WO2001040480A1 - Novel polypeptide - human endopolypeptidase 6 and polynucleotide encoding it - Google Patents

Abstract

The invention concerns human endopolypeptidase 6 and polynucleotide encoding it. The invention also concerns the process of producing the polypeptide by recombinant DNA technique. The methods for treating many diseases e.g. malignant tumor, hemopathy, infection of HIV, immunological diseases and pruritus utilizing the polypeptide are disclosed. The invention discloses the antagonist against the polypeptide and therapeutics thereof. The invention also discloses the uses of the polynucleotide, which encodes the novel human endopolypeptidase 6.

Description

New polypeptide human endopeptidase 6 and polynucleotide encoding the same

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human endopeptidase 6, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique

The egg white albumin endonuclease enzyme CC''llpp is a dimer of egg white albumin, which contains subunits containing one by one hydrolysed egg white albumin. ((Ccll ppPP)) and one or two related AATTPP junctions are one of the regulatory subunits ((ccllppAA and and cc llppXX)). . CCllppPP is a serine amino acid protease enzyme, which has a similar activity to that of pancreatin curd lactoprotease. . Its enzyme-catalyzed active sites are serine amino acid-111111 and histidine amino acid-113366. These two sites are located at CC11 _PP enzymes from different sources are highly conserved, and can be used to identify whether a certain type of protein albumin belongs to CCllpp eggs. Albumen. .

 Its conservative base sequence sequence is: TT--xx ((22))-[[LLIIVVMMFF]] --GG--XX--AA-- [[SSAACC]] -SS-- [[MMSSAA]]-[[PPAAGG]]-[[SSTTAA]] ((wherein SS is the active site of enzymatic activity)) and RR-- xx ((33))-[[EEAAPP]] --XX ((33))-[[LLIIVVMMFFYYTT]] --MM-- [[LLIIVVMM]] --HH--QQ--PP ((the Among them, HH is the active site of enzymatic catalytic activity))

 The active activity of CCllpp is related to the different symbiotic growth and growth state of fine cell cells. . In the early and early stages of the logarithmic growth phase, the activity of CC llpp is relatively low, but the activity of CC llpp increases with the growth and growth of fine cells. The rapid increase in strength is the highest in the activity of the fine-cell cells in the logarithmic growth phase after the long-term period, and in the fine cell cells that enter the stable and regular period. Then it regained its original activity. . These changes in living activity are related to the living activity of the CCll pp AA component of CCllpp, and to the living activity of CC llpp PP and CC llpp XX. Not big. .

 The function of the protein protease endonuclease CCllpp is to cut and cut the protein into small peptides. This process requires AATTPP and magnesium Participation of ionomers. . In the absence of AATTPP, only oligomeric peptides that are shorter than 55 residues will be cut. .

 Another function of the protein endonuclease CCllpp is to act as a bridesmaid as a molecular molecule. . It mediates the process of egg protein albumin insertion into the structure of the cell membrane membrane junction structure, the process of depolymerization or oligomerization of the egg protein complex complex . . The protein protease endonuclease CC''llpp can also repair and repair the protein albumin that has been damaged due to the pressure pressure caused by the unhealthy environment, and And live activated MMUu, λλ, and PPII DDNNAA required for protein reproduction. . Looking at it this way, the protein proteolyzed CCllpp not only not only can hydrolyze the protein proteoglycan with water, but also it is also lifting in the protein proteolytic repair. Important to play a role. . If this loss of viability of egg protein albumen is caused, the function of egg protein repair and restoration of fine cell cells can be affected. . If the protein key protein that regulates the control of the cell cycle cycle is damaged and cannot be repaired and repaired in time, then the fine cell will have a chance It may be able to split and proliferate without limit, and ultimately lead to carcinogenic cancer. .

 CC llpp egg white plays an important role in balancing the balance of egg white protein in fine cell cells. . The decrease in the activity of egg white water hydrolysing enzymes will reduce the partial activity, which will lead to partial fragmentation and increase the concentration of protein albumin in the cells of the fine cells. The osmotic and osmotic pressure of cells induces changes in the shape and shape of fine cell cells, and in the urinary system, it causes various urinary systems. Traditional diseases such as egg albumin, urine, blood, hematuria, edema, uremia, etc.

