WO2001083677A2 - A novel polypeptide- human chiken limb deformity protein fh12-13 and the polynucleotide encoding said polypeptide - Google Patents
A novel polypeptide- human chiken limb deformity protein fh12-13 and the polynucleotide encoding said polypeptide Download PDFInfo
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- WO2001083677A2 WO2001083677A2 PCT/CN2001/000626 CN0100626W WO0183677A2 WO 2001083677 A2 WO2001083677 A2 WO 2001083677A2 CN 0100626 W CN0100626 W CN 0100626W WO 0183677 A2 WO0183677 A2 WO 0183677A2
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- polypeptide
- polynucleotide
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- chicken wing
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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
Definitions
- the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide ⁇ ⁇ chicken wing deforming protein FH2-13, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. -
- Cell division is a basic feature of individual growth and life reproduction. There are two types of cell division: meiosis of germ cells and mitosis of normal cells. The division of a cell includes two processes: nuclear division and cytokinesis. Mitosis and cytokinesis are two closely related processes. Cytokinesis usually occurs after mitosis. Cytokinesis is an essential step for a cell to successfully divide into two identical daughter cells. Failure of cytokinesis often results in various polyploid products.
- the morphogen protein is a protein encoded by the LD gene, and is highly expressed during embryogenesis, especially during mouse wing and organ development. In humans, it also plays an important biological function in the formation and development of embryos. In vivo, morphogen proteins control embryonic and cellular polarity by regulating cytoskeleton formation and cell signal transduction [Peter Uetz, Stefano Fumaga lli et al., The Journa l of Biological Chemi s try, 1996 (271 ): 33525-33530] 0 It is also necessary for cells to complete cytokinesis, and may be a part of the contractile ring during cytokinesis, and regulates the role of the contractile ring [Diego H.
- FH1 a proline-rich domain
- FH2 a highly conserved amino acid sequence containing GxxGNxMN mot if.
- the FH1 domain plays a role in regulating signal transduction during biological morphogenesis and development; at the same time, the FH1 domain plays an important role in the cytokinesis of cells, which may regulate the actin in the cell The completion of the aggregation and cytokinesis process.
- the FH2 domain cooperates with or hinders the role of the FH1 domain in the organism : [Diego H.
- the human chicken wing deforming protein FH2-13 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field
- the human chicken wing deformed protein FH2-1 3 protein during these processes, in particular the amino acid sequence of this protein was identified. Isolation of the novel human chicken wing deformed protein FH2-13 protein encoding 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 it is important to isolate its code for DM.
- 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 the human chicken wing deformed protein FH2-1 3.
- Another object of the present invention is to provide a method for producing human chicken wing morphoprotein FH2-13.
- Another object of the present invention is to provide an antibody against the polypeptide-human chicken wing deforming protein FH2-13 of the present invention.
- Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human chicken wing deforming protein FH2-13.
- Another object of the present invention is to provide a method for diagnosing and treating a disease associated with abnormality of human chicken wing deformin FH2-1 3. 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.
- the 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 1291 to 1641 in SEQ ID NO: 1; and (b) a sequence having 1-1779 in SEQ ID NO: 1 Sequence of bits.
- the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; 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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the human chicken wing morphoprotein FH2-13 protein, which comprises utilizing the polypeptide of the invention.
- the invention also relates to compounds obtained by this method.
- the present invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human chicken wing morphoprotein FH2-13 protein, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Detection of 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 human chicken wing deformable protein FH2-13.
- FIG. 1 is a comparison diagram of gene chip expression profiles of human chicken wing deforming protein FH2-13 and human chicken wing deforming protein FH2 according to the present invention.
- the upper graph is a graph of the expression profile of human chicken wing morphin FH2-13, and the lower graph is the graph of the expression profile of human chicken wing morphin FH2.
