WO2001049738A1 - A novel polypeptide - human fkbp protein 11 and a polynucleotide encoding the same - Google Patents

Abstract

The present invention discloses a novel polypeptide - human FKBP protein 11 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various diseases, such as malignant tumor, blood disease, HIV infection, immunological diseases and various type inflammations. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding human FKBP protein 11.

Description

 A new peptide-human FOP protein 11 and a polynucleotide encoding this polypeptide

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, A FKBP protein 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.

technical background

 Immunavidin is a housekeeping gene expressed in a variety of tissues. Cyclophilin (Cyps) and FK506 binding protein (FKBP) are two of these subfamilies. The former binds to CsA and the latter binds to FK506 or rapamycin. CsA, FK506, and rapamycin are immune system inhibitors and are immunosuppressive prodrugs with diagnostic value. CsA and FK506 can selectively inhibit early T cell activation, leading to failure of early gene transcription, and the cytokines encoded by these genes are involved in the immune response of a variety of cells. This inhibitory effect on T cell activation can be used diagnostically to inhibit allograft rejection. They can form complexes with endogenous intracellular receptors such as immunophilins. This complex acts on protein phosphatase, calcineurin, and calcium ions.

Immunavidin has peptidyl-proline cis-trans isomerase (PPIase) activity. PPIase catalyzes the cis- and trans-transformation of peptidyl-proline in protein substrates. PPIase has a high degree of sequence conservation and is a multifunctional enzyme that exists in multiple cell types and has different physiological divisions of labor. At the cellular level, PPI ase is present at every site, both inlaid on the membrane and free. PPIase regulates protein folding, merger, and channel opening (Ca2 +). Binding of immunophilin to FK506 results in the loss of its own PPI _ase enzyme activity, because the latter contains a structure called "twisted amino compound surrogate" (twisted ami de surroga te), which mimics the protein substrate cis- Intermediate state of trans-peptidylproline conversion. Some immunophilins combine with immunosuppressants to form complexes that are toxic to mammalian cells. Except that the enzyme's active site is a drug-binding site, the PPIase enzyme activity of immunophilin is not related to the role of immunosuppressive drugs. In the absence of immunosuppressive drugs, PPIase mainly interacts with proline-rich proteins in cells. FKBP contains proline-binding pocket regions and isomerase active sites. These structures are related to aromatics The sites are parallel. Therefore, binding to proline-rich proteins at the active site will make proline isomerization easier. It is just a convenient detection of proline binding.

FKBP has an immunosuppressive effect only when combined with the immunosuppressive drug FK506. FK506 itself does not have this type of immunosuppressive activity, so FK506 and CsA are only a prerequisite drug, and they can only be combined with immunophilins FKBP and Cyps It can bind to calcineurin and the like. Immunostatin has the function of molecular glue here, combining FKBP and calcineurin. There are many members of the F BP family, eukaryotic cells, and prokaryotic cells. These enzymes have molecular weights between 12 and 63 kDa, and their isoelectric points are both acidic and basic. FKBP is present in the cytoplasm, membrane, and T cell nuclei. FKBP family members differ greatly in their hydrophobicity. FKBP has PPIase enzyme, but the substrate is very different compared to Cyps. And the biological functions of FKBP family members are also very different. FKBP-12 is a co-factor of calmodulin, and is closely bound to calcium ion release channels, and is related to calcium ion release. FKBP-52 is a multifunctional FK506 binding protein that can bind to 90-Kda heat shock protein and is a component of the cholesterol receptor complex, but has no immunosuppressive activity when combined with FK506 to form a complex. FKBP-25 interferes with the immune system of the host cell. FKBP-25 can also bind to DNA, and this ability is not lost by binding to FK506 or rapamycin. FKBP36 is expressed in many tissues such as liver, kidney, and skeletal muscle, and is missing in Williams syndrome.

 Because PPIase regulates the folding, merging and channel opening of proteins, and the binding of immunophilin to FK506 will lead to the loss of its own PPIase enzyme activity, the abnormal binding of FKBP and FK506 will affect the three-dimensional structure of the protein and affect the function of the protein. If this happens to a key protein that regulates the cell cycle or cell signal transmission, it may lead to uncontrolled cell proliferation and lead to the occurrence of various tumors.

