WO2001031031A1

WO2001031031A1 - Novel polypeptide---human helper t cell growth factor 15 and polynucleotide encoding it

Info

Publication number: WO2001031031A1
Application number: PCT/CN2000/000324
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Shanghai Bio Road Gene Development Ltd.
Priority date: 1999-10-22
Filing date: 2000-10-16
Publication date: 2001-05-03
Also published as: AU1015501A

Abstract

The invention concerns human helper T cell growth factor (15) and polynucleotide encoding it. The invention also concerns the process of producing the polypeptide by recombinant DNA technique. The methods for treating many diseases e.g. malignant tumor, hemopathy, infection of HIV, immunological diseases and pruritus utilizing the polypeptide are disclosed. The invention discloses the antagonist against the polypeptide and therapeutics thereof. The invention also discloses the uses of the polynucleotide, which encodes the novel human helper T cell growth factor (15).

Description

A new polypeptide-helper T cell growth factor 15 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a helper T cell growth factor 15, 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

Many cytokines are composed of polypeptides, which directly or indirectly regulate the immune system of the body, tissue development and cell differentiation. It is now widely believed that cytokines can regulate the body's defense response to cancer and infectious diseases. These cytokines include: interferon (IFN-oc, IFN- (3, IFN- γ), tumor lethal factor (TNF-α), lymphotoxin (TNF-β), interleukin (IL1, 2, 3, 4, 5 and 6), leukocyte regulatory factors, cytotoxic factor (NKCF) of natural killer cells, transforming growth factor (TGF), colony-stimulating factor (CSF) such as macrophages (M-CSF), granulocytes (G-CSF), both macrophages and granulocytes (G, M-CSF) and oncostatin M. Each of these cytokines has different characteristics and specific anti-proliferative, cytostatic, Anti-filtration pathogen, regulating growth activity.

In vitro experiments have shown that some cytokines are produced by microorganisms, antigens, or mitogens that stimulate the natural physiological response of leukocytes. Stimulating cell lines with these cytokines will produce phenomena that will explain the characteristics and functions of this cytokine. Recently, it has been proved by this method that the cytokines that regulate T cell growth include not only interleukin 2, but also interleukin 4 (Fernandez—Botran et al., Proc. Natl. Acad. Sci. USA 83: 9689-9693, 1986; Lichtman et al., Proc. Natl. Acad. Sci. USA 84: 824-827, 1987) G, M-CSF (Woods et al., J. Immunol. 138: 4293-4297,

1987); upper et al., J. Immunol. 138: 4288-4292, 1987) and interleukin 1 and interleukin 6 (Houssiau et al., Eur. J. Immunol. 18: 653-656,

1988). Although interleukin 2 is a potent and widely used T cell growth factor, the other cytokines also mediate the proliferation of non-interleukin 2 dependent T cells, thereby complicating the originally envisioned T cell growth regulatory process.

An important member of the T cell family is helper T cells (T. sub.H). At least two helper T cells with different functions have been found. One of them, T. sub.H1, assists B cells to differentiate into antibody-secreting cells, which is a direct immune response. It has antigen specificity and is an early immune response. Another T. sub.H 2 is related to cellular immunity and the formation of delayed-type hypersensitivity inflammation, which is a delayed immune response.

A few years ago, people collected helper T cells from the lymph nodes of mice injected with the antigen and cultured them. Department (Corrad in etal., J. Immuno l. 119: 1048-1 053, 1977.). Without the increase of exogenous growth factors, these cell lines were cultured in the presence of antigens from the beginning, and antigens and irradiation were regularly added. Most cells produced a large amount of interleukin 3, interleukin 4, interleukin 5 and Interleukin 6, but not interleukin 2, from which T. sub. H 2 type was identified (Mosmann etal., J. Immuno l. 1 36: 2348-2357, 1986.). By changing the experimental method, instead of adding antigen, the above-mentioned rule of helper T cells self-proliferation was used to extract a non-interleukin 2 and interleukin 4 dependent helper T cell growth factor (P40). The invention provides a new T cell growth factor, which has a high degree of homology with the known cytokine P40. This new growth factor can be used in therapeutic drugs to stimulate the proliferation of helper T cells.

