CN1148381C - Novel human chemokine macrophage inflammatory protein, its coding sequence and use - Google Patents

Abstract

The present invention provides a novel human chemotactic factor-macrophage inflammatory protein (MIP)-2 gamma, polynucleotide which codes the polypeptide, and a method for generating the polypeptide by a recombination technique. The present invention also discloses an application of the polynucleotide which codes the novel human MIP-2 gamma protein. The present invention also discloses the expression situations of the polypeptide in normal tissues and various tumour cells, a method used for tumour diagnosis, and a curative method used for a plurality of diseases, such as neutrophilic granulocyte deficiency or hypofunction, tumour, autoimmune diseases, etc. The present invention also provides a medical composition which comprises MIP-2 gamma. The present invention also discloses an antagonist for resisting the polypeptide, and curative effect thereof.

Description

Novel human chemokine macrophage inflammatory protein, its coding sequence and use

technical field

The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to a new encoding human chemokine macrophage inflammatory protein MIP-2γ (macrophage inflammatory protein-2γ, also known as "chemokine MIP-2γ") or "MIP-2γ protein") polynucleotides, and polypeptides encoded by such polynucleotides. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. Specifically, the polypeptide of the present invention is a new chemokine related to tumor and infection.

Background technique

Chemokines and their receptors are a rapidly developing field of research, and new chemokines and their receptors have been discovered in recent years. Chemokines are low-molecular-weight proteins with conserved sequences and chemotactic effects on leukocytes (neutrophils, monocytes, and lymphocytes, etc.), and are a superfamily of cytokines (Ward et al. Immunity, 1998, 9 : 1). The discovery of chemokines has progressed rapidly and has become a hot spot in the field of molecular immunology.

At present, there are at least 50 kinds of chemokines, the size of their mature molecules is 68-120 amino acids, and the chemokines have a conserved Cys structure. According to the characteristics of the Cys domain in its structure, it can be divided into at least four subfamilies, and the chemokines of different families have different functional characteristics.

(a) The C-X-C family, that is, the α subfamily, is characterized by a non-conserved amino acid between the two Cys at the amino terminal, including IL-8, etc., which can chemoattract neutrophils and have no chemotactic activity on monocytes.

(b) The C-C family, namely the β subfamily, is the largest family of chemokines, including more than 20 members in humans. Its structure is characterized by the continuous arrangement of two Cys at the amino terminal, and it mainly chemokines monocytes and lymphocytes , has no chemotactic effect on neutrophils. The vast majority of chemokines expressed in dendritic cells (DCs) belong to this subfamily, such as DC-CK1, which specifically chemoattracts resting T cells.

(c) The C subfamily, also known as the γ family, currently has only one member of the lymphocyte chemokine, which only contains two of the four Cys of the usual chemokine, and can specifically chemoattract lymphocytes and NK cells.

(d) The CX3C family, namely the δ family, has a structural characteristic of three non-conserved amino acids separated by two Cys at the amino terminal, and has chemotactic activity for T cells.

Macrophage inflammatory protein (macrophage inflammatory protein, referred to as "MIP") is a kind of mammalian cells, such as macrophages and lymphocytes, stimulated by G-bacteria, lipopolysaccharide or concanavalin A, etc. protein. Therefore, MIP may play a role in the diagnosis and treatment of infection, tumor, inflammation, inhibition of myeloid proliferation and autoimmune diseases.

The cellular expression spectrum of chemokines is very broad, including monocytes and macrophages, endothelial cells, mesenteric cells, fibroblasts, keratinocytes and lymphocytes, dendritic cells, etc.

Dendritic cells (DC) are important professional antigen-presenting cells in the body, and are the direct initiators and regulators of the body's T cell-specific immune response. In recent years, the differentiation and development of dendritic cells, the mechanism of antigen processing and presentation, and their roles in tumors, infections, autoimmune diseases, and transplant rejection have become the frontiers of immunology (Cao Xuetao et al. Chinese Journal of Immunology. 1998 3:322, Banchereau et al. Nature. 1998;392:245). The in vivo migration of DC is necessary for DC differentiation and maturation and completion of its antigen presentation function. Under the action of chemokines, dendritic cells undergo directional migration in vivo, so as to further develop and mature, and complete their antigen presentation function. The expression of chemokine receptors is regulated by DC differentiation and maturation status. Thus, DCs at different maturation stages have different reactivity to different chemokines (Delgado et al. Immunobiology 1998, 198: 490 and Dieu et al. J. Exp. Med. 1998, 188: 373). Although DCs express CXC chemokine receptors, most CXC chemokines such as IL-8, IP-10 and Gro-β are not chemotactic for DCs. So far only one CXC chemokine, SDF-1, has been found to have a chemotactic effect on DC.