 The protein of the present invention contains a characteristic conserved sequence of FKBP. The expression profile analysis of the gene chip found that the content of this gene in glioma tissue was lower than that in normal tissue, indicating that its function is related to cancer. Therefore, the present invention is considered to be a new human FKBP protein with similar Biological function and named human FKBP protein 11.

 Since human FKBP protein 11 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human FKBP protein 11 proteins involved in these processes, especially The amino acid sequence of this protein was identified. The isolation of the new human FKBP protein 11 protein encoding gene also provides a basis for the study to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs, so isolating its coding DNA is important.

Object of the invention

 It is an object of the present invention to provide an isolated novel polypeptide, human FKBP protein 11, and fragments, analogs and derivatives thereof.

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

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human FKBP protein 11. Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human FKBP protein 11.

Another object of the present invention is to provide a method for producing human FKBP protein 11. Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human FKBP protein 11. Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human FKBP protein 11.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human FKBP protein 11.

Summary of invention

 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. 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 549-860 in SEQ ID NO: 1; and (b) a sequence having 1-953 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 FKBP protein 11 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 susceptibility to disease associated with abnormal expression of human FKBP protein 1 1 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.

 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 FKBP protein 11.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. BRIEF DESCRIPTION OF THE DRAWINGS

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

 Fig. 1 is a comparison diagram of the amino acid sequence homology of the FKBP protein 11 of 47 amino acids and domains of FKBP protein 11 of 56-1 03 in the present invention. The upper sequence is the human FKBP protein 1 1 and the lower sequence is the FKBP protein domain. Ί "and": "and". "Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human FKBP protein 1 1. l l kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.

Summary of the Invention

 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 DM 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 base acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.

An "agonist" refers to a molecule that, when combined with human FKBP protein 1 1, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate or any other molecule that can bind to human FKBP protein 1 1, An "antagonist" or "inhibitor" refers to a molecule that, when combined with human FKBP protein 11, can block or regulate the biological or immunological activity of human FKBP protein 11. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind human FKBP protein 11.

 "Regulation" refers to a change in the function of human FKBP protein 11, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human FKBP protein 11.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human FKBP protein 11 using standard protein purification techniques. Essentially pure human FKBP protein 11 produces a single main band on a non-reducing polyacrylamide gel. The purity of human FKBP protein 11 can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene 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, D. G. and P.M. Sharp (1988)

Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:

The number of residues matching between sequence ^ and sequence β ₌ X, Ι _Λ ϋϋ _Λ number of residues in the sequence-sequence ί ¹ 1 'fi-ij number of residues in the sequence-the number of interval residues in sequence ^ can also be determined by Cluster Determine the percent identity between nucleic acid sequences using methods known in the art, such as Jotun He in (Hein J., (1990) Methods in enzymology 183: 625-645).

"Similarity" refers to the identity or preservation of amino acid residues at corresponding positions in the alignment of amino acid sequences. The extent of conservative substitution. Amino acids used for conservative substitution, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

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

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to an intact antibody molecules and fragments thereof, such as _{Fa, F (a b ')} 2 and F _V, which specifically binds to human FKBP 11 protein antigen determinant.

 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 matter from its original environment (for example, its natural environment if it is naturally occurring). 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 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 FKBP protein 1 1" means that human FKBP protein 1 1 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human FKBP protein 1 1 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 FKBP protein 1 1 polypeptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, human FKBP protein 1 1, 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 produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or Can be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

 The invention also includes fragments, derivatives and analogs of human FKBP protein 1 1. 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 FKBP protein 1 1 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted The amino acid may or may not be encoded by the genetic code; or (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 UV) a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide (such as the leader Sequences or secreted sequences or sequences or proteinogens used to purify this polypeptide. As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 953 bases, and its open reading frame (549-860) encodes 103 amino acids. This peptide has the characteristic sequence of the FKBP protein, and it can be deduced that the human FKBP protein 11 has the structure and function represented by the FKBP protein.

 The polynucleotide of the present invention may be in the form of DNA or RM. 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. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" 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 is known in the wood 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 (with at least two sequences between

50%, preferably 70% identity). 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) added during hybridization Use a denaturant, such as 50 ° /. (V / v) formamide, 0.1% calf serum /0.1°/. Ficoll, 42 ° C, etc .; or (3) 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, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human FKBP protein 11.

