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WO2001047979A1 - Nouveau polypeptide, proteine 11 a particule de reconnaissance du signal 54, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine 11 a particule de reconnaissance du signal 54, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001047979A1
WO2001047979A1 PCT/CN2000/000698 CN0000698W WO0147979A1 WO 2001047979 A1 WO2001047979 A1 WO 2001047979A1 CN 0000698 W CN0000698 W CN 0000698W WO 0147979 A1 WO0147979 A1 WO 0147979A1
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Prior art keywords
protein
polypeptide
polynucleotide
signal recognition
recognition particle
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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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Shanghai Biowindow Gene Development Inc
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Priority to AU24991/01A priority Critical patent/AU2499101A/en
Publication of WO2001047979A1 publication Critical patent/WO2001047979A1/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-signal recognition particle 54 protein 11 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • cytoplasm and cell membrane of eukaryotes there are many organelles with membrane structures. If the protein synthesized in the cytoplasm does not have a transport signal, it will remain in the cytoplasm; to reach proteins in other parts of the cell, there must be a transport signal. Different localized proteins have different signals.
  • Cell surface proteins Secreted proteins and lysosomal proteins (the three are sometimes collectively referred to as secreted proteins) have signal peptides similar to prokaryotic transmembrane proteins. These signal peptides are located at the N-terminus, and most of them are hydrophobic amino acids, which can form a hairpin structure including two ct-helixes.
  • Signal recognition particle is a known cytoplasmic ribonucleoprotein, which acts as a linker between the soluble protein synthesis device and the endoplasmic reticulum membrane transporter to mediate the target-seeking function and the signal sequence of the exported protein Insert the endoplasmic reticulum.
  • the SRP cycle is an ordered sequence of steps that starts by recognizing the specific exported protein by specifically binding the particles to the SRP receptor of the docking protein, triggering a pause in extension.
  • SRP In the endoplasmic reticulum membrane, SRP interacts with membrane integrins or docking proteins that are SRP receptors and releases signal sequences to the transporter. The ribosomal nascent chain complex then forms a tight linker with the membrane protein, and the secreted protein precursor is cotranslated into the endoplasmic reticulum cavity. Finally, the SRP is released through a GTP-dependent process. SRP is a small RNA-protein complex consisting of about 300 nucleotides of 7S R and six tightly bound protein subunits. It is divided into two domains, one is a signal recognition domain and consists of 7SL RNA.
  • the approximately 100th to 250th nucleotides are composed of 19KD, 54KD, 68KD, and 72KD proteins, and the other is the rest of 7SL RNA and 9KD and 14KD proteins. Its function is to suspend the peptide chain extension This is called an extended-acting braking domain. SRP's blocking of translation is temporary. When it encounters the 72KD docking protein on the rough endoplasmic reticulum membrane, it no longer prevents translation of ribosomes.
  • SRP leaves the ribosome to recognize a new signal sequence; the ribosome is connected to the rough endoplasmic reticulum membrane via a signal peptide, and interacts with the ribosomal receptor protein on the membrane by its large subunit.
  • SRP only plays a transient role in the transport of secreted proteins, its concentration in the cytoplasm is high, which is one tenth of the number of ribosomes.
  • a subunit of SRP is a GTP-binding protein that interacts with the signal sequence when it appears in the ribosome.
  • N-terminal 300 residues of SRP54 including GTP-knot The binding site (G-domain) is evolutionarily related to a similar region of the signal recognition particle receptor "subunit (dock protein), which is a membrane-integrated GTP binding protein, which in combination with SRP ensures correct localization of newly secreted proteins
  • the G-domain is located at the C-terminus of the protein.
  • the SRP54 protein plays an important role in both the soluble and membrane-bound phases of the SRP cycle. It recognizes signal sequences in the ribosome and interacts with the endoplasmic reticulum membrane.
  • SRP54 is almost certainly an essential component of the signal recognition particle.
  • the SRP54 protein can be divided into three different regions: the amino terminal region is called the 'X'domain; followed by four including the GTPase family The G domain of the characteristic sequence template and the M domain whose carboxyl terminus is rich in methionine and binds to the signal peptide. There are similar regions in the SR a sequence, although the X and G domains in SR a are at the carboxyl end and not the amino group.