- 1 represents fetal kidney
- 2 represents fetal large intestine
- 3 represents fetal small intestine
- 4 Indicates fetal muscle
- 5 indicates fetal brain
- 6 indicates fetal bladder
- 7 indicates non-starved L02
- 8 indicates L02 +
- lhr As 3+
- 9 indicates ECV304 PMA-
- 10 indicates ECV304 PMA +
- 11 indicates fetal liver
- 12 indicates normal liver.
- 13 indicates thyroid
- 14 indicates skin
- 15 indicates fetal lung
- 16 indicates lung
- 17 indicates lung cancer
- 18 indicates fetal spleen
- 19 indicates spleen
- 20 indicates prostate
- 21 indicates fetal heart
- 22 indicates heart
- 23 indicates muscle
- 24 indicates For the testis
- 25 indicates the fetal thymus
- 26 indicates the thymus.
- Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human chicken wing deformed protein FH2-13.
- OkDa 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 the 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 human chicken wing deforming protein FH2-13, 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 human chicken wing morphoprotein FH2-13.
- An "antagonist” or “inhibitor” refers to a molecule that can block or regulate the biological or immunological activity of human chicken wing morphoprotein FH2-13 when combined with human chicken wing morphoprotein FH2-13.
- Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human chicken wing morphoprotein FH2-13.
- Regular refers to a change in the function of human chicken wing morphoprotein FH2-13, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human chicken wing morphoprotein FH2-13 Change of nature.
- substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
- Those skilled in the art can purify human chicken wing deformed protein FH2-13 using standard protein purification techniques.
- the substantially pure human chicken wing deformed protein FH2-13 can generate a single main band on a non-reducing polyacrylamide gel.
- the purity of human chicken wing deformed protein FH2- 13 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 may 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 imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to target sequences 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 a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
- the MEGALIGN program can compare two or more sequences (Higgins, D. G. and
- Nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein Percentage of identity between them (Hein L, (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 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 DNA or RNA sequence.
- Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
- Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, 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,? (') 2 and?, Which can specifically bind to the epitope of human chicken wing morphoprotein FH2-13.
- 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 a substance 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 thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
- Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain 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 in the natural state .
- isolated human chicken wing morphoprotein FH2- 13 refers to human chicken wing morphoprotein FH2-13 which is substantially free of other proteins, lipids, sugars or other substances naturally associated with it.
- Those skilled in the art can purify human chicken wing morphoprotein FH2-13 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of human chicken wing morphoprotein FH2- 13 peptide can be analyzed by amino acid sequence.
- the present invention provides a novel polypeptide, human chicken wing deformin 13 ⁇ 4-2-13, which is basically composed 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, 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 starting methionine residues.
- the invention also includes fragments, derivatives and analogs of human chicken wing morphoprotein FH2-13.
- fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human chicken wing deforming protein FH2- 13 of the present invention.
- a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 ( ⁇ ) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) like this
- a type in which a mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
- a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences.
- 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 the nucleotide sequence of SEQ ID NO: 1.
- the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue.
- the length of the polynucleotide sequence is 1779 bases, and its open reading frame 1291-1641 encodes 116 amino acids.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DM forms include cDNA, genomic DNA, or synthetic DNA. DM 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); Coding sequence.
- the term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding 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 can 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 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 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, 6 (TC; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences
- the hybridization occurs only when the identity between them is at least 95%, and more preferably 97%.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function as the mature polypeptide shown in SEQ ID NO: 2 and Active.
- 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 human chicken wing morphoprotein FH2-1 3.
- 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 human chicken wing deformable protein FH2-13 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 DM sequence from the genomic MA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
- genome D 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 mRNA extraction. Kits are also commercially available (Qi agene).
- the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, 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.
- 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) the determination of the level of the transcript of human chicken wing deformed protein FH2-13; (4) Detecting the protein product of gene expression by immunological technology or 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 2GG0 nucleotides, preferably within 1000 nucleotides.
- the probe used here is generally 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).
- immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein products expressed by the human chicken wing deformed protein FH2- 13 gene.
