WO2001068685A1 - Nouveau polypeptide, proteine humaine propre au sperme em1, em6-48, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine propre au sperme em1, em6-48, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001068685A1
WO2001068685A1 PCT/CN2001/000193 CN0100193W WO0168685A1 WO 2001068685 A1 WO2001068685 A1 WO 2001068685A1 CN 0100193 W CN0100193 W CN 0100193W WO 0168685 A1 WO0168685 A1 WO 0168685A1
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polypeptide
polynucleotide
human sperm
specific proteins
sequence
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PCT/CN2001/000193
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English (en)
French (fr)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2001239123A priority Critical patent/AU2001239123A1/en
Publication of WO2001068685A1 publication Critical patent/WO2001068685A1/zh

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 new polypeptide, a human sperm-specific protein EM1, EM6-48, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. Background technique
  • Basic sperm proteins of bivalve mollusks can be divided into four classes: PL I, PL II, PLIII, and PLIV [Ausio, J. (1992) Mol. Cell. Biochem. 115, 163-172] 0 PLI protein, containing about 300 Amino acid, with a central globular domain (about 80 amino acids), which is homologous to somatic cell-linked histone and N-terminal and C-terminal domains.
  • the PLII protein which contains approximately 150 amino acids, is also surrounded by terminal sequences on both sides of the globular domain.
  • the PLIII protein contains about 100 amino acids, but has no globular domain, and is rich in lysine and arginine residues.
  • the PLIV protein contains about 5G-6G amino acids and is rich in lysine residues but lacks arginine residues.
  • Ensis minor has three sperm-specific proteins in addition to a small amount of somatic core histones: EM6, EM5 and EMI [Gian coTti, V., Russo, E., Gasparini, M., Serrano, D., Del Pie ro, D , Thorne, AW, Cary, P .. D. & Crane- Robinson, C. (1983) Eur. J. Biochem. 136, 509-516] [Giancotti, V., Burattic, E., Santucci, A.,, Ner i. P & Crane-Rob inson, C. (1992) Biochem. Biophys. Actalll 9, 296-302]. According to the mobility of these proteins in the polyacrylamide gel, EM1 belongs to the PL I protein, while EM6 and EM5 belong to the PLII protein. EM6 and EM5 contain globular domains, while EM1 does not.
  • the remaining EM1 peptide chain consists of seven KRSASKKR repeats, sometimes K is replaced by R. The spacing between these repeats is irregular, and two are overlapping.
  • the C-terminus of EM1 is characterized by several aspartic acids, which are involved in regulatory functions.
  • the 205 amino acids at the N-terminus of the EM6 protein consist of almost identical contiguous repeats. These repeats are KKRSXSRKRSAS, where X indicates K, R, H or D. Primary structure of PL I protein in N The ends are highly repeatable, including the motif KRSASK.
  • the N-terminal repeat of the EM6 protein is followed by a globular domain, which is very similar to the PL1 protein of S. sol idissima and the PL II protein of M. trossulus. Its C-terminal fragment is much shorter than the N-terminus. Its basic residues appear in pairs or in units of three, and alanine and serine residues are distributed among them. Although there are not many prolines in the sequence, the underlying ⁇ -turn structure is very interesting.
  • the EM6 protein does not have a SPX (/ R) cdc2 site. . '
  • the human polypeptide of the present invention has 31% identity and 51% similarity at the protein level with members of the sperm-specific proteins EM1, EM6 family, and the protein sequences of both contain the sperm-specific proteins EM1, EM6 family. Characteristic repeats. Based on the above points, the new gene of the present invention is considered to be a new member of the human sperm-specific protein EM1, EM6 family, and is named human sperm-specific protein EMI, EM6 -48. It was inferred that it is similar to human sperm-specific proteins EM1 and EM6, and is a member of the human sperm-specific proteins EM1 and EM6 family, and has similar biological functions. ''
  • the human sperm-specific proteins EM1 and EM6-48 proteins play 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 more needs to be identified in this field.
  • the isolation of the new sperm-specific proteins EM1, EM6-48 proteins 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 coding DNA. Disclosure of 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 human sperm-specific proteins EM1, EM6-48.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human sperm-specific proteins EM1, EM6-48.
  • Another object of the present invention is to provide a method for producing human sperm-specific proteins EM1, EM6-48.
  • Another object of the present invention is to provide a human sperm-specific protein directed to the polypeptide of the present invention.
  • Another object of the present invention is to provide a human sperm-specific protein directed to the polypeptide of the present invention.
  • EM1, EM6-48 mimic compounds, antagonists, agonists, inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human sperm-specific proteins EM1, EM6-48.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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:
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1 27-1 to 34 in SEQ ID NO: 1; and (b) having a sequence in SEQ ID NO: 1 l -.l 899-bit sequence.
  • the invention further relates to a body, in particular an expression vector containing a polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transfected or transfected host cell; and a method comprising culturing said Preparation of host cells and recovery of expression products.
  • a body in particular an expression vector containing a polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transfected or transfected host cell; and a method comprising culturing said Preparation of host cells and recovery of expression products.
  • 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 human sperm-specific proteins EM1, EM6-48 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of human sperm-specific proteins EM1, EM6-48 protein in vitro, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • 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 sperm-specific proteins EM1, EM6-48.
  • Nucleic acid sequence means an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also be Refers to genomic or synthetic DM or RNA, which 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 acid 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 amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing 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 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.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunological activity is the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in a suitable animal or cell to bind to a specific antibody.
  • An "agonist” refers to a molecule that, when combined with the human sperm-specific protein ⁇ M1, EM6-48, causes a change in the protein to regulate the activity of the protein.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to human sperm-specific proteins EM1, EM6-48. ⁇
  • Antagonist or “Inhibitor”-refers to a type that can block or regulate the biological or immunological activity of human sperm-specific proteins EM1, EM6-48 when combined with human sperm-specific proteins EM1, EM6-48. molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human sperm-specific proteins EM1, EM6-48.
  • Regular refers to changes in the function of human sperm-specific proteins EM1, EM6-48, including the increase or decrease in protein activity, changes in binding characteristics, and any other biological properties and functions of human sperm-specific proteins EM1, EM6- 48 Or changes in immune properties. '-'
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human sperm-specific proteins EM1, EM6-48 using standard protein purification techniques.
  • the substantially pure human sperm-specific proteins EM1, EM6-48 can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of human sperm-specific proteins EM1, EM6-48 can be analyzed by amino acid sequence.
  • “Complementary” or “complementary” refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-GA” can be combined with the 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. The inhibition of such 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 the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods, such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Cluster method arranges groups of sequences by checking the distance between all pairs. Into clusters. 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:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art, such as Jotun He in (Hein J., (1990) Methods in emzumology 183: 625-645). "Similarity” refers to amino acids Degree of identical or conservative substitution of amino acid residues at corresponding positions when aligning between sequences.
  • negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids May include lysine and arginine; Amino acids with similar charge having uncharged head groups 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 DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives encode major organisms that retain natural molecules Peptides with chemical properties.
  • Antibody refers to a complete body molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? , It can specifically bind to the epitopes of human sperm-specific proteins EM1, EM6-48.
  • 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 occurs naturally).
  • 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 means: the substance is separated 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 polynucleotide or polypeptide is separated and purified if it is separated from other substances existing in the natural state. .
  • isolated human sperm-specific proteins EM1, EM6-48 refers to human sperm-specific proteins EM1, EM6-48 that are essentially free of other proteins, lipids, carbohydrates, or other substances naturally associated with them.
  • Those skilled in the art can purify human sperm-specific proteins EM1, EM6-48 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human sperm-specific protein EM1, EM6-48 can be analyzed by amino acid sequence. :.
  • the present invention provides a new polypeptide—human sperm-specific proteins EM 1 and EM 6-48, which are 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, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human sperm-specific proteins EM1, EM6-48.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human sperm-specific proteins EM1, EM6-48 of the present invention.
  • a fragment, derivative or 'analog' of the polypeptide of the present invention may be: (I) two kinds in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and replaced The amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences)
  • 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 cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1899 bases in length and its open reading frame 1 27-1434 encodes 4 35 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 31% homology with sperm-specific proteins EM1 and EM6. It can be inferred that the human sperm-specific proteins EM1 and EM6-48 have similar structures and functions of sperm-specific proteins EM1 and EM6 .
  • 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 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 in the present invention, but which differs 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 comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide 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 present invention also relates to a polynucleotide that hybridizes to a sequence described above 50% less, preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 SSC, 0.1% SDS, 6 (TC; or (2) added during hybridization) Use a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) only the identity between the two sequences is at least Hybridization occurs at 95% or more, and more preferably 97% or more.
  • 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 cores. 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 sperm-specific proteins EM1, EM6-48.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequences encoding the human sperm-specific proteins EM1, EM6-48 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of the CDM sequences.
  • the standard method for isolating 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 extracting mRNA, and kits are also commercially available (Qiagene).
  • CDNA library is constructed in a conventional method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989) 0 may be obtained commercially available cDNA library such as cDNA library from Clontech is different. 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) determination of the levels of transcripts of human sperm-specific proteins EM1, EM6-48 (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for the hybridization is the same as any part of the polynuclear acid of Benmaimin
  • the source has a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein 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).
  • the protein products of human sperm-specific proteins EM1 and EM6-48 can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). .
  • a method using PCR technology to amplify DNA / RM (Saiki, et al. Science 1985; 230: 1350-1354) 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 may 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 determined 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, the sequencing must 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 human sperm-specific proteins EM1, EM6-48 coding sequence, and the recombinant technology to produce the Polypeptide method. '
  • a polynucleotide sequence encoding human sperm-specific proteins EM1, EM6-48 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 a protein containing human sperm-specific proteins EM1, EM6- Expression vector with 48 DM sequences and appropriate transcriptional / translational 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 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: E.
  • coli lac or trp 'promoter Lambda phage PL promoter
  • eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters , Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of the enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • the .polynucleotide 'acid encoding a human sperm-specific protein EM1, EM6-48 or a recombinant vector containing the polynucleotide can be transformed or transferred into a host cell to constitute a gene containing the polynucleotide or the recombinant vector Engineered host cells.
