or Zven2 polypeptide, and an immunoglobulin heavy chain constant region, a typical Fc fragment containing two or three multiregion domains and a hinge region but lacking the variable region Comprising. By way of example, Chang et al., (US Pat. No. 5,723,125) describe a fusion protein comprising human interferon and a human immunoglobulin Fc fragment. The C-terminus of interferon is linked to the N-terminus of the Fc fragment by a peptide linker component. An example of a peptide linker is a peptide that mainly comprises an immunologically inactive T cell inactive sequence. A typical peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO: 7).

  In fusion proteins, the preferred Fc component is a human gamma chain that is stable in solution and has little or no complement that activates activity. Accordingly, the present invention contemplates a Zven fusion protein comprising a Zven1 or Zven2 polypeptide component and a human Fc fragment, wherein the C-terminus of the Zven polypeptide component is derived from a peptide linker, eg, the amino acid sequence of SEQ ID NO: 7. Is attached to the N-terminus of the Fc fragment through the peptide consisting of

In another embodiment, the Zven1 or Zven2 fusion protein comprises an IgG sequence, a Zven polypeptide component covalently linked to the amino acid terminus of the IgG sequence, and a signal peptide covalently linked to the amino acid terminus of the Zven polypeptide component. made, wherein the IgG sequence consists of the following elements in the following order: CH ₂ domain and CH ₃ domain. Thus, the IgG sequence lacks the CH ₁ domain. A Zven polypeptide component exhibits Zven1 or Zven2 activity, eg, the ability to bind to a Zven1 or Zven2 receptor. This general approach for generating fusion proteins comprising both antibody and non-antibody portions is described by LaRochelle et al., European Patent No. 742830 (WO95 / 21258).

  A fusion protein comprising a Zven1 or Zven2 polypeptide component and an Fc component can be used as an in vitro assay tool. For example, the presence of a Zven1 or Zven2 receptor in a biological sample can be detected using a Zven1 or Zven2-antibody fusion protein, wherein the Zven component is a cognate receptor and a macromolecule such as protein A or Used to bind anti-Fc antibody, ie, used to detect bound fusion protein-ligand complex. In addition, antibody-Zven fusion proteins comprising antibody variable domains are useful as therapeutic proteins where the antibody component binds to a target antigen, such as a tumor associated antigen.

  Fusion proteins can be prepared by methods known to those skilled in the art by preparing the individual components of the fusion protein and chemically conjugating them. On the other hand, polynucleotides that correctly align the open reading frame and encode both components of the fusion protein can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical degradation of fusion proteins are described, for example, by Ausubel (1995), p. 16-19-16-25.

6). Production of Zven polypeptide:
The polypeptides of the invention, including full length polypeptides, functional fragments and fusion proteins, can be produced in recombinant host cells according to conventional techniques. In order to express a Zven1 or Zven2 gene, a nucleic acid molecule encoding a polypeptide should control transcriptional expression in the expressed peptide and then be operably linked to regulatory sequences introduced into the host cell. It is. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and marker genes suitable for selection of cells that carry the expression vector.

  Expression vectors that are suitable for production of foreign proteins in eukaryotic cells typically include (1) bacterial origins of replication and antibiotic resistance markers to provide for the propagation and selection of expression vectors in cellular hosts. A coding eukaryotic DNA element; (2) a eukaryotic DNA element that controls the initiation of transcription, such as a promoter; and (3) a DNA element that controls the processing of the transcript, such as a transcription termination / polyadenylation sequence. As discussed above, the expression vector also includes a nucleotide sequence that encodes a secretory sequence that directs the heterologous polypeptide into the secretory pathway of the host cell. For example, a Zven1 expression vector can include a Zven1 gene and a secretory sequence derived from the Zven1 gene or another secreted gene.

  The Zven1 or Zven2 protein of the present invention can be expressed in mammalian cells. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL1587), human embryonic kidney cells (293-HEK; ATCC CRL1573), child hamster kidney cells (BHK-21, BHK-570; ATCC CRL8544, ATCC CRL10314), canine kidney cells (MDCK; ATCC CCL34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al., Som. Cell. Molec. Genet. 12: 555 (1986)], rat Pituitary cells (CH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL1658).

  For mammalian hosts, transcriptional and translational signals can be derived from viral sources such as adenovirus, bovine papilloma virus, simian virus or the like, where the modulating signal is a specific signal with a high level of expression. It is related to genes. Suitable transcriptional and translational regulatory sequences are also obtained from mammalian genes such as actin, collagen, mycosin and metallothionein genes.

  Transcriptional regulatory sequences contain sufficient promoter regions to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the mouse metallothionein I gene promoter (Hamer et al., J. Molec. Appl. Genet. 1: 273 (1982)), the herpesvirus TK promoter (Mcknight, Cell 31: 355 (1982)). , SV40 early promoter (Benoist et al., Nature 290: 304 (1981)), Rous sarcoma virus promoter (Gorman et al., Proc. Natl. Acad. Sci. USA 79: 6777 (1982)), cytomegalovirus promoter (Foecking Gene 45: 101 (1980)) and mouse mammary tumor virus promoter (generally Etcheverry, “Expression of Engineered Protein in Mammalian Cell Culture”, in Protein Engineering: Principles and Practice, Cleland et al., (Eds .), p. 163-181 (see John Wiley & Sons, Inc. 1996)).

  On the other hand, prokaryotic promoters, such as bacteriophage T3 RNA polymerase promoter, can be used to control Zven1 or Zven2 gene expression in mammalian cells when the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10: 4529 (1990), and Kaukaman et al., Nucl. Acids Res. 19: 4485 (1991)).

  Expression vectors can be introduced into host cells using a variety of standard techniques, such as calcium phosphate transfection, liposome-mediated transfection, microproject-mediated delivery, electroporation and the like. Preferably, transfected cells are selected and propagated to provide a recombinant host cell comprising an expression vector that is stably integrated into the host cell genome. Techniques for introducing vectors into eukaryotic cells, and techniques for selecting such stable transformants using dominant selectable markers are described in, for example, Ausubel (1995) and Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

  For example, one suitable selectable marker is a gene that confers resistance to the antibiotic neomycin. In this case, the selection is performed in the presence of a neomycin type drug, such as G-418 or the like. A selection system, a method referred to as “amplification”, is also used to increase the expression level of a gene of interest. Amplification involves culturing transfectants in the presence of low levels of selective agent and then increasing the amount of selective agent to select cells that produce high levels of the product of the introduced gene. Implemented by:

  A preferred amplifiable selectable marker is dihydrofolate reductase that confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Other markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, class I MHC, placental alkaline phosphatase, can be transduced by means such as FACS classification or magnetic bead separation techniques. It can be used to classify uninfected cells and transfected cells.

  Zven1 or Zven2 polypeptides can also be produced by cultured mammalian cells using a viral delivery system. Typical viruses for this purpose include adenovirus, herpes virus, retrovirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, or double-stranded DNA virus, is currently the most studied gene transfer vector for the supply of heterologous nucleic acids (Becker et al., Meth. Cell Bio. 43: 161-89, 1994; and JT Douglas and DT Curiel, Science & Medicine 4: 44-53, 1997). The advantages of the adenovirus system allow for the accommodation of relatively large DNA inserts, the ability to grow to high titers, the ability to infect a wide range of mammalian cell types, and the use of numerous available vectors containing different promoters. Includes flexibility to do.

  By deleting portions of the adenovirus genome, large inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be introduced into the viral DNA by direct ligation or by homologous recombination with the transfected plasmid. If the EI gene is not supplied by the host cell, it is optional to delete the essential EI gene from the viral vector resulting in replication incompetence. For example, adenoviral vector-infected human 293 cells (ATCC No. CRL-1573, 45504, 45505) are used as adherent cells or as suspension cultures at relatively high cell densities to produce significant amounts of protein. (See Garnier et al., Cytotechnol. 15: 145 (1994)).

  The Zven1 or Zven2 gene can also be expressed in other higher eukaryotic cells such as birds, fungi, insects, yeast, or plant cells. The baculovirus system provides an effective means for introducing cloned Zven1 or Zven2 genes into insect cells. Suitable expression vectors are based on the Autographa californica nuclear polyhedrosis virus (AcMNPV) and are well known promoters such as the Drosophila heat shock protein (hsp) 70 promoter, Autographa californica. Carnonica nuclear polyhedrosis virus immediate-early gene promoter (ie-I) and delayed early 39K promoter, baculovirus p10 promoter and Drosophila metallothionein promoter.

A second method for producing recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, VA, et al., J. Virol 67: 4566-79, 1993). This system utilizing transfer vectors is commercially available as a Bac-to-Bac ^™ kit (Life Technologies, Rockville, MD). This system is a large plasmid called “bacmid”, a transfer vector containing the Tn7 transposon, pFastBacI ^™ (Life Technologies). Hill-Perkins, MS and Possee, RD, J. Gen. Virol. 71: 971-6, 1990; Bonning, BC, etc., J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk, GD, and See Rapoport, B., J. Biol Chem. 270: 1543-9,1995.

  In addition, the transfer vector can include an epitope tag at the C- or N-terminus of the expressed Zven polypeptide, eg, an in-frame fusion with DNA encoding a Glu-Glu epitope tag (Grussenmeyer, T. et al. , Peoc. Natl. Acad. Sci. 82: 7952-6, 1985). Using techniques known in the art, E. coli is transformed with a transfer vector containing a Zven1 or Zven2 gene and screened for bacmida containing an intermittent lacZ gene that is representative of a recombinant baculovirus. . Bacmid DNA containing the recombinant baculovirus genome is isolated using conventional techniques.

  Exemplary PFASTBAC vectors can be modified to a considerable degree. For example, the polyhydrin promoter has been removed and expressed early in baculovirus infection, and a baculovirus basic protein promoter known to be advantageous for expressing secreted proteins (also (Also known as Pcor, p6.9 or MP promoter). Hill-Perkins, MS and Possee, RD, J. Gen. Virol. 71: 971-6, 1990; Bonning, BC, etc., J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk, GD, and See Rapoport, B., J. Biol Chem. 270: 1543-9,1995.

  In such transfer vector constructs, short or long versions of the basic protein promoter may be used. In addition, transfer vectors can be constructed in which the native Zven1 / Zven2 secretion signal sequence is replaced by a secretion signal sequence derived from an insect protein. For example, signal sequences from ecdysteroid glucosyltransferase (EGT), bee Melittin (Invitrogen, Carlsbad, Calif.) Or baculovirus gp67 (PharMingem, San Diego, Calif.) Replace the native Zven1 / Zven2 secretion signal sequence. Can be used in the structure.

  Recombinant virus or bacmid is used to transfect host cells. Suitable insect host cells include cell lines derived from IPLB-Sf-21, Spodoptera frugiperda pupa ovary cell lines such as Sf9 (ATCC CRL 1711), Sf21AE and Sf21 (Invitrogen Corporation; Sanvi Diego, CA), and Drosophila Schneider-2 cells, as well as the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (US Pat. No. 5,300,435).

Commercial serum free media can be used for cell growth and maintenance. Suitable media are Sf900II ^™ (Life Technologies) or ESF921 ^™ (Expression Systems) for Sf9 cells; and Ex-cellO405 ^™ (JRH Biosiences, Lenexa, KS) or Express FiveO ^™ (for Trichopulcia ni cells). Life Technologies). When recombinant viruses are used, the cells are typically about 2 at the point where the recombinant virus stock is added at 0.1-10, more typically about 3 multiplicity of infection (MOI). Catalyzed at an inoculation density of ˜5 × 10 ⁵ cells and 1-2 × 10 ⁶ cells.

Established techniques for producing recombinant proteins in baculovirus are Bailey et al., “Manipulation of Baculovirus Vectors”, in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), P. 147-168 (the Humana Press, (by nc 1991), such as Patel, "the buculovirus expression system" , in DNA Cloing2:.... Expression Systems, 2 nd Edition, such as Glover, (eds) p.205 -244 (Oxford University Press 1995), Ausubel (1995) p.16-37-16-57, Richardson (ed.), Baculovirus Expression Proteocols (The Humana Press, Inc. 1995), and Lucknow, “Insect Cell Expression Technology ”, in Protein Engineering: Principles and Practice. Cleland et al. (Eds.), P.183-218 (John Wiley & Sons, Inc. 1996).

  Fungal cells, such as yeast cells, can also be used to express the genes disclosed herein. In this regard, yeast species of particular interest include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerin kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase) and the like Include.

  Methods for transforming S. cerevisiae cells with exogenous DNA and generating recombinant polypeptides therefrom, for example, Kawasaki, US Pat. No. 4,599,311; Kawasaki et al., US Pat. No. 4,931,373; Brake, US Patent No. 4,870,008; Welch et al., US Pat. No. 5,037,743; and Murray et al., US Pat. No. 4,845,075. Transformed cells are selected by a phenotype determined by a selectable marker, usually drug resistance, or the ability to grow in the absence of a particular nutrient (eg, leucine).

  A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (US Pat. No. 4,931,373), which allows selection of cells transformed by growth in glucose-containing media. is there. Suitable promoters and terminators for use in yeast can be found in glycolytic enzyme genes (see, eg, Kawasaki, US Pat. No. 4,599,311; Kingsman et al., US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092). And alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936; and 4,661,454.

  Other enzymes, such as Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis til, D Transformation systems for (Pichia pastoris), Pichia methanolica, Pichia guillermondii, and Candida maltosa are known in the art.

  See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cregg, US Pat. No. 4,882,279. Aspergillus cells can be used according to the method of Mcknight et al., US Pat. No. 4,935,349. A method for transforming Acremonium chrysogenum is disclosed by Sumino., US Pat. No. 5,162,228. A method for transforming Neurospora is disclosed by Lambowitz, US Pat. No. 4,486,533.

  For example, the use of Pichia methanolica as a host for the production of recombinant proteins is described in Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al., Yeast 14: 11-23 (1998), And WIPO publications WO97 / 17450, WO97 / 17451, WO98 / 02536 and WO98 / 02565. DNA molecules for use in transformation of P. methanolica will normally be prepared as double-stranded circular plasmids, preferably linearized, prior to transformation. For the production of polypeptides in P. methanolica, the promoter and terminator in the plasmid are preferably those of the P. methanolica gene, such as the P. methanolica alcohol utilization gene (AUG1 or AUG2).

  Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. In order to promote the integration of the DNA into the host chromosome, it is preferable to have a complete expression segment of the plasmid having the host DNA sequence at both ends. A preferred selectable marker for use in Pichia methanolica is P. methanolica encoding phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC. 4.1.1.21) that allows the growth of ade2 host cells in the absence of adenine. ADE2 gene. For large-scale industrial processes where it is desirable to minimize the use of methanol, it is preferable to use host cells that are deficient in both methanol utilization genes (AUG1 and AUG2).

  For the production of secreted proteins, host cells that lack the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of plasmids containing DNA encoding the polypeptide of interest into P. methanolica cells. An exponentially decaying, pulsed with an electric field strength of 2.5-4.5 kV / cm, preferably about 3.75 kV / cm, and a time constant (t) of 1-40 ms, most preferably about 20 ms Preferably, P. methanolica cells are transformed by electroporation using an electric field.

  Expression vectors can also be introduced into plant protoplasts, intact plant tissue, or isolated plant cells. Methods for introducing expression vectors into plant tissues include direct infection of plant tissues by Agrobacterium tumefaciens, microprojectile-mediated feeding, DNA injection, electroporation and the like. Or co-culture of plant cells with them. For example, Horsch et al., Science 227: 1229 (1995), Klein et al., Biotechnology 10: 268 (1992) and Miki et al., “Procedures for Intoducing Foreign DNA into Plants”, in Methods in Plant Molecular Biology and Biotechnology, Glick (Eds.), P. 67-88 (CRC Press, 1993).

On the other hand, the Zven gene can be expressed in prokaryotic host cells. Suitable promoters which may be used to express the Zven1 or Zven2 polypeptide in prokaryotic cells, are well known to those skilled in the art, and T4, T3, promoter capable of recognizing the Sp6 and T7 polymerases, P _R of the bacteriophage λ And P _L promoter, E. coli trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA and lacZ promoters, B. subtilis promoter, Bacillus bacteriophage promoter, Streptomyces promoter, bacteriophage λ Int promoter, bla promoter of pBR322 and CAT promoter of chloramphenicol acetyltransferase gene are included. Prokaryotic promoter, Glick, J. Ind Microbiol 1: ..... 277 (1987), such as Watson, molecular Biology of the Gene, 4 th Ed (Benjamin Cummins 1987), and Ausubel, such as to be reconsidered by the (1995) Yes.

  Suitable prokaryotic hosts include E. coli and Bacillus subtilis. Suitable strains of E. coli are BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4, DH5, DH51, DH51F, DH51MCR, DH 10B, DH10B / p3, DH11S, C600, HB101 , JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451 and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilis include BR151, YB886, MI119, MI120 and BI70 (eg, Hardy, “Bacillus Cloning Methods”, in DNA Cloning: A Practical Approach, Clover (ed.) (IRL Press 1985) checking ...

  When expressing a Zven polypeptide in bacteria, such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by bacterial secretion sequences. In the former case, the cells are lysed and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The modified polypeptide is then regenerated and dimerized by diluting the variant, for example, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione followed by dialysis against a buffered solution. Can be In the latter case, the polypeptide is soluble and functional from the periplasmic space by destroying the cells to release the contents of the periplasmic space (eg, by sonication or osmotic shock) and recovering the protein. Recovered in the form, thereby avoiding the need for denaturation and regeneration.

Methods for expressing proteins in prokaryotic hosts are well known to those skilled in the art (eg, Williams et al., “Expression of foreign protein in E. coli using plasmid vectors and purification of specific polyclonal antibodies” in DNA Cloning 2 :. (. eds) Expression Systems , 2 nd Edition, such as Glover, P.15 (Oxford University Press 1995 ), Ward , etc., "Genetic Manipulation and Expression of Antibodies " in Monoclonal Antibodies:.. Principles and Applications, p 137 ( Wiley-Liss, Inc. 1995), and Georgiou, “Expression of Proteins in Bacteria”, in Protein Engineering: Principles and Practice; Cleland et al. (Eds.), P101 (John Wiley & Sons, Inc. 1996), and Rudolph , "Successful Refolding on an Industrial Scale", Chapter 10.)

Standard methods for introducing expression vectors into bacterial, yeast, insect and plant cells are provided by Ausubel (1995).
General methods for expressing and recovering foreign proteins produced by mammalian cell systems are, for example, Etcheverry, “Expression of Engineered Proteins in Mammalian Cell Culture“ in Protein Englineering: Principles and Practice, Cleland, etc. .), p163 (Wiley-Liss, Inc. 1996). Standard techniques for recovering protein produced by a bacterial system, such as, for example, Grisshammer, "Purification of Over-Produced proteins from E.coli cells" in DNA Cloning 2:. Expression Systems, 2 nd Edition, like Glover (. eds.), p.59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from baculovirus systems are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995).

On the other hand, the polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or conventional solution synthesis. Methods for their synthesis are well known to those skilled in the art (eg, Merrifield, J. Am. Chem. Soc. 85: 2149 (1963), Stewart et al., “Solid Phase Peptide Synthesis” (2 ^nd Edition)). , (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. 3.3 (1986). Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989). Fields and Colowick, “Solid-Phase Peptide Synthesis ”Methods in Enzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997)).

  Variability in overall chemical synthesis methods such as “natural chemical ligation” and “expressed protein ligation” is also standard (eg, Dawson et al., Science 266: 776 (1994), Hackeng et al., Proc. Natl. Acad. Sci. USA 94: 7845 (1997), Dawson, Methods Enzymol. 287: 34 (1997), Muir et al., Proc. Natl. Acad. Sci. USA 95: 6705 (1998), and Severinov and Muir , J. Biol. Chem. 273: 16205 (1998)).

  The peptides and polypeptides of the invention comprise at least 6, at least 9, or at least 15 consecutive amino acid residues of SEQ ID NO: 2. An exemplary polypeptide of Zven2 comprises 15 consecutive amino acid residues from amino acids 82-105 of SEQ ID NO: 5. A typical polypeptide of Zven2 comprises 15 consecutive amino acid residues of amino acids 1-32 or amino acids 75-108 of SEQ ID NO: 2, and a typical Zven2 polypeptide comprises amino acids 82- Including 105. In one embodiment of the invention, the polypeptide comprises 20, 30, 40, 50, 75 or more consecutive residues of SEQ ID NO: 2 or 5. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.

Examples for the generation of Zven1 are shown in Examples 9, 10, 11 and 12.
The present invention contemplates a composition comprising a peptide or polypeptide described herein. Such compositions can further comprise a carrier. The carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer solutions, alcohol, propylene glycol, macrogels, sesame oil, corn oil and the like.

7). Isolation of Zven polypeptide:
The polypeptides of the present invention are at least about 80% pure, at least about 90% pure, at least about 95% pure, or more than 95% pure relative to contaminating macromolecules, particularly other proteins and nucleic acids. It is preferred to purify and have no infectious and pyrogenic agents. The polypeptides of the present invention can also be purified to a pharmaceutically pure state that is 99.9% or more pure. In certain formulations, the purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

  Fractionation and / or conventional purification methods include Zven1 or Zven2 purified from natural sources (eg, lymph node tissue), and preparations of recombinant Zven polypeptides and fusion Zven polypeptides purified from recombinant host cells. Can be used to get. In general, ammonium sulfate precipitation and acid or chaotropic agent extraction are used for sample fractionation. Typical purification steps include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextran, agarose, cellulose, polyacrylamide, special silica and the like. PEI, DEAE, QAE and Q derivatives are preferred.

  Typical chromatographic media are those derivatized with phenyl, butyl or octyl groups such as phenyl-Sepharose FF (pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), octyl-Sepharrose (Pharmacia) and Or the like; or polyacrylic resins such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports are glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are used. Including. These supports can be modified from reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties.