Object of the invention

It is an object of the present invention to provide an isolated novel polypeptide, helper T cell growth factor 15 and its fragments, analogs and derivatives.

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 helper T cell growth factor 15.

Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a helper T cell growth factor 15.

Another object of the present invention is to provide a method for producing helper T cell growth factor 15.

Another object of the present invention is to provide antibodies against the polypeptide of the present invention, helper T cell growth factor 15.

Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention, helper T cell growth factor 15.

Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of helper T cell growth factor 15.

Summary of invention

In a first aspect of the present invention, a novel isolated helper T-cell growth factor 15 is provided. The polypeptide is of human origin and includes: 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 the isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 78 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the above-mentioned helper T cell growth factor 15; (b) with a polynucleotide (a) Complementary polynucleotide. 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 842-1252 in SEQ ID NO: 1; and (b) a sequence having 1-151 in SEQ ID NO: 1 A sequence of zero bits.

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.

Figure 1 is a comparison diagram of the amino acid sequence homology of helper T cell growth factor 15 and P40 in human tissues of the present invention. The upper sequence is helper T cell growth factor 15, and the lower sequence is P40 in human tissue. 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 helper T cell growth factor 15. 15. OkDa 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 helper T cell growth factor 15" means that helper T cell growth factor 15 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify helper T cell growth factor 15 using standard protein purification techniques. Substantially pure peptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the helper T cell growth factor 15 peptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, helper T cell growth factor 15, 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 helper T cell growth factor 15. 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 helper T cell growth factor 15 of the present invention. 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 1510 bases, and its open reading frame (842-1252) encodes 1 36 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 77% homology with P40 in human tissues, and the polypeptide has a conserved base of the P40 gene family. It can be inferred that the new human helper T cell growth factor has Similar structure and function of the P40 gene family. The polynucleotide of the present invention may be in the form of DNA or RNA. DM forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DM can be coded or non-coded. 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 present invention also relates to a variant of the polynucleotide described above, which encodes the same amino group as the present invention. Acid sequences of polypeptides or fragments, analogs and derivatives of polypeptides. 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%, preferably 70% identity, between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 Ό, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, 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 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding helper T cell growth factor 15.

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 helper T cell growth factor 15 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 D fragment sequence of the present invention can also be obtained by the following methods: 1) separating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.

Of the methods mentioned above, genome D is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qi agene). And the construction of cDNA libraries is also a common method (Sambrook, etal., Mol ecu lar Cloning, A Labora tory Manua, Co. Harbor Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned. These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of transcripts of helper T cell growth factor 15; (4) ) Detection of protein products expressed by genes through 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 homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used herein is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of the helper T cell growth factor 15 gene expression.

A method using PCR technology to amplify DM / 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 full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used, and 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 / R fragment 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 cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cD 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 genetically engineered using the vector of the present invention or directly using a helper T cell growth factor 15 coding sequence, and a recombinant technology for producing a polypeptide of the present invention. method.

In the present invention, a polynucleotide sequence encoding a helper T cell growth factor 15 may 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 expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as For replication and stabilization in the host, 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 known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding helper T cell growth factor 15 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, leg synthesis technology, and in vivo recombination technology (Sambroook, et al. Mol ecu lar Clinging, a Labora tory Manua, 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 mRM 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 Promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral 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 helper T cell growth factor 15 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 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 DM 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 necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following D transfection methods can be used: calcium phosphate co-precipitation method, Or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant helper T cell growth factor 15 (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human helper T cell growth factor 15 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 invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) helper T cell growth factor 15. Agonists increase helper T cell growth factor 15 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing helper T cell growth factor 15 can be cultured together with labeled helper T cell growth factor 15 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

Antagonists of helper T cell growth factor 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of helper T cell growth factor 15 can bind to helper T cell growth factor 15 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.