  • 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; 'animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of 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. Staining method: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human sperm-specific protein EMI, EM6-48 (Science, 1984; 224: L431). Generally have 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. '.'
  • the recombinant polypeptide may be coated intracellularly, or expressed on a cell membrane, or secreted extracellularly.
  • the physical, chemical, and other properties can be used to separate 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, osmosis, 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, osmosis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ' Ion exchange chromatography, high performance liquid
  • Fig. 1 is a comparison diagram of the amino acid sequence homology between the sperm-specific proteins EM1, EM6-48 and the sperm-specific proteins EM1, EM6 of the present invention.
  • the upper sequence is human sperm-specific proteins EM1, EM6-48, and the lower sequence is sperm-specific proteins EM1, EM6.
  • Identical amino acids are represented by 'single-character amino acids' between the two sequences, and similar amino acids are represented by "+”. '
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human sperm-specific proteins EM1, EM6-48. 48KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5cc. The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the inserted cDNA fragment contained in this clone was determined in both directions by synthesizing a series of primers.
  • the 0659g07 clone contained a full-length cDNA of 1899bp (as shown in Seq ID NO: 1), and a 1308bp open reading frame (0RF) from 127bp to 1434bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0659g07 and the encoded protein was named human sperm-specific protein EM1, EM6-48.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human sperm-specific egg 'White EM1, EM6-48 and the protein sequence encoded by the present invention were subjected to a Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.
  • 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'— TAATTGGGTACCCGGGCCCCCCCT -3 '(SEQ ID NO: 3)
  • Primer2 5,-AAAACCATTTTTATTATCATTACC -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, 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 are added. (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • Cx- 32 P dATP with 32 P- DM labeled probe prepared by random priming method.
  • the DM probe used was the PCR amplified human sperm-specific protein EM1, EM6-48 coding region sequence (127bp to 1434bp) shown in FIG.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in lx SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human sperm-specific proteins EM1, EM6-48
  • Primer3 5'- CCCCATATGATGGCGGCTAGTGATACAGAGCGA -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTCAAACTGCATCCATTCCTCGCAT -3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, and the Nde I and BamH I restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Sex endonuclease site.
  • the pBS-0659g07 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- 0659g07 plasmid, primers Primer-3 and Primer-4, and 10 ⁇ l, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • 'Loop parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed with colibacillus DH50 by the calcium chloride method. After the LB plate (final concentration: 30 M g / ml) was cultured overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0659g07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (Novagen product) by the calcium chloride method.
  • the host bacteria BL21 (pET-0659g07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used for chromatography to obtain 6 histidine (6His-Tag). The purified human sperm-specific proteins EM1, EM6-48 were obtained.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human sperm-specific proteins EM1, EM6-48 specific peptides:
  • 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 hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • 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 blotting Trace method, Nor thern blot method, and copy method, etc., all are used to fix the polynucleotide sample to be tested on the filter membrane and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic 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 utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained. .
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and its known genomic sequence and their complementarity Region for homology comparison, if the homology with the non-target molecular region is greater than 85% or there are more than 1 consecutive bases, the primary probe should generally not be used; '
  • Probe 1 belongs to the first type of probe and is completely homologous or complementary to the gene fragment of SEQ ID D 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) : '5'- TGGCGGCTAGTGATACAGAGCGAGATGGACTAGCCCCAGAA -3' (SEQ ID NO: 9)
  • 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) : '5'- TGGCGGCTAGTGATACAGAGCGAGATGGACTAGCCCCAGAA -3' (SEQ ID NO: 9)
  • DNA PROBES GH Ke ll er MM Manak; S tockton Pres s, 1989 (USA) and more commonly used molecular cloning laboratory manuals such as "Molecular Cloning Experiment Guide” U 998 Second Edition) [US] Sambu Luke waiting, Science Press.
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC Yue Mo nitrocellulose of appropriate size
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as 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 method steps have been reported in the literature, for example, refer to the literature DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And the literature Helle, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-21
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs as target DNA, including the present invention Polynucleotide. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and a Cartesian 7500 spotter (purchased from Cartesian, USA) was used to spot the glass medium. The distance is 280 ⁇ ⁇ .
  • the spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on a glass slide to prepare a chip.
  • the specific method steps have been reported in the literature in various ways. The post-spotting processing steps of this embodiment are: '..
  • 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 malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Sperm-specific proteins EM1, EM6 members are mainly used as the basic protein components of sperm, and may be part of their cytoskeleton and movement.
  • the polypeptide of the present invention has the characteristic sequences of the sperm-specific proteins EM1 and EM6 family, is a member of the sperm-specific proteins EM1 and EM6 family, and has similar biological functions. It is mainly related to sperm development and movement in the body, and its abnormal expression can cause sperm disease.
  • the abnormal expression of the human sperm-specific proteins EM1, .EM6-48 of the present invention will produce various diseases, especially sperm diseases, including but not limited to: seminoma, male infertility
  • the polypeptide of the present invention can be directly used in the treatment of diseases, for example, it can treat various diseases, especially sperm diseases and the like.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human sperm-specific proteins EM1, EM6-48. Agonism enhances human sperm-specific proteins EM1, EM6- 4 8 stimulates cell proliferation and other biological functions, and antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human sperm-specific proteins EM1, EM6-48 can be cultured with labeled human sperm-specific proteins EM1, EM6-48 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human sperm-specific proteins EM1, EM6-48 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human sperm-specific proteins EM1, EM'6-48 can bind to human sperm-specific proteins EM1, EM6- 48 and eliminate their functions, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to The polypeptide cannot perform biological functions.
  • human sperm-specific proteins EM1, EM6-48 can be added to bioanalytical assays to determine the effect of compounds on human sperm-specific. Protein EM1, EM6-48 and their receptor interactions To determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. ⁇ Polypeptide molecules capable of binding to human sperm-specific proteins EM1, EM6-48 can be screened for a variety of possible combinations of amino acids bound to the solid phase. Machine peptide library. When screening, the human sperm-specific proteins EM1, EM6- 48 molecules should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human sperm-specific proteins EM1, EM6-48 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human sperm-specific proteins EM1, EM6-48 directly 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 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human sperm-specific proteins EM1, EM6- 48 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256 ': 495-497), triple tumor technology, human beta-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 U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human sperm-specific proteins EM1, EM6-48.
  • Antibodies against human sperm-specific proteins EM1, EM6-48 can be used in immunohistochemistry to detect human sperm-specific proteins EM1, 6-48 in biopsy specimens.
  • Monoclonal antibodies that bind to human sperm-specific proteins EM1, EM6-48 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 sperm-specific protein Human sperm-specific protein
  • E 1, EM6-48 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • SPDP thiol cross-linking agent
  • This hybrid antibody can be used to kill human sperm-specific proteins EM1, EM6-48 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human sperm-specific proteins EM1, EM6-48.
  • the proper dose of antibody can stimulate or block the production or activity of human sperm-specific proteins EM1, EM6-48.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the levels of human sperm-specific proteins EM1, EM6-48.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of human sperm-specific proteins EM1, EM6-48 detected in the test can be used to explain the importance of human sperm-specific proteins EM1, EM6- 8 in various diseases and to diagnose human sperm-specific proteins EM1, EM6-48 A working 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 enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human sperm-specific proteins EM1, EM6-48 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 sperm-specific proteins EM1, EM6-48. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human sperm-specific proteins EM1, EM6-48 to inhibit endogenous human sperm-specific proteins EM1, EM6- 48 activity.
  • a variant human sperm-specific protein EM1, EM6-48 may be a shortened, human sperm-specific protein 'white EM1, EM6-48 lacking a signaling domain, although it can bind to downstream substrates, but lacks Signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human sperm-specific proteins EM1, EM6-48.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding human sperm-specific proteins EM1, EM6-48 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human sperm-specific proteins EM1, EM6-48 can be found in the existing literature (Sambrook, et al.).
  • the polynucleotide encoding human sperm-specific proteins EM1, EM6- 48 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 sperm-specific proteins .EM1, EM6-48 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DM, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RM 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 human sperm-specific proteins EM1, EM6-48 can be used for the diagnosis of diseases related to the human sperm-specific proteins EM1, EM6-48.
  • Polynucleotides encoding human sperm-specific proteins EM1, EM6-48 can be used to detect the expression of human sperm-specific proteins EM1, EM6-48 or the abnormal expression of human sperm-specific proteins EM1, EM6-48 under disease conditions.
  • the DNA sequence encoding human sperm-specific proteins EM1, EM6-48 can be Used to hybridize biopsy specimens to determine the expression status of human sperm-specific proteins EM1, EM6-48.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related 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 DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human sperm-specific proteins EM1, EM6-48 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) amplification in vitro to detect the transcription products of human sperm-specific proteins EM1, EM6-48. ' ⁇ '
  • Detection of mutations in the human sperm-specific proteins EM1, EM6-48 genes can also be used to diagnose human sperm-specific proteins EM1, EM6-48-related diseases.
  • Human sperm-specific proteins EM1, EM6-48 mutations include point mutations, translocations, deletions, recombination, and any other abnormalities compared to the normal wild-type human sperm-specific proteins EM1, EM6-48 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.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments. '
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • 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 physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available through contact with Johns Hopkins University Welch Medical Li brary is available online). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as 'medicines' 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.
  • containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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 sperm-specific proteins EM1, EM6-48 are administered in amounts effective to treat and / or prevent specific indications.
  • the amount and range of human sperm-specific proteins EM1, EM6-48 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.

