WO2002033077A1 - Novel polypeptide--- a regulatory protein 10.01 being related to human development and polynucleotide encoding it - Google Patents

Abstract

The invention concerns a regulatory protein 10.01 being related to human development and polynucleotide encoding it. The invention also concerns the process of producing the polypeptide by recombinant DNA technique. The methods for treating many diseases e.g. malignant tumor, hemopathy, infection of HIV, immunological diseases and a variety of inflammation etc. utilizing the polypeptide are disclosed. The invention discloses the antagonist against the polypeptide and therapeutics thereof. The invention also discloss the uses of the polynucleotide, which encodes a regulatory protein 10.01 being related to human development.

Description

A novel polypeptide-development-regulation related protein 10.01 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide-development-regulation-related protein 10.01, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. technical background

 Many proteins contain a cysteine-rich conserved domain called the LIM domain. This domain combines with regulatory regions of developmental control genes (such as PRD, GSB homeodomain proteins). It consists of 60 Amino acid composition. The LIM protein is divided into two subfamilies: A and B. The A subfamily includes SF3, hCRP, CRIP, ESP-1, and the B subfamily includes 7 other proteins. These proteins usually have a LIM domain at the N-terminus, with the exception of Zyxin and paxillin. They contain 3 and 4 LIM domains, respectively, and are located at the C-terminus. For adapterous, isl-1, LH-2, lin-11, lim-1, lim-3, lmx-1, ceh-14 and niec-3, there is a homology of 50-95 amino acids after the LIM domain Box domain. Among them, the LIM of lin-11 protein is a metal domain containing iron, sulfur, and zinc. The LIM protein containing iron-sulfur clusters may act as a regulator of oxidation-reduction-sensitive transcripts and respond to oxygen-reduction-active molecular signals such as oxygen. Pollen-specific protein SF3 controls the maturation, pollen tube formation, and fertilization of male zygote [Plant Cell 1992, 4: 1465-1466].

 In the LIM domain, there are 7 conserved cysteines and 1 histidine, and the conserved sequences are: Cx (2) -Cx (16, 23) -HX (2)-[CH] -x ( 2) -Cx (2) -Cx (16, 21) -Cx (2, 3)-[CHD]. This domain binds to two zinc ions, but is not a DNA-binding protein. It is more likely to serve as a protein-protein interaction interface. The last residue at the second zinc ion binding site is usually Asp [Plant Cell 1992, 4 : 1465-1466].

 The protein of the present invention contains a LIM domain, has the similar structure as above, and has a consistent conserved sequence. Therefore, it is considered to be a new LIM domain-containing protein, named as a development regulation-related protein 10.01, which is similar to the aforementioned protein Biological functions that regulate development and differentiation. It is widely distributed in various tissues and organs of the organism. If the expression is abnormal, it will destroy the normal physiological development and differentiation of the organism. In addition, it also plays a role in the diagnosis and treatment of related diseases.

As described above, the development regulation related protein 10.01 protein plays an important role in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more development regulation related protein 10.01 proteins involved in these processes Especially the amino acid sequence of this protein Column. New development regulation related protein 10. 01 The isolation of protein-coding genes also provides a basis for the study to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Object of the invention

 An object of the present invention is to provide an isolated novel polypeptide-development-regulation related protein 10.01, and fragments, analogs and derivatives thereof.

 Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

 Another "object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a development regulation-related protein 10. 01 .;

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a development regulation-related protein 10.01.

 Another object of the present invention is to provide a method for producing a protein related to development regulation 10. 01.

 Another object of the present invention is to provide an antibody against the polypeptide-development regulation-related protein 10.01 of the present invention.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, a development regulation-related protein 10.01.

 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities in development regulation-related proteins 10. 01. Summary of invention

 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

 The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

 (b) a polynucleotide complementary to polynucleotide (a);

 The polynucleotide sequences of (c) and (a) or (b) have at least 70 ° /. Identical polynucleotides.