  • the conserved domain located at the C-terminus of the G domain of SRP54 is selected: P- [LIVM] -X- [FYL]-[LIVMAT]-[GS] -x- [GS]-[EQ] - ⁇ (4)-[LIVMF ], Because this region is a putative CDS (GTP dissociation stimulator) binding component, it is called the PGB domain.
  • SRP As a negative regulator of translation, SRP has very important biological significance. Many secreted proteins are degrading enzymes (such as protease nucleases, etc.), and if a small amount leaks into the cytoplasm, it will cause a huge disaster. SRP pauses its synthesis and waits until it finds a safe way out (transmembrane) before letting the ribosome continue to synthesize these secreted proteins.
  • degrading enzymes such as protease nucleases, etc.
  • the signal recognition particle 54 protein 1 1 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 signal recognition particle 54 proteins that participate in these processes. 11 proteins, especially the amino acid sequence of this protein.
  • the new signal recognition particle 54 protein 1 1 The isolation of the protein-coding gene also provides a basis for research to determine the role of the 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. Object of the invention
  • 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 signal recognition particle 54 protein 1 1.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a signal recognition particle 54 protein 1 1.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide-signal recognition particle 54 protein 11 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in signal recognition particle 54 protein 11. 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 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 160-450 in SEQ ID NO: 1; and (b) a sequence having 1-750 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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit signal recognition particle 54 protein 1 1 protein activity, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of the signal recognition particle 54 protein 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 preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other drugs caused by abnormal expression of signal recognition particle 54 protein 1 1 Use.
  • FIG. 1 is a comparison diagram of amino acid sequence homology of the characteristic proteins of the protein 54 of the signal recognition particle 54 of the present invention at a total of 45 amino acids and domains from 18 to 62.
  • the upper sequence is the signal recognition particle 54 protein 11 and the lower sequence is the characteristic protein domain of the signal recognition particle 54 protein.
  • " And ":” and ".” Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated signal recognition particle 54 protein 1 1.
  • l lkDa 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” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • the term “immunologically active” refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with signal recognition particle 54 protein 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 binds to a signal recognition particle 54 protein 11.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of signal recognition particle 54 protein 11 when combined with signal recognition particle 54 protein 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the signal recognition particle 54 protein 11.
  • Regular refers to a change in the function of the signal recognition particle 54 protein 11, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of the signal recognition particle 54 protein 11.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the signal recognition particle 54 protein 11 using standard protein purification techniques.
  • the substantially pure signal recognition particle 54 protein 11 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the signal recognition particle 54 protein 11 polypeptide 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 inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical 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 (Hi gg ins, DG, and PM Sharp (1988) according to different methods, such as the Cluster method. Gene 73: 237-244). The Clus te! 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 following formula: Number of residues that match between the sequence
  • the number of residues in the sequence ⁇ -the number of spacer residues in the sequence-the number of spacer residues X in the sequence S can also be determined by the Clus ter method or by a method 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 substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (') 2 and? ⁇ It can specifically bind to the epitope of signal recognition particle 54 protein 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 signal recognition particle 54 protein 11 means that the signal recognition particle 54 protein 1 1 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify the signal recognition particle 54 protein 11 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the signal recognition particle 54 protein 1 1 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-signal recognition particle 54 protein 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 invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the present invention also includes fragments, derivatives and analogs of the signal recognition particle 54 protein 1 1.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the signal recognition particle 54 protein 1 1 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 UI) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such a type Wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as a leader Sequence or secreted sequence or the sequence used to purify the polypeptide or protease sequence).
  • a leader Sequence or secreted sequence or the sequence used to purify the polypeptide or protease sequence
  • 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 of 750 bases in length and its open reading frame 160-450 encodes 96 amino acids. This peptide has the characteristic sequence of the characteristic protein of the signal recognition particle 54 protein, and it can be deduced that the signal recognition particle 54 protein 11 has the structure and function represented by the characteristic protein of the signal recognition particle 54 protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA 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 the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant means in the present invention that the code has A protein or polypeptide of SEQ ID NO: 2 but a nucleic acid sequence different from the coding region sequence shown in SEQ ID NO: 1.