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Description

一种新的多肽一一人精子特异蛋白 ΕΜ1，ΕΜ6-48和编码这种多肽的多核苷酸 技术领域
本发明属于生物技术领域， 具体地说， 本发明描述了一种新的多肽一一人 精子特异蛋白 EM1，EM6- 48, 以及编码此多肽的多核苷酸序列。 本发明还涉及此 多核苷酸和多肽的制备方法和应用。 背景技术
双壳类软体动物基本精子蛋白可以分为四类： PL I , PL II , PLIII和 PLIV [Ausio, J. (1992)Mol. Cell. Biochem.115, 163-172] 0 PLI 蛋白， 包含大约 300 个氨基酸， 有一个中心球状结构域 （约 80 个氨基酸） ， 该结构域与体细胞连 接组蛋白和 N末端以及 C末端结构域是同源的。 PLII蛋白， 包含大约 150个氨 基酸， 同样地在球状结构域 ^两旁是末端序列。 PLIII蛋白， 包含大约 100 个氨 基酸， 但是没有球状结构域， 富含赖氨酸和精氨酸残基。 PLIV蛋白， 包含大约 5G-6G个氨基酸， 富含赖氨酸残基， 但缺乏精氨酸残基。
Ensis minor除少量的体细胞核心组蛋白以外有三种精子特异性蛋白： EM6, EM5 和 EMI [Gian coTti, V. , Russo, E. , Gasparini, M. , Serrano, D. , Del Pie ro, D. , Thorne, A. W. , Cary， P.. D. &Crane- Robinson, C. (1983) Eur. J. Biochem. 136, 509-516] [Giancotti, V. , Burattic, E. , Santucci, A. , Ner i. P&Crane-Rob inson, C. (1992) Biochem. Biophys. Actalll9, 296—302]。 按照这些蛋白在聚丙 酰胺凝胶的移动性来划分， EM1属于 PL I蛋白， 而 EM6和 EM5则属于 PLII蛋白。 EM6和 EM5包含球状结构域， 而 EM1则没有。
EM1蛋白有几个共同特征 ά 在 Ν末端有几个连 的二肽 S (K/R) 重复结抅。 这个基元在 PLIII蛋白和 PLII蛋白 N 末端中也同样保守 [Carlos, L. , Jutglar, L. 'Borrell. I. , Hunt, D. F. &Ausio, J. - (1993a) J. Biol. Chem.268, 185-194] [Rocchini, C.， Rice, P. &Ausion, J. (1995) FEBS Lett.363, 37-40]。 在这个保守 基元之后是一个包含 6个潜在 cdc2磷酸位点序列（ SPAK, SPKR/K)。 剩下的 EM1 肽链由 7个 KRSASKKR重复序列组成， 有时 K替换成 R。 这些重复序列之间的间 距是不规则的， 并且有两个是重叠的。 EM1 的 C 末端特征是几个天门冬氨酸， 这些天门冬氨酸与调节功能有关。
EM6蛋白的 N末端 205个氨基酸组成 Π个几乎一致的连 重复序列， 这些 重复序列是 KKRSXSRKRSAS, X表示是 K, R， H 或 D。 PL I蛋白的一级结构在 N 末端有高度的重复性， 包括基元 KRSASK。
EM6蛋白 N末端重复序列后紧跟着一个球状结构域，这个球状结构域与 S. sol idissima的 PL1蛋白以及 M. trossulus的 PL II蛋白极其相似。 其 C末端片段比 N 末端要短得多， 它的基本残基成对或以三个为单位出现， 丙氨酸和丝氨酸残 基点缀分布其中。 虽然序列中的脯氨酸不多， 但是潜在的 α-转角结构是非常 有意义的。 EM6蛋白没有 SPX ( /R ) cdc2位点。 .'
本发明的人的多肽与精子特异蛋白 EM1，EM6 家族中的成员在蛋白水平上有 31%的同一性及 51%的相似性， 且两者的蛋白序列均含有精子特异蛋白 EM1，EM6 家族的特征性重复序列。 基于以上各点， 故认为本发明的新基因为人精子特异 蛋白 EM1,EM6家族的新成员， 命名为人精子特异蛋白 EMI, EM6 -48。 并以此推 断其与人精子特异蛋白 EM1，EM6相似， 同为人精子特异蛋白 EM1，EM6 家族的成 员， 并具有相似的生物学功能。 ' '
由于如上所述人精子特异蛋白 EM1，EM6 - 48 蛋白在调节细胞分裂和胚胎发育 等机体重要功能中起重要作用， 而且相信这些调节过程中涉及大量的蛋白， 因 而本领域中一直需要鉴定更多参与这些过程的人精子特异蛋白 ΕΜ1, ΕΜ6-48 蛋 白， 特别是鉴定这种蛋白的氨基酸序列。. 新人精子特异蛋白 EM1，EM6 - 48 蛋白 编码基因的分离也为研究确定该蛋白在健康和疾病状态下的作 ¾提供了基础。 这种蛋白可能构成开发疾病诊断和 /或治疗药的基础， 因此分离其编码 DNA 是 非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一人精子特异蛋白 ΕΜ1, ΕΜ6-48 以及其片段、 类似物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。 '
本发明的另一个目的是提供含有编码人精子特异蛋白 ΕΜ1, ΕΜ6-48 的多核苷 酸的重组载体。
本发明的另一个目的是提供含有编码人精子特异蛋白 ΕΜ1，ΕΜ6-48 的多核苷 酸的基因工程化宿主细胞。
本发明的另一个目的是提供生产人精子特异蛋白 ΕΜ1, ΕΜ6-48的方法。
本发明的另一个目 的是提供针对本发明的多肽一一人精子特异蛋白
EMI, EM6-48的抗体。
本发明的另 一个目 的是提供了针对本发明多肽一一人精子特异蛋白 EM1，EM6- 48的模拟化合物、 拮抗剂、 激动剂、 抑制剂。
本发明的另一个目的是提供诊断治疗与人精子特异蛋白 ΕΜ1 , ΕΜ6-48 异常相 关的疾病的方法。
本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ I D No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多 肽是具有 SEQ I D NO: 2氨基酸序列的多肽。
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或 其变体：
(a)编码具有 SEQ I D No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(c)与 (a)或（b)的多核苷酸序列具有至少 70%相同性的多核苷酸。
^佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ I D NO: 1 中 1 27-1 4 34位的序列； 和（b)具有 SEQ I D NO: 1 中 l -.l 899位的序列。
本发明另外涉及一种含有本发明多核苷酸的,体， 特别是表达载体； 一种 用该载体遗传工程化的宿主细胞， 包括转化、 转 或转染的宿主细胞； 一种包 括培养所述宿主细胞和回收表达产物的制备本发明.多肽的方法。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制人精子特异蛋白 ΕΜ1，ΕΜ6- 48蛋白活性的化合物的方法， 其包括利用本发明的多肽。 本发明还涉 及用该方法获得的化合物。
本发明还涉及一种体外检测与人精子特异蛋白 ΕΜ1，ΕΜ6-48 蛋白异常表达相 关的疾病或疾病易感性的方法， 包括检测生物样品中所述多肽或其编码多核苷酸 序列中的突变， 或者检测生物样品中本发明多肽的量或生物活性。
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮 抗剂或抑制剂以及药学上可接受的载体。
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、 发育性 疾病或免疫性疾病或其它由于人精子特异蛋白 ΕΜ1 , ΕΜ6 - 48表达异常所引起疾病 的药物的用途。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。
本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以 指基因组或合成的 DM或 RNA， 它们可以是单链或双链的， 代表有义链或反义链。 类似地， 术语 "氨基酸序列" .是指寡肽、 肽、 多肽或蛋白质序列及其片段或部 分。 当本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序 列时， 这种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质 分子相关的完整的天然氨基酸。
蛋白质或多核苷酸 "变体" 是指一种具有一'个或 -多个氨基酸或核苷酸改变 的氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸 序列中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其 中替换的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异 亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。 -
"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的 缺失。 .
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存在 的分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸或 核苷酸替换一个或多个氨基酸或核苷酸。
"生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类似 地， 术语 "免疫学活性" 是 天然的、 重组的或合成蛋白质及其片段在合适的动 物或细胞中诱导特定免疫反应以 与特异性抗体 合的能力。
"激动剂" 是指当与人精子特异蛋白 ΈΜ1，ΕΜ6-48结合时， 一种可引起该蛋 白质改变从而调节该蛋白质活性的分子。 激动剂可以包括蛋白质、 核酸、 碳水 化合物或任何其它可结合人精子特异蛋白 ΕΜ1，ΕΜ6-48的分子。 ·
"拮抗剂" 或 "抑制物" -是指当与人精子特异蛋白 ΕΜ1 , ΕΜ6-48结合时， 一 种可封闭或调节人精子特异蛋白 ΕΜ1，ΕΜ6-48的生物学活性或免疫学活性的分 子。 拮抗剂和抑制物可以包括蛋白质、 核酸、 碳水化合物或任何其它可结合人 精子特异蛋白 ΕΜ1，ΕΜ6-48的分子。
"调节" 是指人精子特异蛋白 ΕΜ1，ΕΜ6 - 48的功能发生改变， 包括蛋白质活 性的升高或降低、 结合特性的改变及人精子特异蛋白 ΕΜ1，ΕΜ6- 48的任何其它生 物学性质、 功能或免疫性质的改变。 ' -'
"基本上纯"是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化人精子特异蛋白 ΕΜ1，ΕΜ6 - 48。 基本上纯的人精子特异蛋白 ΕΜ1，ΕΜ6- 48 在非还原性聚丙烯酰胺凝胶上能产生单一 的主带。 人精子特异蛋白 ΕΜ1 , ΕΜ6- 48多肽的纯度可用氨基酸序列分析。 "互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的 多核苷酸天然结合。 例如， 序列 "C- T- G-A" 可与互补的序列 "G- A- C- T" 结合。 两个单链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于 核酸链之间杂交的效率及强度有明显影响。
"同源性" 是指互补的程度， 可以是部分同源或完全同源。 "部分同源" 是指一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂 交。 这种杂交的抑制可通过在严格性程度降低的条件下进行杂交 （ Southern印 迹或 Northern印迹等） 来检测。 基本上同源的序列或杂交探针可竟争和抑制完 全同源的序列与靶序列在的严格性程度降低的条件下的结合。 这并不意味严格 性程度降低的条件允许非特异性结合， 因为严格性程度降低的条件要求两条序 列相互的结合为特异性或选择性相互作用。
"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或 相似的百分率。 可用 电子方法测定相同性百分率， 如通过 MEGALIGN程序 ( Lasergene software package, DNASTAR, Inc. , Madison Wis. ) 。 MEGALIGN 程序可根据不同的方法如 Cluster法比较两种或多种序列（Higgins, D. G. 和 P.M. Sharp (1988) Gene 73: 237-244) 0 Cluster法通过检查所有配对之间的距 离将各组序列排列成簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如序 列 A和序列 B之间的相同性百分率通过下式计算：
序列 A与序列 B之间匹配的残基个数 X 100 序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数
也可以通过 Cluster法或用本领域周知的方法如 Jotun He in 测定核酸序列 之间的相同性百分率（Hein J. , (1990) Methods in emzumology 183: 625-645)„ "相似性" 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或 保守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括 天冬氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷 的头部基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸 和丙氨酸； 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。
"反义" 是指与特定的 DM或 RNA序列互补的核苷酸序列。 "反义链" 是指 与 "有义链" 互补的核酸链。
"衍生物" 是指 HFP或编码其的核酸的化学修饰物。 这种化学修饰物可以是 用烷基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物 学特性的多肽。
"抗体" 是指完整的 体分子及其片段， 如 Fa、 ？（^') 2及？ , 其能特异 性结合人精子特异蛋白 ΕΜ1，ΕΜ6-48的抗原决定簇。
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更 为相似， 但仍保留原始结合活性的抗体。 . ·
"分离的" 一词指将物质从它原来的环境'（例如， 若是自然产生的就指其 天然环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物 中就是没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中 与之共存的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也可能这样的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是 它天然环境的成分， 它们仍然是分离的。 - 如本发明所用， "分离的" 是措:物质从其原.始环境中分离出来 （如果是天 然的物质， 原始环境即是天然环境）'。 如活体细胞内的天然状态下的多聚核苷 酸和多肽是没有分离纯化的， 但同样的多聚核智酸或多肽如从天然状态中同存 在的其他物质中分开， 则为分离纯化的。
如本文所用， "分离的人精子特异蛋白 EM1 , EM6 - 48 " 是指人精子特异蛋白 ΕΜ1 , ΕΜ6-48 基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本 领域的技术人员能用标准的蛋白质纯化技术纯化人精子特异蛋白 ΕΜ1，ΕΜ6-48。 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。 人精子特异蛋 白 EM1 , EM6- 48多肽的纯度能用氨基酸序列分析。 ：.