 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 169-444 in SEQ ID NO: 1; and (b) a sequence having 1-2291 in SEQ ID NO: 1 Sequence of bits.

The invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention; The vector genetically engineered host cell includes a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an expression product.

 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit development regulation related protein 10. 01 protein activity, which comprises using the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a development regulation-related protein 10.01 protein, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.

 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of development-regulation-related protein 10.01.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. BRIEF DESCRIPTION OF THE DRAWINGS

 The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention as defined by the claims.

 Fig. 1 is a comparison diagram of the amino acid sequence homology of a total of 59 amino acids and domain MOTIF in the development regulation-related protein 10.01 of the present invention at 7-65. The upper sequence is the development regulation related protein 10. 01, and the lower sequence is the domain M0TIF. '屮' and ":" and "indicate that the probability that the same amino acid appears between the two sequences decreases in order.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated development regulation related protein 10. 01. 10. OlkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention

The following terms used in this specification and the claims shall have the following meanings unless specifically stated: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic D or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

 "Insertion" or "addition" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.

 An "agonist" is a molecule that, when combined with a protein related to developmental regulation 10.01, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to development-related proteins 1 0.01.

 • "Antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of development-regulating related protein 1.0.01 when combined with development-regulating related protein 1.01. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to development-related proteins 10.01.

 "Regulation" refers to a change in the function of a developmental regulation-related protein 10. 01, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of the developmental regulation-related protein 1. 01. Change.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify development regulation related proteins using standard protein purification techniques

1 0. 01. A substantially pure developmental regulation-related protein 1 0. 01 produces a single main band on a non-reducing polyacrylamide gel. The purity of the development regulation related protein 1 0.01 can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". Two The complementarity between individual single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction. ,

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, D. G. and P. M. Sharp (1988)

Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:

 Number of residues matching between sequence ^ and sequence i?

Residues of a sequence interval residues ^ - ^ interval sequence of residues ^X

 The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in enzymology 183: 625-645).

 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitution, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

 "Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence. The "antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') ₂ F _V , which can specifically bind to the antigenic determinant of development-regulating related protein 10. 01. A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

 The term "isolated" refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system. Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.

 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .

 As used herein, "isolated development regulation related protein 10. 01" means development regulation related protein 10. 01 is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. Those skilled in the art can purify development regulation related proteins using standard protein purification techniques 10. 01. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Development regulation related protein

10. The purity of 01 peptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a development regulation-related protein 10. 01, which basically consists of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The present invention also includes fragments, derivatives and analogs of development-regulating related protein 10. 01. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the development regulation-related protein 10.01 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by the genetic code; or (Π) such a type in which one or more amino acid residues are substituted by other groups to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Leader sequence Or secreted sequences or sequences used to purify this polypeptide or protein sequences). As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 2291 bases, and its open reading frame 169-444 encodes 87 amino acids. This peptide has the characteristic sequence of MOTIF, and it can be deduced that the development regulation related protein 10. 01 has the structure and function represented by MOTIF.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include _C DNA, genomic DNA, or synthetic DM. DNA can be single-stranded or double-stranded. The DM can be a coding chain or a non-coding chain. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6CTC; or (2) during hybridization Add a denaturant, such as 50¾ (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42. C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2. The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding development-related proteins 10.01.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the development regulation-related protein 10.01 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

 Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for raRNA extraction, and kits are also commercially available (Qiagene). Building cDNA libraries is also a common method (Sarabrook, et ai., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of transcripts of development-regulation-related protein 10.01; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used alone or in combination.

 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

In the (4) method, the protein product of the 10.01 gene expression of the development regulation related protein can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.

 A method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

 The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a development regulation related protein 10.01 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .

 In the present invention, a polynucleotide sequence encoding a development regulation related protein 10.01 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding a development regulatory related protein 10.01 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known controllable genes in prokaryotic cells Promoters expressed in cells or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.