In addition to expressing chemokine receptors and being responsive to chemokines, DC also expresses and secretes chemokines, regulates the chemotaxis of other immune cells, and participates in the regulation of T cell development and the formation of immune tolerance. Chemokines expressed on DCs include MIP-1α, MIP-1β, MIP-1γ, PANTES, MCP-1, MIP-3β, and DC-CK1. Most of these chemokines belong to the CC chemokine family (Lore et al. J. Immunol. Methods 1998, 214:97). In addition, DCs also express CXC chemokines, such as IL-8, which have the potential to chemoattract neutrophils.

The relationship between chemokines and HIV infection is a recent breakthrough research progress. It is now believed that when HIV infects cells, in addition to the combination of its coat glycoprotein gp120 and CD4, the participation of auxiliary molecules, namely chemokine receptors, is necessary; chemokines can inhibit HIV from entering cells. The molecular mechanism of chemokines inhibiting HIV may be in the link of chemokine receptors. Chemokine receptors such as CXCR4, CCR3 and CCR5 expressed by DC are related to the entry of HIV into DC (Rubbert et al. J. Immunol. 1998, 160: 3933).

Because chemokines play an important role in the differentiation and development of immune cells and the regulation of the body's immune response, people have paid more and more attention to them. Under the action of chemokines, dendritic cells undergo directional migration in vivo, so as to further develop and mature, and complete their antigen presentation function; at the same time, dendritic cells themselves also produce and secrete chemokines, fine-tuning including T cells in Chemotaxis of other immune cells in the body, more effectively perform its role in the initiation and regulation of the body's immune response. At the same time, chemokine receptors expressed by dendritic cells are related to HIV infection. Therefore, it is of great significance to find new diagnostic and therapeutic strategies for tumors, infections, and immune diseases by enhancing or blocking chemotactic signals of dendritic cells.

However, prior to the present invention, the chemokine MIP-2γ involved in the present invention has not been disclosed.

Contents of the invention

The object of the present invention is to provide a new human chemokine-namely macrophage inflammatory protein MIP-2γ (macrophage inflammatory protein-2γ, referred to as “MIP-2γ”) protein polypeptide and its fragments, analogs and derivatives thing. MIP-2γ has a chemotactic effect on DC, which helps to further understand the migration mechanism of DC.

Another object of the present invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide methods for producing these polypeptides and uses of the polypeptides and coding sequences.

Another object of the present invention is to provide a pharmaceutical composition containing MIP-2γ protein.

In the first aspect of the present invention, a novel isolated MIP-2γ protein polypeptide is provided, which is human-derived, and it comprises: a polypeptide having an amino acid sequence of SEQ ID NO: 2, or a conservatively variant polypeptide thereof, or Active fragments, or active derivatives thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In a second aspect of the present invention, isolated polynucleotides encoding these polypeptides are provided, the polynucleotides comprising a nucleotide sequence having at least 70 degrees of a nucleotide sequence selected from the group consisting of % identity: (a) polynucleotide encoding the above-mentioned human MIP-2γ protein polypeptide; and (b) polynucleotide complementary to polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having the sequence of positions 41-376 in SEQ ID NO: 1; (b) having positions 143-376 in SEQ ID NO: 1 (c) has the sequence of 1-463 positions in SEQ ID NO:1.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In the fourth aspect of the present invention, there is provided a method for preparing a polypeptide having human MIP-2γ protein activity, the method comprising: (a) cultivating the above-mentioned transformed or transduced polypeptide under conditions suitable for expressing human MIP-2γ protein a host cell; (b) isolating a polypeptide having human MIP-2γ protein activity from the culture.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned human MIP-2γ protein polypeptide is provided. Also provided is a nucleic acid molecule useful for detection, which contains consecutive 10-463 nucleotides of the above-mentioned polynucleotides.

In the sixth aspect of the present invention, a method for detecting the presence of MIP-2γ protein in a sample is provided. The sample is contacted with an antibody specific for MIP-2γ to observe whether an antibody complex is formed. The formation of an antibody complex means that the protein exists in the sample. MIP-2γ protein.

In the seventh aspect of the present invention, uses of the polypeptides and coding sequences of the present invention are provided. For example, the polypeptide of the present invention can be used to screen for agonists that promote the activity of human MIP-2γ protein polypeptides, or to screen for antagonists that inhibit the activity of human MIP-2γ protein polypeptides, or to identify peptide fingerprints. The human MIP-2γ protein coding sequence or its fragments of the present invention can be used as primers for PCR amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays.

In the eighth aspect of the present invention, a method for screening or detecting a disease or a disease (especially a tumor) susceptibility related to the expression of a human MIP-2γ protein polypeptide is provided.