  Examples of coupling chemicals include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemicals. These and other solid media are well known in the art and are widely used and available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is routine and is determined in part by the nature of the selected support. See, for example, Affinity Chromatograpy: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988, and Doonan, Protein Purification Protocols (The Humana Press 1996).

  Additional variations in Zven isolation and purification can be adjusted by those skilled in the art. For example, anti-Zven antibodies obtained as described below can be used to isolate large amounts of protein by immunoaffinity purification.

  The polypeptides of the present invention can be isolated from a combination of methods including anion and cation exchange chromatography, size exclusion, and affinity chromatography. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify proteins rich in histidine and those proteins comprising polyhistidine labels. Briefly, the gel is first charged with a divalent metal ion to form a chelate (Sulkowski, Trends in Biochem. 3: 1-7, 1985).

  Proteins rich in histidine are adsorbed to this matrix with different affinities, depending on the metal ion used and can be eluted by competitive elution, reduced pH, or the use of strong chelating agents. I will. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (M. Deutscher, (ed.), Methods Enzymol. 182, 529 (1990)). In a further aspect of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, maltose-binding protein, FLAG tag, Glu-Gku tag, immunoglobulin domain) can be configured to facilitate purification. .

  Zven polypeptides or fragments thereof can also be prepared through chemical synthesis as described below. Zven polypeptides can be monomeric or multimeric; may be glycosylated or non-glycosylated; may be PEGylated or non-PEGylated; and contains an initial methionine amino acid residue May or may not be included.

8). Zven analogs:
As described above, the disclosed polypeptides can be used to construct Zven variants. These polypeptides can be used to identify Zven1 or Zven2 analogs. One type of Zven analog resembles Zven by binding to the Zven receptor. Such analogs are considered Zven agonists when binding of the analog to a Zven receptor stimulates the response by a cell that expresses the receptor. On the other hand, Zven analogs that bind to the Zven receptor but do not stimulate cellular receptors can be Zven antagonists. Such antagonists can reduce Zven or Zven agonist activity, for example, by competitive or non-competitive binding of the antagonist to the Zven receptor.

  One general type of Zven analog is an agonist or antagonist having an amino acid having at least one mutation, deletion (amino- or carboxyl-terminus), or substitution of the amino acid sequences disclosed herein. is there. Another general type of Zven analog is provided by anti-idiotype antibodies or fragments thereof, as described below. In addition, recombinant antibodies comprising anti-idiotype variable domains can be used as analogs (see, eg, Monfardini et al., Proc. Assoc. Ani. Physicians 108: 420 (1996)). Since the variable domains of anti-idiotype Zven antibodies are similar to Zven, those domains can provide either Zven agonist or antagonist activity. By way of example, Langer, J. Interferon Res. 13: 295 (1993) describes anti-idiotype interferon-α antibodies having either interferon-α agonist or antagonist properties.

  A third approach for identifying Zven1 or Zven2 analogs is provided by the use of binding libraries. Methods for constructing and screening phage display and other binding libraries are described, for example, by Kay et al., Phage Display of Peptides and Proteins (Academic Press 1996), Verdine, US Patent No. 5, 783, 384, Kay , Etc. Provided by US Pat. No. 5,747,334, and KauffAman et al., US Pat. No. 5,723,323.

  The activity of a Zven polypeptide, agonist, or antagonist can be determined using standard cell proliferation or differentiation assays. For example, assays that measure proliferation include chemosensitivity to neutral red pigments, incorporation of radiolabeled nucleotides, incorporation of 5-bromo-2'-deoxyuridine in proliferating cell DNA, and the use of tetrazolium salts (Mosmann, J. Inzfnaunol. Metlzods 65: 55 (1983); Porstmann et al., J. Imrz2uuol. Methods 82: 169 (1985); Alley et al., Cancer Res. 48: 589 (1988); Cook et al., Analytical Biochem. 179 : 1 (1989); Marshall, etc., Growth Reg. 5: 69 (1995); Scudiero, etc., Cancer Res. 48: 4827 (1988); Cavanaugh, etc., loivestigational New Drugs 8: 347 (1990)) Various assays.

  For example, assays that measure differentiation include measurement of cell-surface markers associated with stage-specific expression of tissue, enzyme activity, functional activity or morphological changes (Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, pages 161-171 (1989); Watt, FASEB, 5: 281 (1991); Francis, Differentiation 57:63 (1994)). The assay can be used to measure other cellular responses including chemotaxis, adhesion, changes in ion channel influx, modulation of second messenger levels and neurotransmitter release. Such assays are well known in the art (see, eg, Chayen and Bitensky, Cytochemical Bioassays: Tec1miques & Applications (Marcel Dekker 1983)).

The effects of mutant Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, mutants and / or chimeras thereof, also observe the contractility of tissues, such as gastrointestinal tissue, by a tensiometer that measures contraction and relaxation in the tissues (Eg, Dainty et al., J. Pharmacol. 100: 767, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, Endocrinol. 114: 364-8, 1984; and Downing and Sherwood, Endocrinol. 116; 1206-14, 1985). For example, the measurement of aortic ring vasodilation is well known in the art. Illustratively, aortic rings were taken from 4 month old Sprague Dawley rats and buffered solutions such as denatured Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO ₄ .7H ₂ O, 1.2 mM KH ₂ PO ₄ , 2.5 mM CaCl ₂ .2H ₂ O, 24.8 mM NaHCO ₃ and 10 mM glucose).

One skilled in the art will recognize that this method can be used for other animals such as rabbits, other rat strains, guinea pigs, and the like. The wheel is then coupled to an isotonic transducer (Radnoti Inc., monrovia, Calif.) And data is recorded by a Ponemah physiological platform (Gould Instrument Systems, Inc, Valley View, OH) and contains a buffer solution Placed in an oxidized (95% O ₂ , 5% CO ₂ ) tissue bath. Tissue is adjusted to 1 g resting tension and stabilized for about 1 hour prior to testing. Ring integrity can be tested with norepinephrine (Sigma Co., St. Louis, MO) and carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity was checked, the rings were washed 3 times with fresh buffer and allowed to rest for about 1 hour.

  To test the sample for vasodilation or relaxation of the aortic ring tissue, the ring is contracted to 2 g tension and stabilized for 15 minutes. The Zven polypeptide sample is then added to four tanks 1, 2 or 3 without flushing, and the tension on the rings is recorded and compared to a control ring containing only buffer. Enhancement or relaxation of contraction by Zven polypeptides, their agonists and antagonists is measured directly by this method and can be applied to other contractile tissues, such as gastrointestinal tissue.

  As another example, the effect of Zven1 was tested in a standard guinea pig ileum organ bath. The organ bath system is a standard method used to measure contractility in isolated tissues, and the guinea pig ileum is usually used to record the contractile response in the intestine ex vivo (Thomas E., et al., Mol Pharmacol 44: 102-10, 1993). Since the components of the enteric nervous system are located entirely in the intestine, it can be removed from the brain and spinal cord and its reflex behavior is studied. The conventional response observed in gastrointestinal tissue from the guinea pig ileum is longitudinal contraction by smooth muscle fibers oriented along the long axis of the intestine. As shown in Example 6, Zven1 treatment stimulated smooth muscle contraction in the ileum at a pmol concentration as low as 0.75 ng / ml, equal to 75 pM. Furthermore, the best response was observed with a Zven1 dose of 20 ng / ml. Additional examples showing the effects of the Zven molecules of the present invention are shown in Examples 7, 14 and 15.

The effects of mutant Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras, on gastric motility are typically due to gastric emptying through the gastrointestinal tract and subsequent transit time. It is measured in the clinical setting as the time required. Gastric emptying scans are well known to those skilled in the art and briefly include the use of oral contrast agents such as barium or radiolabeled meals. Solids and liquids can be measured independently. In general, test foods or fluids are radiolabeled with isotopes (eg ^99m Tc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using a gamma camera (Meyer et al., Am. J. Dig. Dis. 21: 296 (1976); Collins et al., Gut 24: 1117 (1983); Maughan et al., Diabet. Med. 13: S6 (1996), and Horowitz et al., Arch. Intern. Med. 145: 1467 (1985)).

  Oral administration of phenol red (test meal) to measure gastric emptying and intestinal transit in rodents is a well-detailed model (Martinez V, Cuttitta, Tache Y 1997 Endocrinology 138: 3749 -3755). Briefly, animals are isolated from food for 18 hours, but are free to access water. Animals receive an oral dose of 0.15 ml test meal consisting of a 1.5% aqueous methylcellulose solution containing a non-absorbable dye, ie 0.05% phenol red (50 mg / 100 ml Sigma Chemical Company Catalog # P4758). The effect of Zven1 on gastric emptying in an in vivo mouse model is shown in Examples 4, 8, 21 and 22. Additional studies can be performed before and after administration of a Promotivity agent to quantify the efficacy of the Zven polypeptide.

  A radiolabeled or affinity labeled Zven polypeptide can also be used to identify or locate a Zven receptor in a biological sample (eg, Deutscher (ed.), Methods in Enzynzol , vol. 182, pages 721-37 (Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62: 483 (1993); Fedan et al., Biocherrn. Pharnzacol. 33: 1167 (1984) thing). See also Varthakavi and Minocha, J. Gen. Virol. 77: 1875 (1996), which describes the use of anti-idiotypic antibodies for receptor identification.

9． Generation of antibodies against the Zven protein:
Antibodies against Zven polypeptides are obtained using, for example, Zven expression vector products or Zven isolated from natural sources as an antigen. Particularly useful anti-Zven1 and anti-Zven2 antibodies “specifically bind” Zven1 and Zven2, respectively. An antibody appears to specifically bind if the antibody exhibits at least one of the following two properties: (1) the antibody binds to Zven1 or Zven2 with a limiting level of binding activity, and ( 2) The antibody does not significantly cross-react with a polypeptide related to Zven1 or Zven2.

With regard to the first feature, the antibodies are those that are 10 ⁶ M ⁻¹ or more, preferably 10 ⁷ M ⁻¹ or more, more preferably 10 ⁸ M ⁻¹ or more, and most preferably 10 ^When binding to a Zven polypeptide, peptide or epitope with a binding affinity (Ka) of ⁹ M ⁻¹ or higher, it specifically binds. The binding affinity of an antibody can be readily determined by one skilled in the art by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660 (1949)). Regarding the second feature, antibodies significantly cross with related polypeptides if they detect Zven using standard Western blot analysis, but not the currently known polypeptide. no response. Examples of known related polypeptides include known tumor necrosis factor receptors. Certain anti-Zven1 antibodies bind to Zven1, but not Zven2, and certain anti-Zven2 antibodies bind to Zven2, but bind to Zven1.

  Anti-Zven1 or anti-Zven2 antibodies can be generated using antigenic Zven1 or Zven2 epitope-bearing peptides. The antigenic epitope-bearing peptides and polypeptides of the present invention have a sequence of at least 9, or 15 to about 30 amino acids contained within SEQ ID NO: 2 or 5, or another disclosed herein Contains amino acid sequence. However, a peptide or polypeptide comprising a large portion of an amino acid sequence of the present invention comprising 30-50 amino acids, or amino acids of any length up to the entire amino acid sequence of the polypeptide of the present invention is also It is also useful for inducing antibodies that bind to Zven1 or Zven2. Desirably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues and hydrophobic residues) Are preferably avoided). In addition, amino acid sequences containing proline residues are also desired for antibody production.

  As illustrated, Jameson-Wolf method, Jameson and Wolf, CABIOS 4 as potential antigenic sites in Zven1 or Zven2 are implemented by the LASERGENE (DNASTAR; Madison, WI) PROTEAN program (version 3.14). : Identified using 181, (1988). Default parameters were used for this analysis.

  The Jameson-Wolf method predicts potential antigenic determinants by combining six major subroutines for protein structure prediction. In brief, the Hopp-Woods method, Hopp et al., Proc. Natl. Acad. Sci. USA 78: 3924 (1981) was first used to identify the amino acid sequence representing the largest local hydrophilic region. (Parameter: average of 7 residues). In the second stage, the Emini method, Emin et al., J. Virolgy 55: 836 (1985) was used to calculate the surface establishment (parameter: surface determination limit (0.6) = 1).

  Third, the Karplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72: 212 (1985) was used to predict main chain flexibility (parameter: flexibility limit value (0.2) = 1). In the fourth and fifth stages of the analysis, secondary structure prediction is performed according to the Chou-Fasman method, Chou, “Prediction of Protein Structural Classes from Amino Acid Composition”, in Prediction of Protein Structure and the Principles of Protein Conformation, Applied to the data using Fasman (ed.), P.549-586 (1978) (Chou-Fasman parameter: conformation table = 64 proteins; β region limit = 105; Grrnier-Robson parameter: α And β determination parameter = 0).

  In the sixth subroutine, the flexibility parameter and the water treatment / solvent accessibility factor were combined to determine the surface contour value, referred to as the “antigen index”. Finally, a peak expansion function is applied to the antigen index to calculate 20, 40, 60 or 80% of each peak value to calculate additional free energy derived from the mobility of the surface area relative to the internal area. The main surface peak is expanded by adding. However, this calculation was not applied to any main peak present in the helical region because the helical region has little tendency to become flexible.

  The results of this analysis showed that the appropriate antigenic peptide of Zven1 contains the following components of the amino acid of SEQ ID NO: 2: amino acids 22-27 (“antigenic peptide 1”), amino acids 33-41 (“antigenic” Peptide 2 "), amino acids 61-68 (" antigenic peptide 3 "), amino acids 80-85 (" antigenic peptide 4 "), amino acids 97-102 (" antigenic peptide 5 ") and amino acids 61-85 (" antigen "). Peptidic peptide 4 "), amino acids 97-102 (" highly peptide 5 ") and amino acids 61-85 (" antigenic peptide 6 "). The present invention contemplates the use of any one of the high potency peptides 1-6 to generate antibodies against Zven1. The present invention also contemplates a polypeptide comprising at least one of the antigenic peptides 1-6.

  Similarly, analysis of the Zven2 amino acid sequence showed that the appropriate antigenic peptide of Zven2 contains the following components of the amino acid of SEQ ID NO: 5: amino acids 25-33 ("antigenic peptide 7"), amino acids 53- 66 ("antigenic peptide 8"), amino acids 88-95 ("antigenic peptide 9"), amino acids 98-103 ("antigenic peptide 10"), and amino acids 88-103 ("antigenic peptide 11"). The present invention contemplates the use of any one of the high potency peptides 7-11 to generate antibodies against Zven2. The present invention also contemplates a polypeptide comprising at least one of the antigenic peptides 7-11.

Polyclonal antibodies against recombinant Zven protein, or Zven isolated from natural sources, can be prepared using methods well known to those skilled in the art. For example, Green et al., “Production of polyclonal Antisera,” in Immunochemical Protecols (Manson, ed.), Pages 1-5 (Humana Press 1992), and Williams et al., “Expression of foreign proteins in E. coli using plasmid vectors. and purification of specific polyclonal antibodies, " in DNA Cloning 2:. (. eds) Expression Systems, 2 nd Edition, such as Glover,, page 15 (immunogenicity of things .Zven polypeptide refer to the Oxford University press 1995 is, It can be enhanced through the use of adjuvants such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.

  Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zven or portions thereof with immunoglobulin polypeptides or maltose binding proteins. A polypeptide immunogen can be a sufficiently long molecule or a part thereof. Where the polypeptide moiety is “hapten-like”, such moiety is conveniently attached to a macromolecular carrier (eg, limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Can be combined or linked.

  While polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep, the anti-Zven antibodies of the invention are also useful in humans. It can be derived from near primate antibodies. General techniques for generating diagnostically and therapeutically useful antibodies in baboons are, for example, Goldenberg et al., International Patent Application No. WO 91/11465 and Losman et al., Int. J. Cancer 46: 310, Can be found in 1990.

On the other hand, monoclonal anti-Zven antibodies can be generated. Rodent monoclonal antibodies against specific antigens are obtained by methods known to those skilled in the art (eg, Kohler et al., Nature 256: 495 (1975), Coliga et al., (Eds.), Current Protocols in Immunology, .. Vol 1, page 2.5.1-2.6.7 ( John Wiley & Sons 1991), Picksley such as, "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2 nd Edition, Glover et al. (Eds.), Page 93 (see Oxford University Press 1995).

  Briefly, a monoclonal antibody is injected into a mouse with a composition comprising the Zven gene product, the presence of antibody production is confirmed by removing a serum sample, and the spleen is removed to obtain B-lymphocytes. Fusing the lymphocytes with myeloma cells to produce a hybridoma, cloning the hybridoma, selecting positive clones that produce antibodies to the antigen, culturing clones that produce antibodies to the antigen, and hybridoma cultures Is obtained by isolating the antibody from

  Furthermore, the anti-Zven antibodies of the present invention can be derived from human monoclonal antibodies. Human monoclonal antibodies are obtained from transgenic mice constructed to produce specific human antibodies in response to antigenic attack. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines that contain targeted disruptions of endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to generate human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice include Green et al., Nat. Genet. 7: 13, 1994, Lonberg et al., Nature 368: 856, 1994, and Taylor et al., Inc. Immun. 6: 576. , 1994.

  Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography on protein-A sepharose, size exclusion chromatography and ion exchange chromatography (see, eg, Coligan, pages 2.7.1-2.7.12 and page 2.9.1 ^ 2.9). .3; Baines et al., “Purification of Imunoglobulin G (IgG)”, Methods in Molecular Biology, vol. 10, p. 79-104 (The Humana Press, Inc. 1992)).

For certain applications, it is desirable to prepare fragments of anti-Zven antibodies. Such antibody fragments are obtained, for example, by proteolysis of antibodies. Antibody fragments are obtained by pepsin or papain digestion of complete antibodies by conventional methods. As illustrated, antibody fragments can be generated by enzymatic degradation of antibodies with pepsin to provide a 5S fragment denoted as F (ab ′) ₂ . This fragment can be further degraded using a thiol reducing agent to produce 3.5S Fab ′ monovalent fragments.

  Optionally, the degradation reaction can be performed using a blocking group for the sulfhydryl groups resulting from the degradation of disulfide bonds. As another means, oxygen digestion with pepsin produces two unit Fab fragments and an Fc fragment directly. For example, Goldenberg, U.S. Pat.No. 4,331,647, Nisonoff et al., Arcg Biochem. Biophys. 89: 230, 1960, Porter, Biochem. J. 73: 119, 1959, Edelman et al., In Methods in Enzymology vol. 1, p. 422 (Academic Press 1967), and Coligan, p. 2.8.1-2.8.10 and 2.10-2.10.4.

Other methods of degrading the antibody, such as degrading the heavy chain to form a unitary L-H chain fragment, additional degradation of the fragment, or other enzymatic, chemical or genetic techniques may also be used. As long as it binds to an antigen recognized by the intact antibody, it can be used.
For example, Fv fragments comprise an association of _VH and _VL chains. This meeting may be non-covalent, as described by Inbar et al., Proc. Natl. Acad. Sci. USA 69: 2659, 1972. On the other hand, the variable chains can be linked by intermolecular disulfide bonds or cross-linked by chemicals such as gluteraldehyde (see, eg, Sandhu, Crit. Rew. Biotech. 12: 437, 1992).

Fv fragments comprise V _H and V _L chains linked by a peptide linker. These single chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the _VH and _VL domains linked by an oligonucleotide. The structural gene is subsequently inserted into an expression vector that is introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single chain polypeptide having a linker peptide that bridges the two V domains. Methods for generating ScFv are described, for example, by Whitlow et al., Methods: A Companion to Metheds in Enzymology 2:97, 1991. See Bird, et al., Science 242: 423, 1988, Ladner et al., US Pat. No. 4,946,778, Pack et al., Bio / Technology 11: 1271, 1993 and Sandhu, supra.

  As illustrated, scFv exposes lymphocytes to Zven polypeptides in vitro and selects antibody display libraries on phage or similar vectors (eg, immobilized or labeled Zven proteins or peptides). Through the action of). Genes encoding polypeptides having potential Zven polypeptide binding domains include phage (phage display) or bacteria such as E. coli. It is obtained by screening a random peptide library displayed on E. coli. The nucleotide sequence encoding the polypeptide can be obtained through a number of means such as random mutagenesis and random polynucleotide synthesis. These random peptide display libraries are for screening for known targets, which may be proteins or polypeptides, such as ligands or receptors, biological or synthetic macromolecules, or peptides that interact with organic or inorganic substances. Can be used.

  Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., US Pat. No. 4,946,778; Ladner et al. , US Patent No. 5,403,484 and Ladner, etc., US Patent No. 5,571,698, and Kay et al., Phage Display of Peputides and Proteins (Academic Press, Inc. 1996)), and random peptide display libraries and such live Kits for screening rallies are e.g. Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piccataway, MJ) Commercially available. A random peptide display library can be screened using the Zven sequences disclosed herein to identify proteins that bind to Zven.

  Another form of antibody fragment is a peptide that encodes a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) are obtained by constructing the gene encoding the DCR of the antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize variable regions from antibody-producing cell RNA (eg, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106, (1991), Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., (Eds.), Page 166 (Cambridge University Press 1995), and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (Eds.), Page 137 (Wiley-Liss, Inc. 1995)).

  On the other hand, anti-Zven antibodies can be derived from “humanized” monoclonal antibodies. Humanized monoclonal antibodies are generated by translocating mouse complementary determining regions from mouse immunoglobulin H and L variable chains into human variable domains. Next, typical residues of a human antibody are substituted in the skeletal region of the murine counterpart. The use of antibody components derived from humanized monoclonal antibodies avoids problems that may be associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833, 1989.

