WO2006063518A1 - Multifunctional polypeptides - Google Patents

Multifunctional polypeptides Download PDF

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Publication number
WO2006063518A1
WO2006063518A1 PCT/CN2005/002179 CN2005002179W WO2006063518A1 WO 2006063518 A1 WO2006063518 A1 WO 2006063518A1 CN 2005002179 W CN2005002179 W CN 2005002179W WO 2006063518 A1 WO2006063518 A1 WO 2006063518A1
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Prior art keywords
polypeptide
polynucleotide
cklf1
seq
cells
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PCT/CN2005/002179
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English (en)
French (fr)
Inventor
Ying Wang
Yingmei Zhang
Wenling Han
Dalong Ma
Yanan Liu
Dan Li
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Peking University
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Publication of WO2006063518A1 publication Critical patent/WO2006063518A1/zh
Priority to US11/810,590 priority Critical patent/US7465453B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to the field of genetic engineering, and in particular, the present invention relates to polypeptides having multiple functions and polynucleotides encoding the same, vectors or host cells containing the polynucleotides, and uses thereof. Background technique
  • Chemokines are a class of chemokines that play an important role in the process of immune regulation, inflammation, and stem cell proliferation and differentiation. At present, chemokines have become one of the hotspots of research at home and abroad. Applicants successfully cloned a new chemotactic cytokine, chemokine- l ike factor 1, CKLF1, from PHA-stimulated U937 cells using inhibitory subtractive hybridization. CKLF1 encodes 99 amino acids with broad-spectrum chemotactic activity in vivo and in vitro. Mice with CKLF1 eukaryotic expression plasmid have obvious pulmonary inflammatory lesions, which are very similar to the pathological changes in the late stage of asthma.
  • the inventors constructed a CKLF1 Drosophila expression system, which was expressed and purified to obtain a secreted CKLF1 recombinant protein.
  • This recombinant protein still has chemotactic effects on peripheral blood mononuclear cells, neutrophils, lymphocytes and U937 cells.
  • the CKLF1 protein band was obtained by SDS-PAGE separation, and N-terminal sequencing was performed to obtain two CKLF1 secreted polypeptides named C19 and C27 polypeptides (collectively referred to as C LF1 C-terminal polypeptide); Applicants also used CKLF1 recombinant protein to study their receptors. It was found that CCR4 and CCR5 are receptors for CKLF1.
  • the applicant further chemically synthesized the C19 and C27 peptides and studied the chemically synthesized C19 and C27 peptide interaction receptors. It was found that CCR4, CCR5 and CCR6 are interacting receptors of C19 and C27 peptides, showing stimulation. Or antagonistic activity. For example, in the prevention and/or treatment of HI V (human immunodeficiency virus), when a C19, C27 polypeptide binds to a receptor, it prevents the binding of HIV to the receptor or the interaction of these receptors with a known ligand. In addition, Applicants have surprisingly found that the C-terminal polypeptide of CKLF1 not only has the same chemokine-interacting receptor as CKLF1, but also exhibits antagonism.
  • HI V human immunodeficiency virus
  • the C-terminal polypeptide of CKLF1 can be chemically synthesized, which is more promising.
  • receptor antagonists of the CKLF1 C-terminal polypeptide are more desirable for the prevention and/or treatment of HIV (human immunodeficiency virus), transplant rejection, brain diseases, autoimmune diseases or treatment of allergic diseases. Accordingly, it is an object of the invention to provide a polypeptide.
  • Another object of the invention is to provide a polynucleotide.
  • Another object of the invention is to provide a vector comprising a polynucleotide of the invention.
  • Another object of the invention is to provide a host cell comprising a vector of the invention.
  • Another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide, polynucleotide, vector, or host cell of the present invention, and/or one or more pharmaceutically acceptable salts or pharmaceutically acceptable agents An acceptable carrier or excipient.
  • Another object of the present invention is to provide use of the polypeptide or polynucleotide of the present invention for the preparation of a medicament for preventing and/or treating human immunodeficiency virus infection, allergic disease, transplant rejection, brain disease or autoimmune disease.
  • Another object of the present invention is to provide a method of detecting whether a change in the expression level of a polypeptide or polynucleotide of the present invention in a sample to be tested.
  • Another object of the present invention is to provide a monoclonal or polyclonal antibody which specifically binds to a polypeptide of the present invention or a fragment thereof having an antigen.
  • the invention provides a polypeptide comprising -
  • polypeptide of the amino acid sequence of SEQ ID NO: 2 or a polypeptide of amino acids 9-27 of SEQ ID NO: 2;
  • polypeptide having at least 80% homology with (1) the function of which is the same as or similar to the function of (1).
  • the polypeptide of the amino acid sequence shown in SEQ ID NO: 2 (referred to as C27 in the present invention) is the C-terminal 27 amino acid sequence of the CKLF1 polypeptide (the nucleic acid sequence of the CKLF1 polypeptide is registered in Gen-bankTM under the registration number AF096895). See also International Application: PCT/CN00/00026, Chinese Patent Application: 99107284. 7).
  • a fragment of the sequence of SEQ ID NO: 2 of the present invention is also within the scope of the present invention, such as the sequence of amino acids 9-27 of SEQ ID NO: 2, that is, the C-terminal 19 amino acid sequence of CKLF1 polypeptide (in this In the invention, it is called C19).
  • C27 and C19 are collectively referred to as CKLF1 C-terminal polypeptide.
  • the polypeptide of the present invention further comprises an amino acid sequence as shown in SEQ ID NO: 2, or a sequence (sequence match) of more than 80%, preferably more than 85%, of the sequence of amino acids 9-27 of SEQ ID NO: 2.
  • sequences may have the same, similar or different biological functions as the sequence of SEQ ID NO: 2 of the present invention or the sequence of amino acids 9-27 of SEQ ID NO: 2, preferably having the same or similar functions. .
  • the CKLF1 C-terminal polypeptide of the present invention or a fragment thereof may be naturally, synthetically, semi-synthetically, or recombinantly produced.
  • the CKLF1 C-terminal polypeptide of the present invention can be used according to Steward and Young (Steward, JM and Young, JD, Sol id Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, 111. The method described in (1984)) was synthesized by solid phase chemistry using an Applied Biosystem synthesizer or a PioneerTM peptide synthesizer. Generally, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to the growing peptide chain.
  • the amino or carboxyl group of the first amino acid is protected with a suitable protecting group, and the protected amino acid is then attached to an inert solid support, followed by the addition of the corresponding amino or carboxyl group to the appropriately protected sequence under conditions suitable for the formation of an amide bond.
  • the next ⁇ amino acid is then removed from the newly added amino acid residue, followed by the next amino acid suitably protected if necessary, and the procedure is repeated.
  • any remaining protecting groups and solid support are removed sequentially or simultaneously to yield the final polypeptide.
  • the polypeptide of the invention is prepared using an automated synthesizer.
  • An amino acid in which an alpha amino group is protected by an acid or a base sensitive group is used.
  • Such a protecting group should be stable under peptide bond formation conditions, and can be easily removed without destroying the growing peptide chain, and does not cause any chiral center racemization therein.
  • Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 2-cyano-tert-butoxycarbonyl and the like.
  • polypeptides of the invention can also be produced by recombinant DNA sequences in host cells according to conventional bioengineering methods (see, in particular, Examples 1-4).
  • the applicant inserts the CKLF1 coding sequence into the expression system, and expresses and purifies the secreted CKLF1 recombinant protein, and further obtains the CKLF1 protein band by SDS-PAGE, and performs N-terminal sequencing to obtain the present invention.
  • the CKLF1 C-terminal polypeptide of the invention can be produced directly using a polynucleotide sequence encoding a polypeptide of the invention, for example, a polynucleotide sequence encoding a polypeptide of the invention (as set forth in SEQ ID NO: 1
  • the sequence or fragment thereof is directly inserted into an expression system, and the polypeptide of the present invention is obtained by expression and purification.
  • the desired polypeptide can be produced in a cell-free translation system using mRNA derived from the DNA construct of the invention.
  • polypeptides or fragments thereof of the present invention can form fusion polypeptides with other polypeptides or fragments thereof.
  • Other polypeptides or fragments thereof are generally known, some may be commercially available as a carrier, or may be synthesized by conventional methods or cloned from known organisms.
  • Preferred polypeptides of the invention are:
  • polypeptide of the amino acid sequence of SEQ ID NO: 2 or a polypeptide of amino acid 9 to 27 of SEQ ID NO: 2;
  • the invention also provides a polynucleotide comprising:
  • polynucleotide encoding an amino acid sequence as shown in SEQ ID NO. 2, or a polynucleotide encoding amino acid sequence 9-27 of SEQ ID NO: 2;
  • a polynucleotide having at least 80% homology with (1), the polypeptide encoded by the polynucleotide has the same function as (1) the encoded polypeptide.
  • the amino acid sequence shown as SEQ ID NO: 2 is the C-terminal 27 amino acid sequence of the CKLF1 polypeptide (C27).
  • the sequence shown by the chloro acid at position 9-27 of SEQ ID NO: 2 is the C-terminal 19 amino acid sequence of the CKLF1 polypeptide (C19).
  • the polynucleotide sequence of the present invention may encode only the CKLF1 C-terminal polypeptide, or may add a non-coding sequence based on the coding sequence of the above polypeptide, such as an intron, a non-coding sequence at the 5' or 3' end of the coding sequence, and the like.
  • the polynucleotide sequences of the invention are preferably provided in isolated form.
  • the polynucleotide of the present invention is in the form of "isolation" which has not only been separated from the protein accompanying it in the cell, but has also been isolated from the sequence located on both sides thereof in the natural state.
  • the present invention also encompasses at least 70%, 80% of the polynucleotide encoding the CKLF1 C-terminal polypeptide or a fragment thereof,
  • a polynucleotide sequence of 85%, preferably 90%, 95%, preferably 98% homologous Particularly, it relates to a polynucleotide which hybridizes under stringent conditions to a polynucleotide of a CKLF1 C-terminal polypeptide, and the term "stringent conditions" means that hybridization is carried out under the premise that at least 95% of the homology between the sequences is present.
  • sequences may be naturally occurring or artificially produced, may include allelic variants of the polynucleotide sequence of the CKLF1 C-terminal polypeptide, and may also include deletions, insertions and substitutions of bases in the CKLP1 C-terminal polypeptide polynucleotide sequence.
  • Such a sequence-encoded polypeptide may be functionally identical, similar, or different from the CKLF1 C-terminal polypeptide of the present invention, but preferably encodes a polypeptide having substantially the same biological activity as the CKLF1 C-terminal polypeptide. Therefore, a polynucleotide having at least 80% homology with a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, which polypeptide is encoded by SEQ ID NO: 2, is preferred. Polypeptides have the same function.
  • the polynucleotide sequence of the present invention may be DNA or RNA, wherein the DNA includes cDNA, genomic DNA and synthetic DNA, the DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding strand or a non-coding strand (antisense chain).
  • the antisense strand of the present invention may be the complement of the sequence as shown in SEQ ID NO: 1. It is known to those of ordinary skill in the art that the antisense strand or a portion thereof (antisense oligonucleotide) can be used to inhibit expression of a CKLF1 C-terminal polypeptide of the present invention in a cell.
  • the nucleotide sequence of the CKLF1 C-terminal polypeptide of the present invention may be derived from any species, particularly mammals, including cattle, sheep, pigs, mice, horses, and preferably humans.
  • the polynucleotide sequence shown as SEQ ID NO: 1 is a polynucleotide encoding a C27 polypeptide of the present invention, which is derived from a human.
  • the polynucleotide sequence shown at positions 25-81 of SEQ ID NO: 1 is a polynucleotide encoding a C19 polypeptide of the present invention, which is derived from a human. Therefore, it is preferred that the polynucleotide of the present invention comprises the polynucleotide shown in SEQ ID NO: 1 or a complement thereof, or the polynucleotide as shown at positions 25-81 of SEQ ID NO: 1.
  • the invention also relates to a genetically engineered vector comprising a C-terminal polypeptide encoding the invention Polynucleotide.
  • the genetic engineering vector may be a general vector, an expression vector or the like.
