CN1772897A - Cell factor with several functions - Google Patents

Abstract

The present invention relates to one kind of polynucleotide coding the amino acid sequence of SEQ ID No. 2 or its segment, and gene engineering carrier and host cell containing the polynucleotides. The present invention also relates to one kind of polypeptide with the amino acid sequence of SEQ ID No. 2 and its production process; medicine composition containing the polynucleotides or its pharmaceutically acceptable salt, or the polypeptide or its pharmaceutically acceptable salt; the application of the polynucleotides or polypeptide in preparing medicine for preventing and/or treating immune deficiency, AIDS and hematogenesis dysfunction; the extracorporeal detection method of the polynucleotides or polypeptide expression level in the sample from the testee; and the monoclonal or polyclonal antibody combined specifically with the polypeptide or its active segment.

Description

Cytokine with multiple function

Technical field

The present invention relates to the genetically engineered field, especially, the present invention relates to polynucleotide and polypeptide, the carrier that contains described polynucleotide or host cell, the method for the described polypeptide of production or the application of described polypeptide or polynucleotide of novel cytokine.

Background technology

Cytokine is the micromolecular secreted polypeptide class factor, comprises interleukin-, G CFS, Interferon, rabbit, tumour necrosis factor, somatomedin and chemokine etc.Cytokine is brought into play effect by specificity in conjunction with cell surface receptor, regulates body's immunological function, participates in propagation, differentiation and the functionating of immunocyte.Cytokine in the also extensively existence of each system of body, is brought into play very important physical regulating effect except that being present in immunity system.Kind of a cytokine gene engineering medicine has gone through to go on the market surplus having ten at present, diseases such as treatment tumour, infection, hematopoietic disorder, other has tens of kinds of cytokine medicines in clinical experiment, and the new drug of cytokine inhibitor and clinical study also obtain development fast in recent years.Thereby, find new cytokine, carrying out function and application research is international hot fields, has important significance for theories and practical value.

Chemokine (chemokine, claim the chemotactic element again), be that a class formation is similar, molecular weight 8-12kD, have the micromolecule polypeptide of cell chemotaxis effect, they have vital role at aspects such as the immune defense of body, immunomodulatory, inflammatory reaction, hematopoiesis adjusting, vasculogenesis.(chemokine-like factor CKLF) is the cytokine of being cloned success and name by the inventor at first to chemokine-like factors.Functional study finds, CKLF1 has the chemotactic activity of wide spectrum and multiplication-stimulating activity, and (referring to international application: PCT/CN00/00026), and CKLF2 is the full gene product of CKLF, 152 amino acid of encoding.On the basis of above-mentioned CKLF, the inventor has obtained some new cytokines again successively.

Summary of the invention

An object of the present invention is to provide a kind of polynucleotide.

Another object of the present invention provides a kind of carrier that contains polynucleotide of the present invention.

Another object of the present invention provides a kind of host cell that contains carrier of the present invention.

Another object of the present invention provides a kind of method of producing polypeptide of the present invention.

Another object of the present invention provides a kind of polynucleotide passage of the present invention.

Another object of the present invention provides a peptide species.

Another object of the present invention provides a kind of pharmaceutical composition, this pharmaceutical composition contains polynucleotide of the present invention or the acceptable salt of its medicine, carrier, host cell or polypeptide or the acceptable salt of its medicine, and the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle.

Another object of the present invention provides polynucleotide of the present invention or polypeptide and prevents and/or treats application in the medicament of immune deficiency, hematopoietic disorder or acquired immune deficiency syndrome (AIDS) in preparation.

Another object of the present invention provides the method whether expression level that detects polynucleotide of the present invention in the testing sample changes.

Another object of the present invention provides a kind of mono-clonal or polyclonal antibody, and it has antigenic fragments specific with polypeptide of the present invention or its and combines.

The invention provides a kind of polynucleotide, these polynucleotide comprise:

The polynucleotide of the 198-248 amino acids sequence of (1) polynucleotide of coding aminoacid sequence shown in SEQ ID NO:2, or coding shown in SEQ ID NO:2; Or

(2) polynucleotide that have at least 80% homology with (1), the polypeptide of this polynucleotide encoding has identical functions with (1) encoded polypeptides.

As the described aminoacid sequence of SEQ ID NO:2 is the member of chemokine-like factor superfamily (chemokine-like factor superfamily), and this paper is defined as CKLFSF2, is positioned 16q22.1.The aminoacid sequence of CKLFSF2 shown in SEQID NO:2,248 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 94-111,116-135,142-116,179-198.This albumen of SignalP analysis revealed does not have tangible signal peptide.CKLFSF2 homology with other member of CKLFSF on the albumen primary sequence is not high, but it is similar to most CKLFSF members, have potential tetratransmembrane structure and MARVEL structural domain, and caveolin binding domains (caveolin-binding domain) and AR binding motif (AR-binding motif), point out them on function, to have dependency.