  Techniques for generating humanized monoclonal antibodies include, for example, Jones et al., Nature 321: 522, 1986, Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285, 1992, Sandhu, Crit. Rey Biotech. 12: 437, 1992, Singer et al., J. Tmmun. 150: 2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” in Protein Engineering: Principles and Practice, Cleland et al. (Eds.), Pages 399-434 (John Wiley & Sons, Inc. 1996), and Queen et al., US Pat. No. 5,693,762 (1997).

  Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zven antibodies or antibody fragments using standard techniques. For example, Green et al., “Production of Polyclonal Antisera,” in Met7lods In Molecular Biology: Imiizunochemical Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4. 1-2.4. See page 7. On the other hand, monoclonal anti-idiotype antibodies can be prepared by the techniques described above using an anti-Zven antibody or antibody fragment as an immunogen. Alternatively, humanized anti-idiotype antibodies or near human primate anti-idiotype antibodies can be prepared using the techniques described above. Methods for preparing anti-idiotype antibodies are described, for example, by Irie, US Pat. No. 5,208,146, Greene et al., US Pat. No. 5,637,677, and Varthakovi and Minocha, J. Gen. Virol 77: 1875, 1996. The

10. Therapeutic use of Zven polypeptides:
The present invention relates to proteins, polypeptides and peptides having Zven activity in subjects lacking an appropriate amount of this polypeptide (eg, Zven polypeptides, Zven analogs, active Zven anti-idiotype antibodies and Zven fusion proteins). ). The present invention contemplates both livestock and human therapeutic use. Exemplary subjects include mammalian subjects such as domestic animals, livestock animals and human patients.

  Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants, and / or chimeras thereof, and antagonists thereof have limited contractility, gastric emptying capacity and / or increased contractility, and intestinal It is useful in diseases characterized by gastrointestinal tract dysfunction due to disorders associated with inflammation.

  Gastrointestinal dysfunction due to limited contractility and / or gastric emptying may include postoperative bowel obstruction, postpartum intestinal obstruction, chronic constipation, indigestion, intestinal pseudo-obstruction, gastric paralysis, diabetic gastric paralysis, Of diseases and disorders including but not limited to gastroesophageal reflux, vomiting, opioid and / or narcotic use or consumption, muscular dystrophy, progressive systemic sclerosis, infectious diarrhea, and paralytic gastroparesis Show properties.

  Postoperative inhibition of gastrointestinal motility (postoperative ileus) is induced by laparotomy and intraperitoneal methods. Temporary inhibition of gastrointestinal motility occurring in humans, primarily in the stomach and colon, lasts for several days and can contribute significantly to patient postoperative failure. Oral food intake is delayed until postoperative ileus is resolved, and nasogastric inhalation is prolonged or rarely requires a laparotomy (Livingston, E. et al., Post operative ileu. Dig. Dis Sci 35: 121-132, 1990). Zven1 is a potent stimulator of gastrointestinal contractility, as exemplified next. Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to stimulate gastrointestinal contractility in patients after surgery. For example, patients with disorders such as postoperative gastric paresis, eg postoperative ileus, are good candidates for administration of Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof. Is the body.

  Post-operative intestinal obstruction (POI) is a state of reduced bowel movement, such as delayed gastric emptying, that occurs after surgery as a result of a disrupted muscle condition. It is especially problematic following abdominal surgery. The problem arises from the surgery itself, from the residual effects of anesthetics, and in particular from pain-relieving anesthetic and opiate drugs used during and after surgery.

  Post-operative intestinal obstruction reduces gastrointestinal motility, which also delays the absorption of orally administered drugs. Reduced bowel motility following surgery is a major cause of prolonged hospitalization, which is very expensive and provides an increased opportunity for the progression of other complications. The extended period of POI also requires the use of expensive parenteral nutrition. The rising costs of medical care are expected to further increase the costs associated with hospitalization and parenteral food intake.

  The acute nature of this condition provides an opportunity to process with Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof. In addition, drugs that are administered subcutaneously, intramuscularly or intravenously, such as Zven1, are beneficial because oral drugs exhibit the opposite symptoms during POI. “Prokinetics” has been found to alleviate symptoms associated with POI and is effective in treating POI because Zven1 has prokinetic properties. Currently, there are very few drugs that can effectively treat POI, and those drugs that are available have side effects, cannot be taken with other medications, and / or cannot be administered orally.

  The effects of Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras, on POI are appropriate before or following laparotomy and cecal surgery performed under anesthesia. At that time, it is measured in an in vivo model by administering it to oral (po), intraperitoneal (ip), intravenous intravenous (iv), subcutaneous (sc) or intramuscular (im) to fasted animals. obtain. One of the main models listed below can be used to assess gastric emptying capacity and / or extent of intestinal transit in the range of 10-180 minutes after removal of anesthesia. In the canine model, this time is long (up to 50 hours) and the use of several post-surgical time points allows an assessment of the effect of surgery on gastric emptying capacity and passage almost through the gastrointestinal tract. This model has been used extensively to evaluate the efficacy of prokinetic drugs on gastric emptying and / or intestinal transit as a result of POI (Martinez, Rivier, and Tache. J. Pharnzacol. Experimental. Therap. 290 : 629 (1999) and Furuta et al. Biol. Pharm. Bull. 25: 103-1071 (2002)).

In addition, Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to prevent POI. In this scenario, Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof are preliminarily administered to the patient.
Diabetic gastric palsy is gastric palsy that results in motility abnormalities in the stomach as a complication of type I and II diabetes. It is characterized by delayed gastric emptying, bloating after meals, nausea and vomiting. In diabetes, it is also related to the loss of stromal cells, Cajal, the “pace marker cells” of the intestine, which seems to be due to neuropathy.

  Patients with diabetes mellitus also have disorders related to gastric emptying. For example, patients who are diabetic for at least 5 years have a significant delay in gastric emptying in more than 50% (Horowitz, M, et al., J. Gastoenterology Hepatology: 1: 97-113, 1986). Gastric neuromuscular dysfunction occurs in 30-50% of patients after 10 years of type I or type II diabetes (Koch K., et al., Dig Dis Sci 44: 1061-1075, 1999). ZvenI, Zven2, and / or their agonists can also be used as treatments for diabetic patients.

  The often acute nature of the onset of diabetic gastric paresis symptoms provides an opportunity for treatment with Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras. “Prokinetic” has been found to alleviate the delayed gastric emptying associated with diabetic gastric paresis. Currently, there are very few drugs that can effectively treat diabetic gastroparesis and those available have side effects and / or cannot be taken with other drugs. Oral drugs are not tolerated during severe disease states and therefore require intravenous administration of prokinetics.

  Spontaneous diabetic NOD / LtJ mice (available from Jackson Laboratories) develop delayed gastric emptying, impaired electrical pacemarking, and reduced motor neurotransmission. This is described in Watkins et al. J. Clin. Invest. 106: 373-384 (2000). This strain also appears to be defective in the Cajal stromal cell (ICC) network associated with impaired motility.

  Streptozocin treatment of rats and mice is a well-recognized and acceptable method for inducing diabetes; these animals are characterized by impaired gastric emptying and intestinal transit; and thus diabetes Symptoms of gastric paresis (Yamano et al. Naufzyfi-Schmiedeberg's Arcli. Phar7nacol. 356: 145-150 (1997) and Watkins et al. J. Clin. Invest. 106: 373-384 (2000)). Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras (po, iv, ip, sc, or im) that improve impaired gastric emptying and intestinal transit associated with diabetes The ability to be administered through can also be measured by one of the main models described below.

  Inflammatory reactions cause various clinical symptoms that are sometimes associated with abnormal motility of the gastrointestinal tract, such as nausea, vomiting bowel obstruction or kicking. Bacterial lipopolysaccharide (LPS) exposure induces such an inflammatory condition observed in both humans and laboratory animals, and two-stage changes in gastrointestinal motility, namely increased currency in the early stage and in late stages Characterized by delayed passage. Since Zven1 plays its role in inflammation and has two-step activity at low (prokinetic) and high (inhibitory) doses, it would be beneficial in those inflammatory conditions.

  Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, can also be used to treat gastrointestinal related sepsis. Experimental “sepsis” / endotoxemia is produced in rodents using the method described in Ceregrzyn et al. Neurogastroenterol. Mot. 13: 605-613 (2001). Those animals make a two-step change in the gastrointestinal currency. Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras, can be administered at low (prokinetic) or high (inhibitory) concentrations, depending on the stage of the disease (po, iv , ip, sc, or im). The gastric emptying capacity and / or intestinal transit is then measured using one of the main models described below.

  Morphine and other opioid analgesics are the most common pain relieving agents used, especially following surgery. Individuals taking opioid analgesics often have reduced gastric emptying and intestinal transit because they inhibit the release of acetylcholine from the mesenteric plexus and thereby reduce propulsive activity in the gastrointestinal tract. Since Zven1 stimulates intestinal contractility and has gastrointestinal prokinetic activity, Zven1 may be beneficial in the treatment of opioid-induced movement disorders.

  The effect of Zven1 on opioid-induced gastric failure paralysis in experimental rodents can be measured by well-known models. Suchitra et al. See World J. Gastroenterol. 9: 779-783 (2003) and Asai, Arzneim.-Forscl./Drug Res. 48: 802-805 (1998). Mice or rats are dosed (eg, 1-5 mg / kg BW) known to inhibit gastric emptying and intestinal transit at a set time before administration of the test agent, or gastric emptying And the method used to monitor intestinal transit (use of one of the main models listed below) through the appropriate route of administration (po, ip, iv, sc, im) Morpholine). Zven polypeptides, e.g., Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, are used to evaluate their efficacy in reducing opioid-induced gastric failure / intestinal obstruction. Or for the method, it is administered at the appropriate time (through po, iv, ip, sc, or im).

  Individuals with neuropathy (eg, as seen with diabetes) often have gastric paresis and reduced bowel motility as a result of the dysfunctional nervous system. Zven1 has been shown to be beneficial in individuals with neuropathy-induced gastrointestinal disorders.

  The effect of Zven1 on vagus nerve amputation-induced gastric failure in experimental mammals can be measured in animal models. Thoracic vagus system amputation is described in experimental mammals as described, for example, in Takeda et al. Jpn. J. Pharn7acol. 81: 292-297 (1999) and Hatanaka et al. Neurogastroenterol. Motil. 8: 227-233 (1996). Done. These animals are characterized by induced gastric emptying and intestinal transit. Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras are monitored using one of the major models listed below, this vagus nerve amputation-induced stomach Administered as a means to relieve paresis / intestinal obstruction (through po, ip, iv, sc, or im).

  Because Zven1 has contractile activity in the intestinal tract system in the presence of atropine, Zven1 can also have intestinal activity in vivo in the presence of atropine. Atropine is a compound that blocks cholinergic nerve abilities. Thus, the ability of Zven1 to be active in the presence of atropine indicates that Zven1 acts independently of the cholinergic nervous system and therefore of those treatments that have neuropathy-related gastric failure and / or intestinal obstruction. Can be beneficial for.

  Another indicator that Zven1 and / or Zven2 can be used to treat gastric dysfunction is characterized by the reflux of gastric contents into the esophagus as a result of a decrease in pressure barrier due to failure of the lower esophageal sphincter. It is a gastroesophageal reflux disease. Prokinetics such as bethanechol (Urecholine) and metropramide (Reglan) have been shown to help strengthen the sphincter and accelerate gastric emptying. Metoclopramide also improves muscle action in the gastrointestinal tract, but these drugs sometimes have side effects that limit their usefulness. Accordingly, biological prokinetics such as Zven polypeptides, such as Zven1, Zven2, and their agonists, that improve gastric and gastrointestinal tract contraction with or without improved stability of the esophageal sphincter , Fragments, variants and / or chimeras will be useful for treating gastroesophageal reflux disease.

  Methods for examining the effects of atropine in vivo in laboratory rodents are well known in the art. See Chadhuri et al. Life Sciences 66: 847-854 (2000) and Kaneko et al. Digest. Dis. Sci. 40: 2043-2051 (1995). Mice or rats are dosed (eg, 0.1-2.0 mg / kg BW) known to inhibit gastric emptying and intestinal transit at a set time before administration of the test agent, or gastric emptying. The drug used from the opioid type (po, ip, iv, sc, im) through the appropriate route (po, ip, iv, sc, im), depending on the method used to monitor potency and intestinal transit (use of one of the main models listed below) For example, morpholine) is administered. Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, at an appropriate time with respect to atropine and the test diet or method to assess its efficacy in the presence of atropine. Administered (through po, iv, ip, sc, or im).

  Additional indications that Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras can be used to treat gastric dysfunction are due to gastric paresis, motility abnormalities in the stomach or Gastric palsy, progressive systemic sclerosis, anorexia nervosa or muscular dystrophy resulting as a complication of a disease such as diabetes. Diabetic gastric paresis results in delayed gastric emptying, followed by post-meal bloating, and vomiting, which leads to poor glycemic control. It is often associated with Cajal stromal cell (ICC) loss.

  Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras are also characterized by low interstitial exercise, often due to lack of interstitial muscle tone or interstitial spasm It can also be used to treat gastric dysfunction observed in or associated with chronic constipation. Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to treat gastric dysfunction, which is dyspepsia defined as a defect in digestive power or function. It is a sign of major gastrointestinal dysfunction or symptoms of appendicitis, gallbladder disease or malnutrition complications. Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, are also spontaneously induced as a result of delayed gastric emptying or cancer chemotherapy or radiation therapy It can also be used to treat gastric dysfunction from vomiting characterized by symptoms of nausea and vomiting.

  In yet another indication, Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to treat gastric dysfunction associated with paralytic gastric paralysis . This is gastric paralysis caused by motility abnormalities in the stomach or as a complication of a disease (other than diabetes) such as progressive systemic sclerosis, anorexia nervosa or myotonic dystrophy. It results in delayed gastric emptying, followed by postprandial swelling and vomiting.

For disorders associated with defective gastrointestinal functionality, clinical signs of improved function include increased intestinal transit, increased gastric emptying, release and belly, ability to consume fluids and solids, And / or includes but is not limited to nausea and / or vomiting.
Furthermore, since Zven1 and Zven2 reduce contractility when administered at high doses, Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras may have such effects. If desired, it can be used to reduce or inhibit contractility. This effect may be desirable as a single treatment, for example to treat diarrhea, such as chronic diarrhea and family diarrhea.

  For disorders associated with overactive gastrointestinal contractility, clinical signs of improved gastrointestinal function include, but are not limited to, delayed gastric emptying, delayed bowel passage and / or reduced paralysis It is not limited to.

Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to stimulate chemokine production. Chemokines are small pro-inflammatory proteins with a wide range of activities related to leukocyte recruitment and function. Rat CINC-1, murine KC and GROα are members of the CXC subfamily of chemokines. Chemokines are generally preferentially chemotactic for neutrophils and also have angiogenic activity and can be divided into groups that primarily attract T lymphocytes and monocytes. . See Banks, C. et al., J. Pathology 199: 28-35,2002. The chemokines in the first group exhibit an ELR (Glu-Leu-Arg) amino acid motif at the NH ₂ terminus. For example, GROα contains this motif. GROα also has mitogenic and angiogenic properties and is involved in wound healing and angiogenesis (see, eg, Li and ThornkilS, Cytokine 12: 1409 (2000)).

  As illustrated by Examples 2, 3 and 17, Zven1 and Zven2 stimulate the release of the chemokine CINC-1 (cytokine-induced neutrophil chemoattractor 1) in a rat thoracic aorta-derived cell line, Stimulates the release of chemokine KC from mice and chemokine MIP-2 (mouse macrophage inflammatory protein-2) is up-regulated in response to low doses (intraperitoneal injection) of Zven1. Thus, Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used to stimulate chemokine production in vivo. Chemokines are purified from the culture medium and used in research or clinical settings. Zven variants can also be identified by their ability to stimulate chemokine production in vitro or in vivo.

  Up-regulated chemokine expression correlates with increasing activity of IBD. See Banks, C. et al., J. Pathology 199: 28-35,2002. Chemokines can induce inflammatory cells and are involved in their activation. Similarly, MIP-2 expression was found to be associated with neutrophil influx under various inflammatory conditions. As chemokine-stimulating polypeptides, Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, can reduce inflammation by reducing, inhibiting or preventing chemokine influx in the intestinal fistula. It is useful in the treatment of sexual bowel disease.

  Similarly, as shown in Example 3, Zven1 administration can cause neutrophil infiltration. There are many aspects regarding the mammalian immune response to trauma or infection where neutrophil infiltration is desired. Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof will be useful as agents for inducing neutrophil infiltration.

  As proteins capable of stimulating the production of chemokines, Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras are useful in the treatment of infections such as fungi, bacterial viruses and parasitic infections . Thus, administration of Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be used as immune boosters to specific tissue sites. For example, Zven1 administered to gastrointestinal or lung tissue is useful alone or in combination therapy to treat infections.

  Example 5 shows that Zven1 and Zven2 can stimulate angiogenesis. Thus, Zven1, Zven2, Zven1 agonists and Zven2 agonists can be used to stimulate cardiac stem cell proliferation. These molecules can be administered alone or in combination with other angiogenic factors such as vascular endothelial growth factor.

11. Key models used to measure gastric emptying and intestinal transit:
As described above, there are many in vivo models for measuring gastric function. A few of those models are represented below.

Model 1: Method for measuring gastric emptying capacity and rate and extent of intestinal transit with phenol red / methylcellulose in laboratory mammals :
Fasted animals can be transferred to Zven1 (or other Zven agents, Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or by the appropriate route (po, ip, iv, sc, im). Or a chimera). At the appropriate time, a non-nutritive, semi-solid meal consisting of methylcellulose and phenol is administered by gavage and the animals are killed at a set time following this meal administration. Passage is assessed by recovery and spectrophotometric quantification of phenol red from the area planned along the gastrointestinal tract. The duration of pigment recovery in the gastrointestinal tract can be 10-180 minutes, depending on the signs of interest and the intestinal site. This model has been widely used to assess the efficacy of other prokinetic drugs on gastric emptying and / or intestinal transit.

Model 2: Method for measuring the rate and extent of intestinal transit using a gum arabic / charcoal diet in laboratory animals :
Fasted animals can be transferred to Zven1 (or other Zven agents, Zven polypeptides such as Zven1, Zven2, and their agonists, fragments, variants and / or by the appropriate route (po, ip, iv, sc, im). Or a chimera). At the appropriate time, a semi-solid meal consisting of gum arabic and charcoal is administered by gavage and the animals are killed at a set time following this meal administration (Puig and Pol. J. Phannacol. Experifraent. Therap. 287: 1068 (1998)). Passage is assessed by the distance that the charcoal meal moves as a percentage of the total intestinal distance. The duration of the passage measurement in the gastrointestinal tract can be 10-180 minutes, depending on the signs of interest and the intestinal site. This model has been widely used to assess the efficacy of other prokinetic drugs on intestinal transit.

Model 3: Method for measuring the rate and extent of gastric emptying capacity using polystyrene beads (indigestible solids) in experimental rodents :
Gastric emptying can be achieved with Zven1 (or another Zven, Zven polypeptide such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras) through the po, ip, sc, iv or im route of administration. In response, it is assessed by determining the elimination of polystyrene beads of a specific diameter (1 mm for ratons) from the stomach of fasted (24 hours) male or female experimental rodents. Polystyrene beads are administered by gavage and evaluated for excretion as previously described (Takeuchi et al. Digest. Dis. Sci. 42; 251-258 (1997)). Animals are killed at specific time points after pellet administration (eg, 20-180 minutes) and the stomach is removed. The number of pellets remaining in the stomach is counted. In the control study, 90% pellets are expected to remain in the stomach after 30 minutes, and after 3 hours no more than 10% pellets remain in the stomach. This model has been widely used to evaluate the gastric emptying efficiency of prokinetic drugs in experimental rodents.

Model 4: Method for measuring the rate and extent of gastric emptying capacity of a liquid or solid test meal in laboratory mammals using acetaminophen as a tracer :
Fasted animals are fed a liquid or solid meal containing ace and aminophene as tracers. A test compound (eg, Zven1) is administered po, iv, ip, sc, or im before or after administration of the test meal. Blood samples are obtained at 0-120 minute intervals, and the plasma concentration of acetaminophen (which is a measure of gastric emptying capacity) is measured by HPLC. This is described, for example, in Trudel et al. Peptides 24: 531-534 (2003).

Model 5: Method for measuring gastric emptying capacity of a solid meal in experimental rodents :
Mice or rats (“rodents”) are separated into four groups (Zven1—; positive control— [erythromycin, metoclopramide, or cisapride]; negative control— [frog rain]; and vehicle— Processed group). Each group contains about 10 animals. They were cut off food for 24 hours, but have free access to water during the fast. The animals contain one per cage and the floor grid is placed in the cage to prevent contact with the bed or feces. Fasted animals are treated with one of the above agents through one of the following routes of administration (oral: ip, iv, sc, or im).

  The animals are either pre-weighed Purina meal for a set time (eg 1 hour) in their own sputum (bedding is removed) at the appropriate time following or prior to administration of the test agent. Are introduced individually. At the end of food intake, the animals are housed in their own cage without food and water for an additional set time. They are then euthanized, the abdominal cavity is opened, and the stomach is removed after fixing the pylorus and cardia. The stomach is weighed, opened, and the stomach contents are washed with tap water. The stomach wall is wiped dry and the remaining stomach is weighed again. Gastric lysates are collected, dried and calculated.

  The amount of food contained in the stomach (measured in g) is calculated as the difference between the total weight of the stomach including the contents and the weight of the stomach wall after the contents have been removed. The weight of pellets and debris in the cocoons is also measured at the end of the feeding period. The solid food ingested by the animal is determined by the difference between the weight of the Purina meal before ingestion and the weight of the pellets and debris at the end of the feeding period. For the planned period, gastric emptying capacity is calculated according to the following equation:% gastric emptying capacity = (1−gastric contents / food intake) × 100. This model has been widely used in the literature to evaluate the gastric emptying capacity of solid meals (Martinez et al. J. Phanziacol. Experimeyit. Ther. 301: 611-617 (2002)).