 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 FKBP protein 11 of the present invention can be obtained by various methods. For example, 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) separating the double-stranded DNA sequence from the DM of the genome; 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 mRNA extraction, 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 loss of marker gene function; (3) measuring the level of human FKBP protein 11 transcripts; (4) passing Immunological techniques or measuring biological activity, To detect gene-expressed protein products. 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 here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human FKBP protein 11 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, and the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

 The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated, and sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human FKBP protein 11 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.

 In the present invention, the polynucleotide sequence encoding the human FKBP protein 11 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, 19103, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and defensive virus-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 origins of replication, promoters, marker genes, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct a DNA sequence containing human FKBP protein 11 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 DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. 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 from 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.

 In addition, the expression vector preferably contains one or more selectable marker genes to provide the phenotypic traits used to select transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green 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 human FKBP protein 11 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; 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 CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

Using conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human FKBP protein 11 (Science, 1984; 224: 1431). Generally there are the following steps: (1) using the polynucleotide (or variant) encoding human FKBP protein 1 1 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 necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 The polypeptides of the present invention, as well as the antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.

 Immunavidin is a housekeeping gene expressed in a variety of tissues. Cyclophilin (Cyps) and FK506 binding protein (FKBP) are two of these subfamilies. FKBP can be combined with FK506 or rapamycin. FK506 is an immune system inhibitor and a diagnostic immunosuppressive prodrug. FK506 can choose to inhibit the activation of early T cells, leading to the failure of transcription of early genes, and the cytokines encoded by these genes are involved in the immune response of many cells. This inhibitory effect on T cell activation can be used to medically inhibit allograft rejection.

 In addition, the abnormal binding of FKBP and FK506 will affect the three-dimensional structure of the protein and affect the function of the protein. If this happens to a key protein that regulates the cell cycle or cell signal transmission, it may lead to uncontrolled cell proliferation and lead to the occurrence of various tumors.

 FKBP-specific conserved sequences are required to form its active mot i f. It can be seen that abnormal expression of specific FKBP mo tif will cause abnormal function of the polypeptide containing the mo tif of the present invention, resulting in abnormal regulation of cell cycle or cell signal transmission, resulting in tumor, inflammation, growth and development disorders; T-cell function of the immune system is abnormal, and immune diseases are caused.

It can be seen that abnormal expression of the human FKBP protein 1 1 of the present invention will produce various diseases, especially Wi lli ams syndrome, various tumors, immune diseases, inflammation, and growth and development disorders. These diseases Diseases include but are not limited to:

 Tumors of various tissues: liver cancer, adrenal cancer, gastric cancer, skeletal fibroids, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, goiter '. Blind, uterine fibroids, neuroblastoma, astrocytoma , Ependymal tumor, glioblastoma, colon cancer. Melanoma, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymus Tumor, Nasal and Sinus Tumors, Nasopharyngeal Carcinoma, Laryngeal Carcinoma, Trachea Tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma, Leiomyoma

 Inflammation: allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex Types of glomerulonephritis, acute anterior uveitis, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, chronic active hepatitis, emergency bowel syndrome, atrophy Gastritis, systemic lupus erythematosus, myasthenia gravis, cerebrospinal multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, pancreatitis, myocarditis, and infectious inflammation

 Immune diseases: rheumatoid arthritis, chronic active hepatitis, immune complex glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, polymyositis, primary T lymphocyte immunodeficiency disease, Primary phagocytic cell deficiency disease, primary cell-humoral combined immunodeficiency disease, acquired immunodeficiency syndrome, primary complement system deficiency disease, bronchial asthma, allergic rhinitis

 Growth and development disorders: mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation

 Abnormal expression of the human FKBP protein 1] of the present invention will also produce certain hereditary, hematological and immune system diseases.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially Willi ams syndrome, various tumors, immune diseases, inflammation, growth and development. Obstructive diseases, certain hereditary, blood diseases, etc. In addition, the polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can also be directly used in the treatment of allograft rejection.

The invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human FKBP protein 1 1. Agonists enhance human FKBP protein 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing human FKBP protein 1 1 can be labeled with labeled human FKBP in the presence of a drug. Protein 11 was cultured together. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human FKBP protein 11 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human FKBP protein 11 can bind to human FKBP protein 11 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.

 When screening compounds as antagonists, human FKBP protein 11 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human FKBP protein 11 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 FKBP protein 11 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human FKBP protein 11 molecule should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against human FKBP protein 11 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

 Polyclonal antibodies can be produced by injecting human FKBP protein] 1 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. . Techniques for preparing monoclonal antibodies to human FKBP protein 1] 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-hybridization Chimeric antibodies that combine human constant regions with non-human-derived variable regions, such as tumor technology, 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 FKBP protein 11.

 Anti-human FKBP protein 11 antibodies can be used in immunohistochemical techniques to detect human FKBP protein 11 in biopsy specimens.

 Monoclonal antibodies that bind to human FKBP protein 11 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human FKBP protein 11 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human FKBP eggs. White 11 positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases associated with human FKBP protein 11. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human FKBP protein 11.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human FKBP protein 11 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human FKBP protein 11 detected in the test can be used to explain the importance of human FKBP protein 11 in various diseases and to diagnose diseases in which human FKBP protein 11 plays a role.

 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.

 The polynucleotide encoding human FKBP protein 11 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 FKBP protein 11. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human FKBP protein 11, to inhibit endogenous human FKBP protein 11 activity. For example, a variant human FKBP protein 11 may be a shortened human FKBP protein 11, lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human FKBP protein 11. Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding human FKBP protein 11 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding the human FKBP protein 11 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human FKBP protein 11 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human FKBP protein 11 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RM molecule that can specifically decompose a specific RNA, and 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 RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds. The polynucleotide encoding human FKBP protein 11 can be used for the diagnosis of diseases related to human FKBP protein 11. The polynucleotide encoding human FKBP protein 11 can be used to detect the expression of human FKBP protein 11 or the abnormal expression of human FKBP protein 11 in a disease state. For example, the DNA sequence encoding human FKBP protein 11 can be used to hybridize biopsy specimens to determine the expression of human FKBP protein 11. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. 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 FKBP protein 11 specific primers can also be used to detect human FKBP protein 11 transcripts by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.

 Detection of mutations in the human FKBP protein 1] gene can also be used to diagnose human FKBP protein 11-related diseases. Human FKBP protein 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human FKBP protein 11 DNA sequences. 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. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes. Using the 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 (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 To correlate with genetic map data. These data can be found in V. Mckusick, Mendelian Inheritance in Man (available online with Johns Ho) kins 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 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. 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 topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration, human FKBP protein 11 to effectively treat and / or prevent specific The amount of indication to be administered. The amount and range of human FKBP protein 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Examples

 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. In the following examples, the experimental methods without specific conditions are generally performed according to conventional conditions such as Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

 Example 1 Cloning of human FKBP protein 11

Total RNA from human fetal brain was extracted by guanidine isothiocyanate / phenol / chloroform method. Using Quik mRNA Isolation Kit (Qiegene) Isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. A Smart cDNA cloning kit (purchased from Clontech) was used to insert the 00 ^ fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle react 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 it was found that the cDNA sequence of one of the clones 1019A09 was 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 full-length cDNA contained in the 1019A09 clone was 953bp (as shown in Seq IDN0: l), and there was a 311bp open reading frame (0RF) from 549bp to 860bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-1019A09 and the encoded protein was named human FKBP protein 11. Example 2 Domain analysis of cDNA clones

 The sequence of the human FKBP protein 11 and the encoded protein sequence of the present invention were profiled using the GCG profile scan program (Basic local alignment search tool) [Al tschul, SF et al.

J. Mol. Biol.] 990; 215: 403-10], domain analysis was performed in a database such as Prote. The human FKBP protein 11 of the present invention is homologous to the domain FKBP protein at 56-103. The homology results are shown in Fig. 1. The homology is 0.27, and the score is 12.76; the threshold is 12.63. Example 3 Cloning of a gene encoding human FKBP protein 11 by RT-PCR

 CDM was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification by Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5'- GC.AGTGAGCCGAGACCATGCCATTG -3 '(SEQ ID NO: 3)

 Primer2: 5'- TGATTCACAAAAGATTTATTTCTTG -3 '(SEQ ID NO: 4)

 Primer] 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:].

