WO2001030821A1

WO2001030821A1 - A novel polypeptide-homo rna cyclase 41 and polynucleotide encoding said polypeptide

Info

Publication number: WO2001030821A1
Application number: PCT/CN2000/000352
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: AU1126001A

Abstract

The invention discloses a new kind of polypeptide-HOMO RNA cyclase 41 and polynucleotide encoding said polypeptide and a process for producing said polypeptide by recombinant methods. It also discloses the method of applying the polypeptide for the treatment of various kinds of diseases, such as cancer, hemopathy, HIV infection and immune disease, and phlogosis. The antibody and antagonist and the therapeutic use of the polypeptide is also disclosed. In addition, it refers to the use of polynucleotide encoding said HOMO RNA cyclase 41.

Description

A new polypeptide human RNA cyclase 41 and a polynucleotide encoding the polypeptide

The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human RNA cyclase 41, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.

# 术背景

In the RNA splicing mechanism, many enzymes with catalytic functions have been found. During the splicing process of discontinuous gene transcripts, the splicing enzyme system requires no race specificity for the substrate. The chemical nature of splicing is a continuous phosphate transesterification reaction, in which an RNA cyclase can act as a cyclized RNA 3 'in the transesterification reaction. The release of introns and the connection of exons are two results of this process.

RNA 3 'terminal cyclase can catalyze the 3 phosphate group dependent on the existence of ATP to 1', 3 'cyclized phosphodiester, its acting substrate is different RM terminal fragments. In addition to human tissue, RNA-cyclase-like proteins have also been cloned in organisms such as bacteria. (Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W. (1997) EMBO J. 16, 2955-2967)

No protein functional domains have been found in RM cyclases. (EMB0 J. 1997 May 15; 16 (10): 2955-67) In the presence of Mg2 + and ATP at pH 8.0-9.0, RM cyclase can work. (Eur J Biochem 1988 Sep 15; 176 (2): 431-9) ATP is the best synergistic factor for RNA cyclase. The synergistic effects of GTP and other adenosine triphosphates are much lower. (J Biol Chem 1998 Sep 25; 273 (39): 25516-26)

Although the physiological function of RNA cyclase is not clear, RNA cyclase has been shown to play an important role in the regulation of tRNA cyclization end binding and the 3 'cyclization of U6 snRM. According to the results of Northern hybridization, RNA cyclase is expressed in all mammalian tissues and cells, and the indirect immunofluorescence assay also clarifies the intracellular location of RNA cyclase. RNA cyclase-like proteins have also been found in bacteria and other organisms, and they have RNA cyclase activity. The research results show that RNA cyclase plays an important role in the synthesis and metabolism of RNA. (EMBO J. 1997 May 15; 16 (10): 2955-67)

Adenylate cyclase is activated by hormones and plays a role in the synthesis of cyclic AMP-regulated RNA. The expression of adenylate cyclase in rat mammary gland tissues in late pregnancy increased and reached the highest value one day before delivery. The results show that insulin, prolactin, and hydrocortisone can stimulate adenylate cyclase activity. The synthesis of RNA is regulated by cyclic AMP. At the same time, the above hormones can also play a regulatory role, and the effects between the two can be cumulative. We can think that insulin, prolactin and hydrogen The effect of cortisone on RNA synthesis has nothing to do with the regulation of cyclized AMP, except that insulin, prolactin, and hydrocortisone can independently stimulate the activity of adenylate cyclase, thereby achieving the purpose of acting on RNA synthesis. (Acta Biochim Pol 1978; 25 (1): 29-36)

The role of RNA polymerase and RNA cyclase in the treatment of some diseases has been studied long ago. The results show that both have important roles in the diagnosis and treatment of diseases such as cardiac hypertrophy and primary myocarditis. . (Am J Cardiol. 1973 Sep 20; 32 (4): 423-6) (Recent Adv Stud Cardiac Struct Metab. 1973; 3: 479-87)

The polypeptide of the present invention was inferred and identified as human RNA cyclase 41 (HRNAcyclase41), which is the result of amino acid homology comparison.

Object of the invention

It is an object of the present invention to provide an isolated novel polypeptide, human RNA cyclase 41, 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 RM cyclase 41.