  • the common vector is mainly used for the establishment of various genomic libraries and cDNA libraries. They usually contain two or more marker genes, one of which is used to select transformants and the other is used for examination. Whether there is foreign DNA insertion in the vector.
  • Expression vectors are mainly used to study gene expression or to mass produce some useful transcription products or proteins, and some can also be used for the establishment of cDNA libraries. Such vectors, in addition to the characteristics of a conventional vector, should also contain appropriate promoters, ribosome binding sites, terminators, and the like.
  • an appropriate leader sequence can be added upstream of the polypeptide coding sequence.
  • Selection of suitable vectors and promoters is well known to those of ordinary skill in the art.
  • Methods for constructing vectors comprising the polynucleotides of the invention and suitable transcriptional and translational regulatory elements are well known to those of ordinary skill in the art.
  • commercially available expression vectors suitable for use in prokaryotic cells generally carry a selectable marker and a cell origin of replication, with bacterial promoters such as lacI, T7, APL and trp, and the known cloning vector pBR322 (ATCC 37017). Other genetic components.
  • Such commercially available vectors include pGEM (Promega) and P K223-3 (Pharmacia).
  • the appropriate vector derived from pBR322 can be selected based on the appropriate promoter selected and the structural gene sequence to be expressed.
  • a GST prokaryotic expression system can also be used in the present invention.
  • Vectors suitable for eukaryotic cells carry eukaryotic promoters such as CMV, SV40, etc.
  • Such vectors include pMT-hIL-3 (Ma Dalong, Di Chunhui, Pang Jian et al.
  • the PCR product was digested with EcoR I + Xho I and ligated with pMT/V5-His A vector (Invitrogen) digested with EcoR I + Xho I to obtain pMT/V5-HisA- CKLF1- myc-hi6 Plasmid.
  • the invention also relates to a host cell comprising a polynucleotide encoding a CKLF1 C-terminal polypeptide of the invention.
  • Hosts include, but are not limited to, prokaryotic hosts, such as Escherichia coli, Bacillus, Streptomyces, etc.; eukaryotic hosts, such as: Saccharomyces, Aspergillus, insect cells, Drosophila S2 and Spodoptera frugiperda Sf9 ; animal cells, For example, CH0, COS (monkey kidney fibroblast cell line, Gluzman (Cell 23: 175, 1981)) and other cell lines capable of expressing a compatible vector.
  • prokaryotic hosts such as Escherichia coli, Bacillus, Streptomyces, etc.
  • eukaryotic hosts such as: Saccharomyces, Aspergillus, insect cells, Drosophila S2 and Spodoptera frugiperda Sf9
  • Methods for introducing a construct comprising a polynucleotide of the present invention into the above host cell are well known to those skilled in the art including, but not limited to, calcium chloride mediated transformation, calcium phosphate transfection, DEAE-dextran mediated transduction Dyeing, electroporation, microinjection, particle bombardment or gene gun methods (Sambrook, J. (1989), Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press; - Plainview, NY; Ausubel, FM (1989) Current Protocols In Molecular Biology, John Wiley & Sons, NY; Hobbs, S.
  • the host cell used in Example 1 of the present invention is XU-Blue Escherichia coli
  • the host cell used in Example 2 is Drosophila S2 cells
  • the transfection method is a calcium chloride transfection method
  • the host cell used was HEK293 cells (ATCC CRL-1573) and the transfection method was electroporation.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a CKLF1 C-terminal polypeptide, a polynucleotide encoding the polypeptide of the present invention, a vector comprising the polynucleotide, a host cell, and/or one or a
  • pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients refers to a salt which is suitable for contact with the tissues of a human or animal without excessive toxicity, irritation, allergies and the like.
  • the pharmaceutically acceptable salts of the present invention are conventional components of pharmaceutics in the art.
  • Such salts may be prepared during the final isolation and purification of the polypeptides of the invention, or may be prepared separately by reacting the polypeptide with a suitable organic or inorganic acid or base.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or other formulation excipient.
  • the present invention also provides the use of the CKLF1 C-terminal polypeptide of the present invention for the preparation of a medicament for preventing and/or treating human immunodeficiency virus infection, allergic disease, transplant rejection, brain disease or autoimmune disease.
  • co-receptors that mediate HIV entry into cells include: CCR3, CCR5, CXCR4, CX3CR1, and currently developed inhibitors of human immunodeficiency virus (HIV) infection, including Kaposi's sarcoma-associated herpesvirus from viral proteins.
  • HIV human immunodeficiency virus
  • the encoded chemokine and HHV-8 vMIP bind to a variety of chemokine receptors and inhibit HIV-assisted receptor binding to infected cells; and modified human chemokines and small molecule compounds specifically bind to CCR5 , CXCR4 inhibits HIV-infected cells.
  • the CKLF1 C-terminal polypeptide of the present invention binds to various chemokine receptors and is compatible with HIV-binding helper receptors (see Examples 7-8 for details), and is a polypeptide derived from human body, which is beneficial for long-term and effective use. . Therefore, the CKLF1 C-terminal polypeptide has great advantages in preventing infection and spread of HIV.
  • Atopic dermatitis, asthma, and allergic rhinitis are common allergic diseases associated with the migration of Th2 cells, mast cells, and eosinophils.
  • One therapeutic strategy is to use chemokine receptor antagonists to prevent these effects.
  • CCR3, CCR4, and CCR8 are commonly expressed in Th2 cells, mast cells, and eosinophils.
  • CCR5 is also associated with asthma.
  • the present invention demonstrates by exact experiments that the CKLF1 C-terminal polypeptide is a functional ligand for CCR4 and CCR5. Therefore, the CKLF1 C-terminal polypeptide can also play a role in inhibiting allergic diseases. Studies have shown that CCR5 is associated with brain disease.
  • CCR5 can be detected in activated microglia, infiltrating T cells, and microglia at the brain injury site of Alzheimer's disease (Alzheimer's disease) in multiple sclerosis brain injury sites. Expression, it was demonstrated in Examples 7-8 of the present invention that the CKLF1 C-terminal polypeptide is a ligand for CCR5, and therefore, the CKLF1 C-terminal polypeptide can be used to inhibit damage caused by chronic inflammation in these diseases.
  • CCR5 and its ligands are also involved in transplant rejection, rheumatoid arthritis, and CCR5 is also associated with the development of hepatitis C.
  • CCR6 is associated with psoriasis. It is demonstrated in Examples 7-9 of the present invention that the CKLF1 C-terminal polypeptide has a binding effect with various receptors as described above, suggesting that the CKLF1 C-terminal polypeptide can be used to prevent transplant rejection, rheumatoid arthritis, and hepatitis C. Occurrence and/or prevention of psoriasis.
  • CKLF C-terminal polypeptide of the present invention has the property of binding to a chemokine factor receptor, and it is derived from human itself, so the CKLF1 C-terminal polypeptide has broad application prospects in the treatment of various diseases. .
  • the present invention also provides a method for detecting the expression level of the polypeptide or polynucleotide of the present invention in a sample from a test subject in vitro, which is a reverse transcription-polymerase chain reaction or a Western blot detection method.
  • the level of expression of a polypeptide or polynucleotide of the invention can be detected by any method known in the art.
  • the expression level of the polynucleotide at the nucleic acid level is detected by reverse transcription-polymerase chain reaction (RT-PCR); or the expression of the polynucleotide at the protein level is detected using a specific monoclonal or polyclonal antibody.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the test sample can be obtained from cells from a subject, such as cells from blood, urine, saliva, gastric juice, hair, biopsy, and autopsy material.
  • PT-PCR is mainly divided into the following steps: extracting total RNA, adding a primer complementary to the 3' end of the mRNA, and synthesizing cDNA under the action of reverse transcriptase; the second is to use cDNA as a template, and then add another complementary to cDNA.
  • a primer two primers located on different exons to avoid genomic DNA contamination was subjected to PCR amplification.
  • the Western blot is mainly divided into three stages: the first stage is SDS-polyacrylamide gel electrophoresis for protein samples such as antigen; the second stage is electrotransfer: the separated bands in the gel are transferred to the nitrocellulose membrane; The third stage is color detection: a nitrocellulose membrane (equivalent to a solid phase carrier coated with an antigen), followed by a specific antibody and an enzyme-labeled secondary antibody, and an enzyme reaction substrate capable of forming a chromogenic substance is added. To dye the strip. Enzymes that label secondary antibodies include horseradish peroxidase (HRP) and alkaline phosphate (AP).
  • HRP horseradish peroxidase
  • AP alkaline phosphate
  • Glucose oxidase, ⁇ -D-galactosidase, urease and the like can also be used.
  • horseradish peroxidase is employed as the enzyme to label the secondary antibody.
  • substrates for HRP action include, but are not limited to, phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS.
  • OPD phenylenediamine
  • TMB tetramethylbenzidine
  • ABTS ABTS.
  • the substrate of alkaline phosphatase is generally With p-nitrophenyl phosphate (P-NPP), AP also has a fluorescent substrate (4-methylumbelliferone phosphate).
  • P-NPP p-nitrophenyl phosphate
  • AP also has a fluorescent substrate (4-methylumbelliferone phosphate).
  • Commonly used enzyme-labeled secondary antibodies are commercially available.
  • the invention also provides polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the CKLF1 C-terminal polypeptide of the invention or antigenic fragments thereof.
  • specificity refers to the ability of an antibody to bind to a CKLF1 C-terminal polypeptide gene product or fragment of the invention. Those antibodies which bind to the gene product of the CKLF1 C-terminal polypeptide of the present invention but which do not recognize and bind to other unrelated antigen molecules are preferred.
  • the antibodies of the present invention include those which bind to and inhibit the gene product of the CKLF1 C-terminal polypeptide of the present invention, and also those which do not affect the function of the C-terminal polypeptide of the CKLF1 of the present invention.
  • the present invention also encompasses those antibodies which specifically bind to a protein or active fragment having at least 80% homology to the CKLF1 C-terminal polypeptide of the present invention.
  • the above antibodies include not only intact 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., U.S. Patent No. 4, 946, 778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain antibody portions from humans.
  • the antibodies of the invention can be prepared by a variety of methods known to those of ordinary skill in the art. For example, a purified CKLF1 C-terminal polypeptide gene product of the present invention or an antigenic fragment thereof can be administered to an animal to induce production of a polyclonal antibody. Similarly, cells expressing the CKLF1 C-terminal polypeptide of the present invention or a fragment having antigenicity thereof can be used to immunize an animal to produce an antibody.
  • the antibodies of the invention are preferably monoclonal antibodies which can be prepared using hybridoma technology (see Kohler et al, Nature 256: 495; Kohler et al, Eur. J. Immunol.
  • the antibodies of the present invention can be obtained by conventional immunological techniques using the CKLP1 C-terminal polypeptide gene product of the present invention or a fragment or a functional region thereof, which can be produced by recombinant methods or synthesized using a protein synthesizer.
  • An antibody that binds to an unmodified form of the gene product of the CKLF1 C-terminal polypeptide of the present invention can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (for example, E.
  • a protein or protein that is phosphorylated or phosphorylated can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell, such as a yeast or a mammalian cell, such as a rabbit.
  • Figure 1 shows the results of electrophoresis of PCR to identify chromosomal DNA (including the sequence of hCKLF1-myc coding region) of stably transfected S2 cell line, wherein lane 1 is DL2000 DNA as molecular weight standard and lane 2 is transduced by pMT/V5-HisA.
  • the S2 cell chromosome was used as a template, and the third lane was the S2 cell chromosome transfected with pMT/V5HisAhCKLFl-myc as a template, and the same hCKLF1 (pl, p2) was used as a primer for amplification.
  • Figure 2 shows the transcription of hCKLF1-myc mRNA in stably transfected S2 cell line by RT-PCR.
  • the first lane is molecular weight standard
  • the second, third and fourth lanes are pMT/V5HisAhCKLFl-myc, pMT/V5HisA, S2 empty cells are used as templates
  • CKLF1 (PI, P2) is used as the primer
  • 5, 6, and 7 are the same as the above template
  • RT-PCR is performed with G3PDH ( P 1, P2) as the primer, 5, 6, 7 respectively as an internal standard.