In the ninth aspect of the present invention, a research strategy for the migration, differentiation and maturation of dendritic cells and other immune cells related to the expression of human MIP-2γ protein polypeptide in vivo is provided.

In the tenth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the human MIP-2γ protein polypeptide of the present invention or its antagonist, inhibitor and pharmaceutically acceptable carrier. These pharmaceutical compositions can treat immune diseases, tumors, AIDS and other diseases.

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

Description of drawings

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

Fig. 1A is the cDNA sequence and the full-length amino acid sequence of the human MIP-2γ protein of the present invention. Among them, non-coding sequences are represented by lowercase letters, and coding sequences are represented by uppercase letters. "*" indicates a stop codon. "↓" marks the cleavage site of the signal peptide.

Fig. 1B is a comparison diagram of amino acid sequence homology between human MIP-2γ protein of the present invention and human MIP-2α and MIP-2β proteins.

Fig. 2 is an electrophoresis diagram of Northern hybridization of the expression of human MIP-2γ in normal tissues. From left to right are pancreas, kidney, bone marrow muscle, liver, lung, placenta, brain, heart, peripheral blood lymphocytes, colon, small intestine, ovary, testis, prostate, thymus and spleen.

Fig. 3 is an electrophoresis diagram of RT-PCR analysis of the expression of human MIP-2γ in different tumor cell lines. Among them, from left to right are acute lymphoblastic leukemia cell line Molt-4, skin T lymphoma cell line Hut78, acute T-cell leukemia cell line Jurkat, erythroleukemia cell line K562, acute myeloid leukemia cell line HL-60, human monocytogenes Nuclear cell line THP-1, histiocytic lymphoma cell line U937 and lung cancer cell line A549.

Fig. 4A is a structural diagram of the recombinant expression vector pcDNA3.1/Neo.

Fig. 4B is an electrophoresis image of detecting the expression of human MIP-2γ by 35S metabolic labeling in the culture supernatant of 293 cells transfected with the recombinant expression vector. The sequence from left to right is: the MIP-2γ lane is the expression product of human MIP-2γ; the control lane is the control supernatant of 293 cells transfected with an empty vector. On the left is the molecular weight size (kDa).

Fig. 4C is an electrophoresis diagram of Western blot analysis of the human MIP-2γ expression product in the culture supernatant after the recombinant expression plasmid was transfected into 293 cells. Lanes 1 and 3 are controls, and lane 2 is the expression product of human MIP-2γ. On the left is the molecular weight size (kDa).

Fig. 5A is a graph showing the chemotactic effect of 293 cell supernatant transfected with human MIP-2γ gene on freshly isolated neutrophils from human peripheral blood.

Fig. 5B is a graph showing the chemotaxis effect of 293 cell supernatant transfected with human MIP-2γ gene on freshly isolated T lymphocytes in human peripheral blood.

Fig. 5C is a graph showing the chemotaxis effect of supernatant of 293 cells transfected with human MIP-2γ gene on freshly isolated monocytes from human peripheral blood.

Fig. 5D is a graph showing the chemotaxis effect of supernatant of 293 cells transfected with human MIP-2γ gene on dendritic cells derived from human monocytes.

Contents of the invention

In the present invention, the terms "chemokine MIP-2γ", "MIP-2γ protein" and "MIP-2γ" are used interchangeably, all refer to human macrophage inflammatory protein 2γ, which includes chemokine The amino acid sequence of the full-length form of MIP-2γ amino acid sequence (SEQ ID NO: 2) or the mature form without signal peptide (SEQ ID NO: 3) of the human chemokine MIP-2γ. These include the chemokine MIP-2γ with or without the initial methionine.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated MIP-2γ protein or polypeptide" refers to a MIP-2γ protein polypeptide substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify MIP-2γ protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. Amino acid analysis can be performed on the purity of MIP-2γ protein polypeptide.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of human MIP-2γ protein. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the native human MIP-2γ protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the term "human MIP-2γ protein polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 2 or 3 having human MIP-2γ protein activity. The term also includes variant forms of SEQ ID NO. 2 or 3 that have the same function as human MIP-2γ protein. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human MIP-2 gamma protein.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with human MIP-2γ protein DNA under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human MIP-2γ protein polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human MIP-2γ protein polypeptides or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human MIP-2 gamma protein polypeptides. Usually, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 60 contiguous amino acids, and most preferably at least about 60 contiguous amino acids of the human MIP-2γ protein polypeptide sequence. at least about 70 contiguous amino acids.

The invention also provides analogs of human MIP-2γ protein or polypeptide. The difference between these analogues and the natural human MIP-2γ protein polypeptide may be the difference in amino acid sequence, or the difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "human MIP-2γ protein conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID No.2 or 3, there are at most 10, preferably at most 8, more preferably at most 5, Optimally, up to 3 amino acids are replaced by amino acids of similar or closely related properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid encoding a protein having SEQ ID NO: 2 or SEQ ID NO: 3 in the present invention, but differing from the sequence of the coding region shown in SEQ ID NO: 1 sequence.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present 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 which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the polypeptide it encodes. Function.