The inventor utilizes DNAstar software that CKLFSF2 wetting ability and antigenicity are analyzed, show at the CKLFSF2 two ends and have stronger antigenicity, therefore the present invention has studied fragment and the function thereof of CKLFSF2 for example with 51 amino acid of carboxyl terminal (CKLFSF2 C51, the 198-248 amino acids sequence shown in the SEQ ID NO:2).

Polynucleotide sequence of the present invention can only the encode polypeptide shown in the SEQ ID NO:2 or its fragment, also can be on the basis of aforementioned polypeptides or its segmental encoding sequence, increase non-coding sequence, for example intron, encoding sequence 5 ' or the non-coding sequence of 3 ' end etc.Polynucleotide sequence of the present invention preferably provides with unpack format.Polynucleotide of the present invention are " separation " forms, and it not only separates with the protein of following it in cell, and the sequence that is arranged in its both sides under native state is separated.

Polynucleotide sequence of the present invention can be DNA or RNA, and wherein DNA comprises cDNA, genomic dna and synthetic DNA, and DNA can be two strands or single stranded form, and single stranded DNA can be coding strand or noncoding strand (antisense strand).Antisense strand of the present invention can be the complementary sequence of the sequence shown in SEQ ID NO:1.Those of ordinary skills are known, and antisense strand or its part (antisense oligonucleotide) can be used for suppressing the expression of CKLFSF2 of the present invention in the cell.The nucleotide sequence of CKLFSF2 of the present invention can comprise ox, sheep, pig, mouse, horse from any species, particularly Mammals, and is preferred human.

The present invention also comprises having with coding CKLFSF2 or its segmental polynucleotide to have 70%, 80%, 85% at least, and is better 90%, the polynucleotide sequence of best 95% homology.Be particularly related under stringent condition the polynucleotide with the multi-nucleotide hybrid of CKLFSF2, said " stringent condition " means the prerequisite that hybridization takes place is to possess 95% homology between sequence at least.Such sequence can be natural existence or artificial the generation, can comprise the allelic variation body of CKLFSF2 polynucleotide sequence, also can comprise disappearance, insertion and the displacement of base in the CKLFSF2 polynucleotide sequence.The polypeptide of such sequence encoding can be identical with CKLFSF2 of the present invention on function, similar or different, but preferably encode and the essentially identical polypeptide of CKLFSF2 biologic activity.Therefore, the present invention preferably with the polynucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:2 or the polynucleotide that its fragment has at least 80% homology, the polypeptide shown in the polypeptide of this polynucleotide encoding and the SEQ IDNO:2 has identical functions.

Polynucleotide of the present invention more preferably comprise polynucleotide sequence or its complementary fragment of the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence shown in SEQ ID NO:1 derives from people's TESTIS cDNA library, 1065 Nucleotide of total length, Nucleotide 153-899) and 5 ' non-coding region (Nucleotide 1-152) and 3 ' non-coding region (Nucleotide 900-1065) comprise coding CKLFSF2 proteic sequence, (encoding sequence (CDS) for example:.Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CKLFSF2 protein sequence, for example encoding sequence shown in the SEQ ID NO:1: Nucleotide 153-899.The accession number that the nucleotides sequence of the present invention shown in SEQ ID NO:1 is listed among the Genbank is: AF479260.

The fragment of the polynucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 also within the scope of the invention, polynucleotide or its complementary sequence of described fragment shown in the 752-904 position of SEQ ID NO:1.The polypeptide of described fragment coding has the polypeptide identical functions with the sequence encoding shown in the SEQ ID NO:1.

The present invention also provides a kind of polynucleotide or its segmental engineering carrier that contains code book invention CKLFSF2 polypeptide.Described engineering carrier can be general carrier, expression vector etc.Wherein general carrier is mainly used in the foundation in range gene group library and cDNA library, they contain two or more marker gene usually, one of them gene is used to select transformant (transformant), and another gene then is to be used for checking whether carrier has foreign DNA to insert.Expression vector is mainly used in the research expression of gene or is used for mass production some useful transcription product or protein, the foundation that also can be used for the cDNA library that has.This class carrier should contain suitable promotor, ribosome bind site, terminator etc. in the expression vector except that the feature with coventional type carrier.Locate in cell for the ease of expression product, can add suitable leader sequence in the polypeptid coding sequence upstream.Suitable carrier and promotor to be chosen as those of ordinary skills known.Those of ordinary skills are known to be used to make up and to contain polynucleotide of the present invention and suitable transcribing and the method for the carrier of translational control element.Specifically, but be applicable to that the commercially available expression vector of prokaryotic cell prokaryocyte generally all has selection marker and cellular replication initial point, have bacterium promotors such as lacI, T7, λ PL and trp, and other genetic elements of known cloning vector pBR322 (ATCC 37017).Commercially available carrier like this comprises pGEM (Promega) and pKK223-3 (Pharmacia).Can select to be derived from the suitable carrier of pBR322 according to selected suitable promotor and structural gene sequence to be expressed.The GST prokaryotic expression system also can be used for the present invention.Be applicable to that eukaryotic carrier has eukaryotic cell promotor such as CMV, SV40 etc., such carrier comprises pMT-hIL-3 (horse big dragon, Di Chunhui, Pang Jian etc. (1991) hi-tech communication 11:26-29), pQE-9 (Qiagen), pD10, pNH18A (Stratagene), pKK233-3, pDR540, pRIT5 (Pharmacia), and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL (Pharmacia).(referring to embodiment 3) in one embodiment of the invention, CKLFSF2 carboxyl terminal 51 amino acid (CKLFSF2C51) stronger with antigenicity are example, make up sequencing vector of the present invention and expression vector.Wherein, CKLFSF2 C51 nucleotide sequence is cloned in the pGEM-T easy plasmid (Promega) and carries out the purifying order-checking; The pGEX-4T-3 carrier (Amersham-Pharmacia Biotech company) that adopts EcoRI to handle makes up the expression vector pGEX-4T-3-CKLFSF2 C51 that the present invention carries CKLFSF2 C51.In embodiment 5, with the complete open reading frame of CKLFSF2cDNA, enzyme is cut product and is connected in the pcDNA3.1B carrier (Ivitrogen company) that EcoRI, Cip handle, and obtains pcDNA3.1B-CKLFSF2.In addition, in embodiment 10, also with the cDNA fragment cloning of the complete open reading frame of CCR4 and CCR5 in the pcDI carrier, make it effective expression in HEK 293 cells (ATCC CRL-1573).