Conditions for the amplification reaction: The reaction volume of 5 () _μ 1 contains 50 mraol / L KC1, 10 mmol / L Tris-HCl, pH 8.5, 1.5 mmoi / L gCl ₂ , 200 _μ ηι1 / 1 / dNTP, lOpmol primers, 1U of Taq DNA polymerase (C 1 ontech). The reaction was performed on a PE 9600 DN A thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Inviogen) using a TA cloning kit. DM sequence analysis The results showed that the DNA sequence of the PCR product was exactly the same as the l-953bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of human FKBP protein 11 gene expression

Total RM was extracted in one step [Anal. Biochem 19103, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time 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. , (N- morpholino) propanesulfonic acid (H7 0.) With 20μ ₈ RNA containing 20mM 3- - electrophoresed on ImM EDTA-2.2M formaldehyde-1.2% agarose gel - 5mM sodium acetate. It was then transferred to a nitrocellulose membrane. Preparation ³² P- DNA probe labeled with a- ³² P dATP by random priming method. The DNA probe used was the sequence of the human FKBP protein 11 coding region (549bp to 860bp) amplified by PCR as shown in FIG. 1. The 32P- labeled probe (approximately 2 X 10 ⁶ cpm / ml) and RNA was transferred to nitrocellulose membrane ⁴² "C overnight in a hybridization solution, the solution comprising 50% formamide - 25mM KH ₂ P0 ₄ (pH7.4) -5 x SSC- 5 x Denhardt's solution and 20 (^ g / ml salmon sperm DM. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then Analysis and quantification using Phosphor Imager. Example 5 In vitro expression, isolation and purification of recombinant human FKBP protein 11

 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'- CATGCTAGCATGGAGTCTCTCTCTGTCGCCCAGG -3 '(Seq ID No: 5) Primer4: 5'- CATGGATCCTCATATTTGTATACAAGATAAACAA -3' (Seq ID No: 6) These two primers contain Nhel and BamHI restriction sites, respectively. The coding sequences of the 5 'and 3' ends of the target gene are followed, respectively. The Nhel and BamHI restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3). Sex endonuclease site. The PCR reaction was performed using the pBS-1019A09 plasmid containing the full-length target gene as a template. PCR reaction conditions were: 1 in a total volume of ₅₀ μ plasmid pBS- 1019A09 containing 10pg, primer Primer- 3 and Primer- 4 are 10pmol, Advantage polymerase Mix (Clontech Products) 1μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. NheT 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 into the colibacillus DH5 cx by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 3 (g / ml)), the positive clones were screened by colony PCR method and sequenced. The correct positive clone (pET-1019A09) was used to transform the recombinant plasmid into E. coli BL2] (DE3) plySs (product of Novagen) by calcium chloride method. Kanamycin (final concentration) 30 _μ§ / ιη1) of LB liquid medium, the host bacteria BL21 (pET-1019A09) was cultured at 37 ° C. to the logarithmic growth phase, IPTG was added to a final concentration of 1 mmol / L, and the culture was continued for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The chromatography was performed using an affinity column His. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). The purified human protein FKBP protein 11 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at llkDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of anti-human FKBP protein 11 antibodies

 The following peptides specific to human FKBP protein 11 were synthesized using a peptide synthesizer (product of PE):

NH ₂ -Met-G 1 u-Ser-Leu-Ser-Va 1 -A 1 a- G 1 nA 1 aG 1 y-Va 1-G 1 n- Tr p- Arg- As p- C00H (SEQ ID NO : 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Imi inocheniistry, 1969; 6: 43. Rabbits were immunized with ½ g 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. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human FKBP protein 11. Example 7. Application of the polynucleotide fragment of the present invention as a hybridization probe

 The suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects. For example, the probes can be used to hybridize to the genome or CDM library 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. Further, 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. 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 as follows: 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 Incubation hybridizes the probe to the target nucleic acid. After the crossover step, 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. 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 D NO: 1 of the present invention should follow the following principles and several aspects to be considered:

 1. The preferred size of the probe ranges from 18 to 50 nucleotides;

 2.The GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;

