WO2001062782A1 - Nouveau polypeptide, famille proteique 11 de la rhodopsine, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, famille proteique 11 de la rhodopsine, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001062782A1
WO2001062782A1 PCT/CN2000/000710 CN0000710W WO0162782A1 WO 2001062782 A1 WO2001062782 A1 WO 2001062782A1 CN 0000710 W CN0000710 W CN 0000710W WO 0162782 A1 WO0162782 A1 WO 0162782A1
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polypeptide
polynucleotide
rhodopsin
protein family
sequence
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PCT/CN2000/000710
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU23418/01A priority Critical patent/AU2341801A/en
Publication of WO2001062782A1 publication Critical patent/WO2001062782A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, rhodopsin inhibitor protein family 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • Rhodopsin inhibitory protein is a protein that interacts with photo-activated phosphorylated rhodopsin in vivo to inhibit the interaction of rhodopsin and transductin. All rhodopsin inhibitor proteins constitute a large protein superfamily, the rhodopsin inhibitor protein superfamily. Members of this protein superfamily act as important regulatory proteins in vivo, downstream regulating the expression and role of phosphorylated G protein membrane receptors in vivo.
  • the rhodopsin inhibitor protein is widely distributed in organisms, and a large number of members of this protein family are found in various higher organisms.
  • the rhodopsin inhibitory proteins in spinal and non-spinal animals were studied. Based on their sequence similarity, members of the protein superfamily can be divided into five subfamilies. Seven variable structural motifs and four conserved structural motifs were found in all family members. These structural motifs are all proteins that play important functions in normal biological functions.
  • the rhodopsin inhibitor protein family from all different sources also has high homology. Therefore, members of this protein family are evolutionarily conserved [Craf t CM, Whitmore DH, 1995, FEBS Let t, 362: 247 —255].
  • This sequence fragment contains a large number of highly variable, hydrophobic amino acid residues, which may be the central region of the protein that binds to rhodopsin and performs normal physiological functions. Mutations of some amino acid sites in this region will lead to abnormal expression and function of the protein, which will cause abnormalities in the regulation processes of various related organisms. For example, failure to properly regulate the expression of G protein will cause abnormal proliferation of related tissues and cells or Cell signal transduction is blocked.
  • Rhodopsin inhibitor protein in vivo mainly regulates the expression and role of some G protein membrane receptors by inhibiting the interaction between rhodopsin and transductin.
  • transducin is an important G protein in the body. It involves many important biological functions such as cell proliferation, signal transduction, protein synthesis, and protein localization in the body. The realization of all these functions is Requires various G protein membrane receptors mediate. Therefore, rhodopsin inhibitor protein, as a downstream regulator of G protein membrane receptor, also has very important physiological functions in vivo. It regulates the expression and function of proteins in various related tissues in the body, such as regulating the function of adrenergic receptors.
  • the abnormal expression of members of the protein family will lead to abnormal proliferation of tissue cells and abnormal expression of the protein, and thus disorder of some related metabolic steps. It is usually associated with the occurrence of various malignant tumors and cancers, various development disorders and various immune system diseases in the body. In particular, based on the relationship between the expression of rhodopsin protein and light and its role in the biological visual system, it is believed that this protein is also closely related to some autoimmune uveitis and uveitis in mammals. Related.
  • rhodopsin inhibitor protein family 11 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more rhodopsin inhibitor proteins involved in these processes Family 11 proteins, especially the amino acid sequence of this protein. Isolation of the 11 rhodopsin inhibitor protein family coding gene also provides a basis for research 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 isolating its coding DNA is important.
  • An object of the present invention is to provide an isolated novel polypeptide, the rhodopsin inhibitor protein family 11 and fragments, analogs and derivatives thereof.
  • Another object of the 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 rhodopsin inhibitor protein family 11.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a rhodopsin inhibitor protein family 11.
  • Another object of the present invention is to provide a method for producing rhodopsin inhibitor family 11.
  • Another object of the present invention is to provide antibodies against the rhodopsin inhibitory protein family 11 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, the rhodopsin inhibitor protein family 11.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to rhodopsin inhibitor protein family 11 abnormalities.