本发明提供了一种新的多肽一一人精子特异蛋白 ΕΜ 1 , ΕΜ6-48， 其基本上是由 SEQ I D NO: 2所示的氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学合成 的产物， 或使用重组技术从原核或真核宿主（例如， 细菌、 酵母、 高等植物、 昆虫 和哺乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的多肽可以是糖 基化的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸 残基。
本发明还包括人精子特异蛋白 EM1 , EM6- 48 的片段、 衍生 和类似物。 如 本发明所用， 术语 "片段" 、 "衍生 '物" 和 "类似物" 是指基本上保持本发明 的人精子特异蛋白 ΕΜ1 , ΕΜ6-48 相同的生物学功能或活性的多肽。 本发明多肽 的片段、 衍生物或'类似物可以是： （ I ) 这样二种， 其中一个或多个氨基酸残 基被保守或非保守氨基酸残基 （优选的是保守氨基酸残基） 取代， 并且取代的 氨基酸可以是也可以不是由遗传密码子编码的； 或者 （ I I ) 这样一种， 其中一 个或多个氨基酸残基上的某个基团被其它基团取代包含取代基； 或者 （ I I I ) 这样一种， 其中成熟多肽与另一种化合物 （ 比如延长多肽半衰期的化合物， 例 如聚乙二醇） 融合； 或者 （ IV ) 这样一种， 其中附加的氨基酸序列融合进成熟 多肽而形成的多肽序列 （如前导序列或分泌序列或用来纯化此多肽的序列或蛋 白原序列） 通过本文的阐述， 这样的片段、 衍生物和类似物被认为在本领域技 术人员的知识范围之内。
本发明提供了分离的核酸 （多核苷酸） ， 基本由编码具有 SEQ I D NO: 2 氨 基酸序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SEQ I D NO: 1 的 核苷酸序列。 本发明的多核苷酸是从人胎脑组织的 cDNA 文库中发现的。 它包 含的多核苷酸序列全长为 1899个碱基， 其开放读框 1 27-1434编码了 4 35 个氨 基酸。 根据氨基酸序列同源比较发现， 此多肽与精子特异蛋白 EM1，EM6 有 31 % 的同源性， 可推断出该人精子特异蛋白 EM1，EM6 - 48 具有精子特异蛋白 EM1 , EM6 相似的结构和功能。
本发明的多核苷酸可以是 DNA形式或是 RNA 形式。 DNA 形式包括 cDNA、 基 因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链 或非编码链。 编码成熟多肽的编码区序列可以与 SEQ I D NO: 1 所示的编码区序 列相同或者是简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是 指编码具有 SEQ I D NO: 2 的蛋白质或多肽， 但与 SEQ I D NO: 1 所示的编码区序 列有差别的核酸序列。
编码 SEQ I D NO: 2的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附 加编码序列） 以及非编码序列。
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加 编码和 /或非编码序列的多核苷酸。
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基 酸序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天 然发生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异 体、 缺失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸 的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质 上改变其编码的多肽的功能。
本发明还涉及与以上所描述的序列杂交的多核苷酸 （两个序列之间具有至 少 50%, 优选具有 70%的相同性） 。 本发明特别涉及在严格条件下与本发明所 述多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低 离子强度和较高温度下的杂交和洗脱， 如 0.2 SSC, 0.1%SDS,6(TC;或（2)杂交 时加用变性剂， 如 50%(v/v)甲酰胺， 0.1。/。小牛血清 /0. l%Ficoll, 42°C等； 或（3) 仅在两条序列之间的相同性至少在 95%以上，更好是 97%以上时才发生杂交。 并 且， 可杂交的多核苷酸编码的多肽与 SEQ ID NO: 2 所示的成熟多肽有相同的 生物学功能和活性。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核 酸片段"的长度至少含 10个核苷酸， 较好是至少 20- 30个核苷酸， 更好是至少 50-60 个核苷酸， 最好是至少 100 个核苷酸以上。 核酸片段也可用于核酸的扩 增技术（如 PCR)以确定和 /或分离编码人精子特异蛋白 ΕΜ1,ΕΜ6-48的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码人精子特异蛋白 ΕΜ1，ΕΜ6-48 的特异的多核苷酸序列能用多种 方法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些技术包括但不 局限于： 1)用探针与基因组或 cDNA文库杂交以检出同源的多核苷酸序列， 和 2) 表达文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸片段。
本发明的 DNA片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DNA 序列； 2)化学合成 DNA序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA 最不常用。 DNA 序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDM序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录， 形成质粒或 噬菌体 cDNA 文库。 提取 mRNA 的方法已有多种成熟的技术， 试剂盒也可从商业 途径获得（Qiagene)。 而构建 cDNA 文库也是通常的方法（Sambrook, et al.， Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989) 0 还可得到商业供应的 cDNA 文库， 如 Clontech 公司的不同 cDNA 文库。 当结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括（但不 限于）： （l)DNA-DNA或 DNA-RNA杂交； （2)标志基因功能的出现或丧失； （3)测定 人精子特异蛋白 EM1,EM6- 48 的转录本的水平； （4)通过免疫学技术或测定生物 学活性， 来检测基因表达的蛋白产物。 上述方法可单用， 也可多种方法联合应 用。
在第（1)种方法中， 杂交所用的探针是与本麦明的多核 #酸的任何一部分同 源， 其长度至少 10个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷 酸， 最好是至少 100个核苷酸。 此外， 探针的长度通常在 2000个核苷酸之内， 较佳的为 1000个核苷酸之内。 此处所用的探针通常是在本发明的基因序列信息 的基础上化学合成的 DNA序列。 本发明的基因本身或者片段当然可以用作探针。 DNA探针的标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。
在第（4)种方法中， 检测人精子特异蛋白 ΕΜ1,ΕΜ6-48 基因表达的蛋白产物 可用免疫学技术如 Western 印迹法， 放射免疫沉淀法， 酶联免疫吸附法（ELISA) 等。 .
