WO2001030834A1

WO2001030834A1 - A new polypeptide-zinc finger protein 33 and the polynucleotide encoding it

Info

Publication number: WO2001030834A1
Application number: PCT/CN2000/000350
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Shanghai Bio Road Gene Development Ltd.
Priority date: 1999-10-26
Filing date: 2000-10-23
Publication date: 2001-05-03
Also published as: AU1016301A

Abstract

The present invention discloses a new polypeptide-zinc finger protein 33, the polynucleotide encoding it and a method producing the polypeptide by recombinant DNA technology. The present invention further discloses a method treating various disorders, e.g. malignant neoplasm, hematopathy, HIV infection and immunological disease and various inflammation. The present invention also discloses an agonist of the polypeptide and its therapeutic use. The present invention further discloses the use of the polynucleotide encoding zinc finger protein 33.

Description

A new polypeptide, zinc finger protein 33, and a polynucleotide encoding this polypeptide

The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, zinc finger protein 33, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.

technical background

Transcriptional regulation of eukaryotic genes is very important for the normal expression of genes and exerts biological functions. Usually, transcriptional regulatory factors complete this process. Transcriptional regulatory factors are involved in the body to determine which tissues and developmental stages of genes begin to transcribe. If the genes encoding such proteins are mutated, not only the gene itself cannot be expressed normally, but many genes regulated by it cannot be normal Perform transcription and expression. The regulation of gene expression by transcription factors is mainly accomplished through the binding of transcription factors to specific DNA sequences, the interaction between transcription factors, and the interaction of transcription factors with conventional transcriptional mechanisms. According to the structural motifs, the known DM-binding proteins can be divided into two main categories: proteins containing helix-turn-helix motifs and zinc finger proteins [Kama l Chowdhury, He id i Rohdewohld et a l., Nuc le ic Ac ids Research, 1988, 16: 9995-10011] „

Zinc finger proteins are members of multiple gene families encoding zinc ion-mediated nucleotide binding proteins. Zinc finger proteins can be divided into various families according to their structural characteristics. Various types of zinc finger proteins have been isolated from various organisms such as yeast, fruit fly, rat and human. Drosophila Kruppe l genes with similar zinc finger proteins are the most widely distributed and have important biological functions in the body. These genes all contain the characteristic continuous repeats of the zinc finger protein, the C2-H2 zinc finger protein domain. Studies have found that these proteins are related to the activation and suppression of gene transcription. Abnormal expression of these proteins will cause various developmental disorders, the development of various tumors, various genetic diseases and immune system diseases [Kama l Chowdhury, He idi Rohdewohld eta l., Nuc le ic Ac ids Research, 1988, 16: 9995-10011].

All members of the zinc finger protein Kruppe l family contain a conserved finger repeat (F / Y) XCXXCXXXFXXXXXLXXHXXXHTGE P of 28-30 amino acids in length, and some of the specific amino acid residue sites are highly conserved. This sequence contains multiple copies in many different zinc finger proteins, with different copy numbers (different number of zinc fingers) and different functions. The binding of zinc finger protein to DNA of different lengths depends on the number of finger structures. The multi-finger structure may be related to the binding stability of the complex, which is the site of RNA polymerase transcription. Studies have found that the zinc finger domain of many zinc finger proteins The interconnect region is also highly conserved. This region usually contains the following sequences: H i s-Thr- G ly-G l y-Ly s-Pro- (Tyr, Phe) -X-Cys, where histidine and hemi Cystine is the binding site for metal ions, and X is a variable amino acid residue. This region is necessary for the formation of zinc finger structures. The number of finger structures will directly affect the binding of zinc finger proteins to DNA of different lengths, and the multi-finger structure is related to the binding stability of the complex [Jeremy M. Berg, Annu. Rev. Bi ophys. Chem, 1990, 19: 405-421].

The human gene of the present invention has 53% identity and 67% similarity at the protein level with ZNF1 35, a member of the human zinc finger protein Kruppe l family. Moreover, the protein sequences of both of them contain the characteristic continuous finger repeats of the Kruppe l family of human zinc finger proteins and the structurally connected regions of the fingers. Based on the above points, the new gene of the present invention is considered to be a new member of the human zinc finger protein Kruppe l family and named ZFP33. Based on this, it is inferred that it is similar to ZNF1 35, a member of the Kruppe l family of human zinc finger proteins, and has similar biological functions.

Object of the invention

It is an object of the present invention to provide an isolated novel polypeptide, zinc finger protein 33, 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 a zinc finger protein 33. It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a zinc finger protein 33.