  • 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.
  • 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:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 988 to 1281 in SEQ ID NO: 1; and (b) a sequence having 1-2025 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of rhodopsin inhibitor protein family 11 protein, which comprises utilizing the polypeptide of the present 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 disease susceptibility associated with abnormal expression of rhodopsin inhibitor protein family 11 protein, which comprises 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
  • 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 rhodopsin inhibitor protein family 11.
  • FIG. 1 is a comparison diagram of amino acid sequence homology of a characteristic sequence of the rhodopsin inhibitor protein family 11 with 59 amino acids in 29-87 and a domain of the rhodopsin inhibitor protein family 11 of the present invention.
  • the upper sequence is the rhodopsin inhibitor protein family 11, and the lower sequence is the characteristic sequence domain of the rhodopsin inhibitor protein family.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated rhodopsin inhibitor protein family 11.
  • llKDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • 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 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.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • 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” refers to a molecule that, when combined with rhodopsin inhibitor protein family 11, 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 the rhodopsin inhibitor protein family 11.
  • Antagonist refers to a molecule that, when combined with rhodopsin inhibitor protein family 11, can block or regulate the biological or immunological activity of rhodopsin inhibitor protein family 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to rhodopsin inhibitor protein family 11.
  • rhodopsin inhibitor protein family 11 refers to a change in the function of rhodopsin inhibitor protein family 11, including an increase or decrease in protein activity, a change in binding properties, and any other biological properties of rhodopsin inhibitor protein family 11, Changes in functional or immune properties.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify the rhodopsin inhibitor protein family 11 using standard protein purification techniques.
  • the substantially pure rhodopsin inhibitor protein family 11 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the rhodopsin inhibitor protein family 11 peptides can be analyzed by amino acid sequence. .
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T
  • the complementarity between two 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 blot etc.) to detect.
  • 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 based on different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988)
  • 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:
  • 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 Hein (Hein 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; having an uncharged head group is 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 D or RNA sequence.
  • 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 and Fv, which can specifically bind to the epitopes of rhodopsin inhibitor protein family 11.
  • 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.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • 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.
  • 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).
  • 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. .
  • isolated rhodopsin inhibitor protein family 11 means the rhodopsin inhibitor protein family 11 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the rhodopsin inhibitor protein family 11 using standard protein purification techniques. Essentially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the rhodopsin inhibitor protein family 11 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, rhodopsin inhibitor protein family 11, 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 present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. 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 initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of rhodopsin inhibitor protein family 11.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the rhodopsin inhibitor protein family 11 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) 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 substituted
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) is one in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide (such as Leader or secreted sequence or the sequence used to purify the polypeptide or protease sequence).
  • 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 2025 bases, and its open reading frame 988-1281 encodes 97 amino acids.
  • This polypeptide has a characteristic sequence of the rhodopsin inhibitor protein family characteristic sequence, and it can be deduced that the rhodopsin inhibitor protein family 11 has the structure and function represented by the rhodopsin inhibitor protein family characteristic sequence.
  • the polynucleotide of the present invention may be in the D form or the RNA form.
  • DM forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • 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); and Non-coding sequence.
  • 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.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is a replacement form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change its editing The function of the polypeptide.
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50 ° v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only the same between the two sequences Crosses occur only when the sex is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • 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 the rhodopsin inhibitor family 11.
  • 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 rhodopsin inhibitor protein family 11 of the present invention can be obtained by various methods.
  • 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.
  • 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.
  • Q i agene There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecu lar Cloning, A Labora tory Manua, Coll Spring Harbor Labora tory. 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.
  • genes can be screened 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 a marker gene function; (3) the determination of the level of the rhodopsin inhibitor protein family 11 transcripts; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • 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.
  • 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).
  • the protein product of the rhodopsin inhibitor protein family 11 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method (Sa ik i, et al. Sc; 1985; 230: 1 350-1 354) using PCR technology to amplify DNA / RM is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers 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.
  • 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 rhodopsin inhibitor protein family 11 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a rhodopsin inhibitory protein family 11 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • 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.
  • 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 well known to those skilled in the art can be used to construct expression vectors containing the D sequence encoding rhodopsin inhibitor protein family 11 and appropriate transcription / translation regulatory elements.