Model 6: Method for measuring the rate and extent of gastric emptying capacity of a solid test meal in laboratory mammals :
Fasted animals receive barium sulfate spheroids with a standard diet, followed by test compounds such as Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or before or after the diet. Chimera is administered (through po, iv, ip, sc, or i.m.). Gastric emptying capacity is measured by means of X-ray positioning and the route is monitored at least every 15 minutes to 2 hours. This method is described, for example, in Takeda et al. Jpyz. J. Pharinacol. 81: 292-297 (1999).

Model 7: Method for measuring colon propulsion motility in experimental rodents :
This demonstrates the pharmacological effects of compounds on colonic propulsion motility in experimental rodents, as described in Martinez et al. J. Phannacol. Experi7ment.'Ther. 301: 611-617 (2002) And used for characterizing. This test is based on the propulsion of glass beads from the distal colon, showing the drug effect on the reflective arch. This test is useful in assessing whether diarrhea is a side effect. Mice or rats can be tested by appropriate routes (ip, sc, im, po, iv) of test compounds such as Zven polypeptides, eg Zven1, Zven2, and agonists, fragments, mutants and / or chimeras, or vehicles thereof. One hour prior to administration, fasted, followed by glass beads inserted into the distal colon 30 minutes later (or other suitable time). Rodents are marked for identification and placed in a large glass beaker (or other) for observation. The time required for bead elimination is shown for individual rodents.

Model 8: Model for measuring gastrointestinal motility activity in dogs :
The dog is anesthetized and the abdominal cavity is opened. An extraluminal force transducer (a sensor to measure contraction) is sutured on five sites: the gastric cardia end, 3 cm proximal to the pyloric ring, 5 cm far from the duodenum and pyloric ring Position, jejunum, 70 cm distal to the pyloric ring, ileum, 5 cm proximal to the ileum-colon connection, and 5 cm distal to the ileum-colon connection. Those force conversion leads are taken from the abdominal cavity and then withdrawn through a skin incision made between the scapulas where the connectors are connected. After surgery, a jacket protector is placed on the dog to protect the connector.

  Measurement of gastrointestinal motility activity begins 2 weeks after surgery. For any measurement, a telemeter (radio data transmitter) is coupled to the connector to determine the contraction movement at individual sites in the gastrointestinal tract. Data is stored on a computer through a telemeter for analysis. Test compounds, Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, at the appropriate time (po) to assess their ability to affect gastrointestinal motility activity , iv, ip, sc, im). This can be done in normal dogs or dogs where gastric paresis / intestinal obstruction has been induced. The above methods are modified from those in Yoshida. And Ito. J. Phannacol. Experisnent. Therap. 257, 781-787 (1991) and Furuta et al. Biol. Phare. Bull. 25: 103-1071 (2002). It is.

12. Additional methods for measuring stomach function:
A model for the assessment of pain associated with intestinal distension (rat: rabbit: dog) :
Symptoms: Inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), gastric paresis, bowel obstruction, indigestion.
Animals are surgically supplied with electrodes implanted on the proximal colon and striated muscle and a cathode implanted in the lateral ventricle of the brain. Rectal bloating is performed by inflation of a balloon inserted into the rectum, and the pressure that elicits a characteristic viscometer response is measured. Test compounds, such as Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, through the appropriate pathway (po, ip, sc, iv, or im) and prior to fullness, It is administered at the appropriate nominal time (ie, about 20 minutes for ip or icv). Test compounds are evaluated for their ability to affect colonic motility, abdominal contractions, and visceral pain.

A model for assessing vomiting :
Signs: Vomiting (primarily or as a result of gastric failure).
The anti-emetic activity of the test compound is tested by its ability to inhibit cisplatin- or tocony syrup-induced emesis in staghorn (since mice and rats do not vomit). In this model, the onset of nausea and vomiting occurs approximately 1 hour after administration of cisplatin (200 mg / m.sup.2i.p.). In the first nausea in response to cisplatin, test compounds such as Zven polypeptides, such as Zven1, Zven2, and their agonists, fragments, variants and / or chimeras are administered (eg, ip, po, iv, sc , icv), and its effect on vomiting is determined by comparison with an appropriate control (eg water).

When using a tocon to induce vomiting, the test compound is given at an appropriate time before the tocon supply. The latency to first nausea, the latency to first vomiting and the number of nausea and vomiting conditions are recorded over 60 minutes. Data are based on average latency to first nausea or vomiting (minutes), average number of vomiting states per bark based on animals that did not show vomiting and animals that showed vomiting, and remained in response to Tokon Expressed as the average number of nausea / vomiting exhibited by the animal ("responder"). Staghorn that does not show vomiting is later removed from the calculation.
[Ie (+-) cis-3- (2-methoxybenzylamino) -2-phenylpiperidine showed anti-emetic activity when administered at a dose of 3 mg / kg ip. ]

(Cajal stromal cells) model of ICC deletion :
ICC deficiency results in severe gastrointestinal motility dysfunction and is found in many diseases associated with altered gastrointestinal function. The fluid for Kit provides an opportunity to evaluate the ICC network of gastrointestinal muscles in movement disorders.
Signs: Diabetic gastric paresis: IBD; sham-occlusion, chronic constipation.

  Induction example: BALB / c child mice are treated with a monoclonal antibody against Kit (ACK2) for 4 days after birth. This inhibits the progression of c-kit, resulting in severe impairment of intestinal motility. Test compounds, such as Zven polypeptides, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof are administered to assess their effects on intestinal motility. Isolated segments of the intestine from Kit-treated mice can also be tested for rhythmic contraction and relaxation in vitro in response to test compounds.

  Spontaneous example: Spontaneous diabetic NOD / LtJ mice (Jackson Labs) develop delayed gastric emptying, impaired electrical pacemarking, and reduced motor neurotransmission. To assess test compounds, Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof for gastric emptying (ie through phenol red / methylcellulose) and their effect on intestinal motility Be administered. Isolated segments of the intestine from those mice can also be tested for in vitro rhythmic contraction and relaxation in response to test compounds.

  Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can also be used to enhance sensitization in mammals. For example, the Zven1 and Zven2 ortho, Bv8, was used to stimulate the PK-R1 and PK-R2 receptors in rats resulting in sensitization of peripheral nociceptors to thermal and mechanical stimuli. See Negri, L. et al., Brit. J. Phann. 137: 1147-1154,2002. Thus, the Zven1 and Zven2 polypeptides of the present invention, including agonists, enhance sensitization (pain, heat or mechanical) when delivered locally or locally, systemically or centrally. Can be used. The polypeptides of the invention can also be administered to enhance brain cell sensitivity to improve function from living neurons to neurotransmitters and are therefore effective in Parkinson's disease or Alzheimer's disease. . Zven1 polypeptides and other Zven1 agonists can also be used to relieve pain, such as visceral pain, or severe headache (eg, migraine).

Similarly, antagonists to Zven1 and Zven2 can be used to reduce the sensation of pain in patients with neuropathy if the patient has increased sensitivity to pain. For example, patients with diabetic neuropathy have chronic enhanced pain, and antagonists to Zven1 are useful for limiting, preventing or reducing the pain.
As shown in Example 5, Zven1 and Zven2 can stimulate angiogenesis. Thus, Zven1, Zven2, Zven1 agonists and Zven2 agonists can be used to induce new blood vessel growth. These molecules can be administered to a mammalian subject alone or in combination with other angiogenic factors such as vascular endothelial growth factor.

  Moreover, prolonged gastrointestinal congestion often complicates the course of patients with sepsis (Hemann G, et al., Am. J. Phys. Regul. Integr. Compr. Physio. 276: R59-R68, 1999). Activation of the systemic immune response by trauma, infection, radiation or chemotherapy is often accompanied by gastric congestion which is perceived as nausea, anorexia and vomiting (Emch G., et al., Am. J. Physiol. Gastrointest. Liver Physio. 279: G5582-G586, 2000). Thus, Zven1 and its agonists may be useful in the treatment of sepsis associated with gastrointestinal congestion and bowel obstruction.

In addition, Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof have nausea and vomiting, in particular the nausea and vomiting is related to or a result of bowel obstruction or other gastrointestinal motility disorders Useful in patient treatment. They include, for example, prophylaxis where vomiting is associated with treatment for cancer or are post-administered for chemotherapy.
Using telemetry in conscious male Sprague-Dawley rats, there was no significant change in blood pressure or heart rate in response to an iv dose of 200 μg / kg Zven1. The stool consistency from those rats appeared to remain unchanged for 24 hours after Zven1 administration. Rats do not appear to be affected by their dose of Zven1. No apparent effect has been reported when mice are administered 10,000 μg / kg Zven1 by ip injection.

  The Zven polypeptides of the present invention, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof, can also be used as food supplements. Zven2 polypeptide has been purified from bovine milk. See Masuda Y. et al., Bioc. And Biophys. Res. Cornm. 293: 396-402,2002. Furthermore, increased gastrointestinal contractility helps with improved metabolism and weight gain. A combination of Zven1 or Zven2, or agonists, variants and / or fragments, as a protein that can be administered orally, is useful as a supplement or adjuvant to a feeding program, where the mammalian subject has anorexia and / or weight Have an increase. Such conditions are known, for example, as growth failure, cachexia, and lupus syndrome. The polypeptides of the present invention may be useful in adapting more conventional types of food for digestion by infant mammals.

In general, the dose of polypeptide, protein or peptide administered will vary depending on such factors as the patient's age, weight, height, sex, general medical condition and previous medical history Will. Typically, a dose of a molecule having Zven activity in the range of about 1 pg / kg to 10 mg / kg (amount of drug / patient body weight), although lower or higher doses are also directed by the environment. If so, it is desirable to be able to be administered to the receptor.
A Zven1 polypeptide heated to 56 ° C. for 30 minutes maintained some activity as measured by a reporter assay. See Example 13. Thus, the polypeptides of the present invention can be effectively administered orally.

  Administration of molecules with Zven activity to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, by local catheter, perfusion through inhalation, or as a suppository or by direct intralesional injection. May be intrathecal administration. When administering therapeutic proteins by injection, the administration can be by continuous infusion or by one or more boluses. On the other hand, Zven polypeptides such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof can be administered as a controlled open formulation.

  Additional routes of administration include oral, dermal, mucosal, pulmonary and transdermal. Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres and lipid-based systems (eg, DiBase and Morrel, “Oral Delivery of Microencapsulated Protein”, in Protein Delivery: See Physical Systems, Sanders and Hendren (eds.), P.255-288 (Plenum Press 1977)). The feasibility of intranasal delivery is exemplified by such aspects of insulin administration (see, eg, Hinchcliffe and Illum, Adv. Drug Deliv. Rev. 35: 199 (1997)). Dry or liquid particles comprising Zven1, Zven2, and their agonists, fragments, variants and / or chimeras can be prepared and inhaled with the aid of a dry-powder disperser, liquid aerosol generator or nebulizer ( See, for example, Pettit and Gombotz, TIBTECH 16: 343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35: 235 (1999)).

  This approach is exemplified by the AERX diabetes treatment system, which is an electronic inhaler that delivers aerosolized insulin to the lungs. Studies have shown that proteins as large as 48,000 kDa are delivered through the skin at therapeutic concentrations with the aid of low frequency ultrasound, illustrating the feasibility of transdermal administration (Mitragotri et al., Science 269: 850 (1995)). Transdermal delivery using electroporation provides another means of administering, for example, Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof (Potts et al., Pharm. Biotechnol. 10 : 213 (1997)).

The Zven protein can also be applied locally, for example as a liposome preparation, gel, ointment, as an adhesive, prosthetic or bandage component and similarly.
Because chemokines promote and accelerate tissue repair, Zven1, Zven2, and their agonists, fragments, variants and / or chimeras can have a beneficial role in disease resolution. For example, topical administration is useful for wound healing applications, such as preventing excessive scars and granulated tissue, and preventing healing following surgery.

  A pharmaceutical composition comprising a molecule having Zven1 or Zven2 activity may be supplied in liquid form, aerosol, or solid form. A protein having Zven1 or Zven2 activity can be administered as a conjugate with a pharmaceutically acceptable water-soluble polymer component. As illustrated, Zven1-polyethylene glycol conjugates are useful to increase the circulating half-life of interferon and to reduce the immunogenicity of a polypeptide. Liquid forms including liposome-encapsulated formulations are exemplified by injectable solutions and oral suspensions. Typical solid forms include capsules, tablets and controlled open forms such as mini-osmotic pumps or implants. Other dosage forms include, for example, Ansel and Popovich, Pharnnaceutical Dosage Fonns and Drug Delivery Systen is, 5th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Phannaceutical Sciences, 19th Edition (Mack Publishing Company 1995), and Ranade and Hollinger, Drug Delivery Syste 7ns (CRC Press 1996) can be devised by those skilled in the art.

A pharmaceutical composition comprising a protein, polypeptide or peptide having Zven1 or Zven2 activity can be formulated according to known methods for preparing pharmaceutically useful compositions, whereby the therapeutic protein is pharmaceutically administered. Mixed with an acceptable carrier. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. A sterile phosphate buffer solution is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. For example, Gennaro (ed.), Remington 's Pharmaceutical Sciences, 19 th Edition (Mack Publishing Company 1995) reference.

  For treatment, a molecule having Z Zven1 or Zven2 activity and a pharmaceutically acceptable carrier are administered to the patient in a therapeutically effective amount. A combination of a protein, polypeptide or peptide having Zven activity and a pharmaceutically acceptable carrier is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. Is called. An agent is physiologically significant if its presence results in a detected change in the physiology of the recipient patient.

  For example, the invention includes a step of administering to a patient a composition comprising a Zven polypeptide, such as Zven1, Zven2, and agonists, fragments, variants and / or chimeras thereof. Includes methods for enhancing excretion and / or intestinal transit. In an in vivo approach, the composition is a pharmaceutical composition that is administered to a mammalian subject in a therapeutically effective amount.

  A pharmaceutical composition comprising a molecule having Zven activity can be supplied in liquid or solid form. Liquid forms including liposome-encapsulated formulations are exemplified by injectable solutions and oral suspensions. Typical solid forms include capsules, tablets and controlled open forms such as mini-osmotic pumps or implants. Other dosage forms include, for example, Ansel and Popovich, Pharnnaceutical Dosage Fonns and Drug Delivery Systen is, 5th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Phannaceutical Sciences, 19th Edition (Mack Publishing Company 1995), and Ranade and Hollinger, Drug Delivery Syste 7ns (CRC Press 1996) can be devised by those skilled in the art.

  The Zven1 or Zven2 pharmaceutical composition may be supplied as a kit comprising a container comprising Zven1 or Zven2, a Zven1 or Zven2 agonist, or a Zven1 or Zven2 antagonist (eg, an anti-Zven1 or Zven2 antibody, or antibody fragment). For example, Zven1 or Zven2 can be supplied in the form of an injectable solution for single or multiple doses, or in the form of a sterile powder that will be reconstituted prior to injection. On the other hand, such a kit can include a dry-powder disperser, a liquid aerosol generator or a nebulizer for administration of the therapeutic polypeptide. Such kits further comprise information based on the labeling and usage of the pharmaceutical composition. Further, such information can include information that indicates that the Zven1 or Zven2 composition is contraindicated in patients with a known hypersensitivity to Zven1 or Zven2.

13. Therapeutic use of Zven nucleotide sequences:
The present invention encompasses the use of a Zven nucleotide sequence to supply the Zven amino acid sequence of a protein, polypeptide or peptide having Zven activity to its required subject, as discussed in the previous section. In addition, therapeutic expression vectors that inhibit Zven gene expression, such as antisense molecules, ribozymes or external guide sequence molecules can be provided.

  Use of recombinant host cells that express Zven, supply of naked nucleic acid encoding Zven, use of a cationic lipid carrier with a nucleic acid molecule encoding Zven, and viruses expressing Zven, such as recombinant retroviruses, recombination There are many approaches for introducing the Zven gene into subjects, including the use of adeno-related viruses, recombinant adenoviruses, and recombinant herpes simplex virus (HSV) (eg, Mulligan, Science 260: 926 (1993)). ), Rosenberg et al., Science 242: 1575 (1988), LaSalle et al., Science 250: 988 (1993), Wolff et al., Science 247: 1465 (1990), Breakfield and Deluca, The New Biologist 3: 203 (1991 ) checking). In the ex vivo approach, cells are isolated from the subject, transfected with a vector expressing the Zven gene, and then transplanted into the subject.

  To effect expression of the Zven gene, an expression vector is constructed in which the nucleotide sequence encoding the Zven gene is operably linked to a core promoter and optionally regulatory elements to control gene transcription. The general need for expression vectors is described above.

  On the other hand, the Zven gene may be a recombinant viral vector such as an adenoviral vector (eg Kass-Eister etc., Proc. Nat'l Acad. Sci. USA 90: 11498 (1993), Kolis et al., Proc. Nat'l Acad. Sci. USA 91: 215 (1994), Li et al., Hum. Gene Ther. 4; 403 (1993), Vincent et al., Nat. Genet. 5: 130 (1993), and Zabner et al., Cell 75 207 (1993)), adenovirus-related virus vectors (Flotte et al., Proc. Natl. Acad. Sci. USA 90: 10613 (1993)), alphaviruses such as “Semliki Forest virus and Sindbis virus (Hertz and Huang, J. Vir. 66: 857 (1992), Raju and Huang, J.vir. 65: 2501 (1991), and Xiong et al., Science 243: 1188 (1989)), herpes virus vectors (Koering et al., Hum. Gene. Therap. 5: 457 (1994)), poxvirus vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193: 653 (1993), Panicali and Paoietti, Proc. Natl. Acad. Sci. USA 79: 4927 (1982)) Viruses such as canarypox virus or vaccinia virus (Fisher-Hoch etc., Proc. Hatl. Acad. Sci. USA 86: 317 (1989) and Flexner etc., Ann. NY Acad. Sci. 569: 86 (1989) ), And retroviruses (Baba et al., J. Neurosurg 79: 729 (1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res. 33: 493 (1992) Vile and Hart, Cancer Res. 53: 962 (1993), Vile and Hart, Cancer Res. 53: 3860 (1993) and Anderson et al., US Pat. No. 5,399,346). In various embodiments, either the viral vector itself or a viral particle comprising the viral vector can be used in the methods and compositions described below.

  As an illustration of one system, adenovirus, or double-stranded DNA virus, is a well-characterized gene transfer vector for the supply of heterologous nucleic acid molecules (Becker et al., Meth. Cell Biol. 43: 161 ( 1994); Douglas and Curiel, Science & Medicine 4:44 (1997)). The adenovirus system provides several advantages: (i) ability to accommodate relatively large DNA inserts, (ii) ability to grow to high titers, (iii) infect a wide range of mammalian cell types. Ability, and (iV) the ability to be used with many different promoters, such as ubiquitous, tissue-specific and regulatable promoters. In addition, adenovirus can be administered by intravenous injection since the virus is stable in the bloodstream.

  Using an adenoviral vector in which a portion of the adenoviral genome has been deleted, the insert is integrated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In a typical system, the essential E1 gene is deleted from the viral vector, and the virus will not replicate if the E1 gene is not supplied by the host cell. Adenoviruses primarily target the liver when delivered intravenously to intact animals. If an adenoviral delivery system with an E1 gene deletion does not replicate in the host cell, the host tissue will express and process the encoded heterologous protein. A host cell will also secrete a heterologous protein if its corresponding gene contains a secretory signal sequence. Secreted proteins will enter the circulation from tissues that express heterologous genes (eg, highly vascularized liver).

  In addition, adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate the immune response against the vector. Such adenoviruses are E1-deleted and additionally contain deletions of E2A or E4 (Lusky et al., J. Virol. 72: 2022 (1998); Raper et al., Human Gene Therapy 9: 671 ( 1998)). Deletion of E2b has also been reported to reduce the immune response (Amalfitano et al., J. Virol. 72: 926 (1998)). By deleting the complete adenovirus genome, very large inserts of heterologous DNA can be accommodated. The generation of so-called “gutless” adenoviruses in which all viral genes are deleted is particularly advantageous for the insertion of large inserts of heterologous DNA (Yeh and Perricaudet, FASEB J. 11: 615 (See 1997).

  High titer stocks of recombinant viruses capable of expressing therapeutic genes are obtained from infected mammalian cells using standard methods. For example, recombinant HSV can be used in Vero cells in Brandt et al., J. Gen. Virol. 72: 2043 (1991), Herold et al., J. Gen. Virol. 75: 1211 (1994), Visalli and Brandt, Virology. 185: 419 (1991), Grau et al., Invest. Ophtalmol. Vis. Sci. 30: 2474 (1989), Brandi et al., J. Virol. Meth. 36: 209 (1992), and Brown and MacLean (eds. ), HSV Virus Protocols (Human Press 1997).

  On the other hand, an expression vector comprising the Zven gene can be introduced into the cells of interest by in vivo lipofection with liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of genes encoding markers (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17, 1987; and Mackey Et al., Proc. Natl. Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. It is clear that the orientation of transfection to specific cell types is particularly advantageous in tissues with cellular heterogeneity, such as pancreas, liver, kidney and brain. Lipids are obtained chemically to other molecules for targeting. Targeted peptides (eg hormones or neurotransmitters), proteins such as antibodies or non-peptide molecules can be chemically coupled to liposomes.

Electroporation is another aspect of administration of Zven nucleic acid molecules. For example, Aihara and Miyazaki, Nature Biotechnology 16: 867 (1998) shows the use of in vivo electroporation for gene transfer into muscle.
In another approach to gene therapy, the therapeutic gene can encode a Ztnfr12 antisense RNA that inhibits the expression of Zven. Suitable sequences for antisense molecules can be derived from the nucleotide sequences of Zven disclosed herein.