  • 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.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • 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% Ficol 1, 42 ⁇ , etc .; or (3) only the same between the two sequences Hybridization occurs when the sex is at least 95%, and more preferably 97%. Furthermore, 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 signal recognition particle 54 protein 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 signal recognition particle 54 protein 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 the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate raRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • 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 a marker gene function; (3) determining the level of the transcript of the signal recognition particle 54 protein 11; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used alone 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 products expressed by the signal recognition particle 54 protein 11 gene 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 using PCR technology to amplify DM / RNA 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 a polynucleotide of the present invention, and a vector or a direct use of the vector of the present invention.
  • a host cell produced by genetic engineering using a signal recognition particle 54 protein 11 coding sequence and a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding the signal recognition particle 54 protein 11 can 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 known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding the signal recognition particle 54 protein 11 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • 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 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 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.
  • the recombinant vector 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 Sf 9
  • animal cells such as CH0, COS, or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • 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 M g C l 2.
  • 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 lipid Plastid packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant signal recognition particle 54 protein 1 1 (Scence, 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 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.
  • 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
  • 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.
  • Signal recognition particle is a known cytoplasmic ribonucleoprotein, which is synthesized as a soluble protein.
  • the linker between the device and the endoplasmic reticulum transporter mediates targeting and inserts the signal sequence of the exported protein into the endoplasmic reticulum membrane.
  • the SRP cycle is an orderly series of steps that begin by recognizing a specific exported protein by specifically binding particles to the SRP receptor of a docked protein, triggering a pause in prolongation.
  • SRP54 is a subunit of SRP and is a GTP-binding protein. The SRP54 protein plays an important role in both the soluble and membrane-bound phases of the SRP cycle. It recognizes signal sequences in the ribosome and contacts SRP receptors in the endoplasmic reticulum membrane.
  • the GTP-binding domain of the SRP54-type protein is its characteristic mot if.
  • SRP secreted protein transport
  • SRP As a negative regulator of secreted protein transport, SRP has very important biological significance.
  • the abnormal expression of the specific STP54-type protein GTP binding domain will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, which will cause abnormal transport and distribution of secreted proteins, and then affect the transmission of important cellular signals.
  • the abnormal expression of the signal recognition particle 54 protein 11 of the present invention will produce various diseases, especially degenerative changes of the nervous system, tumors, growth and development disorders, and inflammation. These diseases include, but are not limited to:
  • Nervous system degenerative diseases Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, dementia, multiple sclerosis
  • 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 relaxation, Alzheimer's disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Various tumors 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, thymus tumor, nasal cavity and sinus cancer, nasopharyngeal cancer , Laryngeal cancer, tracheal tumor, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • Inflammation allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex Types of glomerulonephritis, acute anterior uveitis, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, chronic active hepatitis, emergency bowel syndrome, atrophy Gastritis, systemic lupus erythematosus, myasthenia gravis, cerebrospinal spinal multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, pancreatitis, myocarditis, and inflammation caused by infections and trauma
  • Abnormal expression of the signal recognition particle 54 protein 11 of the present invention will also produce certain hereditary, blood Sexually transmitted diseases 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 the degenerative changes of the nervous system, tumors, disorders of growth and development, inflammation, and some Hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) signal recognition particles 54 protein 1 1.
  • the agonist enhances the signal recognition particle 54 protein 11 to stimulate biological functions such as cell proliferation, and the antagonist prevents and treats disorders related to excessive cell proliferation such as various cancers.
  • mammalian cells or a membrane preparation expressing the signal recognition particle 54 protein 11 can be cultured together with the labeled signal recognition particle 54 protein 1 1 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of the signal recognition particle 54 protein 1 1 include screened antibodies, compounds, receptor deletions, and the like.
  • the antagonist of signal recognition particle 54 protein 1 1 can bind to signal recognition particle 54 protein 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 perform biological functions .
  • the signal recognition particle 54 protein 11 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between the signal recognition particle 54 protein 11 and its receptor. .
  • Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • the peptide molecules capable of binding to the signal recognition particle 54 protein 1 1 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to the solid phase. When screening, the signal recognition particle 54 protein 11 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 directed against the signal recognition particle 54 protein 11 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.