  • Fig. 3A and Fig. 3B show the results of examination of the expression of the protein of interest by Western Blotting.
  • the rabbit anti-hCKLF1 polypeptide antibody was added, and the anti-rabbit IgG HRP was used as the secondary antibody reaction. After X-ray development, there were two clear bands at 8-lOkd (shown by arrows in the figure); It can be seen in 3B that after the anti-c-myc antibody is added, the anti-mouse IgG HRP secondary antibody reaction is added, and the X-ray film is developed to have two clear bands at 8-10 kd (arrows in the figure). .
  • Figure 4 shows that the purified protein sample was directly stained with Coomassie brilliant blue by 15% SDS-PAGE electrophoresis.
  • the arrow refers to the CKLF1 myc protein.
  • FIG. 5 shows the results of Western Bloting after the purified protein sample was transferred to the PVDF membrane.
  • Band 1 is the molecular weight standard
  • strip 2 is the S2/pMT supernatant
  • strip 3 is the S2/CKLF1 cell lysate
  • strip 4 is the S2/CKLF1 supernatant.
  • Figures 6A and 6B show the N-terminal sequencing results after the purified protein sample was transferred to the PVDF membrane.
  • Fig. 6A shows a C19 peptide
  • 6B represents a C27 peptide.
  • Figure 7 shows the results of an analysis of calcium flow analysis in which the corresponding ligand for CCR4 is TARC and the corresponding ligand for CCR5 is RANTES.
  • AD is a CCR4 transfected cell;
  • E-H is a CCR5 transfected cell.
  • a-b is ⁇ TARC after stimulation, c-d is 167nM C27; B is a-b is 167nM C27, c-d is lOOnM TARC; C is a-b is lOOnM TARC, cd is 167nM C19 ; A-b is 167n C19 and cd is lOOnM TARC; E is a-b is lOOnM RANTES, cd is 167nM C27; F is a-b is 167nM C27, cd is ⁇ RANTES; G is a-b is ⁇ RANTES, cd 167nM C19; H is 167nM C19, cd is ⁇ RANTES 0
  • Figures 8A and 8B show the relative fluorescence of the calcium flow change image analyzed by Leica confocal software. Among them, Fig. 8A shows CCR4 transfected cells; Fig. 8B shows CCR5 transfected cells.
  • Figure 9A and Figure 9B show the chemotaxis of CKLF1 C-terminal polypeptide to CCR4 or CCR5 transfected cells, wherein, Figure 9A: Transfection of CCR4 untreated HEK293 cells and pretreatment with CKLF1 C-terminal polypeptide, TARC or PTX for 30 min HEK293/CCR4 cells; Figure 9B: HEK293 cells transfected with CCR5 untreated and HEK293/CCR5 cells pretreated with CKLF1 C-terminal polypeptide, RANTES or PTX for 30 min.
  • ⁇ stands for RPMI_1640
  • T stands for TARC
  • 27 stands for C27
  • 19 stands for C19
  • P stands for PTX
  • R stands for RANTES.
  • Figure 10 shows the chemotactic activity of purified pMT/V5HisA-CKLF1-myc protein on U937 cells.
  • Figure 11 shows the chemotactic activity of purified pMT/V5HisA-CKLF1-myc protein on CCR4-transfected HEK293 cells. Sexual and desensitizing effects.
  • Figure 12 shows the effect of CKLF1 C-terminal polypeptide on the internalization of the chemokine receptor CCR6.
  • 1 is a control
  • 2 is BSA
  • 3 is MIP3- ⁇
  • 4 is a C27 polypeptide
  • 5 is a C19 polypeptide.
  • the pMT/V5-His-CKLF1-myc-his6 plasmid was constructed for expression of the CKLF1-myc-h6 fusion protein.
  • the CKLF1 coding sequence (SEQ ID NO: 3) was inserted into the pCDNA3.1-myc-his6 (Invitrogen) expression vector to construct pCDNA3.
  • l-CKLFl-myc-his6 expression plasmid Using the correctly sequenced pCDNA3.1-CKLF1-myc-his6 as a template, T7 was used as the upstream primer (5'-TGTAA TACGA CTCAC TATAG-3, (SEQ ID NO: 4)) and the Xho I cleavage site was used.
  • the downstream primer (5'-CAT TGA GTT TAA ACG GTC TCG AGC GG-3' (SEQ ID NO: 5)) was subjected to PCR to obtain a DNA fragment encoding the CKLF1-myc-his6 fusion protein, and the PCR product was recovered by electrophoresis using EcoR I. After cleavage with Xho I, the pMT/V5-His6 vector (Invitrogen) ligated with EcoR I + Xho I was ligated to obtain the pMT/V5-His-CKLF1-myc-his6 plasmid. The ligation product was transformed into XL1-Blue E. coli, and the positively inserted clone was selected. After sequencing to verify the correctness of the plasmid, plasmid amplification was carried out, and the plasmid was extracted with Qiagen 100 for cell transfection.
  • the logarithmic growth of Drosophila Schneider 2 cells was adjusted to 3 X 10 6 cells/3 ml.
  • the cells were transfected in a 35 cm dish at 28 ° C for 16 hours.
  • 2M CaCl 2 36 1 , p T / V5-HisA hCKLFl - myc DNA 1 g/ 1 19 1 , PCoHyGrol g/ 1 1 1 make up 300 1 volume with water, slowly add to an equal volume of 2XHBS buffer, let stand for 30 minutes at room temperature, and slowly mix the mixture into the prepared cells.
  • pMT/V5-HisA empty vector transfected cells and untransfected S 2 cell control were simultaneously cultured, and cultured at 28'C for 16 hours.
  • the transfected cells were centrifuged, washed twice with PBS, and replaced with 10% FBS.
  • the culture medium was cultured, and after 72 hours, the cells were again centrifuged, and after replacing with fresh medium containing 10% FBS, hygromycin B was added to a final concentration of 300 g/ml to stabilize the cell strain.
  • the normal cultured log phase S2 cells were plated as feeder cells in a 96-well cell culture plate, l. lxl0 6 /ml,
  • the selected and passaged cells were separately aspirated, added to the cell lysate, and the protein chromosomal DNA was extracted after incubation with proteinase K and RNaseA.
  • the chromosomal DNA was used as a template, and CKLF1 upstream 5 ' ATG GAT AAC GTG CAG CCG AAA AT 3 ' (SEQ ID NO: 6), downstream 5' CAA AAC TTC TTT TTT TTC ATG CAC A 3' (SEQ ID NO: 7) is a primer, amplified for 30 rounds, and the target gene integration is selected based on the PCR result and is high copy positive.
  • the cloned cells were further identified by RT-PCR.
  • the protein was transferred to the PVDF membrane by lXCaps electroporation, and anti-c-myc antibody (Sigma) was added, followed by anti-mouse IgG HRP secondary antibody reaction (Promega), using ECL.
  • Western Blotting detection system developed by X-ray film, has two clear bands at 8-10kd. After the PVDF membrane was peeled off, rabbit anti-hCKLF1 polypeptide antibody was added, and anti-rabbit IgG HRP was used as a secondary antibody reaction, and the system was developed by X-ray using an ECL Western Blotting detection system.
  • PCR identification of stable transfection S2 cell line chromosome DNA contains hCKLFl-myc coding region sequence:
  • CKLF1 pi 5'-GCA AGA AGC GGG AAG CCG A-3' (SEQ ID NO: 8)
  • ⁇ 2 5' - CAT TGA GTT TAA ACG GTC TCG AGC GG -3' (SEQ ID NO: 5) was amplified by primers, and 1% agarose was prepared for electrophoresis. See Figure 1 for results.
  • the first lane is based on DL2000 DNA and the second lane is The chromosome of S2 cells was transfected with pMT/V5_HisA as a template, and the third lane was the chromosome of S2 cells transfected with pMT/V5HisA hCKLFl-myc.
  • the same hCKLF1 (pl, p2) was used as a primer to amplify the third lane. There is a specific zone at the 300 bp position.
  • Lane 1 is the molecular weight standard
  • Lanes 2, 3, and 4 are pMT/V5HisA- hCKLFl-myc, pMT/V5HisA, S2 empty cells as templates, and CKLF1 (Pl, P2) Primers
  • 5, 6, and 7 are the same as the above template
  • PG 5' ACCACAGTCCATGCCATCAC 3' (SEQ ID NO: 9)) of G3PDH
  • P2 (5, TCCACCACCCTGTTGCTGTA 3' (SEQ ID NO: 10)
  • Primers were subjected to RT-PCR. RT-PCR results showed that a clear band was visible at 300 bp in the second lane.
  • the positive cells were expanded and transferred to Drosophila-SFM (Invitrogen) to remove hygromycin B to an appropriate concentration, and CuS0 4 was added to induce expression of the target protein.
  • Cells were harvested 30 hours S 2 expression supernatant, 2000rpm / min, 10 minutes, 8000rpm / min, 20 minutes, 0. 4 m membrane filter.
  • 3 ⁇ 4 Chelating Sepharose Fast Flow column 20 volumes of water after equilibration, loading in PBS buffer, 0.5 M NaCl, followed by 10 mM, 50 mM, 500 mM imidazole / 50 mM Tris-HCl 1 pH 8. 0, 20 mM After the EDTA washes the column, the column can be equilibrated with water, and the first pass through the column is again loaded, and the above process is repeated.
  • Figure 5 shows the results of Western Bloti ng identification after the purified protein sample was transferred to the PVDF membrane, wherein the band 1 is the molecular weight standard, the band 2 is the S2/pMT supernatant; the band 3 is the S2/CKLF1 cell lysate; Strip 4 is the S2/CKLF1 supernatant.
  • CKLF1 secretes expression.
  • the results showed that the two bands with larger molecular weights detected two main band types, A-L- 1-Y-R-K-LL and F-N-P. - S-G-P-YQ, the smaller molecular weight bands also have two main band types, and are identical to the N-terminal sequence of the first two bands.
  • Example 5 CKLF1 C-terminal polypeptide synthesis
  • CKLF1 has at least two mature structures; their sequences are ALIYRKL LFNPSGPYQKKPVHE KEVL (C27) (SEQ ID NO: 2) and FNPSGPYQKKPVHEKKEVL (C19) (positions 9 to 27 of SEQ ID NO: 2) .
  • SEQ ID NO: 2 amino acid sequence analysis revealed that CKLF1 has at least two mature structures; their sequences are ALIYRKL LFNPSGPYQKKPVHE KEVL (C27) (SEQ ID NO: 2) and FNPSGPYQKKPVHEKKEVL (C19) (positions 9 to 27 of SEQ ID NO: 2) .
  • the two peptides were chemically synthesized according to standard methods.
  • the lyophilized polypeptide was obtained, dissolved in phosphate buffer, and stored at a concentration of 1 mg/ml at -80 °C.
  • Example 6 Construction of Receptor Expression Plasmid and Cellular Expression
  • CCR4 CCR4 (N1005508. 2), CCR5 (NM_000579), CCR6 (XM-004279).
  • pcDI this modified vector: the Bgl ll - Kpnl fragment of pcDNA3 (Invi trogen) plasmid was replaced with the Bgl ll - Kpnl fragment of pCI (Promega) plasmid.
  • the eukaryotic expression vector) and the pEGFP (CL0NTECH) expression vector were efficiently expressed in ⁇ 293 cells.
  • the sequence of the DNA sequencing coding region was correct and matched the sequence of the Gen-BankTM login. 2.
  • HEK293 cells were cultured with RPMI 1640 (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 g/nil streptomycin.
  • RPMI 1640 Life Technologies, Inc.
  • fetal bovine serum 100 U/ml
  • penicillin 100 g/nil streptomycin.
  • Each 4 ⁇ 10 6 ⁇ 293 fine 3 ⁇ 4/400 ⁇ 1 was transfected with 15 chemokine receptor expression plasmids by transient electroporation, provided that the instrument used was 120 V, 20 ms was an electrical pulse generator (Electro Square porator ECM 830, BTX, San Diego, CA), calcium flow analysis and chemotaxis experiments were performed after 48 h.