Another object of the present invention is to provide a method for producing zinc finger protein 33.

It is another object of the present invention to provide an antibody against the polypeptide of the present invention, a finger protein 33. Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, zinc finger protein 33.

Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of zinc finger protein 33. Summary of invention

In a first aspect of the present invention, a novel isolated zinc finger protein 33 is provided. The polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or its activity Fragments, or their active derivatives, analogs. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In a second aspect of the present invention, there is provided a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Phase Identities: (a) a polynucleotide encoding the aforementioned zinc finger protein 33; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the polynucleotide is a sequence selected from the group: (a) having SEQ ID N0: 1 ^869-1774 in the sequence of bits; and (b) a SEQ ID N0: 1 1- 2004-bit sequence.

In a third aspect of the present invention, there are provided a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide.

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 as defined by the claims.

Fig. 1 is a comparison diagram of amino acid sequence homology of zinc finger protein 33 and human ZNF135 of the present invention. The upper sequence is zinc finger protein 33 and the lower sequence is human ZNF135. Identical amino acids are represented by single character amino acids between the two sequences, and similar amino acids are represented by "+".

FIG. ² is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated zinc finger protein 33. FIG. 33kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.

Summary of the Invention

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 in the natural state .

As used herein, "isolated zinc finger protein 33" means that zinc finger protein 33 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify zinc finger protein 33 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the zinc finger protein 33 polypeptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, zinc finger protein 33, which basically consists of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues. The invention also includes fragments, derivatives and analogs of zinc finger protein 33. 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 zinc finger protein 33 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) 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 substitution The amino acid may or may not be encoded by a genetic codon; or (Π) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (Π Ι) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) 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 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 full-length polynucleotide sequence of 2004 bases, and its open reading frame (869-1774) encodes 301 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide is 53% homologous to human ZNF135, and it can be deduced that the new human zinc finger protein 33 has a similar structure and function as human ZNF1 35.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. 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 comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants Body, deletion variant, and insertion variant. 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, 6 (TC; 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) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more. Moreover, 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 zinc finger protein 33.

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 zinc finger protein 33 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) 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 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 not (Limited to): (l) DNA-DNA or DM-RM hybridization; (2) appearance or loss of marker gene function; (3) determination of zinc finger protein 33 transcript levels; (4) immunological techniques or determination of biological To detect gene expression of 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 Is 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, the protein product of the zinc finger protein 33 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA).

A method for amplifying DNA / RNA by using PCR technology (Sa ik i, e t a l. Science 1985; 230: 1 350-1 354) 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 DM fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

The present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a zinc finger protein 33 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 zinc finger protein 33 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, etal. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors (Lee) expressed in mammalian cells and Na thans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can replicate in the host And stable, any plasmid and vector can be used to construct recombinant expression vectors. 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 well known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding zinc finger protein 33 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, etal. Moleculiar Cloning, a Labora tory Manua l, cold Spring Harbor Labora tory. 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: l ac or trp promoter of E. coli; _PL promoter of lambda phage; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 Promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, 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 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 zinc finger protein 33 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 Sf 9; animal cells such as CH0, COS or Bowes melanoma cells, etc. .

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. The alternative is to use MgC l ₂ . If required, transformation can also be performed by electroporation Method. 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 DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant zinc finger protein 33 (Scence, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human zinc finger protein 33 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 antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

The polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used in the treatment of diseases, for example, it can treat various malignant tumors and cancers; development disorders, various diseases caused by metabolic disorders of the immune system, and the like.

The members of the zinc finger protein family are numerous and widely distributed in organisms, most of which are eukaryotic transcription regulators, which are responsible for activating or inhibiting the expression of various genes in eukaryotes. Studies have found that members of this family are expressed in various human tissues, including hematopoietic cells, brain, nervous system, epidermal tissue, various tissues related to secretion and absorption, and tumor and immortal cell lines. Organization, etc. Therefore, members of this family play a very important role in the differentiation and development of various tissues in the body. They can effectively control the transcription levels of various genes in the body, and their abnormal expression may lead to abnormal differentiation and proliferation of cells, thereby causing various diseases, Such as cancer and various immune system diseases.

As far as the ZFP33 protein is concerned, the polypeptide of the present invention or a fragment or a derivative thereof can be used to prevent and treat various diseases caused by abnormal expression, differentiation and proliferation of cells. These diseases include but are not limited to the following: cancers of various cells and tissues, including leukemia, lymphoma, lymphosarcoma, myeloma, neuroma, glioma, meningiomas, neurofibromas, and astrocytomas; And diseases of various tissues and organs, including adrenal, thyroid, lung, pancreas, liver, prostate, uterus, bladder, kidney, testis, and gastrointestinal tract (small intestine, colon, rectum, and stomach); also include some related to metabolic disorders Diseases include diseases such as hyperthyroidism, hypothyroidism, gastritis, colon polyps, and gastroduodenal ulcers.