应 用 PCR 技 术 扩 增 DNA/RM 的 方 法 （Saiki, et al. Science 1985; 230: 1350-1354)被优选用于获得本发明的基因。 特别是很难从文库中得到 全长的 cDNA 时， 可优选使用 RACE 法（RACE - cDNA末端快速扩增法）， 用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择， 并可用常 规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
如上所述得到的本发明的基因， 或者各种 DNA 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法（Sanger et al. PNAS, 1977, 74： 5463- 5467)测定。 这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的 cDNA序列， 测 序需反复进行。 有时需要测定多个克隆的 cDNA 序列， 才能拼接成全长的 cDNA 序列。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或直接 用人精子特异蛋白 EM1，EM6- 48 编码序列经基因工程产生的宿主细胞， 以及经重 组技术产生本发明所述多肽的方法。 '
本发明中， 编码人精子特异蛋白 ΕΜ1，ΕΜ6-48 的多核苷酸序列可插入到载体 中， 以构成含有本发明所述多核苷酸的重组载体。 术语 "载体" 指本领域熟知 的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转录病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细菌中表达 的基于 T7 启动子的表达载体（Rosenberg, et al. Gene, 1987, 56: 125)； 在哺 乳动物细胞中表达的 pMSXND 表达载体（Lee and Nathans, J Bio Chem. 263: 3521, 1988)和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能 在宿主体内复制和稳定， 任何质粒和载体都可以用于构建重组表达载体。 表达 载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻译调控元 件。
本领域的技术人员熟知的方法能用于构建含编码人精子特异蛋白 EM1,EM6- 48 的 DM 序列和合适的转录 /翻译调控元件的表达载体。 这些方法包括体外重 组 DNA 技术、 DNA 合成技术、 体内重组.技术等（Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mRNA 合成。 这些启动子的代表性例子有： 大肠杆菌的 lac 或 trp'启动子； λ噬菌体 的 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早 期和晚期 SV40 启动子、 反转录病毒的 LTRs 和其它一些已知的可控制基因在原 核细胞或真核细胞或其病毒中表达的启动子。 表达载体还包括翻译起始用的核 糖体结合位点和转录终止子等。 在载体中插入增强'子序列将会使其在高等真核 细胞中的转录得到增强。 增强子是 DNA表达的顺式作用因子， 通常大约有 10到 300 个碱基对， 作用于启动子以增强基因的转录。 可举的例子包括在复制起始 点晚期一侧的 100到 270个碱基对的 SV40增强子、 在复制起始点晚期一侧的多 瘤增强子以及腺病毒增强子等。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择 转化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗 性以及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件 （如启动 子、 增强子等） 和选择性标记基因。 - 本发明中， 编码人精子特异蛋白 ΕΜ1,ΕΜ6-48 的.多核苷'酸或含有该多核苷酸 的重组载体可转化或转导入宿主细胞， 以构成含有该多核苷酸或重组载体的基 因工程化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌细胞； 或是低等真 核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞如酵母； 植物细 胞； 昆虫细胞如果蝇 S2或 Sf9; '动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。
用本发明所述的 DNA序列或含有所述 DM序列的重组载体转化宿主细胞可 用本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能 吸收 DNA 的感受态细胞可在指数生长期后收获， 用 (12法处理， 所用的步骤 在本领域众所周知。 可供选择的是用 MgCl2。 如果需要， 转化也可用电穿孔的方 法进行。 当宿主是真核生物， 可选用如下的 DNA 转.染方法： 磷酸钙共沉淀法， 或者常规机械方法如显微注射、 电穿孔、 脂质体包装等。
通过常规的重组 DNA 技术， 利用本发明的多核苷酸序列可用来表达或生产 重组的人精子特异蛋白 EMI, EM6-48 (Science, 1984; 224： L431)。 一般来说有 以下步骤：
(1) .用本发明的编码人 人精子特异蛋白 ΕΜ1, ΕΜ6-48 的多核苷酸（或变异 体）， 或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基.中培养宿主细胞；
(3).从培养基或细胞中分离、 纯化蛋'白质。
在步骤 （ 2 ) 中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种 常规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当 的细胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将 细胞再培养一段时间。 ' .'
在步骤 （ 3 ) 中， 重组多肽可包被于 胞内、 或在细胞膜上表达、 或分泌到 细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通.过各种分离方法分 离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但 并不限于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波处理、 超离心、 分子筛层析（凝胶过滤）、 吸附层析、' 离子交换层析、 高 效液相层析（HPLC)和其它各种液相层析技术及这些方法的结合。 附图的简要说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所 界定的本发明范围。
图 1是本发明人精子特异蛋白 EM1，EM6 - 48和精子特异蛋白 EM1，EM6的氨基酸 序列同源性比较图。 上方序列是人精子特异蛋白 ΕΜ1，ΕΜ6-48， 下方序列是精子 特异蛋白 EM1，EM6。 相同氨基酸在两个序列间用'单字符氨基酸表示， 相似氨基 酸用 "+" 表示。 '
图 2为分离的人精子特异蛋白 EM1，EM6- 48的聚丙烯酰胺凝胶电泳图 （ SDS- PAGE) 。 48KDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 实现本发明的最佳方式
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法，通常按照常规条件如 Sambrook等人， 分子克隆：实验室手册（New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建 议的条件。 ' ' 实施例 1: 人精子特异蛋白 ΕΜ1，ΕΜ6-48的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolation Kit ( Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录 形成 cDNA。用 Smart cDNA克隆试剂盒（购自 Clontech )将 cDNA片段'定向插入到 pBSK (+) 载体（Clontech公司产品）的多克隆位点上， 转化 DH5cc， 细菌形成 cDNA文库。 用 Dye terminate cycle react ion sequencing ki t (Perkin- Elmer公司产品) 和 ABI 377自 动测序仪（Perkin-Elmer公司）测定所有克隆的 5'和 3'末端的序列。 将测定的 cDNA序 列与已有的公共 DNA序列数据库 （Genebank ) 进行比较， 结果发现其中一个克隆 0659g07的 cDNA序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA片段 进行双向测定。 结果表明， 0659g07克隆所含的全长 cDNA为 1899bp (如 Seq ID NO: 1 所示） ， 从第 127bp至 1434bp有一个 1308bp的开放阅读框架 （ 0RF ) , 编码一个新的 蛋白质 （如 Seq ID NO: 2所示） 。 我们将此克隆命名为 pBS - 0659g07， 编码的蛋白质 命名为人精子特异蛋白 EM1， EM6-48。 实施例 2: cDNA 克隆的同源检索
将本发明的人精子特异蛋 '白 ΕΜ1，ΕΜ6-48的序列及其编码的蛋白序列， 用 Blast 程 序 (Bas iclocal Alignment search tool) [Altschul, SF et al.
J.Mol.Biol.1990; 215: 403-10] , 在 Genbanl 、 Swissport等数据库进行同源检索。 与本发明的人精子特异蛋白 EMI, EM6-48同源性最高的基因是一种已知的精子特异蛋 白 EM1，EM6, 其编码的蛋白在 Genbank的准入号为 L41834。 蛋白质同源结果示于图 1， 两者高度同源， 其相同性为 31%; 相似性为 51%。 实施例 3: 用 RT- PCR方法克隆编码人精子特异蛋白 ΕΜ1,ΕΜ6-48的基因
用胎脑细胞总 RNA为模板， 以 oligo-dT为引物进行逆转录反应合成 cDNA,用 Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5'— TAATTGGGTACCCGGGCCCCCCCT -3' (SEQ ID NO: 3)
Primer2: 5，- AAAACCATTTTTATTATCATTACC -3' (SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列;
Primer2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在.50 μ 1的反应体积中含有 50隱 ol/L KC1, 10匪 ol/L Tris-
CI, (pH8.5), 1.5mmol/L MgCl2, 200 μ mol/L dNTP, lOpmol引物， 1U的 Taq DNA聚合酶 (Clontech公司产品)。 在 PE9600型 DNA热循环仪（Perkin- Elmer公司）上按下列条件 反应 25个周期: 94。C 30sec; 55°C 30sec; 72°C 2min。 在 RT- PCR时同时设 β - actin 为阳性对照和模板空白为阴性对照。 扩增产物用 Q.IAGEN公司的试剂盒纯化， 用 TA克 隆试剂盒连接到 pCR载体上 （ Invitrogen公司产品） 。 DNA序列分析结果表明 PCR产 物的 DNA序列与 SEQ ID NO: 1所示的 1- 1899bp完全相同。 实施例 4: Northern 印迹法分析人精子特异蛋白 EMI, EM6-48基因的表达：
用一步法提取总 RNA [Anal. Biochem 1987, 162, 156-159]。 该法包括酸性硫氰 酸胍苯酚-氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸钠， 0.2M乙酸钠 （ pH4.0 ) 对 组织进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 .（49: 1 ) , 混合后离 心。 吸出水相层， 加入异丙醇 （0.8体积） 并将混合物离心得到 RNA沉淀。 将得到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 20 g RNA, 在含 20mM 3- ( N -吗啉代） 丙磺酸 （pH7.0 ) - 5mM乙酸钠 - ImM EDTA- 2.2M甲醛的 1.2%琼脂糖凝胶上进行电泳。 然后转移至硝酸纤维素膜上。 用 cx- 32P dATP通过随机引物法制备 32P-标记的 DM探 针。 所用的 DM探针为图 1所示的 PCR扩增的人精子特异蛋白 EM1，EM6- 48编码区序列 (127bp至 1434bp)。 将 32P-标记的探针 （约 2 χ 106cpm/ml ) 与转移了 RNA的硝酸纤维 素膜在一溶液中于 42°C杂交过夜， 该溶液包含 50%甲酰胺 - 25mM KH2P04 ( pH7.4 ) -5 χ SSC- 5 χ Denhardt's溶液和 200 μ g/ml鮭精 DNA。 杂交之后， 将滤膜在 l x SSC- 0.1%SDS中于 55°C洗 30min。 然后， 用 Phosphor Imager进行分析和定量。 实施例 5: 重组人精子特异蛋白 EM1，EM6- 48的体外表达、 分离和纯化
根据 SEQ ID N0: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序 列如下：
Primer3: 5'- CCCCATATGATGGCGGCTAGTGATACAGAGCGA -3' ( Seq ID No: 5 ) Primer4: 5'- CATGGATCCTCAAACTGCATCCATTCCTCGCAT -3， (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点， 其后分别为目的基因 5'端 和 3'端的编码序列， Nde I和 BamH I酶切位点相应于表达载体质粒 pET-28b (+) (Novagen 公司产品， Cat. No.69865.3)上的选择性内切酶位点。 以含有全长目的基因的 pBS- 0659g07质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 pBS- 0659g07 质粒 10pg、 引物 Primer- 3和 Primer- 4分另 U为 lOp'mol、 Advantage polymerase Mix (Clontech公司产品） 1 μ 1。 '循环参数： 94。C 20s, 60°C 30s, 68°C 2 min,共 25个 循环。 用 Ndel和 BamHI分别对扩增产物和质粒 pET-28(+)进行双酶切，分别回收大片 段，并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH50 ,在含卡那霉素 (终浓度 30 M g/ml ) 的 LB平板培养过夜后， 用菌落 PCR方法筛选阳性克隆， 并进行 测序。 挑选序列正确的阳性克隆 （pET-0659g07 ) 用氯化钙法将重组质粒转化大肠 杆菌 BL21 (DE3)plySs (Novagen公旬产品）。 在含卡那霉素 （终浓度 30 μ §/η1 ) 的 LB 液体培养基中， 宿主菌 BL21 ( pET- 0659g07 ) 在 37°C培养至对数生长期， 加入 IPTG 至终浓度 lmmol/L, 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心收集上清， 用能与 6个组氨酸（ 6His- Tag)结合的亲和层析柱 His. Bind Quick Cartridge( Novagen 公司产品） 进行层析， 得到了纯化的目的蛋白人精子特异蛋白 ΕΜ1，ΕΜ6-48。 经 SDS- PAGE电泳， 在 48KDa处得到一单一的条带（图 2 )。 将该条带转移至 PVDF膜上用 Edams 水解法进行 N-端氨基酸序列分析， 结果 N-端 15个氨基酸与 SEQ ID NO: 2所示的 N-端 15 个氨基酸残基完全相同。 ' 实施例 6 抗人精子特异蛋白 EM1，EM6- 48抗体的产生
用多肽合成仪 （PE公司产品） 合成下述人精子特异蛋白 ΕΜ1，ΕΜ6-48特异性的 多肽：
NH2-Met-A 1 a- A 1 a-Ser-As -Thr-G 1 u-Arg-Asp-G 1 y- Leu- A 1 a-Pro-G 1 u-Lys-C00H
(SEQ ID NO: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合， 方法参 见： Avrameas, et al. Immunochemistry, 1969; 6: 43。 用 4mg上述血蓝蛋白多肤复合 物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂 加强免疫一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA测 定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 IgG。 将多肽结合于溴化氰活化的 Sephar0se4B柱上， 用亲和层析法从总 IgG中分离抗多肽 抗体。 免疫沉淀法证明纯化的抗体可特异性地与人精子特异蛋白 ΕΜ1,ΕΜ6-48结合。 实施例 7: 本发明的多核苷酸片段用作杂交探针的应用
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。 .