  • TARC transfected receptor
  • RANTES all purchased from peprotech
  • A-D is a CCR4 transfected cell
  • E-H is a CCR5 transfected cell.
  • c-d is 167nM C27; B is a-b is 167nM C27, c-d is ⁇ TARC; C is a-b is ⁇ TARC, c-d is 167nM C19; In D, a-b is 167nM C19, c-d is ⁇ TARC; E is a-b is ⁇ RANTES, c-d is 167nM C27; F is a-b is 167nM C27, cd is ⁇ RANTES; G is a- b is ⁇ RANTES, c-d is 167 nM C19; H is a-b of 167 nM C19, and cd is ⁇ RANTES.
  • Example 7 Effect of CKLF1 C-terminal polypeptide (C19 and C27) on intracellular calcium flux
  • HEK293 cells were cultured with RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin. Each 4x10 e HEK293 cells/400 ⁇ 1 was transfected with 15 expression plasmids by transient electroporation, with a condition of 120 V, 20 ms.
  • the instrument used was an electrical pulse generator (Electro Square porator ECM 830, BTX, San Diego, CA), 48 h Calc flow analysis and chemotaxis experiments were performed.
  • HEK293 cells transfected with pcDI-CCR4 and CCR5 were cultured in glass-bottomed microplates (MatTek corporation, USA), loaded with 10 ⁇ fluo-3/AM (HEPES buffered saline solution), 37 'C 1 h, avoiding Light.
  • the cells were washed with HEPES buffered saline and then stimulated with 167 n C-terminal polypeptide, 100 nM TARC, 100 nM RANTES, respectively.
  • the above operation was carried out at room temperature, randomly selected cells of each field of view . Images were collected every 5 seconds for a total of 240 seconds. Images were analyzed for relative fluorescence intensity using Leica confocal software and the data was processed using Microsoft Excel 2000.
  • C27 can cause CCR4 or CCR5 transfected cells to produce low-intensity signals
  • C19 can cause CCR4-transfected cells to produce low-intensity signals.
  • Pretreatment of CCR4 and CCR5 transfected cells with 100 nM TARC or RANTES also reduced the sensitivity of the transfected cells to the next CKLF1 C-terminal polypeptide.
  • Chemotaxis function assay of CKLF1 C-terminal polypeptide Chemotaxis experiments were performed in 48-well chemotaxis chambers (Neutroprobe; Cabin John, MD, USA). All factors used for chemotaxis detection were diluted with Hepes RPMI 1640 containing 0.1% BSA and added to the lower well of the chamber (28 ⁇ /well).
  • HEK293 cells were transfected with pcDI-CCR4, pcDI-CCR5, pcDI-CCR6 or pcDI, then resuspended in the same medium to 2x10 6 cells/ml, added to the upper well of the chamber (50 ⁇ /well), up and down
  • the pores were separated by a polycarbonate filter without polyvinylpyrrolidone, and the membrane pore size was 8 ⁇ m.
  • the chamber was placed in an incubator 37 'C, 5% C0 2 and incubated for 2 h.
  • the filter was removed from the chamber, washed with a 3-step staining kit, fixed, and stained. Five high-power field counts were randomly selected from the cells migrating per well.
  • HEK293 cells transfected with pcDI empty vector served as controls. All samples were tested twice.
  • the chemotaxis index CI is the number of migrated cells divided by the number of control cells. CI>2 is significant.
  • Some experimental cells were pretreated with 100 ng/ml PTX (purchased from ALEXIS Biochemicals Corporation) for 6 h prior to stimulation with CKLF1 C-terminal polypeptide, TARC, RANTES or ⁇ 3 ⁇ .
  • the present invention uses the CKLF1 C-terminal polypeptide to induce migration of CCR4 or CCR5 transfected cells to detect its activity.
  • CKLF1 C-terminal polypeptide can induce migration of CCR4 and CCR5 transfected cells, and HEK293 cells transfected with pcDI-CCR4 or CCR5 without any stimulation were used as controls.
  • Pretreatment with 0.8 g/ml TARC or RANTES, 37 °C, 30 min can make the receptor sensitive to stimulation of the next CKLF1 C-terminal polypeptide.
  • CKLF1 C-terminal polypeptide may also render the receptor sensitive to the following TARC or RANTES stimulation.
  • the effect of CKLF1 was evaluated by the PTX-sensitive Gi/Go family G protein, and CCR4 and CCR5-transfected HEK293 cells were pretreated with PTX 100 ng/ml for 6 h, and then stimulated with CKLF1 C-terminal polypeptide, TARC or RANTES.
  • Figure 9A and Figure 9B show chemotaxis induced by CKLF1 C-terminal polypeptide, with TARC or RANTES completely blocked by PTX, suggesting a Gi pathway.
  • CKLF1 C-terminal polypeptide is a ligand for CCR4 and CCR5.
  • Chemotaxis activity of CKLF1-myc protein was determined using HEK293 cells transfected with passage cell line U937 cells and CCR4 (results shown in Figures 10 and 11 respectively).
  • 50 imidazole-eluted CKLF1-myc protein 1 125-fold dilution against U937 cells
  • the chemotaxis index was 16.
  • the chemotactic results of CCR4-transfected 293 cells are shown in Figure 11.
  • the sequence of the CCR6 coding region was inserted into the pEGFPN1 vector (CLONTECH) to construct a pEGFPN1-CCR6-EGFP fusion expression plasmid.
  • the pEGFPN1-CCR6-EGFP receptor fusion expression plasmid was transfected into HEK293 cells, and serum was starved for 16-24 hours after 24 hours of culture.
  • CKLF1 C-terminal polypeptide or ⁇ 3 ⁇ was added to the culture supernatant, and the cells were washed with cold PBS for 2 hours at 37 °C, fixed in 4% paraformaldehyde, and photographed under a fluorescence microscope or confocal microscope.
  • the EGFP receptor fusion expression plasmid of pEGFPN1-CCR6 was transfected into HEK293 cells, and CKLF1, C LF1 C-terminal polypeptide or MIP3c was added to the culture supernatant.
  • the C LF1 C-terminal polypeptide was induced to induce CCR6 by fluorescence microscopy or confocal microscopy.