 In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding a development regulation related protein 10.01 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl. The steps used are well known in the art. The alternative is to use MgCU ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 By the conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant development regulation-related protein 1 0.01 (Sc ience, 1 984; 224: 1431). Generally there are the following steps:

 (1) using the polynucleotide (or variant) encoding the human development regulation-related protein 1 0.01 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells.

In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When host cells grow to proper After inducing the cell density, the appropriate promoter (such as temperature conversion or chemical induction) is used to induce the selected promoter, and the cells are cultured for a period of time.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 The polypeptides of the present invention, as well as the antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

 The LIM domain is usually used as a cysteine-rich conserved domain in many proteins. This domain combines with regulatory regions of developmental control genes (such as PRD, GSB homeodomain proteins) and participates in the regulation of developmental gene expression . LIM proteins are divided into two subfamilies: A and B. These family proteins usually have two LIM domains in tandem, and a few LIM proteins, such as the in-11 protein, LIM are metal domains containing iron, sulfur, and zinc. The LIM protein containing iron-sulfur clusters may act as a regulator of oxidation-reduction-sensitive transcripts and respond to oxygen-reduction-active molecular signals such as oxygen. The LIM domain-specific conserved sequence contained in the polypeptide of the present invention is necessary for the formation of its active functional domain, and is mainly involved in the regulation of development. It can also be used as a regulator of oxidation-reduction-sensitive transcripts and plays a role in eliminating free radicals in the body着 效应。 Effect.

 It can be seen that the abnormal expression of the LIM functional domain of the specific LIM family protein will cause the functional abnormality of the polypeptide containing the functional domain of the present invention, resulting in the down-regulation of its developmental regulatory function, as a regulator of redox-sensitive transcript The effect is reduced, and related diseases such as embryonic development disorders, growth and development disorders, tumors and the like are generated.

 It can be seen that the abnormal expression of the development regulation-related protein 10.01 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, and immune diseases. These diseases include but are not Limited to:

 Tumors of various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma

Embryonic disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness Growth and development disorders: mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation

 Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

 The abnormal expression of the development regulation-related protein 10.01 of the present invention will also cause certain hereditary and hematological diseases.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth and development disorders, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc.

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) developmental regulation related proteins 10. 01. Agonists increase development-regulation-related proteins 10. 01 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing a development regulation-related protein 1 0.01 can be cultured together with a labeled development regulation-related protein 10.01 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of development-related proteins 10. 01 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of development regulation related protein 10. 01 can bind to development regulation related protein 10. 01 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.

 When screening compounds as antagonists, developmental regulation-related protein 10.01 can be added to bioanalytical assays to determine whether a compound is a compound by measuring the effect of the compound on the interaction between developmental regulation-related protein 10.01 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Peptide molecules capable of binding to developmental regulation-related proteins 10.01 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the molecule of development regulation related protein 10. 01 should be generally labeled.

The present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides Antibodies against the epitope of development-related protein 10.01 were identified. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

 The production of polyclonal antibodies can be obtained by direct injection of development-related proteins 10. 01 to immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent. Techniques for preparing monoclonal antibodies for development regulation related protein 10. 01 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, and human beta-cell hybridoma technology , EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against development-regulating related proteins 10. 01.

 Antibodies against development regulation related protein 10. 01 can be used in immunohistochemistry to detect development regulation related protein 10. 01 in biopsy specimens.

 Monoclonal antibodies that bind to development-regulating related proteins 10. 01 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, development-related proteins 10. 01 High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill development-regulating related proteins 10. 01 positive cell.

 The antibodies of the present invention can be used to treat or prevent diseases related to development regulation-related protein 10.01. Administration of appropriate doses of antibodies can stimulate or block the production or activity of development-related proteins 10.01.