The multi-polypeptide peptide of the present invention has a conservation-sequence sequence sequence with the protein protease endonuclease CC llpp, and the analysis analysis table showing the expression spectrum shows that The content ratio of this multi-peptide peptide in the hepatoma cells of hepatocellular carcinoma is lower than that in normal tissues of normal liver and liver tissues. Consistent with the function of the endoproteinase CCllpp. . To sum up, the present invention is a novel human CCiipp protease endonuclease with similar biological properties * Because the human polypeptide endonuclease 6 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human polypeptide endonuclease 6 involved in these processes. Protein, especially the amino acid sequence of this protein. Isolation of the novel human peptide endonuclease 6 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is very important. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human endopeptidases 6 as well as fragments, analogs and derivatives thereof.

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

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human polypeptide endonuclease 6. It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a human polypeptide endonuclease 6.

 Another object of the present invention is to provide a method for producing human polypeptide endonuclease 6.

 Another object of the present invention is to provide an antibody against the polypeptide-human human polypeptide endonuclease 6 of the present invention, and another object of the present invention is to provide a mimetic compound against the polypeptide-human human polypeptide endonuclease 6 of the present invention, Antagonists, agonists, inhibitors.

Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human endopeptidase 6. 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 No. 2 or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

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

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

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

 (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).

 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 591 to 761 in SEQ ID NO: 1; and (b) a sequence having 1-1494 in SEQ ID NO: 1 Sequence of bits.

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

 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 polypeptide endonuclease 6 protein, which comprises utilizing the polypeptide of the invention. The present invention also relates to compounds obtained by the method. The present invention also relates to an in vitro detection of a disease or disease associated with abnormal expression of a human polypeptide endonuclease 6 protein. A method for susceptibility to disease, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting the amount or biological activity of the polypeptide of the present invention in a biological sample.

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

 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease, or other diseases caused by abnormal expression of human polypeptide endonuclease 6.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

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

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

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

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

 "Biological activity" refers to a protein having the structural, regulatory, or biochemical function of a natural molecule ', and similarly, the term "immunologically active" refers to a natural, recombinant, or synthetic protein and fragments thereof in a suitable animal or cell The ability to induce specific immune responses and to bind specific antibodies.

 An "agonist" refers to a molecule that, when combined with human endopeptidase 6 can cause a change in the protein to regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human endonuclease 6.

An "antagonist" or "inhibitor" refers to a species that can be blocked or regulated when it binds to human endopeptidase 6. A biologically or immunologically active molecule of human endopeptidase 6. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human polypeptide endonuclease 6.

 "Regulation" refers to a change in the function of human polypeptide endonuclease 6, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional or immune properties of human polypeptide endonuclease 6. .

 "Substantially pure '" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify human endopeptidase 6 using standard protein purification techniques. Basically Pure human endopeptidase 6 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human endopeptidase 6 can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides that are base-paired under conditions of acceptable salt concentration and temperature. For example, the sequence "CTGA" can be combined with the complementary sequence "GACT". Two The complementarity between single-stranded molecules can be partial or full. 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" is a partially complementary sequence that at least partially inhibits the hybridization of a completely complementary sequence to a target nucleic acid. The inhibition of such hybridization can be achieved by hybridization under conditions of reduced stringency (Southern blotting) Or Nor thern blot, etc.) to detect. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (La sergene sof tware package, DNASTAR, Inc., Mad Son Wis.). The MEGALIGN program can compare two or more sequences (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244) according to different methods, such as the Cluster method. The Cluster method arranges groups of sequences into 蔟 by checking the distance between all pairs, and then assigns the clusters in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:

 Number of matching residues between sequence A and sequence B

 X 1 00

 Number of residues in sequence A-number of spacer residues in sequence A-number of spacer residues in sequence B

The percent identity between nucleic acid sequences can also be determined by the C 1 uster method or by methods known in the art such as Jo t un He in (He in J., (1990) Methods in emzumo logy 183: 625-645) . "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

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

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? ^ It can specifically bind to the human epitope 6 epitope.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

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

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

 As used herein, "isolated human endopeptidase 6" means that human endopeptidase 6 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human endopeptidase 6 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human endopeptidase 6 polypeptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, a human endopeptidase 6, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals) using recombinant technology. Cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