  • Figure 12 Internalization of the EGFP receptor ( Figure 12), indicating that the C-terminal polypeptide of CKLF1 interacts with the CCR6 receptor.
  • C LF1 has a carboxy terminus of 16 amino acids and a cysteine residue at its N-terminus to facilitate the coupling of the vector.
  • the amino acid sequence is CSGPYQKKPVHEKKEVL (ie, the C-terminal 16 amino acids of C27 or C19 of the present invention, see the sequence SEQ ID NO: 2 amino acids 12-27, and a cysteine residue at its N-terminus).
  • This polypeptide was coupled to KLH (Pierce Coupling Kit). ,

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Description

具有多种功能的多肽
技术领域
本发明涉及基因工程领域， 特别地， 本发明涉及具有多种功能的多肽及编码其的多 核苷酸， 含有所述多核苷酸的载体或宿主细胞及它们的应用。 背景技术
趋化因子是一类具有趋化作用的细胞因子， 在机体的免疫调节、 炎症反应、 干细胞 增殖分化等过程中发挥重要作用。 目前， 趋化因子已经成为国内外研究的热点之一。 申 请人利用抑制性减数杂交技术， 从 PHA刺激的 U937细胞中， 成功克隆了一个新的具有 趋化活性的细胞因子， 即趋化素样因子 1 (chemokine- l ike factor 1, CKLF1 )。 CKLF1 编码 99个氨基酸， 在体内、 外具有广谱的趋化活性， 注射 CKLF1 真核表达质粒的小鼠 肺部炎性病变明显， 表现与哮喘发病后期的病理改变非常相似。 己申请中国发明专利和 国际专利， 中国专利申请号是 99107284. 7， 国际专利号是 PCT/CN00/00026, 国际专利 公布号是 W0 00/69910。 在 CKLF1 (其核酸序列在 Gen- bank™的注册登记号为 AF096895) 的基础上， 本发明人又陆续得到了一些新的多肽。 发明内容
发明人构建了 CKLF1 果蝇表达系统， 经表达、 纯化， 获得分泌表达的 CKLF1 重组蛋 白。 这种重组蛋白对外周血单核细胞、 中性粒细胞、 淋巴细胞及 U937细胞仍然具有趋化 作用。 进一歩通过 SDS- PAGE分离获得 CKLF1 蛋白条带， 进行 N端测序， 得到两种 CKLF1 分泌多肽， 命名 C19、 C27多肽 (统称 C LF1 C末端多肽）； 申请人也利用 CKLF1重组蛋白 研究其受体， 发现 CCR4、 CCR5是 CKLF1 的受体。 申请人又进一步化学合成了 C19、 C27 多肽，并对化学合成的 C19、 C27多肽的相互作用受体进行了研究，发现 CCR4、 CCR5、 CCR6 是 C19、 C27 多肽的相互作用受体， 表现出刺激或者拮抗的活性。 例如， 在预防和 /或治 疗 HI V (人免疫缺陷病毒)中， 当 C19、 C27多肽与受体结合， 阻止 HIV与受体结合或这些 受体与己知配体的相互作用。 另外， 申请人惊奇地发现， CKLF1 C末端多肽不仅具有与 CKLF1相同的趋化因子相互作用受体， 而且表现具有拮抗作用， 同时， CKLF1 C末端多肽 可化学合成， 更有应用前景。 尤其 CKLF1 C末端多肽的受体拮抗剂作用在预防和 /或治疗 HIV (人免疫缺陷病毒）、 移植排斥、 脑部疾病、 自身免疫病或治疗过敏性疾病中更理想。 因此， 本发明的一个目的是提供一种多肽。
本发明另一个目的是提供一种多核苷酸。
本发明的另一目的是提供一种含有本发明的多核苷酸的载体。
本发明的另一目的是提供一种含有本发明的载体的宿主细胞。
本发明的另一目的是提供一种药物组合物， 该药物组合物含有本发明的多肽、 多核 苷酸、 载体、 或宿主细胞， 和 /或一种或一种以上药物可接受的盐或药学上可接受的载 体或赋形剂。
本发明的另一目的是提供本发明的多肽或多核苷酸在制备预防和 /或治疗人免疫缺陷 病毒感染、 过敏性疾病、 移植排斥、 脑部疾病或自身免疫病的药物中的应用。 本发明的 另一目的是提供检测待测样品中本发明的多肽或多核苷酸的表达水平是否变化的方法。
本发明的另一目的是提供一种单克隆或多克隆抗体， 其与本发明的多肽或其具有抗 原性的片段特异性结合。 本发明提供一种多肽， 该多肽包含-
(1) 如 SEQ ID NO: 2所示的氨基酸序列的多肽， 或如 SEQ ID NO: 2的 9-27位氨 基酸所示的多肽； 或
(2) 与（1)具有至少 80%同源性的多肽， 该多肽的功能与（1)的功能相同或相似。 如 SEQ ID NO: 2所示的氨基酸序列的多肽 (在本发明中称为 C27)， 为 CKLF1 多肽 的 C 末端 27 个氨基酸序列（CKLF1 多肽的核酸序列在 Gen- bank™的注册登记号为 AF096895, 另外还可参见国际申请： PCT/CN00/00026, 中国专利申请： 99107284. 7)。 本发明 SEQ ID NO: 2所示序列的片段也在本发明的范围内， 如 SEQ ID NO: 2的 9-27 位氨基酸所示序列，也就是 CKLF1多肽的 C末端 19个氨基酸序列 (在本发明中称为 C19)。 在本发明中 C27和 C19统称为 CKLF1 C末端多肽。 本发明多肽还包括与如 SEQ ID NO: 2 所示的氨基酸序列， 或如 SEQ ID N0: 2的 9-27位氨基酸所示序列的同源性 (序列匹配） 超过 80%,优选超过 85%,更为优选超过 90%,更进一步优选超过 95%,特别优选超过 98%, 更进一步特别优选超过 99%的序列。 这些序列可以与本发明的 SEQ ID N0: 2所示的序列 或如 SEQ ID N0: 2的 9-27位氨基酸所示序列具有相同、 相似或不同的生物学功能， 优 选具有相同或相似的功能。
本发明 CKLF1 C末端多肽或其片段可以是天然的、 合成的、 半合成的、 或者重组产 生。 本发明 CKLF1 C末端多肽可按照 Steward和 Young (Steward, J. M. 和 Young, J. D.， Sol id Phase Peptide Synthesis, 2nd Ed. , Pierce Chemical Company, Rockford, 111. , (1984) )描述的方法用 Applied Biosystem合成仪或 Pioneer™肽合成仪按固相化 学技术合成。 一般， 这些方法包括向成长的肽链上依次添加一个或多个氨基酸或适当保 护的氨基酸。 通常， 第一个氨基酸的氨基或羧基用适当的保护基保护， 然后将保护的氨 基酸连在惰性固相载体上， 随后在适于形成酰胺键的条件下加入相应氨基或羧基被适当 保护的序列中的下一 ^^氨基酸。 然后从新加入的氨基酸残基上除去保护基， 再加入必要 时适当保护的下一个氨基酸， 如此重复操作。 当所有氨基酸以正确的顺序连接后， 相继 或同时除去任何剩余的保护基和固相支持物， 得到最终的多肽。 通过简单修改此标准程 序， 可能一次向成长链添加一个以上的氨基酸。 在本发明的一个优选实施方案中， 使用 自动合成仪制备本发明的多肽。 其中使用 α 氨基被酸或碱敏感性基团保护的氨基酸。 这种保护基应该在肽键形成条件下稳定， 又容易除去而不破坏成长的肽链、 不会引起其 中的任何手性中心外消旋。 适当的保护基有 9-芴基甲氧基羰基 (Fmoc)、 叔丁氧羰基 (Boc)、 苄氧羰基 (Cbz)、 2-氰基叔丁氧羰基等。 9-芴基甲氧基羰基 (Fmoc)是合成本发明 肽特别优选的。也可按常规的生物工程方法由宿主细胞中的重组 DNA序列编码产生本发 明的多肽 （具体参见实施例 1-4)。在实施例 1-4中， 申请人将 CKLF1编码序列插入表达 系统， 经表达、 纯化, 获得分泌表达的 CKLF1 重组蛋白， 进一步通过 SDS- PAGE分离获 得 CKLF1 蛋白条带， 进行 N端测序， 得到本发明的 C19、 C27多肽。 本领域技术人员已 知， 可以直接使用编码本发明的多肽的多核苷酸序列产生本发明的 CKLF1 C末端多肽， 例如将编码本发明的多肽的多核苷酸序列 (如 SEQ ID NO: 1所述的序列或其片段)直接 插入表达系统， 经表达、 纯化， 获得本发明的多肽。 或者可使用衍生于本发明的 DNA构 建体的 mRNA, 在无细胞翻译系统中产生所需的多肽。
本领域普通技术人员己知， 本发明所述的多肽或其片段可以同其它的多肽或其片段 形成融合多肽。 其它多肽或其片段一般是已知的， 有些可以以载体形式购买得到， 或者 可以按常规方法合成或从已知生物体中克隆得到。
本发明优选的多肽为：
(1) 如 SEQ ID NO: 2所示的氨基酸序列的多肽， 或如 SEQ ID NO: 2的 9- 27位氨 基酸所示的多肽； 或
(2) 与（1)具有至少 90%同源性的多肽， 该多肽的功能与（1)的功能相同。
本发明还提供一种多核苷酸， 该多核苷酸包含：
(1) 编码如 SEQ ID NO.- 2所示氨基酸序列的多核苷酸， 或编码如 SEQ ID NO: 2 所示的 9-27位氨基酸序列的多核苷酸； 或 (2) 与（1)具有至少 80%同源性的多核苷酸，该多核苷酸编码的多肽与（1)编码的多 肽具有相同的功能。
如 SEQ ID NO: 2所示的氨基酸序列为 CKLF1多肽的 C末端 27个氨基酸序列（C27)。 如 SEQ ID NO: 2的 9-27位氯基酸所示的序列为 CKLF1 多肽的 C末端 19个氨基酸序列 (C19)。 本发明的多核苷酸序列可以只编码 CKLF1 C末端多肽， 也可以在上述多肽的编 码序列的基础上， 增加非编码序列， 例如内含子、编码序列 5'或 3' 端的非编码序列等。 本发明的多核苷酸序列最好是以分离形式提供的。本发明的多核苷酸是 "分离 "形式的， 其不仅已经与在细胞中伴随其的蛋白质分开，而且已从天然状态下位于其两侧的序列中 分离出来。
本发明还包括与编码 CKLF1 C末端多肽或其片段的多核苷酸具有至少有 70%、 80%、
85%， 较好 90%、 95%， 最好 98%同源性的多核苷酸序列。特别涉及在严格条件下与 CKLF1 C末端多肽的多核苷酸杂交的多核苷酸， 所说的 "严格条件" 意指发生杂交的前提是序 列间至少具备 95%的同源性。这样的序列可以是天然存在或人工产生的，可以包括 CKLF1 C末端多肽的多核苷酸序列的等位基因变异体、 也可以包括 CKLP1 C末端多肽多核苷酸 序列中碱基的缺失、插入及置换。这样的序列编码的多肽可以在功能上与本发明的 CKLF1 C末端多肽相同、 相似、 或不同， 但最好是编码与 CKLF1 C末端多肽的生物学活性基本 相同的多肽。 因此， 优选与编码 SEQ ID NO: 2所示氨基酸序列的多核苷酸序列或其片 段具有至少 80%同源性的多核苷酸， 该多核苷酸编码的多肽与 SEQ ID NO: 2所示的多肽 具有相同的功能。
本发明的多核苷酸序列可以是 DNA或 RNA, 其中 DNA包括 cDNA、 基因组 DNA以及合 成的 DNA， DNA可以是双链或是单链形式，单链 DNA可以是编码链或是非编码链 (反义链）。 本发明所述的反义链可以为如 SEQ ID NO: 1所示的序列的互补序列。 本领域普通技术 人员已知， 反义链或者其一部分 (反义寡核苷酸)可用于抑制细胞内本发明 CKLFl C末端 多肽的表达。 本发明的 CKLF1 C末端多肽的核苷酸序列可以来自任何物种， 特别是哺乳 动物， 包括牛、 羊、 猪、 鼠、 马， 优选人类。
如 SEQ ID NO: 1所示的多核苷酸序列为一种编码本发明 C27多肽的多核苷酸， 其 来自人类。 如 SEQ ID NO: 1的 25-81位所示的多核苷酸序列为一种编码本发明 C19多 肽的多核苷酸， 其来自人类。 所以， 优选本发明的多核苷酸包含如 SEQ ID NO: 1所示 的多核苷酸或其互补序列， 或如 SEQ ID NO: 1的 25- 81位所示的多核苷酸。