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2 or 3.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding MIP-2γ protein.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The full-length human MIP-2γ protein nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

A method of amplifying DNA/RNA using the PCR technique (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. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or MIP-2γ protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

By conventional recombinant DNA technology (Science, 1984; 224:1431), the polynucleotide sequence of the present invention can be used to express or produce recombinant MIP-2γ protein polypeptide. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding human MIP-2γ protein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

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

In the present invention, the human MIP-2γ protein polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the human MIP-2γ protein coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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 for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be 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: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells animal cells, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another way is to treat with _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed intracellularly, extracellularly or on the cell membrane or secreted extracellularly. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (MIP-2γ protein LC) and various other liquid chromatography techniques and combinations of these methods.

The recombinant human MIP-2γ protein or polypeptide has many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by low or loss of MIP-2γ protein function, and screening for antibodies, polypeptides or other ligands that promote or resist the function of MIP-2γ protein. For example, antibodies can be used to activate or inhibit the function of human MIP-2γ protein. Screening the polypeptide library with expressed recombinant human MIP-2γ protein can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of human MIP-2γ protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific to human MIP-2γ protein DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody can bind to human MIP-2γ protein gene product or fragment. Preferably, it refers to those antibodies that can bind to human MIP-2γ protein gene product or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human MIP-2γ protein, as well as those antibodies that do not affect the function of human MIP-2γ protein. The present invention also includes antibodies that bind to modified or unmodified forms of the human MIP-2 gamma protein gene product.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human MIP-2γ protein gene product, or an antigenic fragment thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human MIP-2γ protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). The antibodies of the present invention include antibodies capable of blocking the function of human MIP-2γ protein and antibodies that do not affect the function of human MIP-2γ protein. All kinds of antibodies of the present invention can be obtained by conventional immunization techniques by utilizing fragments or functional regions of human CMIP-2γ protein gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of human MIP-2γ protein gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g. E. Coli); antibodies that bind to post-translationally modified forms (such as glycosylation or Phosphorylated proteins or polypeptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells (eg, yeast or insect cells).

Antibodies against human MIP-2γ protein can be used in immunohistochemical techniques to detect human MIP-2γ protein in biopsy specimens. In addition, the monoclonal antibody combined with human MIP-2γ protein can also be labeled with radioactive isotopes, and its location and distribution can be tracked when injected into the body. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

The antibody of the present invention can be used to treat or prevent diseases related to human MIP-2γ protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human MIP-2γ protein.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, monoclonal antibodies with high affinity to human MIP-2γ protein can be covalently bonded to bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human MIP-2γ-positive cells. For the production of polyclonal antibodies, human MIP-2γ protein or polypeptide can be used to immunize animals, such as rabbits, mice, rats, etc. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Utilizing the protein of the present invention, substances interacting with the MIP-2γ protein, such as receptors, inhibitors or antagonists, can be screened out through various conventional screening methods.

When the protein of the present invention and its antibody, inhibitor, antagonist, or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The polypeptide of the present invention can be directly used in the treatment of diseases, for example, malignant tumors, neutrophil deficiency or low function, autoimmune diseases and the like. When using the MIP-2γ protein of the present invention, other therapeutic agents can also be used at the same time. The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the MIP-2γ protein polypeptide of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The human MIP-2γ protein of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using a pharmaceutical composition, a safe and effective amount of MIP-2γ protein is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, more Preferably the dosage is about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of human MIP-2γ protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by non-expression of MIP-2γ protein or expression of abnormal/inactive MIP-2γ protein. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated MIP-2γ proteins to inhibit the activity of endogenous MIP-2γ proteins. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the MIP-2γ protein gene into cells. The method for constructing a recombinant viral vector carrying the MIP-2γ protein gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human MIP-2γ protein gene can be packaged into liposomes and then transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human MIP-2 gamma protein mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The polypeptide molecule capable of binding to human MIP-2γ protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the human MIP-2γ protein molecule must be labeled.

The present invention also relates to a diagnostic test method for quantitative and localized detection of human MIP-2γ protein level. These assays are well known in the art and include FISH assays and radioimmunoassays. The human MIP-2γ protein level detected in the test can be used to explain the importance of human MIP-2γ protein in various diseases and to diagnose diseases in which MIP-2γ protein plays a role.

A method for detecting the presence of MIP-2γ protein in a sample is to use a specific antibody for MIP-2γ protein to detect, which includes: contacting the sample with an antibody specific to MIP-2γ protein; observing whether an antibody complex is formed, forming Absence of the antibody complex indicates the presence of MIP-2γ protein in the sample.