- ELISA enzyme-linked immunosorbent assay
- a method for amplifying MA / RNA by PCR is preferably used to obtain the gene of the present invention.
- the RACE method RACE-Rapid Amplification of cDNA Ends
- 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.
- 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 the human chicken wing deforming protein FH2- 13 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
- a polynucleotide sequence encoding human chicken wing deformed protein FH2-13 can be inserted into a vector to form 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 vector (Rosenberg, et al.
- pMSXND expression vector expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and in insects A baculovirus-derived vector expressed in cells.
- 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 expression vectors containing a DM sequence encoding human chicken wing deformed protein FH2-13 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, 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.
- the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for D expression, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
- 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 human chicken wing deformable protein FH2-13 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 such as fly S2 or Sf9
- animal cells such as CH0, COS or Bowes melanoma cells.
- Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence may This is done using conventional techniques well known to those skilled in the art.
- the host is a prokaryote such as E. coli
- competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 2 method used in the step are well known in the art. Alternatively, it is a MgCl 2. If If necessary, transformation can also be performed by electroporation.
- the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging Wait.
- polynucleotide sequence of the present invention can be used to express or produce recombinant human chicken wing deformable protein FH2-13 (Science, 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.
- the physical, chemical, and other properties can be used to isolate and purify the recombinant protein by various separation methods. 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.
- polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
- the morphogen protein encoded by the wing deformin gene is expressed in immune response cells and tissues, as well as in cancer and immortal cell lines. Therefore, it plays an important role in diseases caused by cancer, development and immune system disorders.
- the novel human protein of the present invention is a protein similar to the wing deformin FH2 domain, which may play a role in regulating the function of related proteins in the body. Such as interacting with FH1 domain-containing proteins-regulating the function of these proteins in the body.
- the polypeptide or the fragment or the derivative thereof of the present invention can be used to treat diseases caused by developmental disorders, that is, diseases that affect the growth and differentiation of cells and tissues.
- diseases include but are not limited to the following: renal tubular acidification, anemia, Cushing's syndrome, chondrogenic dwarf dwarf, epilepsy, gonad hypoplasia, hereditary neurological diseases such as neurofibromas, hypothyroidism, brain Hydrocephalus, convulsive disorders such as: cerebral palsy, spina bifida, congenital glaucoma, cataracts and sensorineural hearing loss.
- Human chicken wing deforming protein FH2-13 can also be used to prevent and treat various cancers. These cancers include: adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, malformed cancer, especially adrenal gland, bladder, bone, brain , Breast, uterus, heart, kidney, liver, lung, muscle, ovary, salivary gland, skin, testis, thymus, cervix, pancreas, thyroid, penis and other cancers.
- cancers include: adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, malformed cancer, especially adrenal gland, bladder, bone, brain , Breast, uterus, heart, kidney, liver, lung, muscle, ovary, salivary gland, skin, testis, thymus, cervix, pancreas, thyroid, penis and other cancers.
- the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human chicken wing deforming protein FH2-13.
- Agonists enhance human chicken wing deformed protein FH2-13 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
- a mammalian cell or a membrane preparation expressing human chicken wing morphoprotein FH2-13 can be cultured together with labeled human chicken wing morphoprotein FH2-13 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
- Antagonists of human chicken wing morphoprotein FH2-13 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human chicken wing morphoprotein FH2-13 can bind to human chicken wing morphoprotein FH2-13 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide incapable Play biological functions.
- human chicken wing morphoprotein FH2-13 When screening compounds as antagonists, human chicken wing morphoprotein FH2-13 can be added to a bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between human chicken wing morphoprotein FH2-13 and its receptor. Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human chicken wing deformed protein FH2- 1 3 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 human chicken wing morphin FH2- 13 molecule 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 human chicken wing morphoprotein FH2- 13 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
- Polyclonal antibodies can be produced by direct injection of human chicken wing deformed protein FH2-13 into 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 Adjuvant, etc.