When screening compounds as antagonists, helper T cell growth factor 15 can be added to the organism In analytical assays, whether a compound is an antagonist is determined by measuring its effect on the interaction between helper T cell growth factor 15 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 helper T cell growth factor 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the T-cell growth factor 15 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 helper T cell growth factor 15 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

Polyclonal antibodies can be produced by injecting helper T cell growth factor 15 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 help T cell growth factor 15 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 combine human constant regions with non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851) and existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against helper T cell growth factor 15.

Antibodies to helper T cell growth factor 15 can be used in immunohistochemistry to detect helper T cell growth factor 15 in biopsy specimens.

Monoclonal antibodies that bind to helper T cell growth factor 15 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, helper T-cell growth factor 15 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 disulfide exchange. This hybrid antibody can be used to kill helper T cell growth factor 15 positive cells .

The antibodies of the present invention can be used to treat or prevent diseases related to helper T cell growth factor 15. Administration of an appropriate dose of antibody can stimulate or block the production or activity of helper T cell growth factor 15.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of helper T cell growth factor 15. These tests are well known in the art and include FISH assays and radioimmunoassays. In test The detected helper T cell growth factor 15 levels can be used to explain the importance of helper T cell growth factor 15 in various diseases and to diagnose diseases in which helper T cell growth factor 15 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.

Polynucleotides encoding helper T cell growth factor 15 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 helper T-cell growth factor 15. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant helper T cell growth factor 15 to inhibit endogenous helper T cell growth factor 15 activity. For example, a variant helper T-cell growth factor 15 may be a shortened helper T-cell growth factor 15 lacking a signal transduction functional domain, and although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of helper T cell growth factor 15. Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding helper T cell growth factor 15 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a helper T cell growth factor 15 can be found in the existing literature (Sambrook, et al.). In addition, a polynucleotide encoding the helper T cell growth factor 15 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 helper T cell growth factor 15 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 RM to perform endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides. Antisense R molecules can be obtained by in vitro or in vivo transcription of the DM sequence encoding the RM. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

Polynucleotides encoding helper T cell growth factor 15 are useful in the diagnosis of diseases associated with helper T cell growth factor 15. The polynucleotide encoding helper T cell growth factor 15 can be used to detect the expression of helper T cell growth factor 15 or the abnormal expression of helper T cell growth factor 15 in a disease state. For example, DM sequences encoding helper T cell growth factor 15 can be used to hybridize biopsy specimens to Determine the expression of helper T cell growth factor 15. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissue. T-cell growth factor 15 specific primers can also be used to detect the transcription products of T-cell growth factor 15 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.

Detection of mutations in the helper T cell growth factor 15 gene can also be used to diagnose helper T cell growth factor 15-related diseases. Helper T-cell growth factor 15 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type helper T-cell growth factor 15 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, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, 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 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those 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 (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manua 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 in, for example, V. Mckusick, Mende l ian Inher i tance in Man (available through contact with Johns Hopkins Univers i ty Welch Medica l Library available online). 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 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. Helper T cell growth factor 15 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of dosage of helper T-cell growth factor 15 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 performed according to the general conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Laboratory Pres s, 1989), or according to the conditions Conditions recommended by the manufacturer.

Example 1 Cloning of helper T cell growth factor 15

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 raRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed CDM is formed. The Smart 00 cDNA cloning kit (purchased from Clontech) was used to insert the 00 fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5 cc. The bacteria formed a cD library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined CDM sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0575h06 was new DNA. A series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone. The results show that the 0575h06 clone contains a full-length cDNA of 1510bp (as shown in Seq ID N0: 1), a 136bp open reading frame (0RF) from 842bp to 1252bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0575h06 and named the encoded protein helper T cell growth factor 15. Example 2 Homologous search of cDNA clones