The polynucleotide of MIP-2γ protein can be used for the diagnosis and treatment of diseases related to MIP-2γ protein. In terms of diagnosis, the polynucleotide of MIP-2γ protein can be used to detect the expression of MIP-2γ protein or the abnormal expression of MIP-2γ protein in a disease state. For example, the DNA sequence of MIP-2γ protein can be used for hybridization of biopsy specimens to determine the abnormal expression of MIP-2γ protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with MIP-2γ protein-specific primers can also detect the transcripts of MIP-2γ protein.

Detection of mutations in the MIP-2γ protein gene can also be used to diagnose MIP-2γ protein-related diseases. The form of MIP-2γ protein mutation includes point mutation, translocation, deletion, recombination and any other abnormalities compared with the normal wild-type MIP-2γ protein DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The sequences of the invention are also valuable for chromosome identification. In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA of the MIP-2γ protein of the present invention, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

In one example of the present invention, an isolated polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is provided. The polynucleotides of the invention were isolated from a human dendritic cell cDNA library. Its sequence is as follows:

gagctccggg ccgccgctcc gacgggccag cgccctcccc ATGTCCCTGC TCCCACGCCG 60

CGCCCCTCCG GTCAGCATGA GGCTCCTGGC GGCCGCGCTG CTCCTGCTGC TGCTGGCGCT 120

GTACACCGCG CGTGTGGACG GGTCCAAATG CAAGTGCTCC CGGAAGGGAC CCAAGATCCG 180

CTACAGCGAC GTGAAGAAGC TGGAAATGAA GCCAAAGTAC CCGCACTGCG AGGAGAAGAT 240

GGTTATCATC ACCACCAAGA GCGTGTCCAG GTACCGAGGT CAGGAGCACT GCCTGCACCC 300

CAAGCTGCAG AGCACCAAGC GCTTCATCAA GTGGTACAAC GCCTGGAACG AGAAGCGCAG 360

GTICTACGAA GAATAGggtg aaaaacctca gaagggaaaa ctccaaacca gttgggagac 420

ttgtggcaaa ggaactttgc agattaaaaa aaaaaaaaaa aaa 463

The polynucleotide sequence it contains has a full length of 463 bases, its open reading frame is located at positions 41-376, and it encodes a human MIP-2γ protein (SEQ ID NO: 2) with a full length of 111 amino acids.

MSLLPRRAPP VSMRLLAAAL LLLLLALYTA RVDGSKCKCS RKGPKIRYSD 50

VKKLEMKPKY PHCEEKMVII TTKSVSRYRG QEHCLHPKLQ STKRFIKWYN 100

AWNEKRRFYE E 111

Wherein, amino acid 1-34 is a signal peptide. The mature MIP-2γ protein after removing the signal peptide contains 77 amino acids, and its sequence is shown in SEQ ID NO:3.

SKCKCSRKGP KIRYSDVKKL EMKPKYPHCE EKMVIITTKS VSRYRGQEHC 50

LHPKLQSTKR FIKWYNAWNE KRRFYEE 77

The present inventors inserted the cDNA of MIP-2γ containing the full-length coding region into the pcDNA3.1 eukaryotic expression vector, constructed a vector expressing MIP-2γ protein, and expressed it in transfected 293 cells, 48 hours after transfection Cell culture supernatant detection and ³⁵ S metabolism analysis were carried out after 1 hour, which indicated that the chemokine MIP-2γ gene could be expressed in eukaryotic cells, and the purified protein of MIP-2γ was obtained. Western blot analysis demonstrated the expression of MIP-2γ.

In order to investigate the biological activity of MIP-2γ protein, neutrophils, T lymphocytes, monocytes and monocyte-derived dendritic cells freshly isolated from human peripheral blood cells were subjected to chemotaxis experiments using MIP-2γ. The results showed that MIP-2γ had a chemotactic effect on neutrophils, but a weaker chemotactic effect on dendritic cells, and no chemotactic effect on neutrophils and monocytes.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions specified by the manufacturer. suggested conditions.

Example 1: Cloning of human MIP-2γ protein cDNA

Total RNA of human dendritic cells was extracted by Trizol method (Gibco Company). Then, poly(A) mRNA was isolated from total RNA. After the poly(A) mRNA was reverse-transcribed to form cDNA, the cDNA fragment was directional inserted into the multiple cloning site of the vector using the SuperScript II cloning kit (purchased from Gibco), and transformed into DH5α bacteria to form a cDNA plasmid library. The 5' termini of randomly selected clones were sequenced by the dideoxy method. Comparing the determined cDNA sequence with the existing public DNA sequence database, it was found that the DNA sequence of one cDNA clone was a new full-length cDNA. The DNA sequence contained in the new clone is bidirectionally determined by synthesizing a series of primers. Computer analysis showed that the full-length cDNA contained in the clone was a new cDNA sequence (shown in SEQ ID NO: 1) encoding a new protein (shown in SEQ ID NO: 2). The protein is named human chemokine MIP-2γ or macrophage inflammatory protein 2γ, and its coding gene is named human chemokine MIP-2γ protein gene.