本发明还涉及一种基因工程载体， 该基因工程载体含有编码本发明的 C末端多肽的 多核苷酸。 所述基因工程载体可以是普通载体、 表达载体等。 其中普通载体主要用于各 种基因组文库和 cDNA文库的建立， 它们通常含有两个或两个以上的标记基因， 其中一 个基因用于选择转化体（transformant)， 另一基因则是用于检査载体中是否有外源 DNA 插入。表达载体主要用于研究基因的表达或是用于大量生产一些有用的转录产物或蛋白 质， 有的也可用于 cDNA文库的建立。 这类载体除具有普通型载体的特征外, 还应含有 适当的启动子、 核糖体结合位点、 终止子等。 为了便于表达产物在细胞中定位， 在多肽 编码序列上游可加入适当的前导序列。合适载体和启动子的选择为本领域普通技术人员 周知。本领域普通技术人员周知用于构建含有本发明的多核苷酸以及合适的转录及翻译 调控元件之载体的方法。 具体地说， 适用于原核细胞的市售表达载体一般均带有可选择 标志和细胞复制原点， 带有 lacI、 T7、 A PL和 trp等细菌启动子， 以及已知克隆载体 pBR322 (ATCC 37017)的其他遗传元件。 这样的市售载体包括 pGEM(Promega)和 P K223-3 (Pharmacia)。 可根据所选用的适当启动子和待表达的结构基因序列来选择衍 生于 pBR322的适当载体。 GST原核表达系统也可用于本发明。适用于真核细胞的载体带 有真核细胞启动子如 CMV、 SV40等， 这样的载体包括 pMT-hIL- 3 (马大龙， 狄春辉， 庞健 等（1991)高技术通讯 11： 26-29)、 pQE- 9 (Qiagen)、 pD10、 PNHl8A (Stratagene) 、 pKK233- 3、 pDR540、 pRIT5 (Pharmacia) , 以及 pcDNA3、 pCI、 pWLNE0、 pSG (Stratagene) , pSVL (Pharmacia) 0 在本发明的实施例 1 中 CKLF1 编码序列插入 PCDNA3. l-myc-his6 (Invi trogen公司）表达载体，构建 pCDNA3. l-CKLFl-myc-his6表达质粒。将 PCR产物用 EcoR I + Xho I 双酶切处理后， 与用 EcoR I + Xho I 双酶切的 pMT/V5- HisA 载体 (Invitrogen公司）连接， 得到 pMT/V5-HisA- CKLF1- myc- his6质粒。
本发明还涉及一种宿主细胞， 该宿主细胞含有编码本发明 CKLF1 C末端多肽的多核 苷酸。 宿主包括但不限于： 原核宿主， 诸如大肠杆菌、 芽孢杆菌属、 链霉菌属等； 真核 宿主， 诸如： 酵母属、 曲霉属、 昆虫细胞， 诸如果蝇 S2和草地夜蛾 Sf9; 动物细胞， 如 CH0、 COS (猴肾成纤维细胞系， Gluzman (Cell 23 : 175, 1981) )及其它的能表达相容载 体的细胞系。 本领域技术人员周知将含有本发明的多核苷酸的构建体导入上述宿主细胞 的方法， 包括但不限于： 氯化钙介导的转化、 磷酸钙转染、 DEAE-葡聚糖介导的转染、 电 穿孔、 显微注射、 粒子轰击法或基因枪方法 (Sambrook, J. (1989) , Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press ;— Plainview, N. Y.； Ausubel, F. M. (1989) Current Protocols in Molecular Biology, John Wiley & Sons , N. Y.； Hobbs, S. 等人， McGraw Hill Yearbook of Science and Technology (1992) , McGraw Hi ll, N. Y. 191-196； Engelhard, E. K. 等人， PNAS, 91 : 3224-3227 ; Logan, J. 等人， PNAS, 81 : 3655-3659) 0 在适当的培养条件与培养基中培养经转化的宿主菌株或细胞， 使 其生长到恰当的细胞密度之后， 用适当的方法 (例如温度转变或化学品诱导)诱导所选择 的启动子， 并将细胞再培养一段时间。 针对不同的宿主菌株或细胞以及所表达的目的多 肽的性质选择相应的培养条件和培养基在本领域技术人员知识范围之内。 例如： 在本发 明实施例 1中使用的宿主细胞为 XU- Blue大肠杆菌， 在实施例 2中使用的宿主细胞为果 蝇 S2细胞， 转染方法为氯化钙转染方法， 在实施例 7中使用的宿主细胞为 HEK293细胞 (ATCC CRL-1573) , 转染方法为电穿孔方法。
本发明提供一种药物组合物， 其含有治疗有效量的 CKLF1 C末端多肽、 编码本发明 的多肽的多核苷酸、 包含所述多核苷酸的载体、 宿主细胞， 和 /或一种或一种以上药物 可接受的盐或药学上可接受的载体或赋形剂。 "药物可接受的盐"指适于与人或动物的 组织接触， 而无过多的毒性、 刺激、 变态反应等的盐。 本发明的药物可接受的盐是本领 域药剂学常规组分。 这种盐可以在本发明多肽的最终分离和纯化的过程中制备， 也可以 将多肽与适当的有机或无机酸或碱反应单独制备。药学上可接受的载体或赋形剂指无毒 固态、 半固态或液态填充剂、 稀释剂、 包囊材料或其他制剂辅料。
本发明还提供本发明的 CKLF1 C末端多肽在制备预防和 /或治疗人免疫缺陷病毒感 染、 过敏性疾病、 移植排斥、 脑部疾病或自身免疫病的药物中的应用。
目前研究发现， 介导 HIV进入细胞的辅助受体包括： CCR3、 CCR5、 CXCR4、 CX3CR1 , 目前研制的人免疫缺陷病毒 (HIV)感染的抑制剂包括来自于病毒蛋白的 Kaposi 氏肉瘤 相关泡疹病毒编码的趋化因子和 HHV- 8 vMIP, 能与多种趋化因子受体结合而抑制 HIV 与辅助受体结合感染细胞； 还有经修饰改造的人趋化因子及小分子化合物特异地结合 CCR5、 CXCR4而抑制 HIV感染细胞。 本发明的 CKLF1 C末端多肽与多种趋化因子受体 结合， 并与 HIV结合的辅助受体相吻合（具体参见实施例 7- 8) , 而且是来自于人体的 多肽， 利于长期有效地使用。 因此， CKLF1 C末端多肽在用于防止 HIV的感染与播散 中有很大的优势。
特应性皮炎， 哮喘， 和过敏性鼻炎是常见的过敏性疾病， 与 Th2细胞、 肥大细胞和 嗜酸性粒细胞的迁移有关，一种治疗策略是利用趋化因子受体拮抗剂来防止这些效应细 胞的迁移。 CCR3、 CCR4、和 CCR8通常表达于 Th2细胞、肥大细胞和嗜酸性粒细胞。 CCR5 也与哮喘发生有关。 本发明通过确切的实验证明， CKLF1 C末端多肽是 CCR4和 CCR5的 功能性配体。 因此 CKLF1 C末端多肽还可以在抑制过敏性疾病方面起作用。 研究表明 CCR5与脑部疾病有关。 在多发性硬化症脑损伤部位的活化的小神经胶质 细胞、 侵润的 T细胞， 及 Alzheimer' s病（阿尔茨海墨氏病）脑损伤部位的小神经胶质 细胞均可检测到 CCR5 表达， 在本发明的实施例 7-8中证明， CKLF1 C末端多肽是 CCR5 的配体， 因此， CKLF1 C末端多肽可用于抑制这些疾病中慢性炎症引起的损害。
CCR5及其配体还参与了移植排斥反应、 类风湿关节炎， CCR5还与丙型肝炎的发生 有关。 CCR6与银屑病有关。 在本发明的实施例 7-9中证明， CKLF1 C末端多肽具有与上 述多种受体相结合的作用，提示 CKLF1 C末端多肽可以用于阻止移植排斥反应、 类风湿 关节炎、 丙型肝炎的发生和 /或银屑病的防治。
自身免疫病、 过敏性疾病、 HIV感染等疾病与趋化因子及其受体密切相关， 拮抗剂 是一个主要的治疗手段。 本发明通过实验证明， 本发明的 CKLF C末端多肽具有与趋化 素因子受体结合的特性， 而其又来源于人类本身， 所以 CKLF1 C末端多肽在多种疾病的 治疗方面具有广阔的应用前景。
本发明还提供一种体外检测来自待测者的样品中本发明所述的多肽或多核苷酸的 表达水平的方法， 该方法为反转录-聚合酶链式反应或蛋白质印迹检测方法。
可以采用本领域已知的任何方法检测本发明所述的多肽或多核苷酸的表达水平。 优 选利用反转录 -聚合酶链式反应 (RT-PCR)检测所述多核苷酸在核酸水平的表达水平； 或 利用特异性单克隆或多克隆抗体检测所述多核苷酸在蛋白质水平的表达水平，例如或蛋 白质印迹 (Western blotting)检测方法。 所述待测样品可以从来自受试者的细胞获得， 如来自血液、 尿、 唾液、 胃液、 头发， 活组织检查和尸体解剖材料的细胞。 PT-PCR主要 分以下步骤： 提取总 RNA, 加入一个与 mRNA 3 ' 端互补的引物， 在反转录酶的作用下合 成 cDNA; 第二歩是以 cDNA为模板， 再加入与 cDNA互补的另一引物 (两引物位于不同的 外显子上， 以避免基因组 DNA污染)进行 PCR扩增。 蛋白质印迹主要分三阶段： 第一阶 段为抗原等蛋白样品进行 SDS-聚丙烯酰胺凝胶电泳；第二阶段为电转移：将在凝胶中已 分离的条带转移到硝酸纤维素膜上； 第三阶段为显色检测： 硝酸纤维素膜 (相当于包被 了抗原的固相载体）， 依次与特异性抗体和酶标第二抗体作用后， 加入能形成显色物的 酶反应底物，使条带染色。标记二抗的酶包括辣根过氧化物酶（horseradish Peroxidase, HRP)和碱性磷酸酶（alkaline phosphatease, AP)。 也可以使用葡萄糖氧化酶， β - D -半 乳糖苷酶和脲酶等。 在本发明的一个实施方式中， 采用辣根过氧化物酶作为标记二抗的 酶。 本领域普通技术人员己知， 针对不同的酶， 可使用不同的底物， HRP作用的底物包 括， 但不限于临苯二胺（0PD)、 四甲基联苯胺 (TMB)和 ABTS等。 碱性磷酸酶的底物一般 釆用对硝基苯磷酸酯 (P-NPP)， AP也有发荧光底物 (磷酸 4-甲基伞酮）。 常用的酶标第二 抗体已有市售。
本发明还提供对本发明 CKLF1 C末端多肽或其抗原性片段具有特异性的多克隆和单 克隆抗体，尤其是单克隆抗体。这里所述的"特异性"是指抗体能结合于本发明的 CKLF1 C末端多肽基因产物或片段。 优选那些能与本发明的 CKLF1 C末端多肽的基因产物结合 但不识别和结合于其它非相关抗原分子的抗体。本发明中抗体包括那些能够结合并抑制 本发明 CKLF1 C末端多肽基因产物的抗体， 也包括那些并不影响本发明 CKLF1 C末端多 肽功能的抗体。 本发明还包括那些能与本发明 CKLF1 C末端多肽具有至少 80%同源性的 蛋白或活性片段特异性结合的抗体。 上述抗体不仅包括完整的单克隆或多克隆抗体， 而 且还包括具有免疫活性的抗体片段， 如 Fab' 或 (Fab) 2片段； 抗体重链； 抗体轻链； 遗 传工程改造的单链 Fv分子（Ladner等人， 美国专利 No. 4, 946, 778)； 或嵌合抗体， 如具 有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。
本发明的抗体可以通过本领域普通技术人员己知的各种方法进行制备。 例如， 纯化 的本发明 CKLF1 C末端多肽基因产物或其具有抗原性的片段， 可被施用于动物以诱导多 克隆抗体的产生。 与之相似的， 表达本发明的 CKLF1 C末端多肽或具有其抗原性的片段 的细胞可用来免疫动物来生产抗体。 本发明的抗体优选单克隆抗体， 其可利用杂交瘤技 术制备（见 Kohler等人， Nature 256 : 495； Kohler等人， Eur. J. Immunol. 6 : 511， 1976; Hammer ling 等 人 , In Monoclonal Antibodies and T cell Hybridaomad, Elsevier, N. Y. , 1981)。 本发明的各类抗体可以利用本发明的 CKLP1 C末 端多肽基因产物或其片段或功能区， 通过常规免疫技术获得， 这些片段或功能区可以利 用重组方法制备或利用蛋白合成仪合成。 与本发明 CKLF1 C末端多肽的基因产物的未修 饰形式结合的抗体可以用原核细胞 (例如， E. Coli)中生产的基因产物来免疫动物而产 生； 与翻译后修饰形式结合的抗体 (如糖基化或磷酸化的蛋白或蛋白），可以用真核细胞 (例如酵母或哺乳细胞， 例如兔）中产生的基因产物来免疫动物而获得。 附图说明
图 1显示 PCR鉴定稳定转染 S2细胞株染色体 DNA (包含 hCKLFl-myc编码区序列）的电泳 结果， 其中， 第 1泳道为以 DL2000 DNA为分子量标准， 第 2泳道为以 pMT/V5- HisA转染 S2 细胞染色体为模板， 第 3泳道为以 pMT/V5HisAhCKLFl- myc转染的 S2细胞染色体为模板， 用相同的 hCKLFl (pl， p2)为引物扩增。 图 2 为 RT- PCR鉴定稳定转染 S2细胞株有 hCKLFl- myc mRNA的转录， 其中， 第 1 泳道为分子量标准， 第 2、 3、 4泳道是分别以 pMT/V5HisAhCKLFl- myc、 pMT/V5HisA、 S2 空细胞为模板，以 CKLF1 (PI, P2)为引物，第 5、6、7是与上述模板相同，而以 G3PDH(P1， P2) 为引物进行的 RT-PCR, 其中 5、 6、 7分别作为内部标准。
图 3A与图 3B显示利用 Western Blotting检査目的蛋白的表达结果。 在图 3A中可以看 出， 加入兔 Anti- hCKLFl多肽抗体， 抗兔 IgG HRP为二抗反应， 经 X片显影可见 8-lOkd处 有两条清晰条带（图中箭头所示〉； 在图 3B中可以看出， 加入 Anti-c-myc抗体作用后， 再 加入抗鼠 IgG HRP二抗反应，经 X光片显影，在 8-10kd处有两条清晰条带（图中箭头所示）。