 The present invention also includes fragments, derivatives, and analogs of human polypeptide endonuclease 6. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human endoenzyme 6 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted The amino acid may or may not be encoded by the genetic code; or (Π) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or (Π Ι) like this A type in which a mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or a UV), a polypeptide sequence in which an additional amino acid sequence is fused to a mature polypeptide (such as Leader sequence or secreted sequence or sequence or protease sequence used to purify this polypeptide) As explained herein, such fragments, derivatives and analogs are provided by the present invention with isolated nucleic acids (polynucleotides), which are basically composed of SEQ ID NO: 2 Polynucleotide composition of a polypeptide having the amino acid sequence. 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 1494 bases in length and its open reading frame (591-761) encodes 56 amino acids. This polypeptide has the characteristic sequence of the active site of the peptide endonuclease Clp, and it can be deduced that the human polypeptide endonuclease 6 has the structure and function represented by the active site of the peptide endonuclease C l p.

 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 sequence of the coding region encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO: 1 or it can be a degenerate variant. As used herein, a "degenerate variant" refers to a protein or polypeptide having SEQ ID NO: 2 in the present invention, but a nucleic acid sequence that differs from the coding region sequence shown in SEQ ID NO: 1 encodes SEQ The polynucleotide of the mature polypeptide of ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences .

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

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

 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° /. SDS, 60 ° C; or (2) adding denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) only Hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human polypeptide endonuclease 6.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the human polypeptide endonuclease 6 of the present invention can be obtained by various methods. For example, the polynucleotide can be isolated by hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

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

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

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of the human polypeptide endonuclease 6 transcript; (4) ) Through immunological techniques or determination of biological activity, to Detection of protein products expressed by genes. The above methods can be used singly or in combination. In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used 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. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human endopeptidase 6 gene.

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

 The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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 a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human polypeptide endonuclease 6 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .

 In the present invention, a polynucleotide sequence encoding a human polypeptide endonuclease 6 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct a DNA sequence containing a human polypeptide endonuclease 6 And expression vectors with appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The A sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known 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 enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor enhancers and adenoviral enhancers on the late stage of the origin of replication.

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

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

 In the present invention, a polynucleotide encoding a human polypeptide endonuclease 6 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; 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 described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase, and treated with the ( ₁₂ method, the steps used are well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.

Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human polypeptide endonuclease 6 (Science, 1984; 224: 1431). Generally, the following steps are taken: (1) using the polynucleotide (or variant) encoding human human polypeptide endonuclease 6 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

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

 In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. 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.

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

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

FIG. 1 is a comparison diagram of amino acid sequence homology of a total of 49 amino acids of 2 to 50 of the human endopolypeptidase 6 and the C i _P active site domain of the endopolypeptidase. The upper sequence is the human endopeptidase 6 and the lower sequence is the active site domain of the endopeptidase C lp. Ί "and": "" and "·" indicate that the probability of different amino acids at the same position between two sequences decreases in sequence.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human polypeptide endonuclease 6 {Molecular weight} kl) a is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples are not marked with specific conditions, usually according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Industrial Harbor Labora tory Pres s, 1989), Or as recommended by the manufacturer.

Example 1: Cloning of Human Polypeptide 6 Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total A using 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 the pBSK (ten) vector (Clontech) to transform DH5cc. The bacteria formed a cDNA library. Dye terminate cycle reaction ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 and 3 'ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones, 0094 f 04, was found to be new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 0094f04 clone contained a full-length cDNA of 1494bp (as shown in Seq ID NO: 1), and a 170bp open reading frame (0RF) from 591bp to 761bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0094f 04, and the encoded protein was named human endopeptidase 6.

Example 2: Domain analysis of cDNA clones

 The sequence of the human polypeptide endonuclease 6 of the present invention and the protein sequence encoded by the same were used in a profile scan program (Bas icloca 1 Alignment search tool) in GCG [Altschul, SF et al.

J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote. The human polypeptide endonuclease 6 of the present invention is homologous with the domain polypeptide endonuclease Clp active site at 2-50, and the homology result is shown in FIG. 1. The homology rate is {29%}, and the score is 14.41. Threshold Is 14.21. Example 3: Cloning of a Gene Encoding Human Polypeptide 6 by RT-PCR

 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.