The present invention provides a kind of host who is suitable for expressing polypeptide of the present invention simultaneously, and this host includes but not limited to: prokaryotic hosts, such as intestinal bacteria, bacillus, streptomyces etc.; Eucaryon host, such as: yeast belong, Aspergillus, insect cell, such as fruit bat S2 and fall army worm Sf9; Zooblast can be expressed the clone of compatible carrier as CHO, COS (monkey kidney fibroblast, Gluzman (Cell 23:175,1981)) and other.The known construct that will contain polynucleotide of the present invention of those skilled in the art imports the method for above-mentioned host cell, include but not limited to: transfection, electroporation, microinjection, particle bombardment method or the particle gun method (Sambrook of the conversion of calcium chloride mediation, calcium phosphate transfection, the mediation of DEAE-dextran, 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 ﹠amp; Sons, N.Y.; Hobbs, people such as S., McGraw Hill Yearbook of Science and Technology (1992), McGraw Hill, N.Y.191-196; Engelhard, people such as E.K., PNAS, 91:3224-3227; Logan, people such as J., PNAS, 81:3655-3659).In suitable culture condition and substratum, cultivate through host transformed bacterial strain or cell, it is grown into after the appropriate cell density, induce selected promotor with appropriate means (for example temperature transition or pharmaceutical chemicals are induced), and cell is cultivated for some time again.Character at different host strains or cell and expressed desired polypeptides selects corresponding culture condition and substratum within those skilled in the art's ken.The host cell of Shi Yonging for example in an embodiment of the present invention: intestinal bacteria JM 105, e. coli bl21, HEK293 cell (ATCC CRL-1573) and 293T cell (ATCC CRL-11268) etc.

The present invention also provides a kind of method of producing polypeptide, and it may further comprise the steps:

(1) under the condition that is suitable for expressing, cultivates the acid of multinuclear villous themeda or its segmental host cell of containing code book invention polypeptide;

(2) polypeptide of acquisition polynucleotide encoding from described cell culture.

Specifically, at transformed host cell and after being grown into suitable cell density,, continue then to cultivate with appropriate means (inducing) evoked promoter as temperature variation or chemical speciality by transformed host cells.After cultivation is finished, available centrifuging collecting cell, and with any known method, as freeze-thaw method, supersound process method, N,O-Diacetylmuramidase dissolution method or mechanical crushing method smudge cells.Can reclaim from the host cell culture and polypeptide or its fragment or the fusion polypeptide of purifying CKLFSF2 of the present invention with various known methods, these methods comprise ammonium sulfate or ethanol precipitation, acid extraction method, ultrafiltration process, ion exchange chromatography, phosphoric acid fiber chromatography, hydrophobic interaction chromatography method, gel filtration method, affinity chromatography and high pressure fluid column chromatography.

The fragment of CKLFSF2 polynucleotide sequence of the present invention can also be at least 10 of polynucleotide of the present invention, for example at least 20, the fragment of at least 30, at least 50 continuous nucleotide sequences.These fragments can be used as primer or hybridization probe, are used for detecting coding polynucleotide sequence of the present invention.In addition, the fragment of polynucleotide of the present invention also can be the part as the described polynucleotide of SEQ IDNO:1 or its complementary sequence, and for example antisense oligonucleotide can be used for suppressing the expression of CKLFSF2 of the present invention in the cell.As for these fragments whether coded polypeptide or encoded polypeptides whether have the function of polypeptide of the present invention, for detect, hybridization and/or to suppress to express these purposes be not particularly important.

On the other hand, the invention provides a peptide species, this polypeptide comprises:

(1) aminoacid sequence shown in SEQ ID NO:2, or the polypeptide shown in the 198-248 amino acids of SEQ ID NO:2;

(2) polypeptide that has at least 80% homology with (1), the function of this polypeptide is identical with the function of (1).