图 4显示纯化后的蛋白质样品经 15%SDS- PAGE电泳， 直接考马斯亮蓝染色。 箭头所 指为 CKLF1 myc蛋白质。
图 5 显示纯化后的蛋白质样品转移至 PVDF膜后做 Western Bloting鉴定结果。 条 带 1为分子量标准， 条带 2为 S2/pMT上清液； 条带 3为 S2/CKLF1细胞裂解液； 条带 4 为 S2/CKLF1上清液。
图 6A和图 6B显示纯化后的蛋白质样品转移至 PVDF膜后 N端测序结果。 其中， 图 6A表示 C19肽， 6B表示 C27肽。
图 7显示钙流分析的实验结果， 其中 CCR4的相应配体为 TARC， CCR5的的相应配体 为 RANTES。 A-D为 CCR4转染细胞； E- H为 CCR5转染细胞。 A中 a- b为 ΙΟΟηΜ TARC刺激 后， c- d为 167nM C27 ; B中 a- b为 167nM C27 , c- d为 lOOnM TARC; C中 a- b为 lOOnM TARC, c-d为 167nM C19 ; D中 a- b为 167n C19 , c-d为 lOOnM TARC; E中 a- b为 lOOnM RANTES, c-d为 167nM C27 ; F中 a- b为 167nM C27, c-d为 ΙΟΟηΜ RANTES; G中 a- b为 ΙΟΟηΜ RANTES, c-d为 167nM C19; H中 a-b为 167nM C19， c-d为 ΙΟΟηΜ RANTES 0
图 8A和图 8B 为钙流变化图像的相对荧光用 Leica confocal 软件分析。 其中， 图 8A为 CCR4转染细胞； 图 8B为 CCR5转染细胞。
图 9A与图 9B显示 CKLF1 C末端多肽对 CCR4或 CCR5转染细胞的趋化作用， 其中， 图 9A:转染 CCR4未处理的 HEK293细胞及用 CKLF1 C末端多肽、 TARC或 PTX预处理 30 min 的 HEK293/CCR4细胞； 图 9B: 转染 CCR5未处理的 HEK293细胞及用 CKLF1 C末端多肽、 RANTES或 PTX预处理 30 mi n的 HEK293/CCR5细胞。图中，Μ代表 RPMI_1640; T代表 TARC; 27代表 C27 ; 19代表 C19 ; P代表 PTX; R代表 RANTES。
图 10 显示纯化的 pMT/V5HisA-CKLFl- myc蛋白对 U937细胞的趋化活性。
图 11显示纯化的 pMT/V5HisA-CKLFl-myc蛋白对 CCR4转染的 HEK293细胞的趋化活 性及脱敏效应。
图 12显示 CKLF1 C末端多肽对趋化因子受体 CCR6内在化的影响。 其中， 1为对照， 2为 BSA, 3为 MIP3- α， 4为 C27多肽, 5为 C19多肽。 具体实施方式 实施例 1、 构建 pMT/V5-HisA- CKLF1- myc- his6融合蛋白表达质粒
构建 pMT/V5- HisA-CKLFl- myc-his6质粒， 用于表达 CKLF1- myc- his6融合蛋白。 一、 方法：
将 CKLF1编码序列（SEQ ID NO: 3)插入 pCDNA3. 1- myc- his6 (Invitrogen公司）表 达 载 体 ， 构 建 pCDNA3. l-CKLFl-myc-his6 表 达 质 粒 。 以 测 序 正 确 的 pCDNA3. l-CKLFl-myc-his6为模板， 用 T7为上游引物（5 ' - TGTAA TACGA CTCAC TATAG - 3，（SEQ ID NO: 4) )以及带有 Xho I酶切位点的下游引物（5' -CAT TGA GTT TAA ACG GTC TCG AGC GG-3' (SEQ ID NO: 5) )进行 PCR， 获得编码 CKLFl-myc-his6融合蛋白的 DNA 片段， 电泳回收 PCR产物， 用 EcoR I + Xho I双酶切处理后， 与用 EcoR I + Xho I双 酶切的 pMT/V5- HisA载体（Invitrogen公司）连接， 得到 pMT/V5- HisA- CKLF1- myc-his6 质粒。 连接产物转化 XL1- Blue大肠杆菌， 筛选正向插入的克隆， 测序验证质粒的正确 性后， 进行质粒扩增， 用 Qiagen 100提取质粒， 用于细胞转染。
二、 结果：
经 DNA测序编码区序列正确。 实施例 2、 质粒转染细胞筛选稳定细胞株
一、 方法：
对数生长的果蝇 S2 (Drosophila Schneider 2)细胞调成 3 X 106细胞 /3ml 浓度在 35cm平皿中 28°C培养 16小时进行转染， 2M CaCl2 36 1 , p T / V5-HisA hCKLFl- myc DNA 1 g/ 1 19 1 , PCoHyGrol g/ 1 1 1 , 用水补足 300 1 体积， 缓缓加入到等体积 2XHBS 缓冲液中， 室温静置 30分钟， 混合液慢慢滴入准备好的细胞中， 同时设 pMT/V5-HisA 空载体转染细胞及未转染的 S2细胞对照， 28'C培养 16小时， 将转染后的细胞离心， PBS 离洗两次， 换含 10%FBS 的新鲜培养基培养， 72 小时后再次离心细胞， 换上含 10%FBS 新鲜培养基后， 加入潮霉素 B至终浓度 300 g/ml , 压稳定细胞株。
每 4- 5天换液一次， 持续 21天左右。 一星期后，未经转染的 S2细胞大量死亡，两星期后 98%以上未转染的细胞已经死亡， 而经转染的细胞逐渐形成克隆，这时将 S2细胞，空载体转染和 pMT I V5-HisA hCKLFl-myc 转染并经潮霉素 B筛选的细胞克隆分别提取染色体 DNA, 做 PCR， 结果证实目的基因己 成功整合， 可进一步筛选单克隆。
将正常培养对数期的 S2 细胞作为饲养细胞铺于 96 孔细胞培养板， l. lxl06/ml，
100 1/孔, 内含 300 g/ml 潮霉素 B。 将用 pMT/V5- HisA- hCKLFl- myc转染， 将潮霉素 B 压后得到的 S2细胞用含 10%FBS， 300 g/ml 潮霉素 B的新鲜培养基稀释成 lxl07ral， 1x107ml , lxlOVml , lxlO'/ml系列浓度。 分别加入到已经铺好饲养细胞的 96'孔板中， 每孔 100 1 , 每个稀释度一块 96孔板。 24'C培养， 4-5天半量换液一次。 两星期后， 饲 养细胞全部死亡， 有抗性的细胞已长满 96孔板， 将细胞按对应孔传入 48孔板中， 经两 次传代后， 每孔吸出部分细胞， 提取细胞染色体 DNA， 做 PCR。
将筛选并传代后的细胞分别吸出， 加入细胞裂解液， 蛋白酶 K、 RNaseA孵育后提取 细胞染色体 DNA, 以此染色体 DNA为模板， 以 CKLF1上游 5 ' ATG GAT AAC GTG CAG CCG AAA AT 3 ' (SEQ ID NO: 6)， 下游 5' CAA AAC TTC TTT TTT TTC ATG CAC A 3' (SEQ ID NO: 7)为引物， 扩增 30轮， 根据 PCR结果再挑出目的基因整合并且是高拷贝的阳性克 隆细胞做 RT-PCR进一步鉴定。 首先利用博大公司的 TRIZ0L试剂提取细胞总 RNA, 然后 利用 GIBC0- BRL公司的 RT-PCR试剂盒进行逆转录和 PCR, 根据 RT- PCR结果将阳性克隆 细胞进行扩增。
利用 Western Blotting检查目的蛋白的表达：
经 15%SDS- PAGE电泳，用 lXCaps电转液将蛋白转移至 PVDF膜上，加入 Anti- c-myc 抗体（Sigma 公司）作用后， 再加入抗鼠 IgG HRP 二抗反应（Promega 公司）， 采用 ECL Western Blotting检测系统， 经 X光片显影， 在 8- 10kd处有两条清晰条带。 将 PVDF 膜剥离后加入兔抗 -hCKLFl 多肽抗体， 抗兔 IgG HRP 为二抗反应， 采用 ECL Western Blotting检测系统， 经 X片显影。
二、 结果：
1. PCR鉴定稳定转染 S2细胞株染色体 DNA包含 hCKLFl- myc编码区序列：
分别以未转染的 S2细胞， 和 pMT/V5HisA及 pMT/V5HisA-hCKLFl- myc转染的 S2细胞的 染色体为模板， 以 CKLF1 pi (5' -GCA AGA AGC GGG AAG CCG A-3' (SEQ ID NO: 8) ) , ρ2 (5' - CAT TGA GTT TAA ACG GTC TCG AGC GG -3' (SEQ ID NO: 5)为引物进行扩增, 制备 1%琼脂糖 进行电泳， 结果请参见图 1所示， 第一泳道是以 DL2000 DNA为分子量标准， 第二泳道为 以 pMT/V5_HisA转染 S2细胞染色体为模板， 第三泳道是以 pMT/V5HisA hCKLFl-myc转染的 S2细胞染色体为模板， 用相同的 hCKLFl (pl， p2)为引物扩增， 发现第三泳道在 300bp位 置有一条特异区带。
2. RT- PCR鉴定稳定转染 S2细胞株有 hCKLFl- myc mRNA的转录：
请参见图 2 所示： 第 1 泳道为分子量标准； 第 2、 3、 4 泳道是分别以 pMT/V5HisA- hCKLFl-myc、 pMT/V5HisA、 S2空细胞为模板， 以 CKLF1 (Pl， P2)为引物; 第 5、 6、 7是与上述模板相同，而以 G3PDH的 pi (5' ACCACAGTCCATGCCATCAC 3' (SEQ ID NO: 9) ) , P2 (5， TCCACCACCCTGTTGCTGTA 3' (SEQ ID NO: 10) )为引物进行的 RT- PCR。 RT-PCR 结果显示： 第二泳道 300bp处可见一条清晰带。
3. 利用 Western Blotting检査目的蛋白的表 ¾:
结果请参见图 3A与图 3B所示。 加入 Anti-c-rayc抗体作用后， 再加入抗鼠 IgG HRP二 抗反应， 经 X光片显影， 在 8- 10kd处有两条清晰条带（图 3B)。
加入兔 Anti-hCKLFl多肽抗体， （实施例 10中提供该抗体的制备方法），抗兔 IgG HRP 为二抗反应， 经 X片显影可见 8- 10kd处有两条清晰条带， 且两种抗体反应的结果吻合（图 3A)。 实施例 3、 CKLFl-myc蛋白质的纯化
一、 方法：
将阳性细胞经扩增后转入 Drosophila- SFM(Invitrogen公司）中撤掉潮霉素 B培养 到适当浓度，加入 CuS04诱导目的蛋白表达。收获 30小时 S2细胞表达上清， 2000rpm/min, 10分钟， 8000rpm/min, 20分钟， 0. 4 m滤膜过滤。 ¾ Chelating Sepharose Fast Flow 柱料， 20倍体积水平衡后上样， PBS缓冲液、 0. 5M NaCl分别洗柱，再依次用 10mM、 50mM、 500mM咪唑 /50mM Tris- HC1 pH8. 0洗脱， 20mM EDTA洗柱后可用水平衡柱料， 将第一次 过柱穿过液再次上样， 重复上述过程。
二、 结果：
如图 4 所示， 纯化后的蛋白质样品经 15% SDS-PAGE电泳， 直接考马斯亮蓝染色。 图中用箭头指示的 CKLF myc pro为目的蛋白条带。 实施例 4、 纯化后的蛋白质 N端测序
一、 方法：
取 NiCl2鳌合的 Chelating Sepharose Fast Flow柱料，平衡液平衡，收集经 Western blot 检测的过柱阳性洗脱峰， 经透析去除咪唑后上样， 先用杂蛋白洗液洗柱， 再用含 500mM咪唑的蛋白洗液洗脱。 经二次过柱纯化， 洗脱后样品经 15% SDS-PAGE电泳转移至 PVDF膜上， 用考马斯亮兰染色， 甲醇 /冰乙酸脱色后裁下与 Western blot 检测阳性位 置相同的带进行蛋白质的 N端测序 (委托医科院基础所测序）。
~ ^、 结果 ϊ
图 5为纯化后的蛋白质样品转移至 PVDF膜后做 Western Bloti ng鉴定结果， 其中 条带 1为分子量标准， 条带 2为 S2/pMT上清液； 条带 3为 S2/CKLF1细胞裂解液； 条带 4为 S2/CKLF1上清液。 从图中可以看出， CKLF1可分泌表达。 参见图 6 A和图 6B蛋白 质的 N端测序，结果显示分子量较大条带测出两种主要的带型，分别为 A- L- 1- Y- R- K- L-L 和 F- N- P- S- G- P- Y-Q， 分子量较小的条带也有两种主要的带型， 并且与前两个带型的 N 端序列相同。 实施例 5、 CKLF1 C末端多肽合成
一、 方法：
氨基酸序列分析显示 CKLF1最少有两个成熟的结构； 它们的序列分别是 ALIYRKL LFNPSGPYQKKPVHE KEVL (C27) (SEQ ID NO: 2)和 FNPSGPYQKKPVHEKKEVL (C19) (SEQ ID NO: 2的第 9位至 27位）。 为进一步分析功能在深圳翰宇 (Hybio Engineering)中国有限公司， 按照标准方法化学合成了这两个多肽。