After purification of Qiagene's kit, PCR amplification was performed with the following primers:

 Pr imerl: 5 '-CTTGCCCAGCGCACGTGCATTCTCC-3' (SEQ ID NO: 3) Pr imer2: 5 '-TTTTTCAATAGGTTTTTTTATTGAAAT-3' (SEQ ID NO: 4) Forward sequence;

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

Amplification reaction conditions: 50 mmol / L KC1, 10 mmol / L Tris-CI, (pH 8.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, lOpmol primer, 1U in a reaction volume of 50 μ 1 Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, set β-act in as a positive control and template blank as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit. DNA sequence analysis results indicate PCR The DNA sequence of the product is exactly the same as the 1- 1494bp shown in SEQ ID NO: 1 Example 4: Northern blot analysis of the expression of the human polypeptide endonuclease 6 gene:

Total 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 (49: 1 ) And 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. Using 20 μ _§ RNA, electrophoresis was performed on a 1.2% agarose gel containing 20raM 3- (N-morpholino) propanesulfonic acid (PH7.0) -5mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Ex- ³² P dATP was used to prepare-labeled DNA probes by random primers. The DNA probe used was the PCR amplified human polypeptide endonuclease 6 coding region sequence (591bp to 761bp) shown in FIG. 1. A 32P-labeled probe (approximately 2 x 10 ⁶ cpm / ral) 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 (pH7.4) -5 x SSC-5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, place the filter at 1 x SSO 0.1 »/. Wash in 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 Polypeptide 6

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

Primer3: 5 '-CCCGCTAGCATGCCATCCTGGAGAGAAGCAGAGA-3' (Seq ID No: 5) Primer4: 5 '-CCCGGATCCCACACGGGACAAGTTTTCTTTTTAG-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively. The coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively. The Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site. The PCR reaction was performed using the pBS-0094f04 plasmid containing the full-length target gene as a template. The PCR reaction conditions are as follows: The total volume is 50 μ1. The plasmid contains 10 pg of _P BS-0094f 04 plasmid and primers? 1 ^ 1116]: -3 and 卩]: 11116]: -4 points and another!] Is 10 11101, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 25 cycles at 4 ° C 20s, 60 ° C 30s, 68 ° C 2 min, Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and recover large Fragments and ligated with T4 ligase. The ligated product was transformed into E. coli DH5cc using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 μ _§ / ηι1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0094f04) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB containing kanamycin (final concentration 30 g / ml) In the liquid culture medium, the host strain BL21 (υΕΤ-0094f 04) was at 37. C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mol / L, and continue to cultivate for 5 / j. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected using an affinity chromatography column Hi s. Bind Quick Cartr idge (product of Novagen) capable of binding to 6 histidines (6His-Tag). By chromatography, the purified human protein endonuclease 6 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at (6) kDa (Figure 2). The band was transferred to a PVDF membrane and analyzed by N-terminal amino acid sequence using the Edams hydrolysis method. The amino acid is exactly the same as the N-terminal 15 amino acid residues shown in SEQ ID NO: 2. Example 6 Production of an anti-human polypeptide endonuclease 6 antibody

 Polypeptide synthesizer (product of PE company) was used to synthesize the following specific human endonuclease 6 peptides:

NH2-Met-Pro-Ser-Trp-Arg-G lu-Ala-G lu-Arg-Va l-Leu-Ser-Pro-G l y-Va l-C00H (SEQ II) NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avrameas, et al. Immunochemi s try, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex and complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost the immunity once. 15 Use a 15 g / ml bovine serum albumin peptide complex-coated titer plate for ELISA to determine antibody titers in serum-free serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human endonuclease 6. Example 7: Application of the polynucleotide fragment of the present invention as a hybridization probe

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

The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter. These same steps are: 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 loaded And synthetic polymers. The pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washes. 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. In this embodiment, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

First, the selection of the probe

 The selection of oligonucleotide fragments 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:

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

 2. GC content is 30 »/. -70%, non-specific hybridization increases;

 3, there should be no complementary regions inside the probe;

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

 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

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

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

5 -GGTGTGAATCAGATAGGGTTCTGTCTTCAGGTGTTTTCCAG-3 ¹ 41 (SEQ ID NO: 9) For other common reagents and their preparation methods not listed in the following specific experimental steps, please refer to the literature: PROBES GH Keller; MM Manak; Stockton Pres s, 1989 0JSA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" U 998 Second Edition) [US] Sambrook et al., Science Press.