  • the polyclonal antibody can be produced by signal recognition particle 54 protein 11 directly injected into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to the signal recognition particle 54 protein 11 include, but are not limited to, hybridoma technology (Kohler and Miltein, Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morr et al, PNAS, 1985, 81: 6851). 0
  • Existing techniques for producing single-chain antibodies US Pa t No. 4946778) can also be used to produce anti-signal recognition Single-chain antibody against 54 protein 11
  • Antibodies against signal recognition particle 54 protein 11 can be used in immunohistochemistry to detect signal recognition particle 54 protein 1 1 in biopsy specimens.
  • Monoclonal antibodies that bind to signal recognition particle 54 protein 1 1 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.
  • the signal recognition particle 54 protein 1 1 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the signal recognition particle 54 protein 11 positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to signal recognition particle 54 protein 11.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of the signal recognition particle 54 protein 1 1.
  • the present invention also relates to a diagnostic test method for quantifying and localizing detection signal recognition particle 54 protein 11 levels.
  • These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of signal recognition particle 54 protein 11 detected in the test can be used to explain the importance of signal recognition particle 54 protein 11 in various diseases and to diagnose diseases in which signal recognition particle 54 protein 11 functions.
  • 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.
  • the polynucleotide encoding the signal recognition particle 54 protein 11 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development, or metabolic abnormalities caused by the non-expression or abnormal / inactive expression of signal recognition particle 54 protein 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated signal recognition particle 54 protein 11 to inhibit endogenous signal recognition particle 54 protein 11 activity.
  • a mutated signal recognition particle 54 protein 11 may be a shortened signal recognition particle 54 protein 11 that lacks a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of signal recognition particle 54 protein 1 1.
  • 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 signal recognition particle 54 protein 1 1 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a signal recognition particle 54 protein 11 can be found in the existing literature (Sambrook, etal.). Another heavy A group of polynucleotides encoding signal recognition particle 54 protein 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 signal recognition particle 54 protein 11 mRNA 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 RM or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides 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 the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the signal recognition particle 54 protein 11 can be used for diagnosis of diseases related to the signal recognition particle 54 protein 11.
  • the polynucleotide encoding the signal recognition particle 54 protein 11 can be used to detect the signal recognition particle 54 protein 11 expression or the abnormal expression of the signal recognition particle 54 protein 11 in a disease state.
  • the DNA sequence encoding signal recognition particle 54 protein 11 can be used to hybridize biopsy specimens to determine the expression status of signal recognition particle 54 protein 11.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe and fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Signal recognition particle 54 protein 11 specific primers RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the signal recognition particle 54 protein 11 transcript.
  • Signal recognition particle 54 protein 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type signal recognition particle 54 protein 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 protein expression, so Northern 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. At present, the specificity of each gene on the chromosome needs to be identified Site. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling 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 DM 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 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 cDNA 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).
  • 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body.
  • 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.
  • Signal recognition particle 54 protein 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of signal recognition particles 54 protein 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
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA solat I ion Ki t Qiegene Products
  • isolating poly A) mRNA 0 2ug poly
  • A mRNA from total RNA by reverse transcription form cDNA.
  • a SMART cDNA cloning kit purchased from C1 on tech
  • the cDNA fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 cc, and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0386E12 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the signal recognition particle 54 protein 11 at 18 62 of the present invention is homologous with the domain signal recognition particle 54 protein characteristic protein.
  • the homology result is shown in FIG. 1, the homology rate is 2: '%, the score is 12.23; the threshold value is 10.94 .
  • Example 3 Cloning of a gene encoding signal recognition particle 54 protein 11 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5-GTACCCTGCTCCTCCTCATCGCCT-3 '(SEQ ID NO: 3)
  • Primer2 5-GTCAATATTTTATTAAAAAACACA-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l / L KC1, 10 ⁇ l / L Tris-HC1, pH 8.5, 1.5 mmol / L MgCl 2 , 200 mol / L dNTP, lOpmol primer, 1U in a 50 ⁇ 1 reaction volume Taq DM polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 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 product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-750bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of signal recognition particle 54 protein 11 gene expression
  • the total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159] 0 This method involves acid guanidinium thiocyanate-chloroform extraction.