  Alternatively, an expression vector can be constructed in which regulatory elements are operably linked to a nucleotide sequence encoding a ribozyme. Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in an mRNA molecule (eg, Draper and Macejak, US Pat. No. 5,496,698, McSwiggen, US Pat. No. 5,525,468, Chowerra and McSwiggen, US Patent) No. 5,631,369 and Robertson and Goldberg, US Pat. No. 5,225,337). In the present invention, a ribozyme includes a nucleotide sequence that binds to Zven mRNA.

  In another approach, an expression vector can be constructed that directs the generation of RNA transcripts whose regulatory elements can promote RNase P-mediated cleavage of mRNA molecules encoding the Ztnfr12 gene. According to this approach, an external guide sequence can be configured to direct an endogenous ribozyme, ie, RNase P, to a specific species of intracellular mRNA that is subsequently cleaved by a cellular ribozyme (eg, Altman et al., USA). No. 5,168,053, Yuan et al., Science 263: 1269 (1994), Pace et al., WO96 / 18733, George et al., WO96 / 21731 and Wemer et al., WO97 / 33991). Preferably, the external guide sequence comprises a 10-15 nucleotide sequence complementary to Zven mRNA, and a 3'-NCCA nucleotide sequence, where N is preferably a purine. The external guide sequence transcript binds to the targeted mRNA species by base pairing between the mRNA and the complementary external guide sequence and is therefore located 5 'to the base paired region. Promotes the cleavage of mRNA by RNase P at nucleotides.

  In general, the dosage of a therapeutic vector having a Zven nucleotide acid sequence, such as a composition comprising a recombinant virus, is determined according to the age, weight, height, sex, general medical condition of the control and the medical It will change depending on factors such as history. Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into the cavity containing the tumor.

Compositions comprising the viral vectors, non-viral vectors or combinations of viral and non-viral vectors of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions, according to which The virus is mixed with a pharmaceutically acceptable carrier. As indicated above, a composition, eg, a phosphate buffer solution, is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient subject. Other suitable carriers are well-known to those skilled in the art ^{(e.g., Remington's Pharmaceutical Sciences, 19 th} Ed. (Mack Publishing Co. 1995), and Gilman's the Pharmacological Basis of Therapeutics, 7 th Ed. (MacMillan Publishing Co 1985)).

  For therapy, a therapeutic gene vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier can be administered to the subject in a therapeutically effective amount. A combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the receptor subject.

  If the subject treated with the therapeutic gene expression vector or recombinant virus is a human, the treatment is preferably somatic gene therapy. That is, preferred treatment of humans with therapeutic gene expression vectors or recombinant viruses introduces nucleic acid molecules into cells that form part of the human germline and can be passed on to the next generation (ie, humans). Reproductive gene therapy) is not required.

14. Detection of Zven1 gene expression with a nucleic acid probe:
The nucleic acid molecule can be used to detect the expression of a Zven1 or Zven2 gene in a biological sample. Such probe molecules include double stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1 or fragments thereof, and single stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO: 1 or fragments thereof. Probe molecules can be DNA, RNA, oligonucleotides and the like.

  Exemplary probes comprise the nucleotide sequence of nucleotides 66-161 of SEQ ID NO: 1, the nucleotide sequence of nucleotides 288-389 of SEQ ID NO: 1, or a portion of the complement of such nucleotide sequence. An additional example of a suitable probe is a probe consisting of nucleotides 354-382 of SEQ ID NO: 1 or a portion thereof. As used herein, the term “portion” refers to at least 8 nucleotides to at least 20 or more nucleotides.

For example, a nucleic acid molecule comprising a portion of the nucleotide sequence of SEQ ID NO: 1 can be used to detect activated neutrophils. Such molecules can also be used to identify therapeutic or prophylactic agents that modulate neutrophil responses to pathogens.
In a basic assay, a single-stranded probe molecule is combined with RNA isolated from a biological sample under temperature and ionic strength conditions that facilitate base pairing between the probe and the target Zven RNA species. Incubate. After separating the end-bound probe from the hybridized molecule, the amount of hybrid is detected.

Well-established hybridization methods for RNA detection include Northern analysis and dot / slot blot hybridization (eg, Ausubel supra, p4-1 to 4-27, and Wu et al. (Eds.), “Analysis”. of Gene Expression at the RNA Level ”, in Methods in Gene Biotechnology, p. 225-39, CRC Press, Inc., 1997). The nucleic acid probe can be labeled such that it can be detected by a radioisotope, such as ³² P or ³⁵ S. On the other hand, Zven RNA can be detected by non-radioactive hybridization methods (see, eg, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Inc., 1993). Typically, non-radioactive detection is achieved by enzymatic conversion of a chromogen or chemiluminescent substrate. Exemplary non-radioactive components include biotin, fluorescein and digoxigenin.

Zven1 oligonucleotide probes are also useful for in vivo diagnostics. By way of example, ¹⁶ F-labeled oligonucleotide can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nat. Med. 4: 467, 1998).

  Many diagnostic methods utilize the polymerase chain reaction (PCR) to increase the sensitivity of the detection method. Standard techniques for performing PCR are well known (generally, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; White (ed.), PCR Protocols: Current Methods and Applications, Humana Press, Inc., 1993; Cotter (ed.), Molecular Diagnosis of Cancer, Humana Press, Inc., 1996; Hanausek and Walaszed (eds.), Tumor Marker Protocols, Humana Press, Inc., 1998 Lo (ed.), Clinical Applications of PCR, Humana Press, Inc., 1998 and Meltzer (ed.), PCR in Bioanalysis, Humana Press, Inc., 1998).

  One variation of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR). In RT-PCR techniques, RNA is isolated from a biological sample, reverse transcribed into cDNA, and the cDNA is incubated with a Zven1 primer (eg, Wu et al. (Eds.), “Rapid Isolation of Specific cDNA or Genes by PCR ", in Methods in Gene Biotechnology, P. 15-28, CRC Press, Inc. 1997). Next, PCR is performed and the product is analyzed using standard techniques.

  As illustrated, RNA is isolated from a biological sample using, for example, the guanidinium-thiocyanate cell lysis described above. On the other hand, solid phase techniques can be used to isolate mRNA from cell lysates. Reverse transcription reactions can be sensitized with isolated RNA using random oligonucleotides, short homopolymers of dT, or Zven1 antisense oligomers. Oligo-dT primers offer the advantage that a variety of mRNA nucleotide sequences can be amplified that can provide a control target sequence. The Zven1 sequence is amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.

  PCR amplification methods can be detected using various approaches. For example, PCR products are fractionated by gel electrophoresis and visualized by ethidium bromide staining. On the other hand, the fractionated PCR product can be transferred to a membrane, hybridized with a detectably labeled Zven1 probe, and tested by autoradiography. An additional other approach involves the use of deoxyribonucleic acid trimylic acid labeled with digoxigenin to provide chemiluminescence detection and C-TRAK colorimetric assays.

  Another approach for detection of Zven1 expression is the cycling probe technique (CDT), where a single stranded DNA target binds with excess DNA-RNA-DNA chimeric probe to form a complex, The RNA portion is differentiated by RNase H and the presence of degraded probe is detected (eg, Beggs et al., J. Clin. Microbiol. 34: 2985, 1996, and Bekkuoui et al., Biotechniques 20: 240, (See 1996). Other methods for detection of Zven1 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA), cooperative amplification of templates by cross-hybridization (CATCH) and ligase chain reaction (LCR) ( For example, Marshall et al., US Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60: 161, 1996; Ehricht et al., Eur. J. Biochem. 243: 358, 1997; and Chadwick et al. , J. Viro. Methods 70:59, 1998). Other standard methods are known to those skilled in the art.

  Zven1 probes and primers can also be used to detect and localize Zven1 gene expression in tissue samples. Methods for such in situ hybridization are well known to those skilled in the art (eg, Choo (ed.), In situ Hybridization Protocols, Humana Press, Inc., 1994; We et al. (Eds.), “Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization (RISH),“ in Methods in Gene Biotechnology, pages 259-278, CRC Press, Inc., 1997; and Wu et al. (Eds.), “Localizaion of DNA or Abundance of mRNA by Fluorescence In Situ Hybridization (RISH), ”in Methods in Gene Biotechnology, pages 279-289, CRC Press, Inc., 1997).

  Various additional diagnostic approaches are also well known to those skilled in the art (eg, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics, Humana Press, Inc. , 1996; and Elles, Molecular Diagnosis of Genetics Diseases, Humana Press Inc., 1996).

15． Detection of Zven1 protein with anti-Zven1 antibody:
The present invention contemplates the use of anti-Zven1 antibodies to screen biological samples in vitro for the presence of Zven1 and in particular for upregulation of Zven1. In one type of in vitro assay, anti-Zven1 antibody is used in the liquid phase. For example, the presence of Zven1 in a biological sample involves mixing the biological sample with trace amounts of labeled Zven1 and anti-Zven1 antibodies under conditions that promote binding between Zven1 and the antibody. Can be tested. The Zven1 and anti-Zven1 complex in the sample can be separated from the reaction mixture by contacting the complex with an identified protein that binds to an antibody, such as an Fc antibody or Staphylococcus protein A. The concentration of Zven1 in the biological sample is inversely proportional to the amount of labeled Zven1 bound to the antibody and directly related to the amount of free labeled Zven1. Anti-Zven2 antibodies can be used in the same or similar manner.

  On the other hand, an in vitro assay can be performed in which anti-Zven1 antibody is bound to a solid phase carrier. For example, the antibody can be conjugated to a polymer, such as aminodextran, to bind the antibody to an insoluble support, such as a polymer-coated bead, plate or tube. Other suitable in vitro assays will be apparent to those skilled in the art.

  In another approach, anti-Zven1 antibodies can be used to detect Zven1 in tissue sections prepared from biopsy specimens. Such immunochemical detection can be used to determine a relatively large amount of Zcytor14 and to determine the distribution of Zven1 in the tissue being tested. General immunochemical techniques are well established (eg, Ponder, “Cell Marking Techniques and Their Application.” In Mammalian Development: A Practical Approach, Monk (ed.), Pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley interscience 1990), and Manson (ed.), Methods In Molecular Biology. Vol. 10: Immunochemical See Proteocols (The Humana Press. Inc. 1992).

  Immunochemical detection can be performed by contacting a biological sample with an anti-Zven1 antibody, and then contacting the biological sample with a detectable labeled molecule that binds to the antibody. . For example, the detectable labeled molecule comprises an antibody component that binds to an anti-Zven1 antibody. On the other hand, the anti-Zven1 antibody can be conjugated with avidin / streptavidin (or biotin) and the detectable labeled molecule can include biotin (or avidin / streptavidin). Many variations on this basic technique are well known to those skilled in the art.

On the other hand, anti-Zven1 antibodies can be conjugated with a detectable label to form anti-Zven1 immunoconjugates. Suitable detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels or colloidal gold. Methods for the production and detection of such detectable labeled immunoconjugates are well known to those skilled in the art and are described in more detail below.
The detectable label can be a radioisotope detected by autoradiography. Isotopes that are particularly useful for the present invention are ³ H, ¹²⁵ I, ¹³¹ I, ³⁵ S and ¹⁴ C.

  Anti-Zven1 immunoconjugates can also be labeled with fluorescent compounds. The presence of fluorescently labile antibodies is determined by exposing the immunoconjugate to the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescent label compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

  On the other hand, anti-Zven1 immunoconjugates can be detectably labeled by conjugating an antibody compound to a compound luminescent compound. The presence of a chemiluminescent-labeled immunoconjugate is determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

  Similarly, chemiluminescent compounds can be used to label the anti-Zven1 immunoconjugates of the invention. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins enhance the efficiency of chemiluminescence reactions. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.

  On the other hand, anti-Zven1 immunoconjugates can be labeled such that they can be detected by conjugating an anti-Zven1 antibody compound to the enzyme. When the anti-Zven1-enzyme conjugate is incubated in the presence of a suitable substrate, the enzyme component reacts with the substrate to produce a chemical component that can be detected by spectrophotometric, fluorescent or visible means. Examples of enzymes that can be used to label so that many specific immunoconjugates can be detected include β-galactosidase, glucooxidase, peroxidase and alkaline phosphatase.

  Those skilled in the art will be aware of other suitable labels that can be used in accordance with the present invention. Binding of the marker component to the anti-Zven1 antibody can be accomplished using standard techniques known in the art. A typical methodology in this regard is Kennedy et al., Clin. Chim. Acta 70: 1 (1976), Schurs et al., Clim. Acta 81: 1 (1977), Shih et al., Int'l. J. Cancer 46: 1101 (1990), stein et al., Cancer Res. 50: 1330 (1990), and Coligan, described earlier.

  Furthermore, the convenience and diversity of immunochemical detection can be enhanced by using anti-Zven1 antibodies conjugated with avidin, streptavidin and biotin (eg Wilchek. Et al., (Eds.), “Avidin- Biotin Technology, ”Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“ Immunochemical Applications of Avidin-Biotin Technology, ”in Methods In Molecular Biology, Vol. 10, Manson (ed.) Pages 149- 162 (See The Humana Press. Inc. 1992)).

  Methods for performing immunoassays are well established. For example, Cook and Self, “Monoclonal Antibodies in Diagonostic Immunoassays,” in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), Pages 180-208. (Cambridg University Press, 1995), Perry, “ The Role of Monoclonal antibodies in the Advancement of Immunoassay Technoogy ”, in Monoclonal Antibodies; Principles and applications, Birch and Lennox (eds.), Pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press , Inc. 1996.

  In a related approach, biotin- or FITC-labeled Zven1 can be used to identify cells that bind Zven1. Such binding can be detected using, for example, flow cytometry.

  The present invention also contemplates kits for performing immunological diagnostic assays for Zven1 gene expression. Such a kit comprises at least one container comprising an anti-Zven1 antibody or antibody fragment. The kit can also include a second container comprising one or more reagents that can indicate the presence of the Zven1 antibody or antibody fragment. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, colloidal gold and the like. The kit also comprises a means for communicating to the user that the Zven1 antibody or antibody fragment is used to detect the Zven1 protein. For example, the documentation instructions mention that the encapsulated antibody or antibody fragment can be used to detect Zven1. The document material can be applied directly to the container, or the document material can be provided in the form of a packaging insert.

  Accordingly, the generally described invention will be more readily understood by the following examples which are provided by way of illustration but are not intended to limit the invention. These examples describe studies using the Zven1 protein produced in baculovirus with a C-terminal Glu-Glu label according to the methods generally described above. Zven2 (“Endocrine Gland Vascular Endothelial Growth Factor”) protein was purchased from Peprotech, Inc. (Rocky Hill, NJ).

Example 1 : Stimulation of response in Wky12-22 cells:
Wky 12-22 details were derived from the middle layer of the thoracic aorta of Wistor-Kyoto child rats as described by Lemire et al., American Journal of Pathology 144: 1068 (1994). Those cells respond to both Zven1 and Zven2 in a reporter luciferase assay following transfection with the NFkB / Ap-1 reporter construct. The control cell line Wky 3M-22 derived from the same tissue in adult rats was not shown. Activity was detected with Zven1 or Zven2 (approximately 0.1 nM to 10 nM) in the concentration range of 1-100 ng / ml. These data suggest that Wky12-22 cells carry the Zven1 receptor and that Zven1 and Zven2 activate the NfKb / Ap-1 transcription factor.

  In one experiment, Wky 12-22 cells were loaded with the fluorescent dye Fura. The emission peak of Fura shifts when bound to calcium. Intracellular calcium release is detected by monitoring the wavelength shift. Zven1 induced intracellular calcium release at a concentration of 1-1000 ng / ml. Zven2 elicited a similar response.

Extracellular signal-regulated kinase / mitodiene-activated protein kinase (ERK-Map kinase) activity was measured in Wky 12-22 cells in response to Zven1 treatment. Cells were incubated for 30 minutes in Zven1 at concentrations ranging from 1 to 1,000 ng / ml. Cells were fixed and stained for phosphorylated ERD-Map kinase using an Arrayscan that measures fluorescence intensity in the cytosol and nucleus of the treated cells. Differences in nuclear and cytosolic fluorescence were quantified and plotted. Zven1 induced ERK-Map kinase activity with an EC ₅₀ of 0.50 nM (approximately 5 ng / ml).

Zven1 binding to Wky 12-22 cells was assessed using I ¹²⁵ -labeled Zven1. Wky 12-22 cells were seeded at low cell density and cultured for 3-4 days until they reached approximately 70% confluence. Cells were placed on ice, media was removed, and monolayers were washed. Cells were incubated with increasing amounts of I ¹²⁵ -Zven1 in the absence (total binding) and in the presence (non-specific binding) of a very excess of unlabeled Zven1. After various times at 4 ° C., the binding medium was removed and the monolayer was washed and the cells were lysed with a small volume of 1.0 N NaOH.

Cell associated radioactivity was determined by a gamma counter. Specific binding of I ¹²⁵ -Zven1 was calculated as the difference between the total binding value and the non-specific binding value. The measured radioactivity was normalized to the cell number determined for a set of co-cultures. Nonlinear regression using a two-site model was used to fit the binding data for the determination of Kd and Bmax. The high affinity site showed 1.5 nM Kd and 350 fmol binding / 10 ⁶ cell Bmax, and the low affinity site showed 31 nM Kd and 1025 fmol binding / 10 ⁶ cell Bmax.

  The results of those studies indicate that neonatal rat aortic cells express the Zven1 receptor and not the same grown rat cells. This indicates that Zven1 is involved in heart growth and angiogenesis. Zven1 signals through NFkB / Ap1 and induces chemokine release only in neonatal cells, indicating that it induces a mitogenic response in the fetal or neonatal heart. Zven1 may be a required factor required for induction of angiogenesis / angiogenesis in cardiac stem cells. Zven1 induces intracellular calcium release, an effect consistent with chemokine activity, in the Wky 12-22 cell line. Zven1, consistent with its mitogenic activity, activates a mitogen activated protein kinase.

Example 2 : Zven1 and Zven2 stimulate chemokine release in vitro:
Confluent Wky 12-22 or Wky3M22 cells were incubated with various concentrations of Zven1 for 24 hours. Conditioned media was collected and assayed for chemokine CINC-1 using a commercially available rat chemokine multiplex kit (Linco Research, Inc .; St. Charles, Missouri). CINC-1, which appears to be similar to human growth-related oncogene-α (GRO-α), in cells treated with 0.1-100 ng / ml Zven1 respectively, levels ranging from 1.8-5 ng / ml Detected with. CINC-1 was not detected in either the control Wky3M-22 cell line from the grown rat artery or the untreated control.

Example 3 : Zven1 induces a chemotactic response in vivo and stimulates chemokine release and neutrophil infiltration:
Four groups of 10 mice (BALB57 / BL6 female mice at 8 weeks of age) were not treated or injected with vehicle buffer control, 0.1 μg Zven1 or 1 μg Zven1. After 4 hours, peritoneal lavage fluid was collected, concentrated, and the cell pellet was resuspended. Relative cell populations were counted using Cell Dyne and cytospins were prepared for CBC / diff counting. Untreated and buffer control animals had about 2% neutrophils in their wash fluid, and 0.1 μg treated animals had about 30% neutrophils, which , Showing about a 15-fold increase in neutrophils in the peritoneum of Zven1-treated animals. 1 μg Zven1-treated animals have neutrophil levels consistent with untreated controls, indicating a biphasic Zven1 response. In summary, Zven1 induced neutrophil infiltration into the peritoneum following intraperitoneal injection.

  Murine KC, the ortho form of GROα in mice, was measured in serum and lavage fluid obtained from 4 groups of mice using an ELISA kit (R & D Systems Inc .; MN). 0.1 μg Zven1-treated (low dose) mice had about 45 pg / ml KC in their peritoneal fluid, which was untreated control, vehicle control and 1.0 μg Zven1-treated ( Significantly higher than high dose) mice.

  Serum levels of KC in 0.1 μg Zven1-treated mice were considerably higher than untreated mice, 1.0 μg Zven1-treated mice and vehicle-treated mice. 0.1 μg Zven1-treated mice had a KC level of about 185 pmol / ml, a 6-fold increase.

  These results are consistent with the stimulation of chemokine release in vitro shown in Example 2. Furthermore, these results correlate with the observed neutrophil wetting in the peritoneum in 0.1 μg Zven1-treated (low dose) mice.

Example 4 : Zven1 effect on gastric emptying capacity:
Seven mice received about 200 μg Zven1 (10 μg / g body weight) or vehicle control, followed by intraperitoneal injection of 7.5 mg phenol red. Gastric function was measured after 20 minutes by monitoring the transport of phenol red through the intestine. The general behavior of Zven1-treated animals was observed and was consistent with that of control animals. In Zven1-treated mice, gastric transit time was reduced by approximately 50%.

  These results show that Zven1 reduces gastric transit following high intraperitoneal injection at high doses. Zven1 administration did not seem to have any immediate toxic effects. This reduction in passage can be the result of extensive muscle contraction at such high doses. Zven1 can sufficiently enhance motility in vivo at low doses and inhibit motility at high doses.

Example 5 : Stimulation of angiogenesis by Zven1 and Zven2:
The thoracic aorta was removed from rats 12 days, 5 weeks and 3 months old. The tissue was flushed with Hanks' solution to remove any blood cells and the outer membrane tissue. Aortic rings were prepared and plated on Matrigel-coated plates containing serum-free modified MCDB medium from Clonetics and antibiotics, ie penicillin-streptomycin. Various concentrations of Zven1 and Zven2 were added to the culture dish approximately 30 minutes after the plate. Amplification was measured visually and individual rings were photographed and the results recorded. Both Zven1 and Zven2 elicited a proliferative response at concentrations ranging from 1-100 ng / ml. This mitogenic effect was observed in the aorta from animals at all three ages. Zven1 was also tested in a rat corneal model of angiogenesis and showed no effect here. The angiogenic effect observed in aortic ring culture is due to the mitogenic effect of GROα homologues.