 Sample Preparation:

1. Extract DNA from fresh or frozen tissue Steps: 1) Put fresh or freshly thawed normal liver tissue on ice and hold phosphate buffered saline

(PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the pellet (about 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 ol / L Tris-HCl, pH 7.5; 25 mmol / LnaCl; 25 mmol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4% at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (l-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, phenol extraction method for DNA

Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) Resuspend the pelleted cells (1 X 10 ^s cells / ml) with cold cell lysate and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DNA to a new tube. The DNA was then purified and ethanol precipitated.

3, DNA purification and ethanol precipitation

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

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

8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K with final concentrations of 0.5% and 100ug / raU at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), centrifuge for 10 minutes, 12) Carefully remove the water phase, mix 1/10 volume of 2mol / L sodium acetate and 2.5 volume of cold ethanol, and mix well at -20 ° C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-5. 14) Measure A ₂₆ . And A _2S . To detect the purity and yield of DNA. 15) Store at -20 ° (; after preparation). Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number lightly with a pencil. Two NC membranes are needed for each probe for subsequent experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively.

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

 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

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

 Labeling of probes

1) 3 μl Probe (0.10D / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi γ- ³² P- dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of bromophenol blue indicator (BPB).

 4) Pass through a Sephadex G-50 column.

5) Start collecting the first peak before ³² P-Probe washes out (can be monitored by Monitor;).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) After the collection liquid of the first peak is combined, it is ³² P-Probe (the second peak is free γ- ³² P-dATP).

 Pre-hybridization

 Put the sample film in a plastic bag, add 3-10mg of pre-hybridization solution (lOxDenhardfs; 6xSSC, 0.1mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake at 68 ° C with water 2 hour.

 Cross

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

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice). 3) 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

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

 Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

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

 4) In 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice), and dry at room temperature.

 X-ray autoradiography:

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

 Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above four probe hybrid spots; whereas the hybridization experiments performed under low-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactivity of the hybridization spots of the other three probes. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Example 8 DNA Microarray

 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 large numbers 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, see DeRis i, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. ., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 μm. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature, and the specific method steps have been variously reported in the literature. The post-spot processing steps are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. Wash twice with ddH ₂ 0 for 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 1 minute;

 6. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

 (2) Probe marking

 Total raRNA was extracted from normal liver and liver cancer by a single method, and mRNA was purified by Oligotex mRNA Midi Kit (purchased from QiaGen). The fluorescent reagent Cy3dUTP (5- Amino- propargy 1-2 · -deoxyur i dine 5'-tr iphate coupled to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label mRNA of normal liver tissue. Cy5dUTP (5-Amino-propargy l-2'-deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification. Specific steps refer to and methods Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dari. Davis, RW (1995) Science. 270. (20): 467-480.

(Three) cross

 The probes from the two types of tissues and the chip were hybridized in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1> <SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray. The 3000 scanner (purchased from General Scanning Company, USA) was used to scan. The scanned image was analyzed and processed with Imagene software (Biodi scovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point. Genes considered to be differentially expressed.

The experimental results show that Cy3 signal: 65228.12 (average of four experiments), Cy5signal = 3459.88 (average of four experiments), Cy3 / Cy5 = 18.8527116547395, the polynucleotide of the present invention in the above two tissues Significant differences in expression indicate that the polynucleotide of the present invention is closely related to liver cancer. Industrial applicability

The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, For example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.

 C lp protein plays an important role in the protein balance of cells, and it also plays an important role in protein repair. Therefore, C l p protein can be used to treat or prevent cell damage and cell dysfunction caused by changes in osmotic pressure. Clp protein can also be used to treat or prevent cancer.

 Specifically, in terms of the present invention, the polypeptide of the present invention or a fragment thereof can be used to treat or prevent proteinuria, hematuria, edema, uremia and the like, and can also be used to treat or prevent diseases caused by cell rupture such as hemolysis.