The sequence SEQ ID NO: 1 is 463bp in full length, including a 40bp 5' non-coding region and an 86bp 3' non-coding region. The open reading frame is located at positions 41-376, which encodes a polypeptide of 111 amino acids (Figure 1).

According to Kyte-Doolitte hydrophobicity analysis and GCG software analysis, the N-terminal of MIP-2γ contains a signal peptide consisting of 34 amino acids, which indicates that MIP-2γ protein is a secreted protein. The mature MIP-2γ protein consists of 77 amino acids, and the theoretically calculated molecular weight of the unglycosylated mature molecule is 9.5 kD. Contains a conserved 4 Cyc domain, in which the first two Cys residues are separated by a non-conserved lysine, which indicates that MIP-2γ has typical structural characteristics of CXC chemokines.

BLAST analysis showed that MIP-2γ was different from known genes, and the protein product of this molecule was highly homologous to MIP-2α and MIP-2β, 34% identical and 38% homologous to MIP-2α; similar to MIP-2β 31% identical and 36% homologous; but no ELR domain compared with MIP-2α/2β, which revealed that MIP-2γ is a new chemokine (Fig. 1B).

Embodiment 2: Cloning the coding sequence of human MIP-2γ protein by RT-PCR method

Trizol (Gibco Company) was used to extract the total RNA of human mononuclear cell line THP-1 cells in logarithmic growth phase, and 6 mg of total cell RNA was mixed with 0.5 μg Oligo-dT12-18 for reverse transcription. The reverse transcription system is 20 μl, and 80 μl ddH20 is added to dilute after the reaction. The primers used for PCR amplification of MIP are as follows: sense primer: 5'-ggaattcgccatgtccctgctcc cacg-3' (SEQ ID NO: 4), antisense primer: 5'-gggtacctcattcttcgtagaacctg-3' (SEQ ID NO: 5). The sense primer introduces an EcoRI restriction site before the start codon ATG, and introduces a KpnI restriction site before the stop codon to improve the expression level. The PCR reaction volume was 50 μl, which contained 10 μl of reverse transcription template, 0.4 mM primers, 0.2 mM dNTP and 1U ExTaq DNA polymerase (Takara Inc.), and the amplification parameters were 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Seconds, after 25 cycles, the PCR product was initially confirmed by 2% agarose gel electrophoresis. The DNA sequence analysis results showed that the DNA sequence of the coding region of the PCR product was completely identical to the 41-376 bp coding region shown in SEQ ID NO:1.

Example 3 Northern blot analysis of MIP-2γ

Perform Northern blotting according to the following routine method: put the filter membrane to be tested in 10ml of hybridization solution preheated at 68°C, and pre-hybridize at 68°C for 30 minutes in a hybridization oven (Bellco); put the labeled cDNA probe at 95-100 Denature at ℃ for 2-5 minutes, place on ice to cool quickly, add hybridization solution (cDNA probe final concentration is 2-10ng/ml or 1-2×106cpm/ml), mix well, and hybridize at 68℃ for 2 hours. After the hybridization, the filter membrane was washed several times with 2×SSC and 0.05% SDS at room temperature, followed by shaking and washing for 30-40 minutes, during which the washing solution was changed several times. Then rinse with 0.1×SSC, 0.1% SDS at 50° C. for 20 to 40 minutes with shaking. Finally, the filter membrane was wrapped with plastic cling film, and exposed to X-ray film at -70°C for 24 to 48 hours.

The results of Northern blot hybridization showed that chemokine MIP-2γ was present in normal tissues, including kidney, small intestine, brain, placenta, and skeletal muscle. Extensive and persistent expression was found in the liver, spleen, thymus and pancreas, with highest expression in the kidney (Figure 2).

RT-PCR Analysis of Example 4 MIP-2γmRNA

Use Trizol (Gibco Company) to extract total RNA from cells in the logarithmic growth phase. The cell lines to be tested include acute lymphoblastic leukemia cell line Molt-4, skin T lymphoma cell line Hut78, acute T-cell leukemia cell line Jurkat, erythroleukemia cell line Strain K562, acute myeloid leukemia cell line HL-60, human monocyte cell line THP-1, histiocytic lymphoma cell line U937 and lung cancer cell line A549.