  • the tissue is homogenized with 4M guanidinium 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 ), Mix and centrifuge.
  • the aqueous phase was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an R precipitate.
  • the resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transferred to nitrocellulose.
  • 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with RNA-transferred nitrocellulose membrane at 42 ° C overnight in a solution containing 50% Formamide-25mM KH 2 P0 4 (pH7.4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 200 ⁇ 8 / ⁇ 1 salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC- 0.1% SDS at 55 ° C. 30 min. Phosphor Imager was then used for analysis and quantification.
  • Example 5 In vitro expression, isolation, and purification of recombinant signal recognition particle 54 protein 11 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers were designed. The sequences are as follows:
  • Primer 3 5'-CATGCTAGCATGCAGCCGTCTGATGAGGCCACA-3 '(Seq ID No: 5)
  • Primer4 5'-CATGGATCCCTAGAAGCAGGAAGGCAGAAAACA-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET-28M + (Novagen, Cat. No. 69865.3) Enzyme site.
  • the pBS-0386E12 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0386E12 plasmid, primers Primer-3, and Primer-4 were lOpmol and 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 P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • Ligation products were transformed by the calcium chloride method bacteria Escherichia coli DH5cc, after (final concentration of 30 ⁇ 8 / ⁇ 1) grown overnight in LB plates containing kanamycin, positive clones were screened by colony PCR method, and sequenced. A positive clone (PET-0386E12) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method.
  • the host strain BL21 (pET-0386E12) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 ol / L. , Continue to cultivate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The chromatography was performed using an affinity column His. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). The purified target protein signal recognition particle 54 protein 11 was obtained.
  • a peptide synthesizer (product of PE company) was used to synthesize the following peptides specific to 54 protein 11: NH2-Met-Gln-Pro-Ser-Asp-Glu-Ala-T3 ⁇ 4r-Thr-Prc ⁇ -Arg-Pro- Leu-Arg-Ser-C00H
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the i-cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Using protein A-Sepharose from antibody-positive rabbit serum Total IgG was isolated.
  • 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 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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 genomes The sequences and their complementary regions are compared for homology, if the homology with non-target molecular regions is greater than 85 »/. Or if more than 15 consecutive bases are identical, the primary probe should not be used in general;
  • 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)
  • Probe 2 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 membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after the collection solution of the first peak is combined.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (10xDenhardt-s; 6xSSC, 0.1rag / ml CT DNA (calf thymus DM)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardt-s; 6xSSC, 0.1rag / ml CT DNA (calf thymus DM)
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies.
  • 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 target DNA 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 hereditary diseases .
  • the specific methods and steps have been reported in the literature, for example, see the literature DeRi s i, J. L., Lyer, V. ftBrown, P. 0.
  • 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 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to D to fix the slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer by one-step method, and mRNA was purified with Oligotex mRNA Midi Kit (from QiaGen), and the fluorescent reagent Cy3dUTP -Amino- propargyl-Z'-deoxyur idine 5 'was separately reverse-transcribed.
  • the probes from the above two types of tissues were hybridized with the chip in 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 After scanning with a ScanArray 3000 scanner (purchased from General Scanning, USA), the scanned images were analyzed by Imagene software (Biodi scovery, USA), and the Cy3 / Cy5 ratio of each point was calculated, and the ratio was less than 0. Points greater than 5 are considered genes with differential expression.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 11 à particule de reconnaissance du signal 54, 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 protéine 11 à particule de reconnaissance du signal 54.
PCT/CN2000/000698 1999-12-27 2000-12-25 Nouveau polypeptide, proteine 11 a particule de reconnaissance du signal 54, et polynucleotide codant pour ce polypeptide Ceased WO2001047979A1 (fr)

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CN 99125793 CN1301746A (zh) 1999-12-27 1999-12-27 一种新的多肽——信号识别颗粒54蛋白11和编码这种多肽的多核苷酸

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Citations (1)

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EP0902087A2 (fr) * 1997-09-10 1999-03-17 Smithkline Beecham Histidine kinase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0902087A2 (fr) * 1997-09-10 1999-03-17 Smithkline Beecham Histidine kinase

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CN1301746A (zh) 2001-07-04
AU2499101A (en) 2001-07-09

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