 The polypeptides or fragments thereof of the present invention can also be used to treat or prevent various cancers, including but not limited to: Respiratory system tumors: nasal and sinus tumors, nasopharyngeal cancer, laryngeal cancer, tracheal tumors, lung cancer, pleural mesothelioma

 Digestive system tumors: salivary gland tumors, esophageal cancer, esophageal leiomyosarcoma, primary esophageal small cell carcinoma, gastric cancer, gastric malignant lymphoma, gastric carcinoid, colorectal cancer, colon cancer, intestinal malignant lymphoma, primary liver cancer, Hepatoblastoma, primary gallbladder cancer, pancreatic cancer

 Blood, lymphatic tumors: acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant Histiocytosis

 Nervous system tumors: astrocytoma, ependymal tumor, medulloblastoma, meningiomas, glioblastoma, acoustic neuroma, angiogenic tumor, pituitary adenoma, craniopharyngioma

 Motor system tumors: osteoid osteoma, osteochondroma, chondroma, osteoblastoma, chondroblastoma, etc.), malignant bone tumors such as giant cell tumor of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, myeloma, urinary Reproductive system tumors: Benign tumors such as renal cortical tubular adenoma, eosinophil adenoma, juxtaglomerular cell tumor, polycystic kidney tumor, seminoma, teratoma, testicular stromal tumor, endometrium Interstitial tumors, hydatidiform moles, ovarian tumors, breast fibromas, malignant tumors such as renal cell carcinoma, renal sarcomatoid carcinoma, papillary renal cell carcinoma, nephroblastoma, prostate cancer, testicular tumor chorionic carcinoma, cervical cancer, Endometrial cancer, Endometrial cancer, Tubal cancer, Ovarian cancer, Breast cancer

 Endocrine system tumors: pituitary adenoma, benign thyroid tumor, thyroid cancer, parathyroid adenoma, parathyroid cancer, adrenal myeloma, pheochromocytoma, islet cell tumor, multiple endocrine gland tumor, thymic tumor

Soft tissue tumors: fibroids, fibrosarcoma, fibromatosis, lipomas, liposarcomas, leiomyomas, leiomyosarcomas, rhabdomyosarcoma, rhabdomyosarcoma, synovial tissue tumors, hemangiomas, intramuscular hemangiomas, hemangiomas , Hemangioendothelioma, lymphangioma, lymphangiomyoma, lymphatic endothelial sarcoma, fine tissue Cell tumor, malignant fibrous histiocytoma, soft tissue acinar sarcoma, clear cell sarcoma, myxoma, extraosseous Ewing's sarcoma, soft tissue osteosarcoma, soft tissue chondrosarcoma, mesothelioma, epithelioid sarcoma, schwannomas, nerves Fibroma, Malignant Schwannomas, Neurofibromatosis

 Skin malignancies: dermal Mike cell tumor, Kaposi sarcoma, melanoma

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human polypeptide endonuclease 6. Agonists enhance biological functions such as human endonuclease 6 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human polypeptide endonuclease 6 can be cultured together with labeled human polypeptide endonuclease 6 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human polypeptide endonuclease 6 include selected antibodies, compounds, receptor deletions, and the like. Human polypeptide endonuclease 6 antagonists can bind to human polypeptide endonuclease 6 and eliminate its function, or Inhibiting the production of the polypeptide, or binding to the active site of the polypeptide makes the polypeptide unable to perform biological functions ..

 When screening compounds that act as antagonists, human endoenzyme 6 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human endoenzyme 6 and its receptor. . 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 endonuclease 6 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human 6 peptides should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the human polypeptide endonuclease 6 epitope. These antibodies include (but are not limited to): Doxlon antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

The production of polyclonal antibodies can be obtained by immunizing animals (such as home immunity, mice, rats, etc.) directly with human polypeptide endonuclease 6. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. . Techniques for preparing monoclonal antibodies to human endopeptidase 6 include, but are not limited to, hybridoma technology (Koh l ei- and Mil te in. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morr et al, PNAS, 1985, 81: 685 1). ₀ Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single-chain antibodies against human human endonuclease 6.