Take 6 mg of total cellular RNA and mix it with 0.5 μg Oligo-dT12-18 for reverse transcription. The reverse transcription system is 20 μl, and 80 μl ddH20 is added to dilute after the reaction. The primers used for PCR amplification of MIP were as above. The PCR reaction volume was 50 μl, which contained 10 μl of reverse transcription template, 0.4 mM primers, 0.2 mM dNTP and 1U ExTaq DNA polymerase (Takara Inc.), and the amplification parameters were 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Second, after 25 cycles, the PCR products were analyzed by 2% agarose gel electrophoresis, which showed that except THP-1 cell lines, other tumor cell lines had no expression of MIP-2γ (Fig. 3). This suggests that MIP-2γ may play a role in the occurrence and development of tumors, but it may also be a secondary manifestation of the regulation disorder of gene expression in tumor cells.

Example 5 Construction of cytokine MIP-2γ recombinant expression vector

The structure of the eukaryotic expression vector pcDNA3.1/neo (Invitrogen Company) used in the construction is shown in Figure 4A. It contains CMV promoter, SV40 replication origin, neomycin resistance gene, and can perform transient expression and stable expression of the target gene. Downstream of its foreign gene cloning site, it contains the myc epitope and 6 consecutive histidine (6his) coding sequences and the stop codon of the reading frame, and the two ends of the foreign gene cloning site contain T7 promoter The polyA signal of daughter and bovine growth hormone BGH can be used for direct sequencing of the main exogenous gene with T7 and BGH universal primers.

The target gene was amplified by PCR using the PCR amplification product of Example 2 as a template. Amplify MIP-2γ primers as described in Example 2. After the PCR product was purified by the column, after EcoRI and KpnI double enzyme digestion, it was directly connected into the eukaryotic expression vector pcDNA3.1/Myc-His(-) digested with the same enzyme, and its MIP-2γ gene sequence was sequenced by T7 and BGH primers confirmed.

Example 6: Eukaryotic recombinant expression of chemokine MIP-2γ

The recombinant plasmid DNA constructed in Example 5 was mixed with liposomes at a ratio of 1:5, and reacted at room temperature for 45 minutes; after the transfected cells were washed twice, mixed with DNA, and the culture supernatant was collected 48-72 hours after transfection.

For ³⁵ S metabolic labeling of recombinant expression of MIP-2γ eukaryotic cells, collect the cells in the logarithmic growth phase after transfection, carry out ³⁵ S incorporation labeling of cell metabolism, and collect the culture supernatant for SDS-PAGE electrophoresis after 4 hours of incorporation and autoradiographic analysis.

The culture supernatant of 293 cells stably expressing clones transfected with MIP-2γ or control plasmids was subjected to 15% SDS-PAGE electrophoresis after being concentrated by centricon (molecular weight cut-off 10 kD) ultrafiltration.

48 hours after transfection, the expected expression product was detected in the 293 culture supernatant by ³⁵ S labeling, in which the size of MIP-2γ protein was about 9 kDa ( FIG. 4B ). Western detection was performed on the expression product, and corresponding bands were also found ( FIG. 4C ).

Example 7: Production of anti-human MIP-2γ protein antibody

The recombinant protein MIP-2γ obtained in Example 6 was used to immunize animals to produce antibodies, the specific method is as follows. The recombinant molecules are separated by chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 [mu]g/0.2 ml emulsified protein. Fourteen days later, mice were boosted by intraperitoneal injection of the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 µg/0.2 ml. Give booster immunizations at least three times every 14 days. The specific reactivity of the obtained antiserum was assessed by its ability to precipitate the human MIP-2γ protein gene translation product in vitro. As a result, it was found that the antibody could specifically precipitate the MIP-2γ protein of the present invention.

Example 8: Chemotactic effect of human MIP-2γ protein on neutrophils, T lymphocytes, monocytes and dendritic cells

Fresh peripheral blood was centrifuged at 700×g for 30 minutes with a Histopaque 1077-1119 centrifuge (Sigma Company) to separate neutrophils and mononuclear cells. Then anti-CD14 and anti-CD3 were applied, and the MiniMACS magnetic bead separation method was used to separate monocytes and lymphocytes from mononuclear cells. After isolation, the purity of monocytes was >85%, and the purity of T lymphocytes was >90%. Chemotaxis of MIP-2γ was detected with peripheral blood neutrophils, T lymphocytes, monocytes and dendritic cells derived from monocytes.