- Techniques for preparing monoclonal antibodies to human chicken wing morphoprotein FH2-13 include, but are not limited to Based on hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
- Chimeric antibodies that bind human constant regions to 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 (US Pat No. 4946778) can also be used to produce single chain antibodies against human chicken wing deformed protein FH2-1 3.
- Antibodies against human chicken wing morphin FH2-13 can be used in immunohistochemistry to detect human chicken wing morphin FH2-13 in biopsy specimens.
- Monoclonal antibodies that bind to human chicken wing morphoprotein FH2-13 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.
- human chicken wing morphin FH2-13 high affinity monoclonal antibody 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 crosslinker such as SPDP, and toxin is bound to the antibody through the exchange of disulfide bonds.
- This hybrid antibody can be used to kill human chicken wing morphoprotein FH2-13 positive Cell.
- the antibodies in the present invention can be used to treat or prevent diseases related to human chicken wing morphoprotein FH2-13.
- Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human chicken wing deformin FH2-13.
- the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human chicken wing deformin FH2- 13.
- tests are well known in the art and include FISH assays and radioimmunoassays.
- the level of human chicken wing morphoprotein FH2-13 detected in the test can be used to explain the importance of human chicken wing morphoprotein FH2-13 in various diseases and to play a role in the diagnosis of human chicken wing morphoprotein FH2-13. disease.
- the polypeptide of the present invention can also be used for peptide mapping analysis.
- the polypeptide can be specifically cleaved by physical, chemical or enzyme, and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis .
- the polynucleotide encoding human chicken wing morphoprotein FH2-13 can also be used for a variety of therapeutic purposes.
- Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human chicken wing deformin 'FH2-13.
- Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human chicken wing deforming protein FH2-13 to inhibit endogenous human chicken wing deforming protein FH2-13 activity.
- a variant human chicken wing deforming protein FH2- 13 may be a shortened human chicken wing deforming protein FH2-13, which lacks a signaling functional domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human chicken wing morphoprotein FH2-13.
- Virus-derived expression vectors such as retroviruses, adenoviruses, adenopathies Poison-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human chicken wing deformable protein FH2-13 into cells.
- a recombinant viral vector carrying a polynucleotide encoding human chicken wing morphoprotein FH2- 13 can be found in the existing literature (Sambrook, et al.).
- a recombinant polynucleotide encoding human chicken wing deformable protein FH2-13 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 human chicken wing morphoprotein FH2-13 raRNA are also within the scope of the present invention.
- a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for 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 RM polymerase promoter of the vector. 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 human chicken wing morphoprotein FH2-13 can be used for diagnosis of diseases related to human chicken wing morphoprotein FH2-13.
- the polynucleotide encoding human chicken wing deformable protein FH2-13 can be used to detect the expression of human chicken wing deformable protein FH2-13 or the abnormal expression of human chicken wing deformable protein FH2-13 in a disease state.
- the DNA sequence encoding human chicken wing deforming protein FH2-13 can be used to hybridize biopsy specimens to determine the expression of human chicken wing deforming protein FH2-13.
- Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
- kits are commercially available.
- Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a D chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
- Human chicken wing morphin FH2-13 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of human chicken wing morphin FH2--13.
- Detection of mutations in the human chicken wing morphin FH2-13 gene can also be used to diagnose human chicken wing morphin FH2-13-related diseases.
- Human chicken wing morphin FH2-13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human chicken wing morphin FH2-13 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 protein expression, so Northern blotting, Wes tern Blotting can 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.
- a PCR primer (preferably 15-35b P ) is prepared from the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain 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 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 of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
- FISH Fluorescent in situ hybridization
- 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).
- 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 contains a safe and effective amount of the polypeptide or antagonist and does not affect Pharmaceutically effective carriers and excipients. 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.
- Human chicken wing morphoprotein FH2-13 is administered in an amount effective to treat and / or prevent specific indications.