Example 6 : Contractile stimulation in the guinea pig gastrointestinal organ tank assay:
A 6-week-old male Hartley guinea pig weighing approximately 0.5 kg was euthanized by carbon monoxide. Intestinal tissue was harvested as follows: a 2-3 cm longitudinal section of the 10 cm rostral ileum of the cecum, and a 2-3 cm longitudinal section of the duodenum, jejunum, and proximal and distal colon.

Tissue was treated with Krebs Ringer bicarbonate containing 118.2 mM NaCI, 4.6 mM KC1, 1.2 mm MgS0 ₄ , 24.8 mM NaHC0 ₃ , 1.2 mM KH ₂ P0 ₄ , 2.5 mM CaCl ₂ and lOmM glucose. Washed with salt buffer. Following extensive washing, the tissue was fixed longitudinally in a Rednoti organ bath perfusion system (SDR Clinical Technology, Sydney Australia) containing warmed oxygenated Krebs buffer and maintained at 37 ° C. 1 g of pre-filling was applied and the tissue strip was incubated for about 30 minutes. Next, baseline shrinkage was obtained. Isotonic contraction was measured with a force displacement transducer and recorded on a chart recorder using Po-ne-mah Physiology Platform Software. The neurotransmitter 5-hydroxytryptophan (5HT) (Sigma) at 130 μm and atropine at 5-10 mM were used as controls. Atropine blocks the muscarinic effect of acetylcholine.

Zven1 doses from 1 to 400 ng / ml were tested for activity against ileal strips. Muscle contraction was detected immediately after the addition of Zven1 protein and recorded at concentrations as low as 1 ng / ml or 100 pmol. The EC _{50 for} this response was approximately 10 ng / ml or 1 nM. Zven1 was tested for activity in the presence of 5HT and a second contraction was observed. Zven1 was tested for activity in the presence of 0.1 μM tetrodotoxin (TTX), a potent neuroactive agonist, and no reduction in Zven1 effect was observed. Zven1 was also tested for activity in the presence of 100 nM Verapamil (L-type calcium channel blocker). A significant decrease in the amplitude of the contractile response was observed.
The results of the effect of Zven1 on contractility in the ileum are shown in Table 6.

  The results of the effect of Zven1 on contraction in the duodenum, jejunum, proximal colon and distal colon showed that it did not produce contractions in the duodenum, jejunum, or distal colon when performed at a concentration of 40 ng / ml . However, relaxation of the tissue in the proximal colon was observed when the same concentration of Zven1 was added.

Example 7 : Effect of dose on contractility in guinea pig ileal organ tank assay:
All intestinal sections from guinea pig ileum were obtained and tested using the same protocol and reagents as described in Example 6. A longitudinal strip of guinea pig ileum was fixed in the organ bath and stabilized for about 20 minutes. Acetylcholine (ACH) at a concentration of 10 μg / ml was added to the tissue to confirm contractile activity. Two flush and fill cycles were performed to clean ACH from intestinal tissue. Baseline activity was confirmed for approximately 25 minutes. Zven1 was added to the organ bath at a final concentration of 1.0 ng / ml and a deflection of approximately 0.5 g was recorded.

  A 1.0 ng / ml dose of Zven1 was left on the tissue for 5 minutes to allow return to the baseline level of the tissue and then a 10 ng / ml dose was added. Another contractile response that resulted in a 2.0 g deflection was shown. The 10 ng / ml dose was left on the tissue for an additional 5 minutes before the 20 ng / ml dose of Zven1 was administered to the tissue. Another contractile response was observed that resulted in a deflection of about 2.2 g. After a 5 minute incubation, the tissue was treated with a 40 ng / ml dose of Zven1. The tissue contracted again with a change of about 2.0 g. The best response was observed at a Zven1 dose of 20 ng / ml.

Example 8 : Effect of Zven1 on gastric emptying and intestinal transit:
Eight week old female C57B1 / 6 mice are fed a test diet consisting of methylcellulose solution or control, and both gastric emptying and intestinal transit are determined by the amount of phenol red recovered in different sections of the intestine. Was measured. The test meal consists of a 1.5% aqueous methylcellulose solution containing a non-absorbable dye, ie 0.05% phenol red (50 mg / 100 ml Sigma Chemical Company Catalog # P4758). Has a final viscosity of 400-800 centipoise.

  Medium viscosity methylcellulose from Sigma (Cat. No. C4888) was used. One group of animals was killed immediately after administration of the test meal. They represent a standard group, ie 100% phenol red in the stomach or group VIII. The remaining animals were killed 20 minutes after administration of the test meal. Following killing, the stomach was removed and the small intestine was fragmented into proximal, intermediate and distal intestinal fragments. The proximal intestine consists of approximately the duodenum, the middle intestine consists of approximately the duodenum and the jejunum, and the distal intestine approximately consists of the ileum. All tissues were dissolved in 10 ml of 0.1N sodium hydroxide using a tissue homogenizer. Spectrophotometric analysis was used to determine the OD and thus the level of gastric emptying and intestinal transit.

  Each treatment group consists of 10 animals, except for animals used as standard and froglin control groups (n = 5). The study is divided into two days, so that half of all treatment groups are performed on two consecutive days. The animals were fasted for 18 hours in a lifted cage for 18 hours, with free access to the water. The average body weight of the mice was 16g.

  Baculovirus-expressed Zven1 protein with C-terminal Glu-Glu label, formulated in 20 mM MES buffer, 20 mM NaCl, pH 6.5, 0.9% NaCl + 0. Dilute in 1% BSA. (Sigma sodium chloride solution 0.9% and Sigma BSA 30% sterile TC test solution, Sigma Chemical Co. St. Louis, MO). The protein concentration was adjusted to be in a volume of 0.2 ml per mouse. Vehicle animals received an equal dose of Zven1 formulation buffer based on the highest (775 ng / g) treatment group.

  Two minutes prior to receiving 0.15 ml of phenol red test meal as an oral wash, treatment was performed in a volume of 0.2 ml through IP (intraperitoneal) injection. Twenty minutes after administration of phenol red, animals were euthanized and stomach and intestinal fragments were removed. The intestine was measured and divided into three equal length segments: proximal, middle and distal. The amount of phenol red in individual samples was determined spectrophotometrically and expressed as a percentage of total phenol red in the stomach (Group VIII). These values were used to determine the amount of gastric emptying and intestinal transit per collected tissue. The CCK analog froglin at 40 ng / g is used as a positive control and is administered 5 minutes before lavage, at which concentration it inhibits gastric emptying capacity. Colorimetric analysis of phenol red recovered from individual intestinal segments and stomach was performed as follows.

  After euthanasia, stomach and intestinal segments were placed in 10 ml of 0.1 N NaOH and homogenized using a polytron tissue homogenizer. The homogenate was incubated for 1 hour at room temperature and then pelleted by centrifugation on a tabletop centrifuge at 150 xg for 20 minutes at 4 ° C. The protein was precipitated from 5.0 ml of homogenate by the addition of 0.5 ml of 20% trichloroacetic acid. Following centrifugation, 4 ml of the supernatant was added to 4 ml of 0.5N NaOH. A 200 μl sample was read at 560 nm using a Molecular Devices Spectra Max 190 spectrometer. The amount of gastric emptying capacity was calculated using the following formula:% gastric emptying capacity = (1−Amount of phenol red recovered from stomach of test / average amount of phenol red recovered from stomach of group VII) × 100. The amount of gastric transit was expressed as a percentage of the total phenol red recovered.

  The results are shown in Table 7 below. Since test meals were not detected in the distal gut under any condition, their data are not included. As expected, froglin at 40 ng / ml inhibited gastric emptying (93.8% of the test meal in the stomach after 20 minutes compared to 63.8% with vehicle). In the frog-line treated group, only 2.6% of the meal was measured in the distal gut and 1.2% in the midgut, consistent with an inhibited gastric emptying capacity.

  At the lowest Zven1 concentration, ie 0.78 μg / kg body weight, a slight increase in gastric emptying capacity was observed compared to the vehicle (56.3% of the remaining meal vs. 63.8% with the vehicle). An elevated diet was detected in the distal intestine of Zven1-treated animals compared to vehicle controls, 25.5% and 18.4%, respectively, consistent with increased gastric emptying capacity. Animals treated with Zven1 at a dose of 7.8 μg / kg have 20% or less test meal in the stomach (p = 0.001), 16.6% or more meal in the distal intestine (p = 0.004), and 3.5% or more in the middle intestine Had no meals. The maximum effect was observed with 77.5 μg / kg animals, where gastric emptying capacity was increased approximately 2-fold (37.8% test meal in Zven1-treated animals and 63.8% in vehicle-treated animals, p = 0.0002).

  Intestinal transit was also significantly increased (37.1% compared to 15%, compared to 15%, since a more than two-fold increase in the test meal in the middle intestine was measured in animals treated with Zven1 over the vehicle control. p = 0.004). At the final 77.5 μg / kg dose, enhanced gastric emptying capacity was detected at 46.6% compared to the 63.8% control, but the effect was not as high as the 77.5 μg / kg dose. Enhanced intestinal transit was detected in the middle intestine (26% vs 15%), but the effect was not as significant as the effect observed at the lower 77.5 μg / kg dose. These data indicate that at higher concentrations, Zven1 can show gastric emptying and intestinal transit.

Example 9 : Zven1 baculovirus expression:
An expression vector containing the GLU-GLU tag, pzBV32L: zvenlcee, was designed and prepared to express the zven1cee polypeptide in insect cells.

A. Expression vector:
Expression vector pzBV32L: zven1cee was prepared to express human zven1 polypeptide with a carboxy-terminal Glu-Glu tag in insect cells as follows.
zven1, and a 371 bp fragment containing polynucleotide sequences encoding EcoR1 and Xba1 restriction sites on the 5 'and 3' ends, respectively, from a plasmid containing zven1 cDNA (zven1-zyt-1. contig), primer ZC29463 (sequence No. 23) and ZC29462 (SEQ ID NO: 24) were generated by PCR amplification using PCR SuperMix (Gibco BRL, Life Technologies) and appropriate buffer. (Note: zven1 sequence and Xba1 site were out of reading frame. To place an additional two bases, CC-antisense, in the reading frame encoding additional Gly between the zven1 sequence and the CEE label. Added to.)

The PCR reaction conditions were as follows: 95 ° C. for 3 minutes, 1 cycle, followed by 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 68 ° C. for 30 seconds, 25 cycles, followed by maintenance at 4 ° C. . Fragments were visualized by gel electrophoresis (1% agarose—1 μl EtBr at 10 mg / ml per 10 ml agarose). A portion of the PCR product was digested with EcoR1 and XbaI restriction enzymes in appropriate buffer and then developed on an agarose gel. DNA in response to the EcoR1 / Xba1 digested zven1 coding sequence was excised, purified using the Qiagen Gel Extraction kit (# 28704) and ligated into the EcoR1 / Xba1 digested baculovirus donor vector pZBV32L. The pZBV32L vector is a modification of the pFastBacl ^™ (Life Technologies) expression vector, where the polyhedral promoter has been removed and replaced with a late activated basic protein promoter.

  In addition, a Glu-Glu tag (SEQ ID NO: 10) and a coding sequence for a stop signal are inserted at the 3 'end of the multiple cloning region. About 216 ng of the restriction digested zven1 insert and about 300 ng of the corresponding vector were ligated overnight at 15 ° C. 1 μl of the continuous mixture was electroporated into 35 μl of DH10B cells (Life Technologies) at 2.1 kV. Electroporated DNA and cells were diluted in 1 ml LB medium, grown for 1 hour at 37 ° C., and plated on LB plates containing 100 μg / ml ampicillin. Clones were analyzed by restriction digest and one positive clone was selected and streaked on AMP + plates and one colony was obtained for confirmation by sequencing.

  Sequencing showed the presence of a start codon upstream of the actual start codon which probably prevents correct translation. Therefore, the upstream codon was removed using Qick-modified mutagenesis from Stratagene (La Jolla, CA). This was achieved by creating forward and reverse primers that change to the upstream starting ATC. A new mutagenized plasmid containing Sma1 and Xba1 cleavage sites at the 5 ′ and 3 ′ ends of the zven1 sequence was electroporated into DH10B cells as before and by restriction digest with Sma1 and Xba1. Analyzed and positive clones were selected and streaked on AMP + plates to obtain single colonies for confirmation by sequencing as before. A clone for the zven1 polynucleotide sequence was also cloned without an upstream start codon.

Transform 100 μl of DHlOBac Max Efficiency component cells (GIBCO-BRL, Gaithersburg, MD) with 1-5 ng positive clone donor vector in a 42 ° C. water bath for 45 seconds according to manufacturer's instructions did. The transformed cells were then added to 980 μl SOC medium (2% Bacto Trypton, 0.5% Bacto Yeast Extract, 10 ml 1 M NaCl, 1.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ and 20 mM glucose. ), Grown in shaking incubation at 37 ° C. for 1 hour, and plated on Luria agar plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, IPTG and BLUO-GAL .

  Plated cells were incubated at 37 ° C. for 48 hours. Color selection was used to identify those cells with the zVev1cee-encoded donor insert (referred to as “bacmid”) integrated into the plasmid. Those white colonies were picked for analysis. Bacmid DNA was isolated from positive colonies using standard isolation techniques according to Life Technologies guidelines. Clones were screened for the correct insert by amplifying the DNA by PCR using primers for the transposable element in bacmid.

  PCR reaction conditions were as follows: 94 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 5 minutes (35 cycles); 72 ° C for 10 minutes (1 cycle); followed by soaking at 4 ° C . The PCR product was run on a 1% agarose gel to confirm the insert size. Those with the correct size insert were used to transfect Spodoptera frugiperda (Sf9) cells. The polynucleotide sequence is shown in SEQ ID NO: 25. Its corresponding amino acid sequence is shown in SEQ ID NO: 26.

B. Transfection in insect cells:
Sf9 cells were seeded at 1 × 10 ⁶ cells in a 35 mm plate and allowed to attach for 1 hour at 27 ° C. 5 μg of bacmid DNA was diluted with 100 μl of Sf-900II SFM (Life Technologies, Rockville, MD). 50 μl lipofectamine reagent (Life Technologies) was diluted with 100 μl Sf-900II SFM. The bacmid DNA and lipid solution were mixed gently and incubated for 30-45 minutes at room temperature. Name medium was aspirated from one plate of cells. 800 μl of Sf-900II SFM was added to the lipid-DNA mixture. A DNA-fat mixture was added to the cells. Cells were incubated overnight at 27 ° C. The next morning, the DNA-lipid mixture was aspirated and 2 ml of Sf-900II medium was added to each plate. Plates were incubated for 168 hours at 27 ° C. and 90% humidity and the virus was harvested.

C. Primary amplification:
Sf9 cells were seeded at 1 × 10 ⁶ cells per 35 mm plate and allowed to attach for 1 hour at 27 ° C. They were then infected with 500 μl of virus stock from above and incubated for 4 days at 27 ° C., after which the virus was harvested according to standard methods known in the art.

D. Secondary amplification:
Sf9 cells were seeded at 1 × 10 ⁶ cells per 35 mm plate and allowed to attach for 1 hour at 27 ° C. They were then infected with 20 μl of virus stock from above and incubated at 27 ° C. for 4 days, after which the virus was harvested according to standard methods known in the art.

E. Third amplification:
Sf9 cells were grown in 80 ml Sf-900II SFM in a 250 ml shake flask to an approximate density of 1 × 10 ⁶ cells / ml. They were then infected with 200 μl of virus stock from above and incubated for 4 days at 27 ° C., after which the virus was harvested according to standard methods known in the art.

Expression of F. zven1cee:
A third virus stock was titrated by a growth inhibition curve and a culture showing a MOI of 1 was allowed to proceed for 48 hours. The supernatant was analyzed by Western blot using a primary monoclonal antibody HRP-conjugated Gt anti-Mu secondary antibody specific for the N-terminal Glu-Glu epitope. The result showed the expected molecular weight band.

A large volume of stock solution is produced by the following method: Sf9 cells are grown in 1 L Sf-900II SFM in a 2800 ml shake flask to an approximate density of 1 × 10 ⁶ cells / ml. They are then infected with 10 ml of virus stock solution from the last growth and incubated at 27 ° C. for 72 hours, after which the virus is harvested. Large scale infections can be completed and materials for downstream purification can be supplied.

Example 10 : Expression in E. coli:
A. Generation of native Zven1 expression structure:
The native Zven1 (SEQ ID NO: 11) DNA fragment was isolated using PCR. Primer zc # 40,821 (SEQ ID NO: 12) containing a 41 bp vector flanking sequence and a portion 24 bp corresponding to the amino terminus of Zven1 and primer zc # 40,813 (SEQ ID NO: 13) are the 3 ′ ends of the vector containing the zven1 insert 38 bp corresponding to The template was pZBV32L: zven1cee. PCR conditions were as follows: 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 1 minute, 25 cycles; followed by soaking at 4 ° C. A small sample (2-4 μl) of the PCR sample was run on a 1% agarose gel with 1 × TBE buffer for analysis and the expected band of about 500 bp fragment was seen.

  The remaining 100 μl volume of the reaction was precipitated with 200 μl of absolute ethanol. The pellet was resuspended in 10 μl of water used for recombination into the acceptor vector pTAP238 cleaved by Sma1 to generate the zven1 encoding construct disclosed above. A clone with the correct sequence was named pTAP432. It was digested with Not1 / Nco1 (10 μl DNA, 5 μl buffer 3 New England Biolabs, 2 μl Not1, 2 μl Nco1, 31 μl water in 37 ° C. for 1 hour) and T4 DNA ligase buffer (7 μl The previous digest, 2 μl of 5 × buffer, 1 μl T4 DNA ligase) was religated. This step removed the yeast sequence CEN-ARS and linearized the vector. DNA was diagnostically digested with Pvu2 and Pst1 to confirm the absence of yeast sequences. The E. coli strain W3110 / pRARE was transformed with DNA.

Expression of native Zven1 in BE coli:
E. coli is inoculated into 100 ml Superbroth II medium (Becton Dickinson, Franklin Lakes, NJ) containing 0.01% Antifoam 289 (Sigma), 30 μg / ml kanamycin, 35 μg / ml chloramphenicol, and Incubate overnight at 37 ° C. 5 ml of the inoculum was added to 500 ml of the same medium in a 2 L culture flask, which was shaken at 37 ° C. at 250 rpm until the culture reached an OD ₆₀₀ of 4. IPTG was then added to a final concentration of 1 mM and shaking was continued for an additional 2.5 hours. The cells were centrifuged at 4,000 xg for 10 minutes at 4 ° C. The cell pellet was frozen at -80 ° C.

Example 11 : Codon optimization:
A. Generation of a codon-optimized zven1 expression structure:
The native human Zven1 gene sequence could not be expressed in E. coli strain W3110. A test of the codon used for the Zven1 coding sequence showed that it contained an excess of the minimally used codon in E. coli, with a CAI value equal to 0.211. CAI is a statistical measure of similar codon bias and can be used to predict the level of protein production (Sharp et al., Nucleic Acids Res. 15 (3): 1281-95, 1987).

  Genes encoding highly expressed proteins tend to have high CAI values (> 0.6), and proteins encoded by genes with low CAI values (≦ 0.2) are generally expressed with low efficiency . This suggested a reason for the poor production of Zven1 in E. coli. In addition, rare codons are clustered in the second half of the message leading to a high probability of translational stalling, premature termination of translation and misincorporation of amino acids (Kane JF. Curr. Opin. Biotechnol. 6 (5): 494-500, 1995).

  It has been shown that the expression level of proteins whose genes contain rare codons can be dramatically improved if the level of certain rare tRNAs is increased in the host (danovsky et al., Supra., 2000; Calderone Etc., supra., 1996; Kleber-Janke et al., Supra., 2000; You etc., supra., 1999). The pRARE plasmid carries genes encoding argU, argW, leuW, proL, ileX and glyT for several codons that are rarely used in E. coli. The genes are under the control of their native promoter. Co-expression with pRARE enhanced Zven1 production in E. coli and produced approximately 100 mg / l. Co-expression with pRARE also reduced the level of cleaved Zven1 in E. coli lysates. These data suggest that re-synthesizing the gene encoding zven1 with more appropriate codon usage provides an improved vector for the expression of large amounts of zven1.

  The codon-optimized zven1 coding sequence (SEQ ID NO: 14) was composed of six overlapping oligonucleotides: zc45,048 (SEQ ID NO: 15), zc45,049 (SEQ ID NO: 16), zc45,050 (SEQ ID NO: 17), zc45,051 (SEQ ID NO: 18), zc45,052 (SEQ ID NO: 19) and zc45,053 (SEQ ID NO: 20). Primer extension of these overlapping oligonucleotides, followed by PCR amplification, generated a sufficiently long Zven1 gene with codons optimized for expression in E. coli. The final PCR product was inserted into the expression vector pTAP237 by yeast homologous recombination.

  The expression construct was extracted from yeast and used to transform competent E. coli DH10B for transformation. Clone resistance to kanamycin was identified by colony PCR. Positive clones were confirmed by sequencing and subsequently transformed into production host strain W3110. The expression vector with the optimized zven1 sequence was named by pSDH. The resulting gene was very well expressed in E. coli. The expression level for the new structure increased to about 150 mg / l.

Codon-optimized Zven1 expression in BE coli:
E. coli was inoculated into 100 ml Superbroth II medium (Becton Dickinson) containing 0.01% Antifoam289 (Sigma), 30 μg / ml kanamycin and cultured at 37 ° C. overnight. 5 ml of inoculum was added to 500 ml of the same medium in a 2 L culture flask, which was shaken at 250 rpm, 37 ° C. until the culture reached an OD ₆₀₀ of 4. IPTG was then added to a final concentration of 1 mM and shaking was continued for an additional 2.5 hours. The cells were centrifuged at 4,000 xg for 10 minutes at 4 ° C. Cell pellets were frozen at −80 ° C. until later use.