The sequence of the helper T-cell growth factor 15 of the present invention and the protein sequence encoded by the helper T cell growth factor 15 (Basic local Algorithm 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 helper T cell growth factor 15 of the present invention is a known human tissue P40, and its accession number to Genbank is 1193569. The results of protein homology are shown in Figure 1. The two are highly homologous and their identity is 77%. Example 3 Cloning of a gene encoding helper T cell growth factor 15 by RT-PCR

CDNA was synthesized using fetal brain 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:

Primerl: 5,-GGATCAGGCAGCAATATTTGCC-3 '(SEQ ID NO: 3)

Pr imer2: 5, — TTTGCTTG ATAG ATCTTCCTCC- 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.

Amplification conditions: 50 μl / L KC1, 10 mmol / L Tris-CI, (pH 8.5.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L in a reaction volume of 50 μ 1 dNTP, l Opmol primer, 1U 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 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. DNA sequence analysis results indicate PCR The DNA sequence of the product is exactly the same as the 1-1515bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of helper T cell growth factor 15 gene expression

Total RNA was extracted in one step nal. Biochem 1987, 162, 156-159] _c 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 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ), 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. Perform electrophoresis on 2 (g RNA) on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5raM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer Onto a nitrocellulose membrane. A- ³² P dATP was used to prepare a ³² P-labeled DNA probe by a random primer method. The DNA probe used was the PCR-encoded helper T cell growth factor 15 coding region shown in FIG. 1 Sequence (842bp to 1252bp). A 32P-labeled probe (approximately 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight, the solution containing 50% formazan Amide-25mMKH ₂ P0 ₄ (pH7.4)-5 x SSC-5 x Denhardt solution and 200 g / ral salmon sperm DM. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then analysis and quantification was performed using Phosphor Imager. Example 5 In vitro expression, isolation and purification of recombinant helper T cell growth factor 15

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

Primer3: 5,-CCCCATATGATGCCACAAAGATACTCCTCG- 3, (Seq ID No: 5)

Primer4: 5'- CCCGGATCCTTACAATTTGGCATGTTTTTGC- 3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BaraHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). The pBS-0575h06 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: 10 pg of pBS- 0575h06 plasmid in a total volume of 50 μ1, primers Primer-3 and Primer-4 were lpmol, Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Escherichia coli DH50C by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR and sequenced. A positive clone (pET-0575h06) 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 kanamycin-containing (final concentration 30 g / ml) of LB liquid medium, host strain BL21 _(P ET-0575h06) incubated at 37 ° C to logarithmic phase, IPTG was added to a final concentration of lmmol / L, incubation was continued for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using His s. Bind Quick Cartridge (product of Novagen) which can bind to 6 histidines (6His-Tag). The purified target protein helper T cell growth factor 15 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 15. OkDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of anti-helper T cell growth factor 15 antibodies

The following peptides specific for helper T cell growth factor 15 were synthesized using a peptide synthesizer (product of PE company): NH ₂ -Met-Pro-Gln-Arg-Tyr-Ser-Ser-Arg-Arg-Ala-Thr-Pro- Arg-Hi sIle- 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 immunity once.釆 Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate 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 helper T cell growth factor 15.

Claims

Rights request

1. An isolated polypeptide-helper T cell growth factor 15, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof.

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

3. The polypeptide according to claim 2, 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 78% 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 a sequence of positions 842-1252 in SEQ ID NO: 1 or a sequence of positions 1-1510 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 T-cell growth factor 15 activity, characterized in that the method includes:

(a) culturing the engineered host cell according to claim 8 under the condition of expressing helper T cell growth factor 15;

(b) Isolating a polypeptide having T-cell growth factor 15 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 helper T cell growth factor 15.

1 1. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of helper T cell growth factor 15.

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. Use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of helper T cell growth factor 15 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 helper T cell growth factor 15; 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 1-6 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 helper T cell growth factor 15.

18. 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 malignant tumors, blood diseases Drugs for HIV infection and immune diseases and various types of inflammation.