Chemotaxis experiments were performed in a Boyden chamber (NeuroProbe, Cabin John). 200ul of different dilutions of MIP-2γ was placed in the lower part of the small chamber, and 200ul (2×106/ml) of different test cells were added to the upper part of the small chamber. The upper and lower chambers were separated by 5um (neutrophils, lymphocytes, monocytes) or 3um (dendritic cells) polycarbonate filters with a pore size of 13mm (Poretics, Livermore). The chemotaxis chamber was incubated at 37°C for 1 hour (neutrophils), 2 hours (monocytes, dendritic cells) or 4 hours (lymphocytes), and the filter membrane was removed, fixed and stained. Observed under a light microscope, those with a chemotaxis index greater than 2 indicated chemotaxis. (The chemotactic index is the ratio of the number of chemotactic cells in the experimental group to the number of chemotactic cells in the application medium).

The results are shown in Figure 5A-5D, indicating that MIP-2γ has obvious chemotactic effects on neutrophils, but not on T lymphocytes or monocytes, which is similar to the chemotactic effects of other CXC families , but MIP-2γ also has a certain chemotactic effect on dendritic cells derived from monocytes, suggesting that MIP-2γ also plays an important role in the migration and distribution of dendritic cells in vivo.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

(1) General information:

(ii) Title of invention: new human chemokine macrophage inflammatory protein, its coding sequence and use

(iii) Number of sequences: 5

(2) Information on SEQ ID NO: 1:

(i) Sequential features:

(A) Length: 463bp

(B) type: nucleic acid

(C) chain: double chain

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

gagctccggg ccgccgctcc gacgggccag cgccctcccc ATGTCCCTGC TCCCACGCCG 60

CGCCCCTCCG GTCAGCATGA GGCTCCTGGC GGCCGCGCTG CTCCTGCTGC TGCTGGCGCT 120

GTACACCGCG CGTGTGGACG GGTCCAAATG CAAGTGCTCC CGGAAGGGAC CCAAGATCCG 180

CTACAGCGAC GTGAAGAAGC TGGAAATGAA GCCAAAGTAC CCGCACTGCG AGGAGAAGAT 240

GGTTATCATC ACCACCAAGA GCGTGTCCAG GTACCGAGGT CAGGAGCACT GCCTGCACCC 300

CAAGCTGCAG AGCACCAAGC GCTTCATCAA GTGGTACAAC GCCTGGAACG AGAAGCGCAG 360

GTTCTACGAA GAATAGggtg aaaaacctca gaagggaaaa ctccaaacca gttgggagac 420

ttgtggcaaa ggaactttgc agartaaaaa aaaaaaaaaa aaa 463

(2) Information on SEQ ID NO: 2:

(i) Sequential features:

(A) Length: 111 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 2:

MSLLPRRAPP VSMRLLAAAL LLLLLALYTA RVDGSKCKCS RKGPKIRYSD 50

VKKLEMKPKY PHCEEKMVII TTKSVSRYRG QEHCLHPKLQ STKRFIKWYN 100

AWNEKRRFYE E 111

(2) Information on SEQ ID NO: 3:

(i) Sequential features:

(A) Length: 77 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 3:

SKCKCSRKGP KIRYSDVKKL EMKPKYPHCE EKMVIITTKS VSRYRGQEHC 50

LHPKLQSTKR FIKWYNAWNE KRRFYEE 77

(2) Information on SEQ ID NO: 4

(i) Sequential features

(A) Length: 27 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 4:

GGAATTCGCC ATGTCCCTGC TCCCACG 27

(2) Information on SEQ ID NO: 5

(i) Sequential features

(A) Length: 26 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 5:

GGGTACCTCA TTCTTCGTAG AACCTG 26

Claims (12)

1. isolating people MIP-2 γ protein polypeptide is characterized in that it is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 or 3 aminoacid sequences;

(b) replacement, disappearance or the interpolation through 1-10 amino-acid residue of SEQ ID NO:2 or 3 aminoacid sequences formed, and have neutrophil leucocyte chemotactic function by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is to have the polypeptide that is selected from down the group aminoacid sequence: SEQ ID NO:2 or SEQ ID NO:3.

3. a separated coding has the polynucleotide of the polypeptide of neutrophil leucocyte chemotactic function, it is characterized in that it contains a nucleotide sequence, and this nucleotide sequence is selected from:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:3.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 41-376 position among the SEQ ID NO:1;

(b) has the sequence of 143-376 position among the SEQ ID NO:1;

(c) has the sequence of 1-463 position among the SEQ ID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. preparation method with polypeptide of people MIP-2 γ protein-active is characterized in that this method contains in steps:

(a) under the proteic condition of suitable expressing human MIP-2 γ, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people MIP-2 γ protein-active.

9. energy and the described people MIP-2 of claim 1 γ protein-specific bonded antibody.

10. whether there is the proteic method of MIP-2 γ in a test sample, it is characterized in that, comprising:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MIP-2 γ albumen.

11. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people MIP-2 γ protein-active, and perhaps screening suppresses the antagonist of people MIP-2 γ protein-active or is used to the peptide finger print identification.

12. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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