Example 12 : Purification and regeneration of Zven1 produced in E. coli:
A. Inclusion body isolation:
Following induction of protein expression in either batch fermentation or shaker flask culture, E. coli was centrifugated in a 1 L bottle at 3000 RM using a Sorvall rotating bucket rotor. Further washing of the cell paste to remove bouillon contaminants was performed with a 50 mM Tris (pH 8.0) solution containing 200 mM NaCl and 5 mM EDTA until the supernatant was clear.

  The cell pellet is then ice-cold lysis buffer (50 mM Tris pH 8.0; 5 mM EDTA; 200 mM NaCI, 10% sucrose (w / v) to an optical density of 10-20 at 600 nm. 5 mM DTT; 5 mM benzamidine). The slurry was then passed 2-3 times at a quenched APV200Lab homogenizer 12, 8500-9000 psi, producing a disrupted cell lysate. The insoluble fraction (inclusion bodies) was collected by centrifugation of the cell lysate at 20,000 xg for 1 hour at 4 ° C.

  Inclusion body pellets (derived from 20,000 × g rotation) are suspended in wash buffer (50 mM EDTA containing 200 mM NaCl, 5 mM DTT, 5 mM benzamidine) with 10 ml wash buffer per gram of inclusion bodies, And using OMNI international rotor fixed generator, it was completely dispersed. This suspension was centrifuged at 20,000 × g for 30 minutes at 4 ° C. The wash cycle was repeated 3-5 times until the supernatant was clear.

  The washed final pellet was dissolved in 8 M urea, 50 mM borate buffer (pH 8.6) containing 0.1 M sodium sulfite and 0.05 M sodium tetrathionate (pH 8.2). The dissolution and sulfite decomposition reactions were allowed to proceed overnight at 4 ° C. with gentle shaking. The resulting pinkish solution was centrifuged at 35,000 × g for 1 hour at 4 ° C., and the clear supernatant containing soluble Zven1 was filtered through a 0.45 μm filter.

B. Zven1 playback:
Dissolved Zven1 with 55 mM borate (pH 8.6), 1.0 M arginine, 0.55 M guanidine HCL, 10.56 mM NaCl, 0.44 mM KCl, 0.055% PEG, 10 mM reduced glutathione and 1.0 Regeneration was performed by the drop-mode dilution method at a final Zven1 concentration of 100-150 μg / ml in ice-cold regeneration buffer containing mM oxidized glutathione. Once diluted, the mixture was stirred slowly for 48-72 hours at cold room temperature.

C. Product recovery and purification:
After regeneration, the solution was clarified by centrifugation at 22,000 × g for 1 hour at 4 ° C. and filtration using a 0.45 μm membrane. The clarified supernatant containing the regenerated Zven1 was adjusted with 50 mM acetate and the pH was adjusted to 4.5 by the addition of HCl. The pH adjusted material was captured by cation exchange chromatography on a Pharmacia Streamline SP column (33 mm ID x 65 mm length) equilibrated with 50 mM acetate buffer (pH 4.5). The loading flow rate was 10 ml / min with in-line dilution adjusted to 1: 5 in 50 mM acetate buffer at pH 4.5. This dilution reduces the ionic strength and allows for effective binding of the target to the matrix. After sample loading was complete, the column was washed to baseline absorbance with equilibration buffer before elution step with 50 mM acetate buffer (pH 4.5) containing 1 M NaCl.

  The eluate pool from the cation exchange step was introduced into 1% acetic acid (pH 3.0) and equilibrated in 1% acetic acid (pH 3.0), a column containing TOSO Hass Amberchrom CG71m reverse phase medium (22 mm × 130 mm) ) At a flow rate of 10 ml / min. Based on washing to baseline absorbance, the column was eluted with a 20 column volume gradient formed between equilibration buffer and 99% (v / v) acetonitrile, 1% (v / v) acetic acid solution.

  The eluate pool from the reverse phase step was subjected to another step of cation exchange chromatography. The pool was loaded directly onto a Toso Hass SP650S column (10 mm × 50 mm) equilibrated with 50 mM acetate buffer (pH 4.5) at a flow rate of 3 ml / min. After completion of sample loading and washing to baseline absorbance, the column was eluted stepwise with 50 mM acetate buffer (pH 3.0) containing 1.0 M NaCl. The protein eluate pool was concentrated against a 3 KDa cut-off ultrafiltration membrane using Amicon concentration units in preparation for final purification and buffer exchange size exclusion steps.

D. Size exclusion buffer exchange and formulation:
The concentrated cation pool was injected onto a Pharmacia Superdex Peptide size exclusion column (Pharmacia, now Pfizer, La Jolla, California) equilibrated with a solution of 25 mM histidine, 120 mM NaCl (pH 6.5). Symmetrical eluate peaks containing the product were pooled, sterile filtered through a 0.2 μm filter, aliquoted and stored at −80 ° C.

Example 13 : Activity of Zven1 and Zven2 in a reporter assay:
A. Cell lines:
Rat2 fibroblasts (ATCC # CRL-1764, American Type Culture Collection, Manassass, VA) were transfected with the SRE luciferase reporter construct and stable clones were selected. They were then transfected with constructs for either the GPCR73a receptor (SEQ ID NO: 21) or the GPCR73b receptor (SEQ ID NO: 22).

B. Assay method:
Cells were trypsinized and seeded in a cloning 96-well white plate containing medium containing 1% serum at 3,000 cells / well and incubated overnight at 37 ° C. and 5% CO ₂ . The medium was removed and samples were added to the cells in triplicate in medium containing 0.5% BSA and incubated for 4 hours at 37 ° C. and 5% CO ₂ . After removing the medium, the cells were lysed and luciferase substrate was added according to the Promega luciferase assay system (Promega Corp., Madison, Wis.).

C. Data and conclusions:
All data were reported as fold-induction of RLU (relative light units) from the fermentor divided by the base signal (medium only). Zven1 was prepared in the laboratory. Zven2 used in the assay was purchased from PeproTech Inc. (Rocky Hill, NJ).
Tables 8 and 9 show that Zven1 was more active than Zven2 in a dose-dependent manner with respect to cells expressing the GPCR73a receptor.

  Tables 10 and 11 show that Zven1 and Zven2 are actively similar for cells expressing the GPCR73b receptor. The activity of both molecules was low in cells expressing the GPCR73b receptor. It is not known whether the number of GPCR73b receptors is equivalent in both cell lines.

  Table 12 shows that baculovirus-expressed Zven1 heated at 56 ° C. for 30 minutes has a lower activity than the new Zven1.

Example 14 : Activity of Zven1 in the organ bath:
Organ bath tests were performed with Zven1 using various tissues obtained from guinea pigs. A force transducer was used to record mechanical contraction using IOX software (EMKa technologies, Falls Church, VA) and data analysis software (EMKa technologies, Falls Church, VA). The tissues analyzed included: duodenum, jejunum, ileum, trachea, esophagus, aorta, stomach, gallbladder, bladder and uterus.

A. Organ bath method:
A 2-month-old male guinea pig (Hartley, Charles River Labs) weighing approximately 250-300 g was fasted (but with free access to drinking water) for approximately 18 hours and euthanized by CO ₂ nitrogen. All tissues were treated with Krebs buffer (1.2 mM MgS0 ₄ , 115 mM NaCl, 11.5 mM glucose, 23.4 mM NaHCO ₃ , 4.7 mM KC1, 1.2 mM NaH ₂ PO ₄ , and 2.4 mM CaCl _2. was oxygen or by _{oxygenated with 95% 0 2 -5%} C0 2, pH 7.4, temperature 37 ° C.) by rinsing, then suspended in an organ bath of 5 ml, and pre - and tensing. All tissues were tested with positive controls to establish their viability prior to implementation. The positive controls used were CCK-8, acetylcholine (ACH), histamine or 5HT and purchased from Sigma (Saint Louis, MO). All tissues were treated with a vehicle control, ie phosphate buffered solution, to prevent potential vehicle effects.

1) Tissue that did not give a response to Zven1 in the organ bath:
-Bronchial ring: A 3 mm wide bronchial ring (3 cm away from the heel organ branch) was collected and equilibrated with 5 g tension before any treatment. The positive control was 20 μg / ml ACH giving about 1 g of deflection. No effect of Zven1 was seen at 80 ng / ml.
-Aortic ring: A 3 mm wide aortic ring (immediately adjacent to the aortic arch) was collected and equilibrated with 4 g tension prior to any treatment. The positive control was 2 mg / ml KCl giving an average of 1 g of deflection. Zven1 at 80 ng / ml did not cause a visible effect.

-Esophagus: The 2 cm long esophagus (2 cm away from the cardia) was suspended and equilibrated with 1 g tension before any treatment. 2 mg / ml 5HT gave a deflection of about 1.4 g. Zven1 at 20 ng / ml had no visible effect.
-Gallbladder: The luminal fluid was aspirated with a 1 ml syringe, then suspended for a long time and equilibrated with 1 g tension prior to any treatment. 5 ng / ml ACH gave a deflection response of 0.4 g. 20 ng / ml of Zven1 had no effect.
Bladder: A 1.5 cm × 0.3 cm longitudinal strip was suspended and equilibrated to 0.5 g tension prior to any treatment. The positive control elicited a contractile response, but no activity was found at a Zven1 dose of 80 ng / ml.

2) Organizations responding to Zven1:
Stomach / pyloric sinus: A 1.5 cm × 0.3 cm long strip was suspended and equilibrated to 0.5 g tension prior to any treatment. Treatment with either 5 ng / ml ACH or 80 ng / ml CCK8 resulted in approximately 1 g of deflection. 80 ng / ml Zven1 also produced a contractile response with a deflection of approximately 0.5 g.
Duodenum: 2 cm long duodenum (2 cm away from the pylorus) was suspended and equilibrated with 1 g of stringency before any treatment. ACH gave a deflection of about 0.75 g. 20 ng / ml Zven1 also gave a deflection contraction response of about 0.5 g.

-Jejunum: 2 cm long jejunum (midpoint between pylorus and ileum-cecum junction) was suspended and equilibrated with 1 g tension prior to any treatment. ACH gave about 1.6 g deflection and 20 ng / ml Zven1 gave about 0.5 g deflection contraction response.
-Ileum: Collect the ileum 8 cm long (2 cm away from the ileum-cecum continuous part) and flush with Krebs buffer to remove any feces, cut into four equal pieces if present did. All tissues were suspended and equilibrated with 1 g tension prior to any treatment. The ileum was tested simultaneously to compare the Zven1 effect on the small intestine. ACH gave about 1.5 g of deflection and 20 ng / ml Zven1 also gave 1.5 g of deflection.

-Distal colon-A 2 cm long colon (2 cm away from the cecum) was suspended and equilibrated with 0.5 g tension prior to any treatment. Zven1 at 20 ng / ml induced a relaxing effect with decreased muscle tone and contraction amplitude.
The contractile effect of Zven1 is specific to the gastrointestinal tract. The best contractile response was seen in the ileum and lower contractility was seen in the duodenum, jejunum and pylorus. The relaxation effect in the distal colon is an indication of a coordinated effect on intestinal motility. Since smooth muscle contraction is contracted in the pylorus and small intestine, the large intestine is adjusted to accommodate an approaching meal due to relaxation. Consistent contractile activity between different parts of the intestine will lead to improved gastrointestinal function.

Example 15 : Comparative activity of Zven1 and Zven2 in tracheal tank:
Both Zven1 and Zven2 have a contractile effect on intestinal tissue in the organ bath. Side-by-side comparisons were made to compare activity in tissues from the same animal.
Ileum strips from guinea pigs were tested for contractility using the method described above. Zven2 was purchased from Peprotech Inc. (Rocky Hill, NJ). Activity was compared at concentrations of 40, 12 and 3 ng / ml. ACH at 5 ng / ml was used as a positive control. Contractile responses were normalized to ACH responses in individual tissues. All three doses were tested on separate longitudinal ileal tissue strips obtained from the same animal.
Results: Contractile effects are normalized to the ACH positive control and are expressed as the Zven1 or Zven2: ACH ratio in the table below.

結論：Zven1は、回腸における収縮性を比較する場合、Zven2の約２倍の活性である。

Conclusion: Zven1 is approximately twice as active as Zven2 when comparing contractility in the ileum.

Example 16 : Synergistic effect of Zven1 and Zven2:
To determine the combined effect of Zven1 and Zven2 on contractile activity, ileal tissue was pretreated with various doses of Zven2 followed by increasing doses of Zven1.
All tissues were stabilized, treated with ACH, and stabilized again, followed by pretreatment with Zven2 at a concentration of 0.8, 3.0, or 12 ng / ml. Zven2 was allowed to stand for approximately 20 minutes before administering 20 ng / ml of Zven1.

Results : A large 3 g deflection with Zven1 was observed when the tissue was pretreated with 0.8 ng / ml Zven2. Their contraction is greater than that normally observed with a 20 ng / ml dose of Zven1, where a contraction effect of about 1.5-2.0 g of polarized light is usually observed. Zven2 at 0.8 ng / ml alone has negligible contractile effects.
Conclusion : These data suggest that enhanced motor effects can be observed by pretreatment with low doses of Zven2 and then treatment with Zven1.

Example 17 : MIP-2 detection in lavage fluid and serum of mice following IP (intraperitoneal) injection of Zven1:
As discussed in Example 3 above, mouse KC is a mouse homologue of human GROα and CINC-1 is a rat homologue. Similarly, increased MIP-2 expression was found to be associated with neutrophil influx in various inflammatory conditions. See Banks, C. et al., J. Path. 199: 28-35,2003.

  Similar to the method used in Example 3, 4 groups of 10 mice were injected with vehicle control with Zven1 at 5 and 50 μg / kg or were not treated. Those mice weighed approximately 20 g, resulting in a dose of 5 μg / kg. MIP-2 levels were measured in both peritoneal lavage fluid and serum using the Quantikine M Murine mouse MIP-2 ELISA kit (R and D Systems, Minneapolis, Minnesota). The test results are shown in Table 14.

  Conclusion: MIP-2 is up-regulated in serum and lavage fluid in response to low (5 μg / kg) IP injection of Zven1. The concentration in serum is about 2-fold higher in animals treated with Zven1. There was a low effect in the wash fluid, but it is due to the fact that some activity occurs in the organism treated with the vehicle than in the untreated control animal. At higher doses (50 μg / kg dose) no effect is observed, suggesting that there is no chemotactic effect at elevated doses. These results correlated with neutrophil count, where it was observed only in animals that received a dose of Zven1 at a low neutrophil infiltration (5 μg / kg).

Example 18 : Generation of Zven1 polyclonal antibody:
Polyclonal antibodies were prepared by immunizing two female New Zealand white rabbits with the purified recombinant protein huzven1-CEE-Bv (SEQ ID NO: 24). Rabbits were prepared by initial intraperitoneal (ip) injection of 200 μg purified protein in complete Freund's adjuvant followed by ip booster every 3 weeks with 100 μg peptide in incomplete Freund's adjuvant. Seven to ten days after administration of the second additional immunization (total of three injections), the animals were bled and serum was collected. The animals were then boosted and exsanguinated every 3 weeks.

  Polyclonal antibodies were purified from immunized rabbit serum using a 5 ml protein A sepharose column (Pharmacia LKB). Following purification, the polyclonal antibody was dialyzed 20 times with 4 changes of antibody volume in PBS for at least 8 hours. Huzven1-specific antibodies were characterized by ELISA using 500 ng / ml of purified recombinant protein huzven1-CEE-Bv (SEQ ID NO: 24) as an antibody target. The low limit of detection (LLD) of rabbit anti-huzven1 purified antibody was 1 ng / ml based on its specific purified recombinant antigen huzven1-CEE-Bv.

Example 19 : Detection of Zven1 protein:
Purified polyclonal huzven1 antibodies were characterized for their ability to bind recombinant human Zven1 polypeptides using the ORIGEN ((E)) Immunoassay System (IGEN Inc, Gaithersburg, MD). In this assay, antibodies were used to quantitatively determine the level of recombinant huzven1 in rat serum samples. An immunoassay type consisting of a detector antibody that is labeled with a biotinylated capture antibody and a ruthenium (II) tris-bipyridal chelate, thereby sandwiching the antigen in solution and forming an immune complex did.

  Streptavidin-coated paramagnetic beads were bound to the immune complex. In the presence of tripropylamine, the ruthenylated Ab was quenched and this was measured by an ORIGEN analyzer. A concentration curve from 0.1 to 50 ng / ml of huzven1 allowed quantification using 50 μl of sample. The resulting assay showed a low limit of detection of 200 pg / ml huzven1 in 5% normal rat serum.

Example 20 : Effect of Zven1 in the ileum after surgery in vivo:
5-25 male Sprague-Dawley rats (approximately 240 g) per treatment group were used for their POI studies. The animals were fasted for about 22-23 hours (two floor grids are placed on their folds to prevent them from approaching their bed), but water is free to drink. Under gas isoflurane anesthesia, the abdominal hairs of the rats were shaved and wiped with betadine / 70% ethanol. A central line incision was then made through the skin and abdominal white lines (3-4 cm long), so that the intestine was visible and accessible.

  The cecum was manipulated with sterile salt solution-soaking gauze for 1 minute, using light beating pressure. This method is consistent from animal to animal to reduce ileal variability between animals. The white line was sutured with silk and the skin was closed with a wound clip. The animals were maintained on a water-coated healing pad during recovery from surgery and returned to their cages as soon as they recovered full consciousness.

  If fully conscious, the animals are given 1.0 ml of the test meal 15 minutes after completion of the cecal procedure (CM); after 1 or 20 minutes, the rats are 0.8 or 5 μg / kg BWE Colli-generated Zven1, or saline / 0.1% (w / v) BSA was administered through an endogenous carotid catheter. Zven1 was diluted with salt solution / 0.1% BSA to the desired concentration just before the study using a silicone-treated microcentrifuge tube (average BV of rats (about 240 g) and 0.1 ml injection volume (iv )On the basis of the).

  The test meal consisted of 1.5% (w / v) aqueous methylcellulose solution (400 centipoise medium viscosity methylcellulose from Sigma: catalog number M-0262), and non-absorbable dye, 0.05% (50 mg / 100 ml) Made of phenol red (Sigma catalog number P-4758; lot number 120k3660). Twenty minutes after administration of the test meal, the animals were anesthetized under isoflurane and killed by cervical dislocation. Gastric and intestinal segments were removed and the amount of phenol red in each segment was determined by spectrophotometric analysis (see below) and expressed as a percentage of total phenol red recovered per rat. These values were used to determine the amount of gastric emptying and intestinal transit per collected tissue.

  Colorimetric analysis of phenol red recovered from individual intestinal segments and stomach was performed according to a modification of the method outlined by Scarpinato and Bertaccini (1980) and Izbeki et al. (2002). For surgery, following euthanasia, stomach and intestinal fragments were placed in 20 ml of 0.1 N NaOH and homogenized using a Polytron tissue homogenizer. The Polytron was then rinsed with 5 ml of 1N NaOH and added to the previous 20 ml along with an additional 15 ml of 0.1N NaOH. The homogenate was allowed to settle for at least 1 hour at room temperature. The protein was precipitated from 5 ml supernatant by addition of 0.5 ml 20% trichloroacetic acid. Following centrifugation (3000 rpm for 15 minutes), 1 ml of the supernatant was added to 1 ml of 0.5N NaOH. A 0.2 ml sample (in a 96-well plate) was read at 560 nm using a Molecular Devices Spectra Max 190 spectrophotometer. Gastric emptying capacity and degree of intestinal passage were expressed as% of total phenol red recovered per rat.

  Data show that Zven1 (0.8 and 5.0 μg / kg, iv) showed gastric emptying and upper intestinal transit of this semi-solid non-nutritive diet compared to the excretion and passage observed in vehicle-treated rats. , Approximately 1.6 to 2 times, significantly increased. Efficacy in this model was observed when those doses of Zven1 were administered either 1 minute or 20 minutes after meal administration.

Example 21 : Effect of iv and ip BV- and E. coli-generated Zven1 on gastric emptying capacity and intestinal transit of a phenol red semi-solid diet in rats:
Male Sprague-Dawley rats (approximately 240 g) are used for this study and there are 6-12 animals per treatment group. The animals were fasted for about 22-23 hours (two floor grids are placed on their folds to prevent them from approaching their bed), but water is free to drink.

  One minute after administration of the 1.0 ml test meal, rats were administered various doses of Zven1 (0.01-30 μg / lg BW) or saline / 0.1% (w / v) BSA through an endogenous jugular vein catheter. For i.p. doses, Zven1 (0.1-100 μg / lg BW) or saline / 0.1% BSA was administered 1 or 10 minutes before or 1 minute after the meal. Zven1 was used with a siliconized microcentrifuge tube just prior to the study for the desired concentration (average BW of rats (about 240 g) with saline / 0.1% BSA, and 0.1 ml injection volume or ip for iv. Diluted based on an injection volume of 0.5 ml).

The test meal consisted of 1.5% (w / v) aqueous methylcellulose solution (400 centipoise medium viscosity methylcellulose from Sigma: catalog number M-0262), and non-absorbable dye, 0.05% (50 mg / 100 ml) Made of phenol red (Sigma catalog number P-4758; lot number 120k3660). Twenty minutes after administration of the test meal, the animals were anesthetized under isoflurane and killed by cervical dislocation.
Gastric and intestinal fragments were removed and the amount of phenol red in individual samples was determined by spectrophotometric analysis (see below) and expressed as a percentage of total phenol red recovered per rat. These values were used to determine the amount of gastric emptying and intestinal transit per collected tissue.