Wherein the aminoacid sequence of CKLFSF2 is shown in SEQ ID NO:2,248 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 94-111,116-135,142-116,179-198.The fragment of sequence shown in the SEQ ID NO:2 of the present invention also within the scope of the invention, sequence shown in the 198-248 amino acids of SEQ ID NO:2.This sequence is identical with the function of full length sequence shown in the SEQ ID NO:2 of the present invention, but because it is shorter than full length sequence, so it is more convenient to operate.Polypeptide of the present invention also comprises and the aminoacid sequence shown in SEQ ID NO:2, or the homology (sequences match) of sequence surpasses 80% shown in the 198-248 amino acids of SEQ ID NO:2, preferably surpass 85%, more preferably surpass 90%, further preferably surpass 95%, especially preferably surpass 98%, further especially preferably surpass 99% sequence.These sequences can have identical or different function with the sequence shown in the SEQ ID NO:2 of the present invention, preferably have identical functions.

CKLFSF2 polypeptide of the present invention or its fragment can be that natural, synthetic, semisynthetic or reorganization produce.CKLFSF2 polypeptide of the present invention can biological engineering method routinely produce polypeptide product (specifically referring to embodiment) by the coding of the recombinant DNA sequence in the host cell.Also can be according to Steward and Young (Steward, J.M. and Young, J.D., Solid PhasePeptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, I11., (1984)) method described press the solid state chemistry technology with Applied Biosystem synthesizer or PioneerTM peptide synthesizer and synthesizes, perhaps can use the mRNA that is derived from DNA construct of the present invention, in cell free translation system, produce required polypeptide.

Those of ordinary skills are known, and polypeptide of the present invention or its fragment can form fusion polypeptide with other polypeptide or its fragment.Other polypeptide or its fragment generally are known, and some can be bought with carrier format and obtain, and perhaps can synthesize according to a conventional method or clone from the known organism body to obtain.Gene of the present invention, perhaps the nucleotide sequence of various dna fragmentations can be used ordinary method, as dideoxy chain termination (people such as Sanger, PNAS, 1977,74:5463-5467) measure.This class nucleotide sequencing is available commercial sequencing kit etc. also.

The invention provides a kind of pharmaceutical composition, it contains polynucleotide, carrier, host cell or polypeptide or the acceptable salt of its medicine that the present invention treats significant quantity; Pharmaceutically acceptable in case of necessity carrier or vehicle.

In described pharmaceutical composition, can contain CKLFSF2 polynucleotide or its function fragment (fragment that has identical function with it), the carrier that contains it, the host cell that contains it or polypeptide that the present invention treats significant quantity, also can use with the acceptable salt form of the medicine of described polynucleotide or polypeptide." the acceptable salt of medicine " refers to be suitable for contact with human or animal's tissue, and does not have the salt of too much toxicity, stimulation, transformation reactions etc.The acceptable salt of medicine of the present invention is the conventional component of this area pharmaceutics.This salt can be in the final separation of polypeptide of the present invention and the process of preparing of purifying, also polypeptide and suitable organic or inorganic acid or alkali reaction can be prepared separately.Pharmaceutically acceptable carrier or vehicle refer to nontoxic solid-state, semi-solid state or liquid weighting agent, thinner, coating material or other pharmaceutical adjuncts.

The invention also discloses polynucleotide of the present invention or polypeptide (comprising its function fragment) and prevent and/or treat application in the medicine of immune deficiency, acquired immune deficiency syndrome (AIDS) or hematopoietic disorder in preparation.

The contriver utilizes pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant and recombinant human CKLFSF2 C51 albumen to carry out functional study, and the chemotactic experimental result of mouse boosting cell shows that CKLFSF2 has stronger chemotactic activity; The visible recombinant C KLFSF2 C51 of microscopically promotes the adhesion of Turnover of Mouse Peritoneal Macrophages, Flow cytometry is found, CKLFSF2 can raise the expression of Turnover of Mouse Peritoneal Macrophages surface adhesion molecule CD11b, and prompting CKLFSF2 plays a role in immunne response; So CKLFSF2 polypeptide or polynucleotide or their function fragment can be used for the treatment of immune deficiency.

On this basis, in the calcium current experiment, find that CKLFSF2 can make transfection have the HEK293 cell (HEK293/CCR5) of CCR5 that Ca takes place ²⁺Discharge, the effect that pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant can seal CCR5 part CCL5/RANTES confirms that CCR5 is one of CKLFSF2 functional receptor; CCR5 is that HIV-1 infects the necessary co-receptor of mononuclear macrophage, so CKLFSF2 combines meeting inhibition HIV-1 and combines with CCR5 with CCR5, and then suppresses HIV-1 and infect.Therefore, polypeptide of the present invention or polynucleotide or its function fragment also can be used for treating acquired immune deficiency syndrome (AIDS).