本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针， 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核 苷酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印 迹法、 Nor thern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的步骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被合有标记探针的杂交缓冲液替换， 并 保温使探针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除 掉。 本实施例利用较高强度的洗膜条件 （如较低盐浓度和较高的温度）， 以使杂交 背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类： 第一类探针 是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探 针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施 例选用斑点印迹法将样品固定在滤膜上， 在较高强度的的洗膜条件下， 第一类探 针与样品的杂交特异性最强而得以保留。 .
一、 探针的选用
从本发明的多核苷酸 SEQ ID NO: 1 中选择寡核苷酸片段用作杂交探针， 应遵 循以下原则和需要考虑的几个方面：
1 , 探针大小优选范围为 18- 50个核苷酸； -'
2， GC含量为 30%- 70%, 超过则非特异性杂交增加；
3， 探针内部应无互补区域；
4， 符合以上条件的可作为初选探针， 然后进一步作计算机序列分析， 包括将该 初选探针分别与其来源序列区域 （即 SEQ ID NO: 1 ) 和其 已知的基因组序列 及其互补区进行同源性比较， 若与非靶分子区域的同源性大于 85%或者有超过 1 个连续碱基完全相同， 则该初选探针一般就不应该使用;'
5， 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定： 完成以上各方面的分析后挑选并合成以下二个探针：
探针 1 ( probel );. 属于第一类探针， 与 SEQ I D NO: 1 的基因片段完全 同源或互补 （ 41Nt ): '
5'- TGGCGGCTAGTGATACAGAGCGAGATGGACTAGCCCCAGAA C- 3' ( SEQ ID NO: 8 ) 探针 2 ( probe2 ), 属于第二类探针， 相当于 SEQ ID NO: 1 的基因片段 或其互补片段的替换突变序列 （41Nt ): ' 5'- TGGCGGCTAGTGATACAGAGCGAGATGGACTAGCCCCAGAA -3' ( SEQ ID NO: 9 ) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文 献： DNA PROBES G. H. Ke l l er; M. M. Manak; S tockton Pres s, 1989 (USA)以及更常用 的分子克隆实验手册书籍如 《分子克隆实验指南》 U 998 年第二版） [美]萨姆布 鲁克等著， 科学出版社。
样品制备：
1 , 从新鲜或冰冻组织中提取 DNA
步骤： 1 ) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2 ) 以 lOOOg离心切碎组织 10分钟。 3)用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 25誦01/ Tris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl2 ) 悬浮沉淀 （大约 10ml/g )0 4 ) 在 4°C用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5 ) lOOOg 离心 10分钟。 6) 用重悬细胞沉淀 （每 0. lg最初组织样品加 l-5ml ), 再以 1000g离心 10分钟。 7)用裂解缓冲液重悬沉淀 （每 O. lg最 组织样品加 lml ), 然后接以下 的苯酚抽提法。
2, DNA的苯酚抽提法 ·.
步骤： 1 ) 用 l-10ml冷 PBS洗细胞， 1 OOQg离心 10分钟。 2 ) 用冷细胞裂解液 重悬浮沉淀的细胞（l x lO8细胞 /ml ) 最少应用 lOOul 裂解缓冲液。 3) 加 SDS 至 终浓度为 1%, 如果在重悬细胞之前将 SDS:直接加入到细胞沉淀中， 细胞可能会形 成大的团块而难以破碎， 并降低的总产率。 这一牟在抽提〉107细胞时特别严重。 4 ) 加蛋白酶 K至终浓度 200ug/ml。 5) 50°C保温反应 1小时或在 37 C轻轻振摇过夜。 6)用等体积苯酚： 氯仿： 异戊醇 （ 25: 24： 1 )抽提， 在小离心机管中离心 10分 钟。 两相应清楚分离， 否则重新进行离心。 7 ) 将水相转移至新管。 8) 用等体积 氯仿： 异戊醇 （24: 1 ) 抽提， 离心 10分钟。 9) 将含 DNA的水相转移至新管。 然 后进行 DNA的纯化和乙醇沉淀。 '.'
3, DNA的纯化和乙醇沉淀
步骤： 1 )将 1/10体积 2mol/L醋酸钠和 2倍体积冷 100%乙醇加到 DNA溶液中， 混匀。 在- 20。C放置 1小时或至过夜。 2 ) 离心 10分钟。 3) 小心吸出或倒出乙醇。 4 )用 70%冷乙醇 500ul洗涤沉淀， 离心 5分钟。 5 )小心吸出或倒出乙醇。 用 500ul 冷乙醇洗涤沉淀， 离心 5分钟。 6) 小心吸出或倒出.乙醇， 然后在吸水纸上倒置使 残余乙醇流尽。 空气干燥 10-15 分钟， 以使表面乙醇挥发。 注意不要使沉淀完全 干燥， 否则较难重新溶解。 7) 以小体积 TE或水重悬 DNA沉淀。 低速涡旋振荡或 用滴管吹吸， 同时逐渐增加 TE，. 混合至 DNA充分溶解， 每 1-5 X 106细胞所提取的 大约加 lul。
以下第 8-13步骤仅用于必须除去污染时， 否则可直接进行第 14步骤。
8) 将 RNA酶 A加到 DNA溶液中， 终浓度为 100ug/ml, 37"C保温 30分钟。 9)加 入 SDS和蛋白酶 K, 终浓度分别为 0.5%和
Figure imgf000018_0001
。 37QC保温 30分钟。 10 )用 等体积的苯酚： 氯仿： 异戊醇 （ 25: 24： 1 )抽提反应液， 离心 10 分钟。 11 ) 小 心移出水相， 用等体积的氯仿： 异戊醇 （24: 1 ) 重新抽錄， 离心 10 分钟。 12 ) 小心移出水相， 加 1/10体积 2mo.l/L醋酸钠和 2. '5体积冷乙醇， 混匀置 -20°C 1小 时。 13 )用 70%乙醇及 100%乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3 - 6 步骤。 14 ) 测定 A26Q和 A2S。以检测 DNA的纯度及产率。 15 ) 分装后存放于 -20°C。 样膜的制备：
1 )取 4 x 2 张适当大小的硝酸纤维素膜（NC 月莫）， 用铅笔在其上轻轻标出点样 位置及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度条件 和强度条件洗膜 。 ， ，
2 ) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。
3 ) 置于浸润有 0. Imol/LNaOH, 1.5mol/LNaCl 的滤纸上 5分钟 （两次）， 晾干置 于浸润有 0. 5mol/L Tris-HCl ( pH7.0 ), 3mol/LNaCl 的滤纸上 5分钟 (两次）, 晾 干。
4 ) 夹于干净滤纸中，. 以铝箔包好， 60- 80QC真空干燥 2小时。 .
探针的标记
1 ) 3 μ lProbe ( 0. IOD/Ιθ μ 1 ), 加入 2 μ IKinase缓冲液， 8- 10 uCi γ- 32P- dATP+2U Kinase, 以补加至终体积 20 μ 1。
2 ) 37 °C 保温 1小时。
3 )加 1/5体积的溴酚蓝指示剂 （BPB)。
4 ) 过 Sephadex G-50柱。 ，
5 ) 至有 32P-Probe洗出前开始收集第一峰 （可用 Monitor监测：)。
6 ) 5滴 /管， 收集 10- 15管。
7 )用液体闪烁仪监测同位素量
8 ) 合并第一峰的收集液后即为所需制备的 ，32P-Probe (第二峰为游离 γ-32Ρ- dATP )。
预杂交 '
将样膜置于塑料袋中，加入 3- 10mg预杂交液 ( lOxDenhardt's; 6xSSC, 0. lmg/ml CT DNA (小牛胸腺 DNA)。）， 封好袋口后， 68°C水浴摇 2小时。
杂交
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42°C水洛摇过夜。 洗膜： 高强度洗膜：
1 ) 取出已杂交好的样膜。
2 ) 2xSSC, 0.1%SDS中， 40。C洗 15分钟 ( 2次）。
3 ) 0. lxSSC, 0.1%SDS中， 40°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0.1%SDS中， 55°C洗 30分钟 （ 2次）， 室温晾干。 低强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0.1%SDS中 37°C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0.1°/。SDS中， 37 C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0.1%SDS中， 40 洗 15分钟 （ 2次）， 室温晾干。
X -光自显影：
-70°C, X-光自显影 （压片时间根据杂交斑放射性强弱而定）。
实验结果：
采用低强度洗膜条件所进行的杂交实验， 以上两个探针杂交斑放射性强弱没 有明显区别； 而采用高强度洗膜条件所进行的杂交实验， 探针 1 的杂交斑放射性 强度明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分 析本发明的多核苷酸在不同组织中的存在和差异表达。 实施例 8 DNA Microarray
基因芯片或基因微矩阵 （DNA Microarray ) 是目前许多国家实验室和大制药 公司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、 高密度 地排列在玻璃、 硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分 析， 以达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作 为靶 DNA 用于基因芯片技术用于高通量研究新基因功能； 寻找和筛选组织特异性 新基因特别是肿瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法 步骤在文献中已有多种报道， 如可参阅文献 DeRisi, J. L. , Lyer, V. &Brown, P.0. (1997)Science278, 680-686.及文献 Helle, R. A. , Schema, M. , Chai, A. , Shalom, D. , (1997) PNAS 94: 2150-2155.