  • the D 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.
  • 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.
  • 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.
  • 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.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a rhodopsin inhibitory protein family 11 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute 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 such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the D sequence can be performed using conventional techniques well known to those skilled in the art.
  • 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 the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following D transfection methods can be used: calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used for expression or production Recombinant rhodopsin inhibitor protein family (USc ience, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • 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 compound treatment, protein precipitant treatment (salting out method), centrifugation, osmotic bacterium, 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.
  • conventional compound treatment protein precipitant treatment (salting out method), centrifugation, osmotic bacterium, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, Ion exchange chromatography, high performance liquid chromatography (HP
  • polypeptides of the present invention 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 immunological diseases.
  • Rhodopsin inhibitor protein in vivo mainly regulates the expression and role of some G protein membrane receptors by inhibiting the interaction between rhodopsin and transductin.
  • transducin is an important G protein in the body. It involves many important biological functions such as cell proliferation, signal transduction, protein synthesis, and protein localization in the body. The realization of all these functions is Mediation of various G protein membrane receptors is required. Therefore, rhodopsin inhibitor protein, as a downstream regulator of G protein membrane receptor, also has a very important physiological function in vivo. It regulates the protein expression and function of various related tissues in the body, such as the function of adrenergic receptors.
  • the abnormal expression of members of the rhodopsin inhibitory protein family will lead to abnormal proliferation of tissue cells and abnormal expression of the protein, thereby disordering some related metabolic steps. It is usually related to the occurrence of various malignant tumors and cancerous tumors, various developmental disorders and various immune system diseases in the body. In particular, based on the relationship between the expression of rhodopsin protein and light and its role in the biological visual system, it is believed that this protein is also closely related to some autoimmune uveitis and uveitis in mammals. Related.
  • the rhodopsin inhibitory protein-specific conserved sequence is required to form its active mot if. It follows that The abnormal expression of the specific rhodopsin inhibitory protein mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, thereby causing the G protein-receptor pathway signal to be abnormal, and abnormally causing the eye's photoreceptor system to be abnormal. Produce related diseases such as eye diseases, tumors, embryonic developmental disorders, growth and development disorders, etc.
  • the abnormal expression of the rhodopsin inhibitor protein family 11 of the present invention will produce various diseases, especially eye diseases, tumors, embryonic development disorders, and growth and development disorders.
  • These diseases include, but are not limited to: eye diseases: autoimmunity Uveitis, uveitis, retinitis, macular degeneration, optic atrophy, night blindness, pyramidal dysfunction
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, double ureter, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, suburethral Fissure, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma , Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, Nasopharyngeal carcinoma, laryngeal carcinoma, tracheal tumors, fibromas, fibrosarcomas, lipomas, liposarcomas, leiomyomas
  • Abnormal expression of the rhodopsin inhibitor protein family 1 1 of the present invention will also cause certain hereditary, hematological and immune system 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 eye diseases, tumors, embryonic development disorders, growth and development disorders, certain heredity, Blood diseases and immune system diseases.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) rhodopsin inhibitor protein family 11.
  • Agonists enhance biological functions such as rhodopsin inhibitory protein family 11, which stimulates cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing rhodopsin inhibitor family 1 1 can be cultured with labeled rhodopsin inhibitor protein family 11 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of rhodopsin inhibitor protein family 11 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of rhodopsin inhibitor protein family 11 can bind to rhodopsin inhibitor protein family 11 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.
  • rhodopsin inhibitor protein family 11 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 rhodopsin inhibitor protein family 11 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to the rhodopsin inhibitor protein family n can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 11 molecules of the rhodopsin inhibitor protein family should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the rhodopsin inhibitor protein family 11 epitope. 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.
  • polyclonal antibodies can be obtained by direct injection of rhodopsin inhibitor protein family 11 in immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing monoclonal antibodies to rhodopsin inhibitor protein family 11 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology EBV-hybridoma technology.
  • Inlay antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (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 rhodopsin inhibitor protein family 11.