Mtt assay and marrow get rid of sheet dyeing and find that CKLFSF2 has the function that promotes bone marrow cells in mice propagation.In experiment in vitro, MTT result shows that CKLFSF2 promotes the propagation of bone marrow cells in mice.Medullary cell gets rid of the bone marrow cells in mice that the former nucleoprotein of sheet cleer and peaceful CKLFSF2 C51 on the visible CKLFSF2 eukaryotic cell transfection of microscopically handles and obviously bigger cell occurs, cleer and peaceful PBS no abnormality seen then in the control group pCDNA3.1B transfection.Thus, polypeptide of the present invention or polynucleotide or its function fragment can be used in the treatment hematopoietic disorder, comprising illnesss such as primary or Secondary cases hematopoietic disorders.

CKLFSF2 of the present invention is a small protein, can be produced by various kinds of cell such as reproductive system and hemopoietic system cell, white corpuscles; Though CKLFSF2 does not have typical signal peptide sequence, it exists secretion or soluble form; CKLFSF2 has multiple functions such as chemotaxis, immunostimulant and hematopoiesis promotion; CCR5 is one of its functional receptor.Therefore, CKLFSF2 has possessed the 26S Proteasome Structure and Function characteristics of cytokine.

The method whether invention also provides polynucleotide of the present invention in the sample of a kind of vitro detection from person to be measured or polypeptide expression level to change, this method comprises: (1) detects polynucleotide or polypeptide expression level described in the testing sample; (2) polynucleotide or the polypeptide expression level with polynucleotide described in the testing sample or polypeptide expression level and normal specimens compares; (3) determine in the testing sample whether polynucleotide or polypeptide expression level change.

Described normal specimens can obtain from known not ill normal people's cell, and what this cell should be with the testing sample cell is tissue-derived consistent; The expression level of the polynucleotide of normal specimens can be the expression level from the polynucleotide with statistical significance of described normal people's cell acquisition.The method of polynucleotide level can be above-mentioned any detection method in the wherein said detection testing sample, preferably utilizes RT-PCR to detect the expression level of described polynucleotide in nucleic acid level; Or utilize specific monoclonal or polyclonal antibody to detect the expression level of described polynucleotide at protein level, for example immunohistochemistry detects.Described testing sample can obtain Tathagata autoblood, urine, saliva, gastric juice, hair, the cell of examination of living tissue and necrotomy material from the cell from the experimenter.Be preferably person's to be measured testis tissue sample or seminal fluid sample.

In specific embodiments of the invention, utilize anti-CKLFSF2 polyclonal antibody, in the organization chip that comprises 6 kinds of tissues, normal testis tissue and tumor of testis organization chip, detected the expression of CKLFSF2, immunohistochemical results suggest is not seen positive expression at the healthy tissues and the tumor tissues of mammary gland, lung, stomach, colon and rectum, only has than strong positive in the keratinocyte of normal esophagus epithelium and expresses; CKLFSF2 can be secreted in the seminiferous tubule in the normal testis tissue; And in tumor of testis, seminomatous CKLFSF2 expresses abundance apparently higher than healthy tissues, mainly is present in cytoplasm, cytolemma and nuclear membrane, and it is lower to express abundance in testis B lymphoma and the big B lymphoma.This is consistent with our result of expression pattern analysis, and the express spectra of explanation CKLFSF2 is narrower on protein level, has natural secreted form; Compare with the expression of CKLFSF2 in the normal testis tissue, in different tumor of testis tissues, the expression level of CKLFSF2 and cellular localization all have obvious change, prompting CKLFSF2 not only participates in normal production of sperm process, and has vital role in the generation of male reproductive system tumour and development.

The present invention also provides has specific polyclone and monoclonal antibody to CKLFSF2 polypeptide of the present invention or its fragment." specificity " described here is meant that antibody capable is incorporated into CKLFSF2 gene product of the present invention or fragment.Preferred those can combine with the gene product of CKLFSF2 of the present invention but nonrecognition not with other irrelevant antigen molecule bonded antibody.Among the present invention antibody comprise those can in conjunction with and suppress the antibody of CKLFSF2 gene product of the present invention, comprise that also those do not influence the antibody of CKLFSF2 polypeptide function of the present invention.Above-mentioned antibody not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can prepare by the known the whole bag of tricks of those of ordinary skills.For example, the CKLFSF2 polypeptide gene product of the present invention of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.(referring to embodiment 5) in one embodiment of the invention, with stronger 51 amino acid of CKLFSF2 carboxyl terminal of antigenicity (the aminoacid sequence 198-248 of SEQ ID NO:2 (being called for short CKLFSF2 C51)) is example, the preparation polyclonal antibody, detect antibody titer through the ELISA method, Western blot identifies antibodies specific, confirmation obtain tiring height, antibody that specificity is good can be further used for this antibody expression pattern analysis and the functional study of CKLFSF2.Antibody of the present invention can also be monoclonal antibody, and it can utilize the hybridoma technology preparation (to see people such as Kohler, Nature 256:495; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Hammerling, In Monoclonal Antibodies and T cell Hybridaomad, Elsevier, N.Y., 1981).Each antibody-like of the present invention can utilize CKLFSF2 gene product of the present invention or its fragment or functional zone, obtains by the routine immunization technology, and these fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of the gene product of CKLFSF2 polypeptide of the present invention; With posttranslational modification form bonded antibody (as the polypeptide or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

Description of drawings

The distribution of CKLFSF2 mRNA in tissue analyzed in Figure 1A and Figure 1B demonstration by experiment.Wherein, 1: negative control; 2: colon; 3: brain; 4: marrow; 5: ovarian cancer; 6: colorectal carcinoma; 7: mammary cancer; 8: fetal thymus; 9: the fetus spleen; 10: fetus muscle; 11: fetus liver; 12: fetal kidney; 13: heart of fetus; 14: thymus gland; 15: tonsilla; 16: testis; 17: spleen; 18: skeletal muscle; 19: placenta; 20: ovary; 21: lymphoglandula; 22: liver; 23: white cell; 24: kidney; 25: heart.