Abnormal expression of ZFP33 may also cause diseases caused by various acquired and hereditary diseases and metabolic disorders of the immune system, such as: split hand, congenital genital tract malformation, Bezier syndrome, etc.

The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) zinc finger protein 33. Agonists enhance biological functions such as zinc finger protein 33 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing zinc finger protein 33 can be cultured together with labeled zinc finger protein 33 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

Antagonists of zinc finger protein 33 include antibodies, compounds, receptor deletions, and the like that have been screened. An antagonist of zinc finger protein 33 can bind to zinc finger protein 33 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 a biological function.

When screening compounds as antagonists, zinc finger protein 33 can be added to the bioanalytical assay, and the effect of the compound on the interaction between zinc finger protein 33 and its receptor can be determined to determine whether the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to zinc finger protein 33 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 33 molecules of zinc finger protein 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 zinc finger protein 33 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

Polyclonal antibodies can be produced by injecting zinc finger protein 33 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. Wait. Techniques for preparing monoclonal antibodies to zinc finger protein 33 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, and human beta-cell hybridoma technology , EBV-hybridoma technology, etc. A chimeric antibody that binds a human constant region and a non-human-derived variable region can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against zinc finger protein 33.

Anti-zinc finger protein 33 antibodies can be used in immunohistochemical techniques to detect zinc finger protein 33 in biopsy specimens.

Monoclonal antibodies that bind to zinc finger protein 33 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, zinc finger protein 33 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 zinc finger protein 33 positive cells.

The antibodies of the present invention can be used to treat or prevent diseases related to zinc finger protein 33. Administration of an appropriate dose of antibody can stimulate or block the production or activity of zinc finger protein 33.

The invention also relates to a diagnostic test method for quantitative and localized detection of zinc finger protein 33 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of zinc finger protein 33 detected in the test can be used to explain the importance of zinc finger protein 33 in various diseases and to diagnose diseases where zinc finger protein 33 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.

Polynucleotides encoding zinc finger protein 33 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 non-expression or abnormal / inactive expression of zinc finger protein 33. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated zinc finger protein 33 to inhibit endogenous zinc finger protein 33 activity. For example, a variant zinc finger protein 33 may be a shortened zinc finger protein 33 lacking 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 zinc finger protein 33. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfect the polynucleotide encoding zinc finger protein 33 Move into cells. Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a zinc finger protein 33 can be found in existing literature (Sambrook, eta l.). In addition, a recombinant polynucleotide encoding zinc finger protein 33 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 zinc finger protein 33 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 using any existing RM or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.

The polynucleotide encoding zinc finger protein 33 can be used for the diagnosis of diseases related to zinc finger protein 33. The polynucleotide encoding zinc finger protein 33 can be used to detect the expression of zinc finger protein 33 or the abnormal expression of zinc finger protein 33 in a disease state. For example, the DNA sequence encoding zinc finger protein 33 can be used to hybridize biopsy specimens to determine the expression of zinc finger protein 33. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and the relevant 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 known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Zinc finger protein 33 specific primers can also be used to detect the transcription products of zinc finger protein 33 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.

Detection of zinc finger protein 33 mutations can also be used to diagnose zinc finger protein 33-related diseases. Zinc finger protein 33 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type zinc finger protein 33 DNA sequence. Mutations can be detected using existing techniques such as Sou thern blotting, 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. Currently, specific sites for each gene on the chromosome need to be identified. Currently, few chromosome markers based on actual sequence data (repeating polymorphisms) are available. Used to mark chromosome locations. 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 according to cDM, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.

PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes. Using the oligonucleotide primers of the present invention, by a similar method, 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 hybrid pre-selection to construct chromosome-specific cDNA libraries.

Fluorescent in situ hybridization (FISH) of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manu l of Basic Techniques, Pergamon Pres s, 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, for example, in V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopk ins Universe Wetch Medica l 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 of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body. 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. Zinc finger protein 33 is administered in an amount effective to treat and / or prevent a particular indication. The amount and range of zinc finger protein 33 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. The experimental methods without specific conditions in the following examples are generally in accordance with conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Labora tory Pres s, 1989), Or as recommended by the manufacturer.