It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding human RM cyclase 41.

Another object of the present invention is to provide a method for producing human RNA cyclase 41.

Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human RNA cyclase 41. Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human RNA cyclase 41.

Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human RNA cyclase 41.

Summary of invention

In a first aspect of the present invention, a novel isolated human RNA cyclase 41 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 active fragment, or its active derivative, analog. 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 99 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human RNA cyclase 41; (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 sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 122-1247 in SEQ ID NO: 1; and (b) having a sequence of 1-2 in SEQ ID NO: 1 035-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 between the RM cyclocyclase 41 and human RNA cyclozyme of the present invention. The upper sequence is human RNA cyclase 41 and the lower sequence is human RM cyclase. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human RNA cyclase 41. 41 kDa 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 human RNA cyclase 41" means that human RNA cyclase 41 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human RM cyclase 41 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human RNA cyclase 41 peptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, human RNA cyclase 41, 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 may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of human RNA cyclase 41. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to the human RNA circularity that substantially maintains the present invention W enzyme 41 has the same biological function or activity as a polypeptide. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind 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 polynucleotide sequence with a total length of 2035 bases, and its open reading frame (126-1247) encodes 373 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide is 99% homologous to human RNA cyclase. It can be inferred that the new human RNA cyclase 41 has similar structure and function to human RNA cyclase.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DM, or synthetic DNA. DNA can be single-stranded or double-stranded. D can be a coded or non-coded chain. 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, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes . The present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50% identity between the two sequences, and preferably having a sequence identity of 70%). The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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) the identity between the two sequences is at least 95% Above, more preferably 97% or more hybridization occurs. 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 RNA cyclase 41.

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 RNA cyclase 41 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 DM 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. Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene). The construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; Q) the presence or absence of marker gene functions; (3) determination of the level of human RNA cyclase 41 transcripts; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.

In the method (1), the probe used for hybridization is any part of the polynucleotide of the present invention Homologous, at least 10 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, most 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 the protein product expressed by the human RNA cyclase 41 gene.

A method using PCR technology 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 cDNA end rapid amplification method) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RM 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. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human RNA cyclase 41 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology .

In the present invention, a polynucleotide sequence encoding human RNA cyclase 41 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chera. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human RNA cyclase 41 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Labora tory Manua 1, cold Spr ing 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: 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, and 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 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 human RNA cyclase 41 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.

Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l ₂ method used in steps well known in the art. The alternative is to use MgC l ₂ . 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 DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human RNA cyclase 41 (Scence, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human human RNA cyclase 41 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 polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

The polypeptide of the present invention can catalyze the 3 'phosphate group dependent on the presence of ATP to 2', 3 'cyclic phosphodiester, and its acting substrate is different RM terminal fragments. Therefore, the polypeptide of the present invention plays an important role in the process of RNA synthesis, metabolism and RNA splicing, and is an indispensable regulatory factor in the process of DNA transcription.

The polypeptide of the present invention can be used to diagnose and treat many diseases, such as malignant tumors, immune diseases, human acquired immune deficiency syndrome (AIDS), endocrine system diseases, nervous system diseases and the like.

The polypeptides of the present invention can be used for the diagnosis and treatment of malignant tumors, including leukemias and lymphomas; tumors of epithelial cell origin; tumors of mesenchymal origin, such as sarcomas; central nervous system tumors and the like.

The polypeptides of the present invention also have effects on damage, defects or disorders of immune tissues, especially for hematopoietic diseases (such as malignant anemia), skin diseases (such as psoriasis), autoimmune diseases (such as rheumatoid arthritis), and radioactivity. Disease and the production and regulation of immune lymphocytes are extremely closely related.

The role of RNA polymerase and RNA cyclase in the treatment of some diseases has been studied long ago. The results show that both have important roles in the diagnosis and treatment of diseases such as cardiac hypertrophy and primary myocarditis. . (Am J Cardi o l. 1973 Sep 20; 32 (4): 423-6) (Recent Adv Stud Card i ac Struc t Me tab. 1973; 3: 479-87.

The invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human RNA cyclase 41. Agonists enhance biological functions such as human RNA cyclase 41 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, The mammalian cells or membrane preparations expressing human RNA circularase 41 are cultured with labeled human RNA circularase 41 in the presence of. The ability of the drug to increase or block this interaction is then determined.

Antagonists of human RM cyclase 41 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human R N A cyclase 41 can bind to human R N A cyclase 41 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 that act as antagonists, human RNA cyclase 41 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human RNA cyclase 41 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 RNA cyclase 41 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 RNA cyclase 41 molecule should generally be labeled.

The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the human RNA cyclase 41 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.

Polyclonal antibodies can be produced by injecting human RNA cyclase 41 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 human RNA cyclase 41 include, but are not limited to, hybridoma technology (Kohl er and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single-chain antibodies (US Pat No. ⁴ 9 ⁴ 6778) can also be used to produce single-chain antibodies against human RNA cyclase 41.

Antibodies against human RNA cyclase 41 can be used in immunohistochemistry to detect human RM cyclase 41 in biopsy specimens.

Monoclonal antibodies that bind to human RNA cyclase 41 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 RNA cyclase 41 high affinity monoclonal antibodies can interact with bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine). Etc.) Covalent bonding. 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 RNA cyclase 41 positive cells .

The antibodies of the present invention can be used to treat or prevent diseases related to human RNA cyclase 41. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human RNA cyclase 41.

The invention also relates to a diagnostic test method for quantitative and localized detection of human RNA cyclase 41 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human RNA cyclase 41 detected in the test can be used to explain the importance of human RNA cyclase 41 in various diseases and to diagnose diseases in which human RNA cyclase 41 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.

The polynucleotide encoding human RNA cyclase 41 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 RM cyclase 41. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human RNA cyclase 41 to inhibit endogenous human RNA cyclase 41 activity. For example, a mutated human RNA cyclase 41 may be a shortened human RNA cyclase 41 that lacks a signaling domain. Although it can bind to a downstream substrate, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human RNA cyclase 41. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human RNA cyclase 41 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding human RNA cyclase 41 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human RNA cyclase 41 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 RNA cyclase 41 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DM, and ribozymes can be obtained by any existing RNA or DM synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in various ways, such as To increase the sequence length on both sides, the linkage between ribonucleosides uses phosphothioester or peptide bonds instead of phosphodiester bonds.

The polynucleotide encoding human RM cyclase 41 can be used for the diagnosis of diseases related to human RNA cyclase 41. The polynucleotide encoding human RNA cyclase 41 can be used to detect the expression of human RNA cyclase 41 or the abnormal expression of human RNA cyclase 41 in a disease state. For example, the DNA sequence encoding human RNA cyclase 41 can be used to hybridize biopsy specimens to determine the expression of human RNA cyclase 41. Hybridization techniques include Sou thern blotting, Nor thern 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 (Mic Roa ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and Genetic diagnosis. Human RNA cyclase 41 specific primers can also be used to detect human RNA cyclase 41 transcripts by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).

Detection of mutations in the human RNA cyclase 41 gene can also be used to diagnose human RM cyclase 41-related diseases. Human RNA cyclase 41 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human RNA cyclase 41 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. Therefore, the Nor thern blotting and Western blotting methods 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, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be 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 1-35 bp) can be prepared from cDNA to locate the sequence 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 DNA 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 (FI SH) of cDNA clones and metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosome s: a Manua l of Bas ic 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 in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

Next, the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all 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 disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

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

The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human RNA cyclase 41 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of doses of human RM cyclase 41 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 human RM cyclase 41

Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. A Smart cDM cloning kit (purchased from Clontech) ^ cDM fragment was inserted into the multiple cloning 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 Dye terminate cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). The determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0834D09 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 0834D09 clone contained a full-length cDNA of 2035bp (as shown in Seq ID N0: 1), and a 1122bp open reading frame (0RF) from 126bp to 1247bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0834D09 and the encoded protein was named human MA cyclase 41. Example 2 Homologous search of cDNA clones