Fig. 2 shows the distribution of CKLFSF2 mRNA in tranquillization and activatory hemocyte, wherein, and 1: monocyte (B-, T-cell, and monocyte); 2: the CD4+ cell of tranquillization (T-helper/induce); 3: activation CD4+ cell; 4: the CD8+ cell of tranquillization (T-suppresses cell/cytotoxicity); 5: activation CD8+ cell; 6: the monocyte of tranquillization; 7: activated monocyte; 8: the CD19+ cell of tranquillization (B-cell); 9: activation CD19+ cell;-: negative control; +: positive control.

Fig. 3 is the structure synoptic diagram of prokaryotic expression plasmid pGEX-4T-3-CKLFSF2C51.

Fig. 4 shows that SDS-PAGE analyzes expression and the purification result of CKLFSF2.Wherein, 1: inductive E.coli BL21/CKLFSF2 C51 not; 2: inductive E.coli BL21/CKLFSF2 C51; 3: with the CKLFSF2 C51 behind gsh-sepharose 4B purifying; 4: the GST contrast of purifying.

Fig. 5 shows with the monoclonal antibody of anti-GST and identifies GST-CKLFSF2 C51 fusion rotein result, wherein, and 1: inductive E coli.BL21/CKLFSF2 C51 not; 2: inductive E coli.BL21/CKLFSF2 C51.

Fig. 6 shows that Western blot identifies anti-CKLFSF2 antibodies specific result, wherein, and A: with anti--CKLFSF2 antibody test; B: with anti--Myc antibody test, as positive control; 1: contrast pcDNA3.1 transfection supernatant; 2:pcDNA3.1-CKLFSF 2 transfection supernatants; 3:pcDNA3.1-CKLFSF 2/Myc-His6 transfection supernatant.

The expression of Fig. 7 display organization chip detection CKLFSF2 in multiple tissue, wherein, A: normal esophageal tissue's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); B: esophagus cancer tissue (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); C: NBT's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); D: breast cancer tissue's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); E: normal lung tissue's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); F: cancerous lung tissue (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); G: normal gastric mucosa (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); H: stomach organization (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); I: normal colonic tissue's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); J: colon cancer tissue (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); K: normal rectal tissue (last figure is 10 multiplying powers, and figure below is 40 multiplying powers); L: rectum cancer tissue's (last figure is 10 multiplying powers, and figure below is 40 multiplying powers).

Fig. 8 shows that immunohistochemical methods detects the expression of CKLFSF2 in the normal testis tissue, wherein, uses two anti-contrast (A:10 multiplying powers; The B:40 multiplying power), use normal rabbit igg contrast (C:10 multiplying power; The D:40 multiplying power) and use CKLFSF2 specific polyclonal antibody (E:10 multiplying power; The F:40 multiplying power).

Fig. 9 shows that the tumor of testis organization chip detects the expression of results of CKLFSF2, and 40 multiplying powers are used the CKLFSF2 specific polyclonal antibody, A: spermocytoma (blood plasma); B: spermocytoma (barrier film and blood plasma); C:B cell lymphoma (+); D:B cell lymphoma (-); E: large B cell lymphoid tumor (+); F: large B cell lymphoid tumor (-); G: moderate atrophy testis tissue; H: normal testis tissue.

Figure 10 shows the chemotaxis of pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant to mouse boosting cell, wherein, and (A, C, E, G:10 multiplying power; B, D, F, H:40 multiplying power) A, B:CKLFSF2 transfection supernatant; C, D: dilute 5 times CKLFSF2 transfection supernatant; E, F: dilute 25 times CKLFSF2; G, H:pCDNA3.1B transfection supernatant.

Figure 11 is for showing the column synoptic diagram of CKLFSF2 C51 to the chemotaxis of mouse boosting cell.

Figure 12 shows CKLFSF2 C51 to the adherent influence of Turnover of Mouse Peritoneal Macrophages, wherein, and the Turnover of Mouse Peritoneal Macrophages that A:CKLFSF2 C51 handled; The Turnover of Mouse Peritoneal Macrophages that B:GST handled, in contrast; C: the Turnover of Mouse Peritoneal Macrophages that Thrombin treatment is crossed, in contrast.

Figure 13 shows the expression of Flow cytometry Turnover of Mouse Peritoneal Macrophages CD11b, wherein, and A:PBS; B:pCDNA3.1B transfection supernatant; C:CKLFSF2 transfection supernatant; D:PBS; E:PBS (comprising zymoplasm); F:CKLFSF2 C51.