Example 1 Cloning of zinc finger protein 33

Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) m 2ug poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene) to form cDNA by reverse transcription. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5α, and the bacteria formed a cDNA library. The sequences at the 5 'and 3' ends of all clones were determined using a Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and an ABI 377 automatic sequencer (Perkin-Elmer). The determined cD sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 079 Oc 11 was new DNA. The inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers. The results showed that the full-length cDNA contained in the 0790cl l clone was 2004bp (as shown in Seq ID N0: 1), and there was a 906bp open reading frame (0RF) from 869bp to 1774bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0790cll and the protein encoded was zinc finger protein 33. Example 2 Homologous search of cDNA clones

The sequence of the zinc finger protein 33 of the present invention and the protein sequence encoded by the zinc finger protein 33 were analyzed using the Blas t program (Basicloca l Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403 -10], homology search in databases such as Genbank, Swiss spor t. Most homologous to zinc finger protein 33 of the present invention The high gene is a known human ZNF135, which encodes a protein with accession number P52742 in Genbank. The protein homology results are shown in Figure 1. The two are highly homologous, with 53% identity; 67% similarity. Example 3 Cloning of a gene encoding zinc finger protein 33 by RT-PCR

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

Primerl: 5 '-GGAGGCCCTGCTGAGGACTCCGG-3 ^/ (SEQ ID NO: 3)

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

Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;

Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification conditions: 50 mmol / L KC1, 10 mraol / L Tris-CI, (pH 8.5), 1.5 ramol / 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) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2rain ₀ β-act in was set as positive during RT-PCR Controls and template blanks are negative controls. 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 showed that the DNA sequence of the PCR product was exactly the same as the 1-2004bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of zinc finger protein 33 gene expression

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-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. Using 20 g of 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. Preparation cc- ³² P dATP with ³² P- DNA probe labeled by the random primer method. The DNA probe used was the PCR-amplified zinc finger protein 33 coding region sequence (869bp to 1774bp) shown in FIG. 1. The 32P- labeled probes (about 2 x l0 ⁶ cpm / ml) and RNA was transferred to a nitrocellulose membrane overnight at 42 ° C in a hybridization solution, the solution comprising 50% formamide -25raMKH ₂ P0 ₄ ( pH7.4) -5 x SSC-5 X Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 5 In vitro expression, isolation and purification of recombinant zinc finger protein 33

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:

Pr imer 3: 5 '-CCCGGATCCATGGAAAAAGTTGTAAAACAAAG-3' 32 (Seq ID No: 5) Pr imer4: 5 '-CATGCGGCCGCCTAAACTTCAAACGGTTTTTCTTC-3 ^/ (Seq ID No: 6) The 5' ends of these two primers contain BamHI and Not l Enzymatic digestion sites, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively. The BamHI and Not l digestion sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865). 3) Selective endonuclease sites. PCR was performed using the pBS-0790cl1 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μl containing 10 pg of pBS-0790cl l plasmid, Primer-3 and Primer-4 primers were 1 Opmol, Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. The amplified product and plasmid pET-28 (+) were double-digested with BamHI and Not l, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5c by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (PET-0790cl l) 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 liquid medium containing kanamycin (final concentration 30 M g / ml), the host strain BL21 (pET-0790cl l) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L , Continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The chromatography was performed using an affinity chromatography column His s. Bind Quick Cartridge (product of Novagen) capable of binding 6 histidines (6His-Tag) The purified target protein zinc finger protein 33 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 33 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the 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-zinc finger protein 33 antibodies

A peptide synthesizer (product of PE company) was used to synthesize the following zinc finger protein 33-specific peptides:

NH2-Met-Glu-Lys-Va l-Va l-Lys-Gln-Ser-Tyr-Glu-Phe-Ser-Asn-Ser-Asn-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. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin peptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin peptide complex was added incomplete Freund's adjuvant boosts 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 zinc finger protein 33.

Claims

Rights request

1. An isolated polypeptide-zinc finger protein 33, 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 1, further comprising 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 an 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 the polynucleotide comprises the sequence at positions 869-1774 in SEQ ID NO: 1 or the sequence at positions 1-2004 in SEQ ID NO: 1. .

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 and a plasmid, virus or vector 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 zinc finger protein 33 activity, characterized in that the method includes:

(a) culturing the engineered host cell according to claim 8 under conditions of expression of zinc finger protein 33;

(b) Isolating a polypeptide having zinc finger protein 33 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 zinc finger protein 33.

11. 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 zinc finger protein 33.

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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 zinc finger protein 33 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. The 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 zinc finger protein 33; 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 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 abnormality of zinc finger protein 33.

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.

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