(一） 点样
各种不同的全长 cDNA共计 4000条多核苷酸序列作为靶 DNA，其中包括本发明 的多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul左右, 用 Cartesian 7500点样仪（购自美国 Cartesian公司）点于玻璃介 质上， 点与点之间的距离为 280 μΐη。 将点样后的坡片进行水合、 干燥、 置于紫外 交联仪中交联， 洗脱后干燥使 DNA 固定在玻璃片上制备成芯片。 其具体方法步骤 在文献中已有多种报道， 本实施例的点样后处理步骤是： ' ..
1- 潮湿环境中水合 4小时；
2. 0.2%SDS洗涤 1分钟；
3. ddH20洗涤两次， 每次 1分钟；
4. NaBH4封闭 5分钟；
5. 95°C水中 2分钟；
6. 0.2%SDS洗涤 1分钟；
7. ddH20冲洗两次；
8. 凉干， 25°C储存于暗处备用。
(二）探针标记
用一步法分别从正常肝与肝癌中抽提总 mRNA, 并用 Oligotex mRNA Midi
Kit (购自 QiaGen 公司）纯化 mRNA，通过反转录分別将荧光试剂 Cy3dUTP (5-Amino- propargy 1-2' -deoxyuri dine 5'-tr iphate coupled to Cy3 fluorescent dye , 购 自 Amersham Phamacia Biotech 公司）标记正常肝组织的 mRNA , 用荧光试剂 Cy5dUTP (5-Amino-propargy 1-2· -deoxyuri dine 5'-tr iphate coupled to Cy5 fluorescent dye, 购自 Amersham Phamacia Biotech 公司）标记肝癌组织 mRNA, 经纯化后制备出探针。 具体步骤参照及方法见：
Schena, M. , Shalon, D.， Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol.93: 10614- 10619. Schena, M. , Shalon, Dari. , Davis, R. W. (1995) Science.270. (20) : 467-480.
(三） 杂交
分别将来自 以上两种组织的探针与芯片一起在 UniHyb™ Hybridization
Solution (购自 TeleChem 公司）杂交液中进行杂交 16 小时， 室温用洗涤液 （ 1 x SSC, 0.2%SDS ) 洗涤后用 ScanArray 3000扫描仪 （购自美国 General S anning公 司） 进行扫描， 扫描的图象用 Imagene软件 （美国 Biodiscovery公司）进行数据 分析处理， 算出每个点的 Cy3/Cy5 比值， 该比值小于 0.5大于 2 的点被认为是表 达有差异的基因。 实验结果表明 , Cy3signal=10704.8(取四次实验的平均值 ), Cy5signal=8382.43 (取四次实验的平均值 ) , Cy3/Cy5=l. 27705,本发明的多核苷酸在以上两种组织中 的表达无明显差异。 工业实用性
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV 感染和免疫 性疾病等。
精子特异蛋白 EM1，EM6 家族中的成员主要作为精子的基本蛋白成分， 可能 是其细胞骨架与运动的一部分。
本发明的多肽具有精子特异蛋白 EM1，EM6 家族的特征性序列， 为精子特异 蛋白 EM1 , EM6 家族成员， 具有相似的生物学功能。 它在体内主要与精子发育运 动相关， 其表达异常可引起精子疾病。
由此可见， 本发明的人精子特异蛋白 ΕΜ1，.ΕΜ6-48 的表达异常将产生各种疾 病尤其是精子疾病， 这些疾病包括但不限于： 精原细胞瘤， 男性不孕症
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗各种疾病尤其是精子疾病等。 本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂）人精子特 异蛋白 ΕΜ1，ΕΜ6-48 的药剂的方法。 激动 提高人精子特异蛋白 ΕΜ1，ΕΜ6-48 刺 激细胞增殖等生物功能， 而拮抗剂阻止和治疗与细胞过度增殖有关的紊乱如各 种癌症。 例如， 能在药物的存在下， 将哺乳动物细胞或表达人精子特异蛋白 ΕΜ1 , ΕΜ6-48的膜制剂与标记的人精子特异蛋白 EM1 , EM6- 48—起培养。 然后测定 药物提高或阻遏此相互作用的能力。
人精子特异蛋白 ΕΜ1，ΕΜ6-48 的拮抗剂包括筛选出的抗体、 化合物、 受体缺 失物和类似物等。 人精子特异蛋白 ΕΜ1，ΕΜ'6-48 的拮抗剂可以与人精子特异蛋白 EM1，EM6- 48 结合并消除其功能， 或是抑制该多肽的产生， 或是与该多肽的活性 位点结合使该多肽不能发挥生物学功能。
在筛选作为拮抗剂的化合物时， 可以将人精子特异蛋白 ΕΜ1 , ΕΜ6-48加入生 物分析测定中， 通过测定化合物对人精子特异.蛋白 ΕΜ1 , ΕΜ6-48 和其受体之间相 互作用的影响来确定化合物是否是拮抗剂。 用上述筛选化合物的同样方法， 可 以筛选出起拮抗剂作用的受体缺失物和类似物。 ·· 能与人精子特异蛋白 EM1，EM6 - 48 结合的多肽分子可通过筛选由各种可能组合的氨基酸结合于固相物组成的随 机多肽库而获得。 筛选时， 一般应对人精子特异蛋白 EM1,EM6- 48分子进行标记。 本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原 以生产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供 了针对人精子特异蛋白 ΕΜ1，ΕΜ6-48 抗原决定簇的抗体。 这些抗体包括（但不限 于）： 多克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab 片段和 Fab 表达文 库产生的片段。
多克隆抗体的生产可用人精子特异蛋白 EM1,EM6- 48直接注射免疫动物 （如 家兔， 小鼠， 大鼠等） 的方法得到， 多种佐剂可用于增强免疫反应， 包括但不 限于弗氏佐剂等。 制备人精子特异蛋白 EM1，EM6- 48 的单克隆抗体的技术包括但 不限于杂交瘤技术（Kohler and Milstein. Nature, 1975, 256': 495-497) , 三瘤 技术， 人 Β-细胞杂交瘤技术， EBV-杂交瘤技术等。 将人恒定区和非人源的可变 区结合的嵌合抗体可用已有的技术生产（Morrison et al ， PNAS, 1985, 81: 6851)。 而已有的生产单链抗体的技术（U.S. Pat No.4946778)也可用于生产抗人精子特 异蛋白 ΕΜ1,ΕΜ6-48的单链抗体。
抗人精子特异蛋白 ΕΜ1,ΕΜ6-48 的抗体可用于免疫组织化学技术中， 检测活 检标本中的人精子特异蛋白 ΕΜ1， 6-48。
与人精子特异蛋白 ΕΜ1,ΕΜ6-48 结合的单克隆抗体也可用放射性同位素标 记， 注入体内可跟踪其位置和分布。 这种放射性标记的抗体可作为一种非创伤 性诊断方法用于肿瘤细胞的定位和判断是否有转移。
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如人精子特异蛋白
Ε 1,ΕΜ6-48 高亲和性的单克隆抗体可与细菌或植物毒素（如白喉毒素， 蓖麻蛋 白， 红豆碱等）共价结合。 一种通常的方法是用巯基交联剂如 SPDP， 攻击抗体 的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交抗体可用于杀灭 人精子特异蛋白 ΕΜ1,ΕΜ6-48阳性的细胞。
本发明中的抗体可用于治疗或预防与人精子特异蛋白 ΕΜ1，ΕΜ6-48相关的疾 病。 给予适当剂量的抗体可以刺激或阻断人精子特异蛋白 EM1,EM6 - 48 的产生或 活性。
本发明还涉及定量和定位检测人精子特异蛋白 EM1,EM6- 48水平的诊断试验 方法。 这些试验是本领域所熟知的， 且包括 FISH测定和放射免疫测定。 试验中 所检测的人精子特异蛋白 ΕΜ1，ΕΜ6-48 水平， 可以用作解释人精子特异蛋白 ΕΜ1,ΕΜ6- 8在各种疾病中的重要性和用于诊断人精子特异蛋白 ΕΜ1，ΕΜ6-48起作 用的疾病。 本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行 特异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分 析。
编码人精子特异蛋白 ΕΜ1 , ΕΜ6-48的多核苷酸也可用于多种治疗目的。 基因 治疗技术可用于治疗由于人精子特异蛋白 EM1 , EM6- 48 的无表达或异常 /无活性 表达所致的细胞增殖、 发育或代谢异常。 重组的基因治疗载体（如病毒载体）可 设计用于表达变异的人精子特异蛋白 EM1，EM6 - 48 , 以抑制内源性的人精子特异 蛋白 EM1，EM6- 48 活性。 例如， 一种变异的人精子特异蛋白 ΕΜ1，ΕΜ6-48 可以是 缩短的、 缺失了信号传导功能域的人精子特异蛋'白 ΕΜ1 , ΕΜ6-48 , 虽可与下游的 底物结合， 但缺乏信号传导活性。 因此重组的基因治疗载体可用于治疗人精子 特异蛋白 EM1 , EM6- 48表达或活性异常所致的疾病。 来源于病毒的表达载体如逆 转录病毒、 腺病毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病毒等可用于将编 码人精子特异蛋白 EM1 , EM6- 48 的多核苷酸转移至细胞内。 构建携带编码人精子 特异蛋白 EM1，EM6 - 48 的多核苷酸的重组病毒载体的方法可见于已有文献 (Sambrook, e t a l. )。 另外重组编码人精子特异蛋白 EM1，EM6- 48 的多核苷酸可 包装到脂质体中转移至细胞内。
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织 中； 或在体外通过载体（如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再将细胞移植到体内等。
抑制人精子特异蛋白 .EM1 , EM6- 48 mRNA 的寡核苷酸（包括反义 RNA 和 DNA) 以及核酶也在本发明的范围之内。 核酶是一种能特异性分解特定 RM的酶样 RNA 分子， 其作用机制是核酶分子与互补的靶 RNA特异性杂交后进行核酸内切作用。 反义的 RNA和 DM及核酶可用已有的任何 RNA或 DNA合成技术获得， 如固相磷 酸酰胺化学合成法合成寡核苷酸的技术已广泛应用。 反义 RM 分子可通过编码 该 RNA 的 DNA序列在体外或体内转录获得。 这种 DNA序列已整合到载体的 RNA 聚合酶启动子的下游。 为了增加核酸分子的稳 性， 可用多种方法对其进行修 饰， 如增加两侧的序列长度， 核糖核苷之间的连接应用磷酸硫酯键或肽键而非 磷酸二酯键。
编码人精子特异蛋白 ΕΜ1，ΕΜ6-48 的多核苷酸可用于与人精子特异蛋白 ΕΜ1 , ΕΜ6-48的相关疾病的诊断。 编码人精子特异蛋白 EM1 , EM6- 48的多核苷酸可 用于检测人精子特异蛋白 ΕΜ1 , ΕΜ6-48 的表达与否或在疾病状态下人精子特异蛋 白 EM1 , EM6 - 48 的异常表达。 如编码人精子特异蛋白 ΕΜ1，ΕΜ6-48 的 DNA序列可 用于对活检标本进行杂交以判断人精子特异蛋白 ΕΜ1，ΕΜ6-48 的表达状况。 杂交 技术包括 Southern印迹法， Northern印迹法、 原位杂交等。 这些技术方法都是 公开的成熟技术， 相关的试剂盒都可从商业途径得到。 本发明的多核苷酸的一 部分或全部可作为探针固定在微阵列（Microarray)或 DNA芯片（又称为 "基因芯 片" ）上， 用于分析组织中基因的差异表达分析和基因诊断。 用人精子特异蛋白 ΕΜ1,ΕΜ6-48 特异的引物进 RNA-聚合酶链反应（RT-PCR)体外扩增也可检测人精 子特异蛋白 ΕΜ1，ΕΜ6-48的转录产物。 '·'