  • Antibodies to rhodopsin inhibitor protein family 11 can be used in immunohistochemistry to detect rhodopsin inhibitor protein family 11 in biopsy specimens.
  • Monoclonal antibodies that bind to the rhodopsin inhibitor protein family 11 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.
  • rhodopsin inhibitor protein family 11 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (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 retinol Cells positive for erythrostatin family 11.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the rhodopsin inhibitor family 11.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of rhodopsin inhibitor protein family 11.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of rhodopsin inhibitor protein family 11 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of rhodopsin inhibitor protein family 11 detected in the test can be used to explain the importance of rhodopsin inhibitor protein family 11 in various diseases and to diagnose diseases in which rhodopsin inhibitor protein family 11 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • 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.
  • Polynucleotides encoding the rhodopsin inhibitor protein family 1 1 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of rhodopsin inhibitor protein family 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant rhodopsin inhibitor protein family 11 to inhibit endogenous rhodopsin inhibitor protein family 11 activity.
  • a variant rhodopsin inhibitor protein family 11 may be a shortened rhodopsin inhibitor protein family 11 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of rhodopsin inhibitor protein family 11.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a rhodopsin inhibitory protein family 11 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a rhodopsin inhibitor protein family 11 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding the rhodopsin inhibitor protein family 11 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.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit rhodopsin inhibitor protein family 11 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 RNA and performs endonucleation.
  • Antisense R and DNA and ribozymes can be obtained using any existing RNA or DNA synthesis techniques, such as solid phase phosphorus The technology of synthesizing oligonucleotides by acid amide chemical synthesis has been widely used.
  • 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 a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding rhodopsin inhibitor protein family 11 can be used for the diagnosis of diseases related to rhodopsin inhibitor protein family 11.
  • a polynucleotide encoding rhodopsin inhibitor protein family 11 can be used to detect the expression of rhodopsin inhibitor protein family 11 or the abnormal expression of rhodopsin inhibitor protein family 11 in a disease state.
  • the DNA sequence encoding rhodopsin inhibitor protein family 11 can be used to hybridize biopsy specimens to determine the expression of rhodopsin inhibitor protein family 11.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Rhodopsin inhibitor protein family 11 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the rhodopsin inhibitor protein family 11 transcription products.
  • Detection of mutations in the rhodopsin inhibitor protein family 11 gene can also be used to diagnose rhodopsin inhibitor protein family II-related diseases.
  • the forms of rhodopsin inhibitor family 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type rhodopsin inhibitor family 11 DNA 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 the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • 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.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. 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.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome can be utilized Or a large number of genomic clones 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 hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • 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 ian Inheritance in Man (available online from Johns Hopk ins University Wetch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, 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 cDNA 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.
  • suitable pharmaceutical carrier 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.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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.
  • 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.
  • the rhodopsin inhibitor protein family 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of rhodopsin inhibitor family 11 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
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smar t cDNA cloning kit purchased from C1 on tech) was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5ct.
  • the bacteria formed a cDNA library.
  • the Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0045g03 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the sequence of the rhodopsin inhibitor protein family 11 of the present invention and the protein sequence encoded by the rhodopsin inhibitory protein family 11 of the present invention were profiled using the GCG profile scan program (Basic local alignment search tool) [Altsc ul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in a database such as prosite.
  • the rhodopsin inhibitor protein family 11 of the present invention is homologous with the domain rhodopsin inhibitor protein family characteristic sequence at 29-87.
  • the homology result is shown in FIG. 1, the homology rate is 0.24, and the score is 13.87; the threshold value is 13.40 .
  • Example 3 Cloning of a gene encoding the rhodopsin inhibitory protein family 11 gene by RT-PCR method
  • the total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA.
  • the following primers are used for PCR amplification:
  • Primerl 5'- ATTCTGCAGATTTTAACCACTGTC-3 '(SEQ ID NO: 3)
  • Primer2 5-ACGGAGTCTCGCTCTGTCACCAGG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • ⁇ 612 is the 3 'end reverse sequence in 3 £ () ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of 50mmol / L C1, 10mmol / L Tris-HCl, pH8.5, 1.5mmol / L MgCl 2, 20 ( ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT -PCR set P-actin as a positive control and template blank as a negative control at the same time.