 The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of development regulation-related protein 10.01. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of development-regulated protein 10.01 detected in the test can be used to explain the importance of development-regulated protein 10.01 in various diseases and to diagnose diseases where development-regulated protein 10.01 plays a role.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

The polynucleotide encoding the development-regulation-related protein 10.01 can also be used for a variety of therapeutic purposes. Genetic Therapeutic techniques can be used to treat cell proliferation, development, or metabolic abnormalities caused by the non-expression or abnormal / inactive expression of development regulation-related protein 10.01. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated development regulation related protein 10.01 to inhibit endogenous development regulation related protein 10.01 activity. For example, a mutated development regulation related protein 10.01 may be shortened and lack a signal transduction domain development regulation related protein 10.01. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat development-related proteins

10.01 Diseases caused by abnormal expression or activity. Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer a polynucleotide encoding a development-regulation related protein 10.01 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a development regulation-related protein 10.01 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding a development-regulating protein 10.01 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit development regulation related protein 10.01 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

The polynucleotide encoding the development regulation related protein 10.01 can be used for the diagnosis of diseases related to the development regulation related protein 10.01. The polynucleotide encoding the development regulation related protein 10.01 can be used to detect the expression of development regulation related protein 10.01 or the abnormal expression of development regulation related protein 10.01 in a disease state. For example, the DM sequence encoding the development regulation related protein 10.01 can be used to hybridize biopsy specimens to determine the expression of development regulation related protein 10.01. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also called a "gene chip") for analysis. Analysis of differential expression of genes in tissues and genetic diagnosis. Development-specific related protein 10.01 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the transcriptional products of development-related related protein 10.01.

 Detection of mutations in the development regulation related protein 10.01 gene can also be used to diagnose development regulation related protein 10.01 related diseases. Developmental regulation-related protein 10.01 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type developmental regulation-related protein 10.01 DM sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Vern et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals, and the mutation is not observed in any normal individual, The mutation may be the cause of the disease. Comparing diseased and oncoming individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cD accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Developmental regulation related protein 10. 01 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of the developmental regulation-related protein 10.01 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually performed according to the general conditions such as Sambrook et al., Molecular Cloning: Laboratory conditions (New York: Cold Spr ng Harbor Labora tory Press, 1989), or Follow the conditions recommended by the manufacturer.

 Example 1 Cloning of Development Regulation Related Protein 1 0.01

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. A Smart cDNA cloning kit (purchased from Clontech) was used to orient the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a, and the bacteria formed a cDNA library. Dye terminate cyc le react ion sequenc ing kit (Perk in-Elmer) and ABI 377 An automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones. Comparing the determined cDNA sequence with the existing public DM sequence database (Genebaiik), it was found that the cDNA sequence of one of the clones 2296f05 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 2296f05 clone contained a full-length cDNA of 2291bp (as shown in Seq ID NO: l), and a 264bp open reading frame (0RF) from 169bp to 444bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-2296f 05, and named the encoded protein as a development regulation related protein 10. 01. Example 2 Domain analysis of cDNA clones

 The sequence of the development-regulation-related protein 10.01 of the present invention and the protein sequence encoded by the same were applied to a profiling scan tool (Basic local al ignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote. The development-regulating related protein 10.01 of the present invention is homologous to the domain MOTIF, and the homology result is shown in Fig. 1. The homology rate is 0.19, the score is 10.92; the threshold value is 10.44. Example 3 Cloning of a gene encoding a development regulation related protein 10.01 by RT-PCR

 CDNA was synthesized using fetal brain total RM as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5 -CATCCTGAGAACTGAAATTGATCGC-3 '(SEQ ID NO: 3)

 Primer2: 5 -ATAAAATTTTTGAATTTATGTTCAA-3 '(SEQ ID NO: 4)

 Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

 Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

Amplification conditions: 50 mmol / L KC1, 10 mraol / L Tris-HCl, pH 8.5, 1. 5 ramol / L MgCl ₂ , 200 μ mol / L dNTP, lOpmol primer, 1U in 50 μ 1 reaction volume Taq DM polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the l-2291bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of the expression of development-regulation-related protein 10.01 gene Total RM was extracted in one step [Anal. Biochem 1987, 162, 156-159]. The method includes acid sulfur Guanidinium cyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Perform electrophoresis on a 1.2% agarose gel containing 2 (ig RNA) on 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5 mM sodium acetate-IfflM EDTA-2.2M formaldehyde. Then transfer Onto a nitrocellulose membrane. A- ³² P dATP was used to prepare a ³² P-labeled DNA probe by a random primer method. The DNA probe used was the PCR-encoded development regulatory related protein 10.01 coding region sequence shown in FIG. 1 (169bp to 444bp). 32P-labeled probes (approximately 2 x 10 ⁶ cpm / ml) were hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide -25mM KH ₂ P0 ₄ (pH7.4)-5 x SSC-5 x Denhardt's solution and 200 g / mL salmon sperm DNA. After hybridization, the filter was washed in i _x SSC-0.1% SDS at 55 "C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 5 In vitro expression, isolation and purification of recombinant development regulation related protein 10.01

 Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Primer 3: 5'- CCCCATATGATGCTCTGTCACCTTCAAAGGATGG- 3 '(Seq ID No: 5) Primer4: 5-CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively. The coding sequences of the 5 'and 3' ends of the target gene are followed, respectively. The Ndel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3). Enzyme site. The PCR reaction was performed using pBS- ² 296f05 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-2296f 05 plasmid, primers Primer-3 and Primer- 4 points, and 'J is lOpniol, Advantage polymerase Mix

(Clontech) 1 μ1. Cycle parameters: 94 ° C 20s, 60 »C 30s, 68 ') C 2 min, a total of 25 cycles. The amplified product and plasmid pET-28 (+) were double-digested with Mel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into colibacillus DH5a by the calcium chloride method, containing kanamycin

After the LB plate (final concentration 30 μ _§ / _Π11 ) was cultured overnight, the positive clones were selected by colony PCR method and sequenced. A positive clone ( _P ET-2296f05) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg / ml), the host bacteria BL21 (pET-2296f05) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge was used which could bind 6 histidines (6His-Tag).

(Product of Novagen) was chromatographed to obtain purified target protein development regulation related protein 10.01. By SDS-PAGE electrophoresis, a single band was obtained at 10.10 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of Anti-Development Regulation Related Proteins 10. 01 Antibody

A peptide synthesizer (product of PE company) was used to synthesize the following specific peptides for development regulation 10.01: NH2-Met-Leu-Cys-H i s-Leu-G 1 n-Arg-Met-Va l-Ser -G 1 uG 1 n-Cys-H i s-Leu-COOH (SEQ ID NO: 7). The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Av rameas, et al. Immunochemi stry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ral bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The polypeptide bound to cyanogen bromide activated Se _P h _a rose4B column, by affinity chromatography from total IgG isolated anti-polypeptide antibody. 01。 The immunoprecipitation method proved that the purified antibody could specifically bind to development-related protein 1 0.01. Example 7 Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ. ID NO: 1 of the present invention as a hybridization probe, and to use a membrane hybridization method to identify whether some tissues contain the multicore of the present invention. Nucleotide sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Sou thern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals. The probes used in this embodiment include two types: The first type of probe Are oligonucleotide fragments that are completely identical or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are oligonucleotides that are partially identical or complementary to the polynucleotide SEQ ID NO: 1 of the present invention Acid fragments. In this embodiment, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

 First, the selection of the probe

 The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:

 1. The preferred range of probe size is 18-50 nucleotides;

 2.GC content is 30% -70%, non-specific hybridization increases when it exceeds;

 3. There should be no complementary regions inside the probe;

 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements For homology comparison of the regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally;

 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments. After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5'-TGTATATTTTTAAACCTAGGTCTATAAGTGGAAAGAAGCTG-3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

 5 -TGTATATTTTTAAACCTAGGCCTATAAGTGGAAAGAAGCTG-3 '(SEQ ID NO: 9) For other commonly listed reagents and their preparation methods related to the following specific experimental procedures, please refer to the literature: DNA PROBES GH Kel ler; MM Manak; Stockton Press, 1989 (USA (USA) ) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with a cold homogenate buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 mraol / L NaCl; 25 mmol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (add 1-5ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

 2, DNA phenol extraction method

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000 g for 10 minutes. 2) Resuspend the pelleted cells (1 × ^{10 8} cells / ml) with cold cell lysate and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. The DNA was then purified and ethanol precipitated.