Figure 14 A and Figure 14 B are that mtt assay detects the short proliferation function interpretation of result figure of CKLFSF2 to bone marrow cells in mice, wherein, and Figure 14 A:CKLFSF2 and pCDNA3.1B transfection supernatant; Figure 14 B:CKLFSF2 C51 and PBS (contrast).

Figure 15 shows the short proliferation function of CKLFSF2 to bone marrow cells in mice, wherein, and A:pCDNA3.1B transfection supernatant; B:CKLFSF2 transfection supernatant; The C:PBS contrast; D:100ng/L CKLFSF2C51.

Figure 16 shows pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant inducing cell Ca ²⁺Discharge to reach CCL5/RANTES is induced Ca ²⁺The sealing process that discharges is to utilize laser scanning confocal microscope to detect Ca in the cell ²⁺The variation of stream.Wherein, cleer and peaceful 100nM CCL5/RANTES induces HEK293/CCR5 in the A:PCDNA3.1B transfection; Cleer and peaceful 100nM CCL5/RANTES induces HEK293/CCR5 in the B:CKLFSF2 transfection; Cleer and peaceful 100nM CCL5/RANTES induces HEK293/PCDI in the C:PCDNA3.1B transfection; Cleer and peaceful 100nMCCL5/RANTES induces HEK293/PCDI in the D:CKLFSF2 transfection.

Figure 17 shows CKLFSF2 C51 inducing cell Ca ²⁺Discharging, is to utilize laser scanning confocal microscope to detect Ca in the cell ²⁺The variation of stream.Wherein, A:PBS and 100nM CCL5/RANTES induce HEK293/CCR5; B:CKLFSF2 C51 and 100nM CCL5/RANTES induce HEK293/CCR5; C:PBS and 100nM CCL5/RANTES induce HEK293/PCDI; D:CKLFSF2 C51 and 100nM CCL5/RANTES induce HEK293/PCDI.

Figure 18 shows that cleer and peaceful 100nM CCL5/RANTES induces HEK293/CCR5 cell Ca in the pCDNA3.1B transfection ²⁺Discharge.

Figure 19 shows that pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant induces HEK293/CCR5 cell Ca ²⁺Discharge to reach CCL5/RANTES is induced Ca ²⁺The sealing process that discharges.

Figure 20 shows that CCL5/RANTES induces HEK293/CCR5 Ca ²⁺Discharge to reach CKLFSF2 is induced Ca ²⁺The sealing process that discharges is to utilize laser scanning confocal microscope to detect Ca in the cell ²⁺The variation of stream, wherein, A: handle then with 100nMCCL5/RANTES earlier and handle HEK293/CCR5 with 100nM CCL5/RANTES; B: handle then with 100nMCCL5/RANTES earlier and handle HEK293/CCR5 with CKLFSF2 C51.

Figure 21 shows that CCL5/RANTES induces HEK293/CCR5 cell Ca to pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant ²⁺The sealing process that discharges.

Figure 22 shows that cleer and peaceful CCL17/TARC induces HEK293/CCR4 cell Ca on the pCDNA3.1B-CKLFSF2 eukaryotic cell transfection ²⁺Discharging, is to utilize laser scanning confocal microscope to detect Ca in the cell ²⁺The variation of stream, wherein, cleer and peaceful 100nM CCL5/RANTES induces HEK293/CCR4 on the A:PCDNA3.1B eukaryotic cell transfection; Cleer and peaceful 100nM CCL5/RANTES induces HEK293/CCR4 on the B:CKLFSF2 eukaryotic cell transfection.

Embodiment

The splicing and the amplification of embodiment 1:CKLFSF2 full length cDNA sequence

1, the EST of CKLSF2 full-length cDNA splicing:

Utilize CKLF2 protein sequence (Genbank accession number: AF135380) to Protein Data Bank (blastp of NCBI) and people's expressed sequence tag (Expression Sequence Tag, abbreviation EST) carries out data retrieval and homology relatively, find many group est sequences (seeing Table 1), carry out the EST splicing by EST Assembly machine (network address is http://www.tigem.it), form contig (contig) and carry out the BLAST splicing once more, find complete single open reading frame.Obtain people chemokine-like factor super family 2 (CKLFSF2) full length cDNA sequence (referring to SEQ ID NO:1).

The est sequence of CKLFSF2 full length cDNA sequence splicing

BG826826，AA778552，BG212551，AI201364，AW663435，AI223025

2.PCR amplification

The full length cDNA sequence of the CKLFSF2 that splicing obtains according to EST designs special primer, and sequence is

P1 5’-TCGGTGGTCCTGGCAGTTAG-3’(-143～-124)(SEQ ID NO：3)

P2 5’-CAAGCAAAGGTGGTAATGAAAATG-3’(845～868)(SEQ ID NO：4)

Make template with people's testis tissue strand cDNA library that Multiple Tissue cDNA Panels test kit (BD Biosciences Clontech company) provides, carry out pcr amplification, amplification condition is as follows: 94 ℃, and 2 minutes; 94 ℃, 15 seconds, 59 ℃, 15 seconds, 72 ℃, 30 seconds, 35 circulations; 72 ℃, 7 minutes.