一、 结果：
获得冻干的多肽， 溶于磷酸盐缓冲液中， 浓度为 1 mg/ml于- 80°C保存。 实施例 6、 受体表达质粒的构建及细胞表达
一、 方法：
1、 受体表达质粒的构建：
含各种人趋化因子受体开放阅读框的 cDNA片段通过如下方法获得： CCR4、 CCR5和 CCR6受体通过聚合酶链反应（PCR)从 K562细胞的 cDNA文库克隆。 引物设计根据 Gen- Bank™ 登录的序列： CCR4 (N1005508. 2)， CCR5 (NM_000579) , CCR6 (XM— 004279)。 各种受体开放 阅读框的 cDNA片段分别插入 pcDI (本室改造的载体： 用 pCI (Promega公司）质粒的 Bgl l l - Kpnl 片段置换 pcDNA3 (Invi trogen公司） 质粒的 Bgl l l - Kpnl片段所得到的真 核表达载体）和 pEGFP (CL0NTECH公司）表达载体使之在 ΗΕΚ293细胞有效表达。 经 DNA测序 编码区序列正确， 与 Gen- Bank™ 登录的序列相符。 2、 细胞培养：
HEK293细胞用含 10%热灭活的胎牛血清、 lOO U/ml青霉素、 100 g/nil链霉素的 RPMI 1640 (Life Technologies, Inc. )培养。 每 4χ106 ΗΕΚ293 细 ¾/400μ1 用 15 趋化因子 受体表达质粒瞬时电穿孔转染， 条件是 120 V, 20 ms使用的仪器是电脉冲发生器 (Electro Square porator ECM 830, BTX, San Diego, CA) ， 48 h后进行钙流分析和 趋化实验。
二、 结果：
进行钙流分析和趋化实验时， 在转染受体的培养上清中， 分别加入各自受体的己知 配体： TARC：、 RANTES (皆购自 peprotech公司）， 可诱导受体转染细胞钙流或趋化。 参见 图 7- 12中己知配体的阳性对照，即图 7中 CCR4的相应己知配体为 TARC， CCR5的为 RANTES。 A- D为 CCR4转染细胞； E- H为 CCR5转染细胞。 A中 a- b为 100nM TARC刺激后， c- d为 167nM C27; B中 a- b为 167nM C27， c- d为 ΙΟΟηΜ TARC; C中 a- b为 ΙΟΟηΜ TARC, c- d为 167nM C19; D中 a- b为 167nM C19, c- d为 ΙΟΟηΜ TARC; E中 a- b为 ΙΟΟηΜ RANTES, c- d为 167nM C27; F中 a - b 为 167nM C27， c-d为 ΙΟΟπΜ RANTES; G中 a- b为 ΙΟΟηΜ RANTES, c- d为 167nM C19; H中 a- b 为 167nM C19， c-d为 ΙΟΟηΜ RANTES。 实施例 7、 CKLF1 C末端多肽 (C19和 C27)对细胞内钙流的影响
一、 方法：
HEK293细胞用含 10%热灭活的胎牛血清、 100 U/ml青霉素、 100 g/ml链霉素的 RPMI 1640培养。 每 4xl0e HEK293 细胞 /400μ1 用 15 表达质粒瞬时电穿孔转染， 条件是 120 V, 20 ms , 使用的仪器是电脉冲发生器 （Electro Square porator ECM 830， BTX, San Diego , CA) ， 48 h后进行钙流分析和趋化实验。
转染了 pcDI- CCR4、 CCR5的 HEK293细胞用玻璃底的微孔皿（MatTek corporation, U. S. A. )培养， 用 10 μΜ fluo-3/AM (HEPES缓冲盐溶液配制）负载， 37 'C 1 h，避光。 HEPES 缓冲盐溶液洗细胞， 然后分别用 167 n C末端多肽， 100 nM TARC, 100 nM RANTES刺激。 荧光用 Leica TCS- NT荧光共聚焦显微镜 40 X油镜（Wetzler, Heidelberg, Germany) , 每 5秒检测一次， 激发波长 488 nm, 发射波长 530 nm0 上述操作在室温进行， 随机选 取各个视野的细胞。 每 5秒收集一次图象， 共 240秒。 图象用 Leica共聚焦软件分析相对 荧光强度， 数据用微软 Excel 2000处理。
二、 结果： - CCR4 (图 7-8)和 CCR5 (图 7-8)转染的细胞中诱导钙流 如图 7- 8所示， TARC或 RANTES 诱导表达了各自受体的 HEK293的钙流。 CKLFl C末端多肽可诱导转染 CCR4和 CCR5的 HEK293 细胞的钙流， 尤其通过 CCR5引起的钙流最显著。 167nM CKLFl C末端多肽可以使受体对 接下来的 100 nM TARC或 RANTES的刺激敏感性降低。 C27可以使 CCR4或 CCR5转染细胞产生 低强度信号， 而 C19可以使 CCR4转染细胞产生低强度信号。 同样用 100 nM TARC或 RANTES 预处理 CCR4和 CCR5转染细胞可以使转染细胞对接下来的 CKLFl C末端多肽的敏感性降低。 这些结果清晰地显示 CKLFl C末端多肽是 CCR4和 CCR5的功能性配体。 实施例 8、 CKLFl C末端多肽 (C19和 C27)的趋化功能
一、 方法：
CKLFl C末端多肽的趋化功能检测： 趋化实验在 48孔的趋化小室（Neutroprobe; Cabin John, MD, U. S. A. )里进行。用作趋化检测的所有因子用含 0. 1% BSA的 Hepes RPMI 1640稀释后加到小室的下孔里（28 μΐ/孔）。 用 pcDI- CCR4、 pcDI- CCR5、 pcDI- CCR6或 pcDI转染 HEK293细胞， 然后用同样的培养基重悬为 2xl06个细胞 /ml， 加到小室的上孔 中（50 μΐ/孔）， 上下孔用无聚乙烯吡咯垸酮的聚碳酸酯滤膜相隔， 滤膜孔径为 8 μπι。 将小室放入培养箱 37 'C ， 5% C02 , 孵育 2 h。 从小室上移去滤膜， 用 3步染色试剂盒 洗漆， 固定， 染色。 每孔迁移的细胞随机选取 5个高倍镜视野计数。 转染 pcDI 空载体 的 HEK293细胞作为对照。所有样品检测 2次。趋化指数 CI为迁移细胞数除以对照组细 胞数。 CI>2有显著性意义。 某些实验细胞在 CKLFl C末端多肽， TARC、 RANTES或 ΜΙΡ3α 刺激前用 100 ng/ml PTX (购自 ALEXIS Biochemicals Corporation)预处理 6 h。
二、 结果：
本发明用 CKLFl C末端多肽诱导 CCR4或 CCR5转染的细胞的迁移来检测它的活性。 如 图 9A与图 9B所示， CKLFl C末端多肽可以诱导 CCR4和 CCR5转染细胞的迁移，转染 pcDI- CCR4 或 CCR5的不用任何刺激的 HEK293细胞作为对照。用 0. 8 g/ml TARC或 RANTES, 37 °C , 30 min 预处理可以使受体对接下来 CKLFl C末端多肽的刺激去敏感。 类似地， 用 2 μ§/Γη1 CKLFl C 末端多肽预处理也可以使受体对下面 TARC或 RANTES刺激去敏感。 通过对 PTX敏感的 Gi/Go 家族 G蛋白来评价 CKLFl发挥的效应， 用 PTX 100 ng/ml预处理 CCR4和 CCR5转染的 HEK293 细胞 6h, 然后用 CKLFl C末端多肽， TARC或 RANTES刺激。 图 9A与图 9B 显示 CKLFl C末端 多肽诱导的趋化， TARC或 RANTES被 PTX完全阻断， 提示包含一个 Gi途径。 这些结果进一步 证实 CKLFl C末端多肽是 CCR4和 CCR5的配体。 与前期实验相吻合： 利用传代细胞系 U937细 胞和 CCR4转染的 HEK293细胞测定了 CKLFl-myc蛋白的趋化活性（结果请分别参见图 10和图 11所示）。 从图 10中可以看出， 50 咪唑洗脱的 CKLFl- myc蛋白 1 : 125倍稀释对 U937细胞 的趋化指数为 16， 对 CCR4转染的 293细胞趋化结果如图 11所示， 趋化因子 RANTES封闭了 HEK293细胞 CCR4后能够抑制这种趋化作用， 表明两者有交叉脱敏效应。 实施例 9、 C LF1 C末端多肽对趋化因子受体 CCR6内在化的影响
一、 方法：
将 CCR6编码区序列插入 pEGFPNl载体 (CLONTECH) , 构建成 pEGFPNl-CCR6- EGFP融合表 达质粒。 将 pEGFPNl- CCR6- EGFP受体融合表达质粒转染进入 HEK293细胞， 继续培养 24小 时后血清饥饿 16-24小时。 培养上清中加入 CKLF1 C末端多肽或 ΜΙΡ3α, 37 °C 2小时， 细 胞用冷 PBS冲洗， 4%多聚甲醛固定， 荧光显微镜或共聚焦显微镜下观察并拍照。
二、 结果：
利用 pEGFPNl- CCR6的 EGFP受体融合表达质粒转染进入 HEK293细胞， 培养上清中 加入 CKLF1、 C LF1 C末端多肽或 MIP3c， 通过荧光显微镜或共聚焦显微镜观察， 发现 C LF1 C末端多肽能诱导 CCR6- EGFP受体内化（图 12)， 说明 CKLF1 C末端多肽能与 CCR6 受体相互作用。 实施例 10、 兔抗- hCKLFl多肽抗体的制备
一、 方法：
C LF1羧基端 16个氨基酸， 并在其 N端增加一个半胱氨酸残基， .以利于偶联载体， 氨基酸序列为 CSGPYQKKPVHEKKEVL (即本发明的 C27或 C19的 C端 16个氨基酸， 序列参 见 SEQ ID NO: 2的 12-27位氨基酸， 并在其 N端增加一个半胱氨酸残基）。此多肽与 KLH 偶联 (Pierce公司偶联试剂盒）。 、
2. 5kg重成年雄性新西兰兔， 初次免疫用 1 mg CKLF羧基端 16个氨基酸多肽 -KLH 偶联物与等体积弗氏完全佐剂 (FCA)充分乳化后，于背部皮下多点注射。初次免疫后 21、 42、 63天， 用弗氏不完全佐剂 (FIA)完全乳化的抗原 lmg， 进行加强免疫。
~ -、 结果 J
每次免疫后 7-10天， ELISA方法检测血清效价， 达到 1 Χ 1(Τ时， 取血分离血清， 用 Protein G亲和层析法， 纯化抗肽抗体 IgG， 并保存于 0. 01M PBS中， 用于其它实验。

Claims

权利要求书
1、 一种多肽， 该多肽包含：
(1) 如 SEQ ID NO: 2所示的氨基酸序列的多肽， 或如 SEQ ID NO: 2的 9-27位氨 基酸所示的多肽； 或
(2) 与（1)具有至少 80%同源性的多肽， 该多肽的功能与（1)的功能相同或相似。
2、 如权利要求 1所述的多肽， 该多肽为：
(1) 如 SEQ ID NO: 2所示的氨基酸序列的多肽， 或如 SEQ ID NO: 2的 9-27位氨 基酸所示的多肽； 或
(2) 与（1)具有至少 90%同源性的多肽， 该多肽的功能与（1)的功能相同。
3、 一种多核苷酸， 该多核苷酸包含：
(1) 编码如 SEQ ID NO: 2所示氨基酸序列的多核苷酸， 或编码如 SEQ ID NO: 2 所示的 9-27位氨基酸序列的多核苷酸； 或
(2) 与（1)具有至少 80%同源性的多核苷酸，该多核苷酸编码的多肽与（1)编码的多 肽具有相同的功能。
4、 如权利要求 3所述的多核苷酸， 该多核苷酸包含如 SEQ ID NO: 1所示的多核苷 酸或其互补序列， 或如 SEQ ID NO: 1的 25-81位所示的多核苷酸。
5、 一种基因工程载体， 该基因工程载体含有权利要求 3或 4所述的多核苷酸。
6、 一种宿主细胞， 该宿主细胞含有权利要求 3或 4所述的多核苷酸。
7、 一种药物组合物， 该药物组合物含有权利要求 1或 2所述的多肽、 权利要求 3 或 4所述的多核苷酸、 权利要求 5所述的载体、 权利要求 6所述的宿主细胞， 和 /或一 种或一种以上药物可接受的盐或药学上可接受的载体或赋形剂。
8、 如权利要求 1或 2所述的多肽或如权利要求 3或 4所述的多核苷酸在制备预防 和 /或治疗人免疫缺陷病毒感染、 过敏性疾病、 移植排斥、 脑部疾病或自身免疫病的药 物中的应用。
9、 如权利要求 8所述的应用， 其中所述的药物用于预防和 /或治疗特应性皮炎， 哮 喘、 过敏性鼻炎、 阿尔茨海墨氏病、 类风湿关节炎、 丙型肝炎、 多发性硬化和 /或银屑 病。
10、一种体外检测来自待测者的样品中本发明所述的多肽或多核苷酸表达水平的方 法， 该方法为反转录-聚合酶链式反应或蛋白质印迹检测方法。
11、 一种单克隆或多克隆抗体， 该抗体与权利要求 1所述的多肽或其抗原性片段特 异性结合。
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