  • the amplified product was purified with a QIAGEN kit and connected to a pCR vector with a TA cloning kit (Invitrogen product). DNA sequence analysis results showed The DNA sequence of the PCR product is exactly the same as 1-2025bp shown in SEQ ID NO: 1.
  • Example 4 Analysis of the expression of rhodopsin inhibitor protein family 11 gene by Northern blotting method Total RNA was extracted by one step method [Anal. Biochem 1987, 162 156-159] 0 This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and added 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1). Aspirate the aqueous phase layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain the RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA 20 ⁇ B RNA was used in a mixture containing 20raM 3- (N -Morpholine) Propanesulfonic acid (pH 7.0)-5mM sodium acetate-IraM EDTA-2.2M formaldehyde on a 1.2% agarose gel for electrophoresis. Then transfer to a nitrocellulose membrane.
  • Use o- 32 P dATP A 32 P-labeled D probe was prepared by a random primer method.
  • the DM probe used encodes the PCR-enhanced rhodopsin inhibitor protein family 11 shown in Figure 1. The region sequence (988bp to 1281bp).
  • Primer 3 5'-CCCCATATGATGCCTACAACTTTAATTCATTCA-3 '(Seq ID No: 5)
  • Primer4 5-CATGGATCCCTAGTGATAGACCTTTGCATTGGC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, Ndel And BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (product of Novagen, Cat. No. 69865.3).
  • the pBS-0045g03 plasmid of the target gene was used as a template for PCR reaction.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS-0045g03 containing 10pg, primer Pr imer- 3 and Pr imer - 4 were l Opmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E. coli DH5 CC using the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following peptides specific for rhodopsin family 11:
  • Selecting suitable oligonucleotide fragments from the polynucleotides of the present invention has various uses as hybridization probes, such as using the probes to hybridize to genomic or cDNA libraries of normal tissues or pathological tissues from different sources.
  • the probe may further be used to detect the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in normal tissue or Whether the expression in pathological 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 identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses 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 probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • 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.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • 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 The regions are compared for homology. 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;
  • 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-TGCCTACAACTTTAATTCATTCATCTAGAGGATTTTCACTG-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:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • 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.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of pre-hybridization solution (1 OxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After closing the bag, 68. C water bath for 2 hours.
  • pre-hybridization solution 1 OxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as Targeting DNA for gene chip technology for high-throughput research on new gene functions; finding and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases.
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification. The spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRM was extracted from normal liver and liver cancer in one step, and mRNA was purified using Oligotex mRNA Midi Ki "purchased from QiaGen".
  • the fluorescent reagent Cy3dUTP (5-Amino- propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) mRM labeled with normal liver tissue, using a fluorescent reagent Cy5dUTP (5-Amino-propargy 1-2 '-deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and probes were prepared after purification. For specific steps and methods, see

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Abstract

L'invention concerne un nouveau polypeptide, une famille protéique 11 de la rhodopsine, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la famille protéique 11 de la rhodopsine.
PCT/CN2000/000710 1999-12-27 2000-12-25 Nouveau polypeptide, famille proteique 11 de la rhodopsine, et polynucleotide codant pour ce polypeptide WO2001062782A1 (fr)

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CN 99125391 CN1301726A (zh) 1999-12-27 1999-12-27 一种新的多肽——视紫红质抑制蛋白家族11和编码这种多肽的多核苷酸
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WO1998055635A1 (fr) * 1997-06-05 1998-12-10 Duke University Procedes d'analyse de l'activite de recepteurs et produits de recombinaison utiles dans de tels procedes
CN1249341A (zh) * 1998-09-28 2000-04-05 复旦大学 新的人蛋白激酶c抑制蛋白编码序列、其编码的多肽及制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998055635A1 (fr) * 1997-06-05 1998-12-10 Duke University Procedes d'analyse de l'activite de recepteurs et produits de recombinaison utiles dans de tels procedes
CN1249341A (zh) * 1998-09-28 2000-04-05 复旦大学 新的人蛋白激酶c抑制蛋白编码序列、其编码的多肽及制备方法

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