- the amount and range of human chicken wing morphoprotein FH2-13 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
- 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. Smart cDM cloning kit (purchased from Clontech
- Dye terminate cycle react ion sequencing Kit Perkin-Elmer
- ABI 377 automatic sequencer Perkin-Elmer
- the determined CDM sequences were compared with the existing public D sequence database (Genebank)
- the comparison revealed that the cDNA sequence of one of the clones 0215C12 was new DNA.
- a series of primers were synthesized to determine the inserted cDNA fragment in both directions.
- Primer2 5'- TAACAATGAAATTTTATTTAGCCA-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.
- Conditions for the amplification reaction 50 mmol / L KCl, 10 mmol / L Tri s-HC1 pH 8.5, 1.5 mmol / L MgCl 2 , 200 mol / L dNTP, 1 Opmol primer, 1U Taq in a 50 ⁇ 1 reaction volume MA 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.
- ⁇ -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 l-1779bp shown in SEQ ID NO: 1.
- Biochem 1987, 162, 156-159] involves acid guanidinium thiocyanate-chloroform extraction
- the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), mixed and centrifuged. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RM precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water.
- the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
- PCR was performed using the PBS-0215C12 plasmid containing the full-length target gene as a template.
- the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 PBS-0215C12 plasmid, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. 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.
- Ligation products were transformed by the calcium chloride method Escherichia bacteria DH5 cx, after (final concentration of 30 ⁇ ⁇ / ⁇ 1) LB plates incubated overnight positive clones by colony PCR method containing kanamycin, and sequenced.
- a positive clone (PET-0215C12) 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.
- the host bacteria BL21 (pET-0215C12) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1mmol / L, and continued. Incubate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an His. Bind Quick Cartridge (product of Novagen) which can bind 6 histidines (6His-Tag). The purified human chicken wing deformed protein FH2_13 was obtained.
- the following peptides specific to human chicken wing deforming protein RI2-13 were synthesized using a peptide synthesizer (product of PE company): -Arg-Arg-Lys-Ala-C00H (SEQ ID NO: 7).
- the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
- Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
- 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.
- 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 identify whether some tissues contain the polynucleoside of the present invention by using 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), so that the hybridization background is reduced and only strong specific signals are retained.
- 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 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:
- 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 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;
- Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
- Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
- 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 to 10 mg of prehybridization solution (10xDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
- prehybridization solution 10xDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)
- probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
- Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. 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 fast, efficient, and high-throughput analysis of biological information.
- the polynucleotide of the present invention can be used as target D for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; 'diagnosis of diseases, such as heredity disease. The specific method steps have been reported in the literature.
- 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. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DM on the glass slide to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
- Total mRM was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRM was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
- Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Araersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino- propargyl- 2 ' -deoxyuridine 5'-triphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech The company) labeled the body's specific tissues (or stimulated cell lines) with mRM, and purified them to prepare probes.
- 's specific tissues or stimulated cell lines
- the probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
- the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung and fetal heart.
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AU76235/01A AU7623501A (en) | 2000-04-29 | 2001-04-28 | A novel polypeptide, a cockerel wing-deforming human protein fh2-13 and the polynucleotide encoding the polypeptide |
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CN 00115540 CN1321668A (en) | 2000-04-29 | 2000-04-29 | Novel polypeptide-human chicken wing deformation protein FH2-13 and polynucleotide for coding this polypeptide |
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CN116768996A (en) * | 2023-05-30 | 2023-09-19 | 华南农业大学 | Application of morphogenic protein gene OsFH2 in plant breeding regulation and control |
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Non-Patent Citations (2)
Title |
---|
DATABASE GENBANK [Online] 12 May 1997 CONNELL M. Retrieved from NCBI, accession no. GI:2078475 Database accession no. (AC002070.1) * |
DATABASE GENBANK [Online] 23 November 1999 DEADMAN R. Retrieved from NCBI, accession no. GI:2887277 Database accession no. (Z98750.1) * |
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CN116768996A (en) * | 2023-05-30 | 2023-09-19 | 华南农业大学 | Application of morphogenic protein gene OsFH2 in plant breeding regulation and control |
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