 3, DNA purification and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70 ° / »cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DM pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper while gradually increasing TE, mix until the DNA is fully lysed, and add approximately lul per 1-5 X 10 ^s cells.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / ral _o 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase and add ^: ^ volume? !! ^ Sodium acetate and? ^ Volume of cold ethanol, mix and let stand at -20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A _28Q to check the purity and yield of DM. 15) Store at -20 after packing. C.

Preparation of sample film: 1) Take 4x2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe, so that they can be used in the following experimental steps. The film was washed with high-strength conditions and strength conditions, respectively.

 2) Aspirate and control 15 microliters each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper infiltrated with 0.1mol / L NaOH, 1.5mol / L NaCl for 5 minutes (twice), dry and place in 0.5mol / L Tris-HCl (pH7.0), 3mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry.

 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

 Labeling of probes

1) 3 μl Probe (0. IOD / Ιθμ 1), add 2 μ IKinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass Sephadex G-50 column.

5) Collect the first peak before ³² P-Probes are washed out (monitorable).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator.

8) After combining the collection solutions of the first peak, the ³² P-Probe (the second peak is free γ- ³² P-dATP) is prepared.

 Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (lOxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DM)) was added. After sealing the bag, shake at 68 ° C for 2 hours.

 Cross

 Cut a corner of the plastic bag, add the prepared probe, and seal the bag, 42. C water bath overnight. Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0. Γ / oSDS, 40. C Wash for 15 minutes (twice).

 3) 0.1xSSC, 0.1% SDS, 40. C Wash for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 55 ° C for 30 minutes (twice), and dry at room temperature.

 Low-intensity washing film:

1) Take out the hybridized sample membrane. 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS at 37 ° C for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1 ° / »SDS at 40 ° C for 15 minutes (twice), and dry at room temperature.

 X-ray autoradiography:

 -70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).

 Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger To the radioactive intensity of the hybridization spot of another probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1.

Claims

Rights request

I. An isolated polypeptide-development-regulation-related protein 10.01, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof.

 .

2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

 (b) a polynucleotide complementary to the polynucleotide; or

 (c) A polynucleotide that is at least 70% identical to (a) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 169-444 in SEQ ID NO: 1 or the sequence of positions 1-2291 in SEQ ID NO: 1. .

 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is constructed from a polynucleotide, a plasmid, a virus or a vector expression vector according to any one of claims 4-6. Recombinant vector.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with the polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having development regulation-related protein 10.01 activity, characterized in that the method includes:

 (a) cultivating the engineered host cell according to claim 8 under the condition of expressing development regulation-related proteins 10.01;

 (b) Isolating a polypeptide with development-related protein 10.01 activity from the culture.

 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to a protein related to development regulation 10.01.

II. A class of compounds that mimic or regulate the activity or expression of a polypeptide, which is characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of development regulation related protein 10. 01.

12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

 13. The use of the compound according to claim 11, characterized in that the compound is used for regulating the activity of a protein related to development regulation 10. 01 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

 15. The use of the polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimics, agonists, antagonists or inhibitors of development-regulating related proteins 10. 01; or Identification of peptide fingerprints.

 1. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1 to 6 and 11, characterized in that the polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with a developmental regulation-related protein 10.01 abnormality.

 1S, the use of the polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is prepared for the treatment of malignant tumors, Hematological diseases, HIV infection and immune diseases and drugs of various inflammations.