  Colorimetric analysis of phenol red recovered from individual intestinal segments and stomach was performed using Scarpinato et al. Arch If2t. Phannacodyn. 246: 286-294 (1980) and Piccinelli et al. Naufzyn-Schmiedeberg's Arch. Pharmacol 279: 75-82 (1973 ) According to a variant of the method outlined by For surgery, following euthanasia, stomach and intestinal fragments were placed in 20 ml of 0.1 N NaOH and homogenized using a Polytron tissue homogenizer. The Polytron was then rinsed with 5 ml of 1N NaOH and added to the previous 20 ml along with an additional 15 ml of 0.1N NaOH. The homogenate was allowed to settle for at least 1 hour at room temperature. The protein was precipitated from 5 ml supernatant by addition of 0.5 ml 20% trichloroacetic acid. Following centrifugation (3000 rpm for 15 minutes), 1 ml of the supernatant was added to 1 ml of 0.5N NaOH. A 0.2 ml sample (in a 96-well plate) was read at 560 nm using a Molecular Devices Spectra Max 190 spectrophotometer. Gastric emptying capacity and degree of intestinal passage were expressed as% of total phenol red recovered per rat.

  The gastric emptying capacity and intestinal transit of this semi-solid meal was enhanced approximately 2-fold following i.v. administration of 0.1-1.0 μg / kg BW BV- or E. coli-generated Zven1. Inhibitory effects on gastric emptying and intestinal transit were observed using high doses (10-100 μg / kg BW for ip dose; 30 μg / kg BW for iv dose) of BV- and E. coli-Zven1 . The observation of inhibition was particularly evident when those high doses of Zven1 were administered i.v. 1 minute after administration of the test meal or i.p. 10 minutes after administration of the test meal. Similar results were observed when Zven2 was administered at 30 μg / kg i.v.

Example 22 : Effect of iv BV- and E. coli-generated Zven1 on gastric emptying capacity and intestinal transit of a phenol red semi-solid diet in mice:
Female C57BJ / 6 mice 8-10 weeks old (consisting of 8 treatment groups and about 9 mice per group) were used for the study. The animals were fasted for about 20 hours in a cage containing a floor screen and access to water was free. Animals were weighed to determine the correct dose and their average weight was used to adjust protein concentration. Zven1 protein (20 mM Mes buffer / 20 mM NaCl, pH 6.5, or PBS, pH 7.2 stock solutions) dilutions were prepared in silicone-treated tubes just prior to injection.

  The dose was based on the average weight of the study animals (approximately 20 g) and was adjusted with saline / 0.1% (w / v) BSA to an injection volume of 0.1 ml per mouse. Zven1 and vehicle were administered by i.v. injection into the tail vein 1-2 minutes before receiving 0.15 ml of phenol red test meal as an oral lavage. The test meal has a 1.5% (w / v) aqueous methylcellulose solution (400-800 centipoise final viscosity) containing a non-absorbable dye, ie 0.05% phenol red (Sigma catalog number P-4758: lot number 120K3660), Medium viscosity carboxymethylcellulose from Sigma; catalog number C-4888; lot number 108H00052).

  Twenty minutes after administration of the test meal, animals were euthanized and stomach and intestinal fragments were removed. The small intestine was measured and divided into three equal pieces: proximal, middle, and distal gut. The amount of phenol red in each sample is determined by spectrophotometric analysis (as described in Examples 20 and 21 above) and expressed as a percentage of the total phenol red recovered per mouse. These values were used to determine the amount of gastric emptying and intestinal transit per collected tissue.

  The results showed that there was an increase in gastric emptying capacity and intestinal transit in mice treated with i.v. Zven1 at a dose of about 1-10 μ / kg BW. A tendency to inhibit gastric emptying and intestinal transit was observed with higher doses (50 μg / kg: i.v. or higher in mice) of Zven1.

Example 23 : Effect of BV- and E. coli-generated Zven1 on the general morphology of the stomach and intestine of urethane anesthetized rats:
Whether iv administration of BV- or E. coli Zven1 (dose up to 30 μg / kg BV and its dose; doses known to induce intestinal motility) affects the overall appearance of the stomach and small intestine To determine, studies were conducted in urethane-anesthetized male Sprague-Dawley rats.

  Rats were fasted for about 19 hours on a clean double floor ridge (with free access to water). Between 7:00 and 8:30 am, rats received an i.p. injection of urethane (0.5 ml / 100 g BW in 25% solution) and a jugular vein catheter was inserted. Anesthetized rats were returned to their pupae and maintained on a warm pad (maintained at 37 ° C.) throughout the day with additional i.p. doses of urethane administered as needed. Appropriate levels of anesthesia were monitored using the toe-pinning response test.

  About 5 minutes between animals, saline solution was passed through the jugular vein followed by vehicle (PBS) or BV- or E. coli-generated Zven1 at increasing doses (3.10 and 30 μg / kg BW; 0.1 ml injection volume) every 3 hours (total 43 μg / kg BW). A dilution of Zven1 protein was prepared just prior to injection. The dose was based on the body weight of the study animal (approximately 22 g) and was prepared to be included in 0.1 ml total volume of diluent (saline solution / 0.1% BSA). The protein was diluted using a siliconized microcentrifuge tube. Rats also received salt solution infusion through a Harvard pump at a rate of 0.5 ml / hr. Approximately 8-9 hours following the initial urethane, the rats were killed by cervical dislocation (under anesthesia) and their stomach and small intestine were removed for investigation and morphological evaluation.

  There was no evidence of gastric or intestinal trauma in any rat. Vehicle-treated rats had some black fluid in small pieces of the intestinal lumen; no black fluid was observed in Zven1-treated rats. There was a significant amount of mucus in the intestinal lumen in all treatment groups as a result of the urethane anesthesia and fasting protocol.

Example 24 : Effect of BV-generated Zven1 on in vivo gastrointestinal contractility in anesthetized laboratory mammals:
“Sonomicrometry” is a technique that uses piezoelectric crystals to measure gastrointestinal swellability, compliance and tension in vivo (Sonometrics, Corp. Ontario, Canada). The crystals are placed anywhere along the gastrointestinal tract of the laboratory mammal. Next, peristalsis and fragmentation contractions in the stomach and / or intestine can be accurately quantified and assayed in detail in response to administration of Zven1. This system provides a very detailed and elaborate measurement of gastric motility / contraction.

  This method of digital ultrasonography was followed by iv infusion of vehicle (saline / 0.1% (w / v) BSA) and increasing doses of BV-generated Zven1 followed by 10 rats (5 Motility and / or contraction in the ileum, duodenum, cecum and proximal colon as described in Adelson et al. Gastroenterology 122, A-554. (2002). Used to examine sex. For those experiments, piezoelectric crystals were bound to the appropriate intestine location using a drop of cyanoacrylate adhesive (Vetbond, 3M Animal Care, St. Paul, MN). After abdominal wall resection, urethane anesthetized rats were maintained at 37 ° C. through a feedback-regulated heater. Sonographs distance signals are transmitted through a digital sonometer (TRX-13, Sonometrics Corp, London ONT) connected to a Pentium III class computer running SonoLAB software (Sonometrics Corp, London, Ontario, Canada) Obtained continuously at a rate of 50 samples / second.

  Digitally obtained distance data was recorded simultaneously as a similar signal through an installed 4-channel DAC. Their sonic measurement-like signals, together with all the similar physiological data (rectal temperature, blood pressure, EKG, heart rate), to enable simultaneous observation and analysis of the experimental progress, Spike 2 (Cambridge Electronic Design, Ltd , Cambridge) was obtained using a Micro1401 A / D interface (Cambridge Electronic Design, Ltd, Cambridge) linked to a Pentium II class computer running data acquisition software. This method allows simultaneous observation of distance measurements for four crystal pairs. Baseline levels were obtained between individual vehicles and Zven1 injections. Both circular and longitudinal motions were monitored using a 1 mm diameter piezoelectric crystal ternary (Sonometrics Corp.), so that two of the three were oriented parallel to the longitudinal axis and 3 The second was oriented vertically.

  The motor response to the applied stimulus was analyzed separately for the persistent and gradual components of the response comprising a persistent and / or gradual component. The persistent component of the trace was obtained by replacing individual points in the trace by the middle value of the trace over 10 surroundings. The stepped component was obtained by applying a smooth function inverse operation with 10 windows to the original trace, ie removing the “DC component” with a time constant of 10. Sustained response was analyzed by average during one response, i.e. 1 min max range from baseline, duration of response, and integrated response (average standardized response duration). . Graded activity was analyzed by its speed and amplitude. Changes in the relationship between motility in different gut regions measured simultaneously were analyzed using continuous signal cross-correlation and peak position development correlation.

  A strong contractile response is observed in the ileum of Zven1-treated rats at iv doses as low as 3 μg / kg BW; contractility is also not as strong as that observed for the ileum but is also shown in the jejunum and duodenum It was done. Responses related to relaxation were observed in the proximal colon.

Example 25 : Effect of ip administration of BV-generated Zven1 on proximal colon transit in conscious mice:
Adult male C57 / BL6 (6-8 weeks old; Hurlan, San Diego, Calif.) Was used for this study and there were 6-10 animals per treatment group. Mice are maintained at a controlled temperature (21-23 ° C) and humidity (30-35%), maintained on a 12:12 hour light-dark cycle, and are free to food (Purina Chow) and tap water Contained in a group that can approach. Mice were fasted from food for 18-20 hours with free access to water prior to the experiment. BV-generated Zven1 was stored at −80 ° C. in a stock solution of 20 mmol MES and 20 mmol NaCl at pH 6.5. On the day of the experiment, Zven1 was diluted in 0.9% NaCl and 0.1% BSA. The pH for both vehicle and Zven1 at various doses was 6.5.

  Distal colon transit was measured as previously described (Martinez V, et al. J Pharmacol Exp Ther 301: 611-617 (2002.)). Fasted mice can be freely anesthetized with Enflurane (1-2 minutes; Ethrane-Anaquest, Madison, WI), single 2 mm, with free access to water and a pre-weighed Purina chew for 1 hour. Glass beads were inserted into the distal colon, 2 cm from the anus. Bead insertion was performed with a glass rod having a flame-disinfected end to avoid tissue damage. After bead insertion, mice were placed in their cages without food and water, respectively. Mice recovered consciousness within 1-2 minutes and then showed normal behavior. The distal colon transit was determined to be the nearest 0.1 minute by monitoring the time required for glass bead elimination (bead latency).

  At the end of the 1 hour meal period, mice were anesthetized with enflurane for bead insertion into the colon followed by intraperitoneal injection of vehicle or Zven1 (3, 10, 30 or 100 μg / kg). The animals were returned to their original cage without food or water and the bead elimination time was monitored. Results were expressed as mean ± S.E. And analyzed using one-way ANOVA.

  BV-Zven1 (3, 10 and 30 μg / kg) in mice that have been fasted for 18-20 hours and fed a re-meal for 1 hour and injected ip with Zven1 (3, 10, 30 and 100 μg / kg) No significant change in bead elimination time in response to ip injections of 32.7 ± 6.1, 23.1 ± 4.5 and 34.2 ± 5.6 minutes, respectively, compared to 21.1 ± 3.9 minutes in the ip vehicle injected group It was. In a second group of mice treated in the same manner but receiving a high dose of BV-Zven1, the measurement of distal colon transit was determined by ip injection of BV-Zven1 (30 and 100 μg / kg) beads. Showed a dose-related trend that increased elimination time (29.8 ± 7.8 minutes and 35.1 ± 3.7 minutes, respectively, compared to 22.3 ± 5.7 minutes after vehicle ip injection), but the changes were statistically significant Did not reach.

Example 26 : Expression of GPR73a and GPR73b in rat gastrointestinal tract:
Rats were fasted and killed overnight. The intestine and stomach were separated and a 4 cm tissue fragment from the stomach through the end of the colon was immediately frozen in liquid nitrogen. An acid-phenol extraction method was used for RNA isolation. Briefly, tissue fragments were ground in liquid nitrogen and then acid guanidinium-based lysis buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7), 0.5% sarcosyl), NaOAc (0.1 M final concentration) ) + ΒME (1: 100). The lysate was spun down; the supernatant was mixed with an equal volume of acid phenol and 1/10 volume of chloroform. After rotary sedimentation, an equal volume of isopropanol was added to the aqueous layer. Samples were incubated at −20 ° C. and then pelleted by rotary sedimentation. The pellet was washed with 70% ethanol and then resuspended in DEPC treated water.

  GPR73a and GPR73b receptor expression levels were determined using Taqman EZ RT-PCR Core Reagent Kit (Applied biosystems, Foster City, CA). According to the manufacturer's instructions, a standard curve was prepared with one of the RNA isolates that had high quality RNA and showed expression of both receptors at the same level. The standard curve dilution of this RNA sample was adjusted to the following concentrations: 500 ng / μl, 250 ng / μl, 100 ng / μl and 12.5 ng / μl. These standard curve dilutions were used to test primers designed for the GPR73a and GPR73b genes and for the housekeeping gene, ie, rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). . Once primer and standard curve working conditions were established, RNA samples isolated from rats were tested.

  RNA samples were thawed on ice and diluted to 100 ng / μl in RNase free water (Invitrogen, catalog number 750023). Diluted samples were kept on ice during the experiment. Master mix was prepared for GPR73a, GPR73b receptor and housekeeping gene using TaqMan EZ RT-PCR Core Reagent Kit (Applied Biosystems, Cat # N808-0236). To assay the samples in triplicate, 3.5 μl of individual RNA samples were aliquoted. For positive controls, 3.5 μl of individual standard curve dilutions were used instead of sample RNA. For the negative control, 3.5 μl RNase free water was used for the templateless control. For the endogenous control (rodent GAPDH message), 3.5 μl of both standard curve dilution and sample RNA were aliquoted. Next, 84 μl of PCR master mix was added and mixed well by pipette.

A MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems catalog number N801-0560) was placed on ice and 25 μl of RNA / master mix was added in triplicate to the appropriate virus. All plates were then coated with MicroAmp 12-Cap Strips (Applied Biosystems catalog number N801-0534). The plates were then spun for 2 minutes at 3000 RPM in a Qiagen Sigma 4-15 centrifuge.
Samples were spun on PE-ABI 7700 (Perldn Elmer, now EG & G, Inc. Wellesley, MA). A sequence detector was set up and the default was set to Real Time PCR. The fluorescent dye was set to FAM. A plate mold was set up to show where standard and unknown test samples were present.

  Expression for individual samples is reported as Ct values. The Ct value is the point at which fluorescent dyes or RT-PCR products (a direct opposite of RNA multiplicity) are amplified to a level above the limit or background level. RT-PCR of highly expressive samples results in a higher accumulation of fluorescent dye / product that quickly crosses the limit, so lower Ct values result in higher expression levels. A Ct value of 40 means that no product is present and should yield an average expression value of zero. Ct is converted to a relative expression value based on comparison to a standard curve. For the individual samples tested, the amount of GPR73a, GPR73b and GAPDH expression levels was determined from the appropriate standard curve. The calculated expression of those GPR73a and GPR73b was then divided by the GAPDH expression of the individual samples to obtain a normalized expression for the individual samples. Individual normalized expression values were divided by the normalized-calibration values to obtain relative expression levels. Using the GraphPad Prism software, these normalized values were converted to the rate where the highest expression level is shown as 1.

Example 27 : Zven1 and monoclonal antibodies:
Rat monoclonal antibodies were prepared by immunizing 4 female Sprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.) With the purified recombinant protein from Examples 6 or 7 above. Rats were injected with 25 μg of purified recombinant protein in the initial intraperitoneal (IP) in complete Freund's adjuvant (Pierce, Rockford IL), followed by purified recombinant protein in incomplete Freund's adjuvant every two weeks. Each additional 10 μg IP injection is given. Seven days after administration of the second boost, the animals are exsanguinated and serum is collected.

Zven1-specific rat serum samples are characterized by ELISA using 1 μg / ml purified recombinant protein Zven1 as a specific antibody target.
Spleen cells are harvested from a single high titer rat and fused to SP2 / 0 (mouse) myeloma cells using PEG1500 by a single fusion method (4: 1 fusion, spleen cells: Myeloma cells, Antibodies: A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Press).

After 9 days-after fusion growth, the specific antibody-producing hybridoma pool was purified by radioimmunoprecipitation (RIP) using I ¹²⁵ -labeled recombinant protein Zven1 as specific antibody target and specific Identification by ELISA using 500 ng / ml of recombinant protein Zven1 as a target antibody target. Cell-growth of purified recombinant protein Zven1 on Baf3 cells expressing the receptor sequence of GPR73a (SEQ ID NO: 27) and / or GPR73b (SEQ ID NO: 28) positive hybridoma pools in either assay protocol Further analysis is made of their ability to block activity (“neutralization assay”).

Hybridoma pools that produce positive results by RIP alone or by RIP and “neutralization assay” are cloned at least twice by limiting dilution.
Monoclonal antibodies purified from tissue culture media are characterized by their ability to block the cell-proliferative activity ("neutralization assay") of the purified recombinant protein Zven1 on Baf3 cells expressing the receptor sequence . “Neutralizing” monoclonal antibodies are identified in this manner.

According to a similar method, the monoclonal antibody against Zven2 is identified using the amino acid sequence in SEQ ID NO: 5.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described for purposes of illustration, various modifications may be made within the scope of the invention.

Claims (34)

  A method for regulating gastrointestinal contractility in a mammal comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2.   A polypeptide comprising the amino acid sequence of amino acid residues 20 to 105 shown in SEQ ID NO: 5 is administered to the mammal in need thereof, followed by comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2. A method of stimulating gastrointestinal contractility comprising administering a polypeptide.   A method for regulating gastric emptying in a mammal, comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2.   A method for regulating gastric transit in a mammal comprising administering to the mammal in need thereof a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2.   5. A method according to claim 1, 3 or 4, wherein said adjustment is inhibition.   5. The method of claim 1, 3 or 4, wherein the polypeptide is administered in one or more administrations.   One or more administrations of the polypeptide stimulate gastrointestinal contractility, gastric emptying or intestinal passage, and one or more administrations of the polypeptide inhibit gastrointestinal contractility, gastric emptying or intestinal passage The method according to claim 1, 3 or 4.   Claims 1, 3 wherein the one or more administrations that stimulate gastrointestinal contractility, gastric emptying or intestinal passage are performed prior to one or more administrations that inhibit gastrointestinal contractility, gastric emptying or intestinal passage. Also, the method according to 4.   Claims 1, 3 wherein the one or more administrations that inhibit gastrointestinal contractility, gastric emptying or intestinal passage are performed prior to one or more administrations that stimulate gastrointestinal contractility, gastric emptying or intestinal passage. Also, the method according to 4.   The method of claim 1, 3 or 4 wherein therapeutic control of intestinal transit is achieved.   The method according to claim 1, 3 or 4, wherein the polypeptide is administered continuously for a certain period of time.   A method of treating gastric paresis in a mammal in need thereof, comprising administering the polypeptide to a mammal, said polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2, And a method in which gastrointestinal contractility, gastric emptying capacity or intestinal passage is improved.   13. The method of claim 12, wherein the gastric paresis is associated with surgery.   14. The method of claim 13, wherein the polypeptide is administered to the mammal before or after surgery.   14. The method of claim 13, wherein the polypeptide is administered to the mammal before or after the mammal is fed a post-operative meal.   13. The method of claim 12, wherein the treatment is characterized by increased contractility in intestinal obstruction.   14. The method according to claim 13, wherein the gastric insufficiency is post-surgical or paralytic intestinal obstruction.   13. The method of claim 12, wherein the gastric paresis is not associated with surgery.   13. The method of claim 12, wherein the gastric failure is associated with diabetes, intestinal pseudo-obstruction, chronic constipation, reduced digestive function, gastroesophageal reflux disease, vomiting, paralytic gastric failure, sepsis, or drug consumption.   In a mammal comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5 to the mammal in need thereof A method of stimulating chemokine release.   In a mammal comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5 to the mammal in need thereof A method of stimulating neutrophil infiltration.   In a mammal comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5 to the mammal in need thereof A method of inducing or increasing appetite or weight gain.   In a mammal comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5 to the mammal in need thereof A method to increase sensitization to heat, mechanical or painful stimuli.   In a mammal comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28-108 of SEQ ID NO: 2 or the amino acid sequence of amino acid residues 20-105 of SEQ ID NO: 5 to the mammal in need thereof A method of reducing sensitization to heat, mechanical or painful stimuli.   25. The method of claim 24, wherein the antagonist is an antibody that specifically binds to the polypeptide.   A method of inducing angiogenesis in cardiac stem cells, comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2.   27. The method of claim 26, wherein said angiogenesis is induced ex vivo or in vitro.   A method of inducing angiogenesis in cardiac stem cells, comprising administering a polypeptide comprising the amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO: 2.   30. The method of claim 28, wherein the neoplasia is induced ex vivo or in vitro. a) amino acid residues 28-208 of SEQ ID NO: 2;
b) amino acid residues 20 to 105 of SEQ ID NO: 5; and c) amino acid residues 28 to 129 of SEQ ID NO: 29
A method for regulating gastrointestinal contractility, gastric emptying or intestinal transit in a mammal, comprising administering to the mammal in need thereof a polypeptide comprising an amino acid sequence selected from:   32. The method of claim 30, wherein the polypeptide is administered orally, intraperitoneally, intravenously, intramuscularly or subcutaneously.   An isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO: 14.   A method for producing a polypeptide comprising the step of growing a recombinant host cell comprising an expression vector comprising the isolated nucleic acid, transcription promoter and transcription terminator of claim 32, wherein the promoter comprises A method wherein the nucleic acid is operably linked by the nucleic acid, the nucleic acid is operably linked by the transcription terminator, and the protein encoded by the nucleic acid is produced by the recombinant cell.   34. A polypeptide produced by the method of claim 33.