Anti-human polypeptide endonuclease 6 antibody can be used in immunohistochemistry to detect biopsy specimens Human polypeptide endonuclease 6.

 Monoclonal antibodies that bind to human endonuclease 6 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, the human polypeptide endonuclease 6 high affinity monoclonal antibody can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human polypeptide endonuclease 6 positive cells .

 The antibodies in the present invention can be used for the treatment or prevention of diseases related to human endonuclease 6. Administration of an appropriate amount of the antibody can stimulate or block the production or activity of human endonuclease 6.

 The invention also relates to diagnostic test methods for quantitative and localized detection of human polypeptide endonuclease 6 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human endopeptidase 6 detected in the test can be used to explain the importance of human endopeptidase 6 in various diseases and to diagnose diseases in which human endopeptidase 6 functions.

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

 Polynucleotides encoding human polypeptide endonuclease 6 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by the non-expression or abnormal / pre-active expression of human endonuclease 6. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human endopeptidases 6 to inhibit endogenous human polypeptide endase 6 activities. For example, a mutated human polypeptide endonuclease 6 may be a shortened human polypeptide endonuclease 6 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human endopeptidase 6. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human endopeptidase 6 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human polypeptide endonuclease 6 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human endonuclease 6 can be packaged into liposomes and transferred into cells.

Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on. Oligonucleotides (including antisense RM and DM) and ribozymes that inhibit human endonuclease 6 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.

 Polynucleotide encoding human polypeptide endonuclease 6 can be used for diagnosis of diseases related to human polypeptide endonuclease 6 The polynucleotide encoding human polypeptide endonuclease 6 can be used to detect the expression of human polypeptide endonuclease 6 or Abnormal Expression of Human Polypeptide 6 in Disease Conditions. For example, the DNA sequence encoding human polypeptide endonuclease 6 can be used to hybridize biopsy specimens to determine the expression of human polypeptide endonuclease 6. Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly known and mature, and related kits are commercially available. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a micro array or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue. Human polypeptide 6 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human polypeptide 6 endonuclease 6 transcription products.

 Detecting mutations in the human polypeptide endonuclease 6 gene can also be used to diagnose human polypeptide endonuclease 6-related diseases. The forms of human polypeptide endonuclease 6 mutations include those compared to normal wild type human polypeptide endonuclease 6 DNA sequences Point mutations, translocations, deletions, recombinations, and any other abnormalities. Mutations can be detected using existing techniques such as Sou thern imprinting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern 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. At present, it is necessary to identify the specific loci of each gene on the chromosome. At present, only few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark the chromosome position. According to the present invention, in order to associate these sequences with disease-related genes, the important first step is to locate these DNA sequences on a chromosome.

In short, the PCR primers (preferably 15-35 b _P ) are prepared according to the CDM, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those 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. Make Using the oligonucleotide primers of the present invention, sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific C library.

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

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

 Next, the 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 diseased and diseased 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. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

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

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human endopeptidase 6 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human endopeptidase 6 administered to a patient will depend on a number of factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

Claim

1. An isolated polypeptide-human polypeptide endonuclease 6, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or an active fragment, analog or derivative thereof.

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

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

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

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

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

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

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

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 591-761 in SEQ II) NO: 1 or the sequence of positions 1-1494 in SEQ ID NO: 1 sequence.

 7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Vector.

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

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

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

9. A method for preparing a polypeptide having human polypeptide endonuclease 6 activity, characterized in that the method includes:

(a) culturing the engineered host cell according to claim 8 under the condition of expressing human polypeptide endonuclease 6;

(b) Isolating a polypeptide having human endoenzyme 6 activity from the culture.

 10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to a human polypeptide endonuclease 6.

 11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, which are characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of a human polypeptide endonuclease 6.

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

13. The use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human endopeptidase 6 in vitro and in vivo.

 14. A method for detecting a disease or susceptibility to a polypeptide related to any one of claims 1 to 3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide, Alternatively, a polynucleotide variation in the polynucleotide that causes abnormal expression or activity of the polypeptide is detected.

 15. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human polypeptide endonuclease 6; or for peptides Fingerprint identification.

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

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with an abnormality of human endopeptidase 6

 18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.