 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 For homology comparison of the regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally;

 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 (41 N t)

 5'- CAATGCAACCCCCACCTCCCTGGTTCAAGCAATTCCCCTGC -3 '(SEQ ID NO: 8) Probe 1 (probe2), 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 (41 Nt) :

 5 '-CAATATCGCCCCCACCTCATCGGTTCAAGCAATTATCGTGC -3' (SEQ ID NO: 9) For other common reagents not listed below and their preparation methods, please refer to the literature: DNA PROBES GH Ke ll er;. M. Manak; S tock ton Pres s, 1989 (USA) and more commonly used molecular cloning experiment manuals such as "Molecular Cloning Experiment Guide" (1998 Second Edition) [US] Sambrook et al., Science Press.

 Sample Preparation:

1. Extract DNA from fresh or frozen tissue Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with 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 precipitate (about 10 ml / g) with a cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 nol / L NaCl; 25 mmol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C 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 (add 1-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1000 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, DNA phenol extraction method

Steps: 1) Wash the cells with cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the precipitated cells with cold cell lysate U x 10 ^s cells / ml) Apply a minimum of 100ul lysis buffer. 3) Likou SDS to a final concentration of 1%. If SDS is added directly 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 200 u _g / 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:)) 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, and then invert on the absorbent paper to drain the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to precipitate Completely dry, otherwise it is difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Vortex slowly or suck with a dropper while gradually increasing TE, mix until the DNA is fully dissolved, every 1-5 χ 10 Approximately lul was extracted from ⁶ cells.

 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 100 ug / ml, 37. C was held for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug / mi. 37 ^U 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, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 10 volumes of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well-20. C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A _2S . To detect the purity and yield of DNA. 15) Store at -20 after packing. C.

 Preparation of sample film:

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

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

 3) Place on filter paper infiltrated with 0.1mol / L NaOH, 1.5mol / L NaCl for 5 minutes (twice), dry and put in 0.5mol / L Tris-HCl (pH7.0), 3mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry.

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

 Labeling of probes

1) 3yl Probe (0.1 IOD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase, to make up to a final volume of 20μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass Sephadex G-50 column.

5) Collect the first peak before ³² P-Probes are washed out (monitorable).

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

 7) Monitor the amount of isotope with a liquid scintillator.

8) The ³² P-Probe (the second peak is free γ- ³² P-dATP) after the collection of the first peak is combined.

 Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3-lOmg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.

 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) Wash in 0.1 x SSC, 0.1% SDS at 40 ° C for 15 minutes (twice).

 4) 0.1 x SSC, 0.1% SDS, wash 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) Wash at 37 "C for 15 minutes (twice) in 0.1 x SSC, 0.1% SDS.

4) 0.1 x SSC, 0.1% SDS, wash at 40 ^U C for 15 minutes (twice), and dry at room temperature.

X-ray autoradiography:

 -70 ° C, X-ray autoradiography (compression 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 two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than that of hybridization spots. The radioactive intensity of the hybridization spot of the other probe. 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.

Claims

1. An isolated polypeptide-human FKBP protein 1 1 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, characterized in that 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 an amino acid sequence represented by SEQ ID D 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 (a) or (b).

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

 6. The polynucleotide according to claim 4, characterized in that the sequence of said polynucleotide comprises the sequence of positions 549-860 in SEQ ID NO: 1 or the sequence of positions 1-95 in SEQ ID NO: 1 sequence.

 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant constructed from the polynucleotide according to any one of claims 4 to 6 with a plasmid, virus or a carrier expression vector Carrier.

_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 according to any one of claims 4-6.

 9. A method for preparing a polypeptide having human FKBP protein 11 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under the condition of expressing human FKBP protein 11;

(b) Isolating a polypeptide having human FKBP protein 11 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 human FKBP protein 11 1.

 1 1. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human FKBP protein 1 1.

12. The compound according to claim 11, characterized in that it is a multi-core as shown in SEQ ID NO: 1. Antisense sequence of a nucleotide sequence or a fragment thereof.

 13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human FKBP protein 11 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

 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 FKBP protein 11; or for peptide fingerprinting 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 1], characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose and a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with human FKBP protein 11 abnormality.

 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.