检测人精子特异蛋白 ΕΜ1,ΕΜ6-48基因的突变也可用于诊断人精子特异蛋白 ΕΜ1,ΕΜ6-48相关的疾病。 人精子特异蛋白 ΕΜ1,ΕΜ6-48突变的形式包括与正常野 生型人精子特异蛋白 EM1,EM6- 48 DNA序列相比的点突变、 易位、 缺失、 重组和 其它任何异常等。 可用已有的技术如 Southern 印迹法、 DNA 序列分析、 PCR 和 原位杂交检测突变。 另外， 突变有可能影响蛋白的表达， 因此用 Northern 印迹 法、 Western印迹法可间接判断基因有无突变。
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体 位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用 于标记染色体位置。 根据本发明， 为了将这些序,列与疾病相关基因相关联， 其 重要的第一步就是将这些 DNA序列定位于染色体上。 - 简而言之， 根据 cDNA制备 PCR引物（优选 15- 35bp)， 可以将序列定位于染色 体上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只 有那些含有相应于引物的人基因的杂合细胞会 生扩增的片段。 '
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。 .
将 cDNA克隆与中期染 ^体进行荧光原位杂交（FISH) , 可以在一个步骤中精 确地进行染色体定位。 此技术的综述， 参见 V.erma等， Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, ' New York(1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联。 这些数据可见于例如， V.Mckusick,Mendelian Inheritance in Man (可通过与 Johns Hopkins University Welch Medical Li bra ry联机获得）。 然后可通过连锁分析， 确定基因与业已定位到染色体区域 上的疾病之间的关系。
接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如果在一 些或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该突变可能是疾病的病因。 比较患病和未患病个体， 通常涉及首先寻找染色 体中结构的变化， 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺 失或易位。 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与 疾病有关的染色体区域的 cDNA , 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分辨能力和每 20kb对应于一个基因）。
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与 合适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐类、 缓冲 液、 甘油以及它们的组合。 组合物包含安全有效量的多肽或拮抗剂以及不影响 药物效果的载体和赋形剂。 这些组合物可以作为'药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒， 容器中装有一种或多 种本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药 品或生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用 或销售的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它 的治疗化合物结合使用。 .
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 人精子特异蛋白 ΕΜ1，ΕΜ6-48 以有效地治疗和 / 或预防具体的适应症的量来给药。 施用于患者的人精子特异蛋白 ΕΜ1 , ΕΜ6-48 的 量和剂量范围将取决于许多因素 ·， 如给药方式、 待治疗者的健康条件和诊断医 生的判断。

Claims

权 利 要 求 书
1、一种分离的多肽-人精子特异蛋白 EM1 , EM6- 48，其特征在于它包含有： SEQ ID N0: 2所示的氨基酸序列的多肽、 或其多肽的活性片段、 类似物或衍生物。
2、 如权利要求 1 所述的多肽， 其特征在于所 ii多肽、 类似物或衍生物的氨基 酸序列具有与 SEQ I D NO: 2所示的氨基酸序列至少 95%的相同性。
3、 如权利要求 2所述的多肽， 其特征在于它包含具有 SEQ I D NO: 2所示的氨基 酸序列的多肽。
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种： (a) 编码具有 SEQ I D NO: 2 所示氨基酸序列的多肽或其片段、 类似物、 衍生 物的多核苷酸；
(b) 与多核苷酸 ) 互补的多核苷酸； 或
(c) 与 （a ) 或 （b ) 有至少 70%相同性的多核苷酸。 ：
5、 如权利要求 4 所述的多核苷酸， 其特征在于所述多核苷酸包含编码具有 SEQ I D NO: 2所示氨基酸序列的多核苷酸。
6、如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸的序列包含有 SEQ I D NO: 1 中 127- 1434位的序列或 SEQ I D NO: 1 中 1 -1 899位的序列。
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由权利要求 4-6 中的 任一权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载 体。 '
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下 列一种宿主细胞： '
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用权利要求 4-6 中的任一权利要求所述多核苷酸转化或转导的宿主细 胞。
9、 一种具有人精子特异蛋白 ΕΜ1，ΕΜ6-48 活性的多肽的制备方法， 其特征在于 所述方法包括：
(a) 在表达人精子特异蛋白 EM1，EM6- 48 条件下， 培养权利要求 8 所述的工 程化宿主细胞；
(b) 从培养物中分离出具有人精子特异蛋白 EMI , EM6-48活性的多肽。
1 0、 一种能与多肽结合的抗体，其特征在于所述抗体是能与人精子特异蛋白 ΕΜ1 , ΕΜ6-48特异性结合的抗体。
11、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促进、 拮抗或抑制人精子特异蛋白 ΕΜ1, ΕΜ6-48的活性的化合物。 ·
12、 如权利要求 11所述的化合'物， 其特征在于它是 SEQ ID 'N0: 1所示的多核苷 酸序列或其片段的反义序列。 ：
13、 一种权利要求 11 所述化合物的应用， 其特征在于所述化合物用于调节人 精子特异蛋白 ΕΜ1, ΕΜ6-48在体内、 体外活性的方法。
14、 一种检测与权利要求 1-3 中的任一权利要求所述多肽相关的疾病或疾病易 感性的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的 活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。
15、 如权利要求 1-3 中的任一权利要求所述多肽的应用， 其特征在于它应用于 筛选人精子特异蛋白 EM1, EM6- 48 的模拟物、 激动剂， 拮抗剂或抑制剂； 或者 用于肽指紋图谱鉴定。 '
16、 如权利要求 4-6 中的任一权利要求所述的核酸分子的应用， 其特征在于它 作为引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者甩于制造基因 芯片或微阵列。 ；
17、 如权利要求 1-6 及 11 中的任一权利要求所述的多肽、 '· 多核苷酸或化合物 的应用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑 制剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与人精子特异 蛋白 ΕΜ1, ΕΜ6-48异常相关的疾病的药物组合物。 ·
18、 权利要求 1-6 及 11 中的任一权利要求所述的多肽、 多核苷酸或化合物的 应用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血液病， HIV感染和免疫性疾病和各类炎症的药物。
PCT/CN2001/000193 2000-03-02 2001-02-26 Nouveau polypeptide, proteine humaine propre au sperme em1, em6-48, et polynucleotide codant pour ce polypeptide WO2001068685A1 (fr)

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AU2001239123A AU2001239123A1 (en) 2000-03-02 2001-02-26 A novel polypeptide-human sperm specific protein em1,em6-48 and the polynucleotide encoding said polypeptide

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CN00111837.4 2000-03-02
CN00111837A CN1311228A (zh) 2000-03-02 2000-03-02 一种新的多肽——人精子特异蛋白em1,em6－48和编码这种多肽的多核苷酸

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0516386A1 (en) * 1991-05-29 1992-12-02 Merck & Co. Inc. Eimeria mitis DNA probes
WO1999029855A1 (en) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Mutants of endostatin, 'em 1' having anti-angiogenic activity and methods of use thereof
WO1999053021A1 (en) * 1998-04-09 1999-10-21 Bresagen Limited Cell differentiation/proliferation and maintenance factor and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0516386A1 (en) * 1991-05-29 1992-12-02 Merck & Co. Inc. Eimeria mitis DNA probes
WO1999029855A1 (en) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Mutants of endostatin, 'em 1' having anti-angiogenic activity and methods of use thereof
WO1999053021A1 (en) * 1998-04-09 1999-10-21 Bresagen Limited Cell differentiation/proliferation and maintenance factor and uses thereof

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AU2001239123A1 (en) 2001-09-24

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