The sequence of the human RNA cyclase 41 of the present invention and the protein sequence encoded by the human RNA cyclase 41 were coded using the Blas t program (Basiclocal Alignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403 -10], perform homology search in databases such as Genbank, Swissport, etc. The gene with the highest homology to the human RNA cyclase 41 of the present invention is a known human RNA cyclase, and the accession number encoded by the protein in Genbank is AF067172. The results of protein homology are shown in Figure 1. The two are highly homologous and their identity is 99%. Example 3 Cloning of a gene encoding human RNA cyclase 41 by RT-PCR

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

Pr imerl: 5 '-GGCTGGAGGCAGCCCGAGCCGC-3 ^r (SEQ ID NO: 3)

Pr imer2: 5 '-TAAATGAGGAGATTTAATGTCT-3' (SEQ ID NO: 4)

Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.

Conditions for the amplification reaction: 50 mmol / L KC1, 10 mmol / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech). On the PE9600 DNA thermal cycler (Perkin-Elmer) Piece reaction 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-actin was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit. DM sequence analysis results showed that the DM sequence of the PCR product was exactly the same as that of 1-2035bp shown in SEQ ID NO: 1. Example 4 Northern Blot Analysis of Human RNA Cyclase 41 Gene Expression

Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M 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 ), Mix and centrifuge. 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 RM, electrophoresis was performed on a 1.2% agarose gel containing 20raM 3- (N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. ³² P dATP Preparation ³² P- DNA probe labeled by the random primer Method - with o. The DM probe used was the PCR amplified human RM cyclase 41 coding region sequence (126bp to 1247bp) shown in FIG. 1. A 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH ₂ P0 ₄ (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1x SSC-0.1% SDS at 55. C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 5 In vitro expression, isolation and purification of recombinant human RM cyclase 41

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 '-CCCGCTAGCATGGCGACTCATGCGCACTCCCTC-3' (Seq ID No: 5) Primer4: 5 '-GCCCGATCCTCACTTGAGGGTCTTGCTAAGGTTGG-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ncol and BamHI restriction sites, The coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively. The Nhel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site. PCR was performed using the PBS-0834D09 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0834D09 plasmid, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ncol 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 coliform bacteria DH5a by the calcium chloride method, After culturing overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive colonies were selected by colony PCR method and sequenced. A positive clone (PET-0834D09) 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-0834D09) was at 37. C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mmol / L, and 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, and the layer was layered with an affinity chromatography column His s. Bind Quick Cartr idge (product of Novagen) capable of binding to 6 histidines (6His-Tag). Analysis to obtain purified human RNA cyclase 41. After SDS-PAGE electrophoresis, a single band was obtained at 41 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-human RNA cyclase 41 antibody

A peptide synthesizer (product of PE company) was used to synthesize the following human RNA cyclase 41-specific peptides:

NH2-Met-A 1 a-Thr-H i s— A 1 a-H i s-Ser-Leu-Ser-Tyr-Ala-G 1 y- Cy s-Asn-Phe— 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. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. 15A 15 g / ml bovine serum albumin peptide complex-coated titer plate was used for ELISA to determine the antibody titer in rabbit serum. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum. 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 demonstrated that the purified antibody specifically binds to human RM cyclase 41.

Claims

W

1. An isolated polypeptide-human RNA cyclase 41, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID D 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 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 99% 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 of positions 126-1247 in SEQ ID NO: 1 or the sequence of positions 1-2035 in SEQ ID NO: 1. .

7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector 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 RNA cyclase 41 activity, characterized in that the method includes:

(a) culturing the engineered host cell according to claim 8 under the condition of expressing human RNA cyclase 41;

(b) Isolating a polypeptide having human RNA cyclase 41 activity from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to human RNA cyclase 41.

Π, a class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are mimics, substitution pages (Article 26 of the Regulations) Compounds that promote, antagonize or inhibit the activity of human RNA cyclase 41.

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

13. The use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human RNA cyclase 41 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 RNA cyclase 41; or for peptides Fingerprint identification.

16. The use of a nucleic acid molecule according to any one of claims 4 to 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 to 6 and 11, 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 abnormality of human RNA cyclase 41.

18. The use of the polypeptide, polynucleotide or compound according to any one of claims 1-6 and 1 1, characterized in that the polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor Drugs for blood diseases, HIV infection and immune diseases and various types of inflammation.

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