The detection of embodiment 2:CKLFSF2mRNA in multiple tissue and clone

Above-mentioned information science analysis provides reference for CKLFSF2 expression situation, analyzes and verifies with experiment below.

1. the extraction of cell total rna

The TRIZOL of the extraction and application GIBCO-BRL company of total RNA ^TMReagent.Getting well-grown various kinds of cell is, in the culture dish of 60mm, add 1ml TRIZOLTM (Gibico company) reagent, blow and beat smudge cells repeatedly, transfer to 1.5ml and do not have in the Eppendorf pipe of RNA enzyme, room temperature leaves standstill and adds the 0.2ml chloroform after 5 minutes, fierce vibration is after 15 seconds, 12000rpm in 4 ℃ centrifugal 15 minutes, collect supernatant, with 0.7 times of volume isopropanol precipitating, 70% ethanol is washed one time, and after the drying at room temperature, total RNA is suspended from the H that DEPC handles again ₂Among the O, be used for reverse transcription after spectrophotometer (Beckman 640) is quantitative.

2. the synthesizing single-stranded cDNA of reverse transcription

Utilize the SMART of CLONTECH company ^(TM)Powerscript reversed transcriptive enzyme that the RACE test kit provides and conversion primer (switching primer) carry out reverse transcription, synthesizing single-stranded cDNA.The total RNA of 5 μ g, oligo (dT) (10 μ M) 1 μ l, SMARToligo (10 μ M) 1 μ l adds H ₂O to 5 μ l reaction volume, mixing, 70 ℃ 2 minutes, fast the ice bath cooling is 2 minutes, of short duration centrifugal after, add the synthetic damping fluid (First Strand synthesis Buffer) of 2 μ l, 5 * the first chains, 1 μ l 10mM dNTP Mix, 1 μ lH ₂O, 1 μ l Powerscript reversed transcriptive enzyme behind the mixing, places 42 ℃ of airbath incubators gently, reacts 90 minutes, and ice bath stops the first chain cDNA building-up reactions.Synthetic back cDNA first chain is frozen in-80 ℃ and preserves or directly carry out the PCR reaction.

3.PCR amplification

The cDNA of the various kinds of cell system that the multiple tissue cDNA that provides with Multiple Tissue cDNA Panels test kit (BD Biosciences Clontech company), the multiple hemocyte cDNA that Human Blood Fractions cDNA Panels test kit (BD Biosciences Clontech company) provides and above-mentioned reverse transcription obtain is a template, the design primer, sequence is as follows:

P3 5’-AGTCATGGCACCTAAGGCGG-3’(-4～16)(SEQ ID NO：5)

P4 5’-CCTCCAAGTCATTTCTTTCCC-3’(735～755)(SEQ ID NO：6)

Carry out the pcr amplification first time, amplification condition is as follows: 94 ℃, and 2 minutes; 94 ℃, 15 seconds, 59 ℃, 15 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃, 7 minutes.

With hemocyte and 10 times of dilutions of clone pcr amplification product, get 1 μ l as template, the same pcr amplification second time that carries out of other condition.The PCR product carries out agarose gel electrophoresis.

4.CKLFSF2mRNA the distribution in tissue

Adopting has been the standardized in advance multiple tissue cDNA of multiple house-keeping gene library (Multiple Tissue cDNAPanels), detects the distribution situation of CKLFSF2 mRNA in tissue.The result as can be seen from the figure, just is followed successively by testis, marrow and white corpuscle according to expressing abundance referring to Figure 1A and Figure 1B, in addition, at placenta, ovary, tire liver and brain a small amount of expression is arranged also, and other organizes the expression that does not detect CKLFSF2.

The result is consistent with bioinformatic analysis, and visible CKLFSF2 mainly is expressed in reproduction, immunity and hemopoietic system, and the present invention will be based on this, further studies the function of CKLFSF2.

5.CKLFSF2 the expression of mRNA in tranquillization, activated leukocyte cell

CKLFSF2 is high expression level in peripheral blood leucocyte, therefore adopt Human Blood Fractions cDNA library (BDBiosciences Clontech company) to increase, see also Fig. 2, be the distribution results of CKLFSF2 mRNA in tranquillization and activatory hemocyte, as can be seen from the figure, in peripheral blood mononuclear cell, T lymphocyte and monocyte, all detect the expression of CKLFSF2 mRNA, then do not see its expression at bone-marrow-derived lymphocyte; Have more the fragment of an about 600bp in the CD8+T lymphocyte of tranquillization and the monocyte of tranquillization, in corresponding active cells, then do not have this band, infer that it may be a kind of varient of CKLFSF2, but the sequence verification of still needing.

6.CKLFSF2 the distribution of mRNA in clone

Extract total RNA of 8 kinds of cells (comprising normal cell system, tumor cell line and human peripheral leucocytes), detect the expression of CKLFSF2 through RT-PCR.Because the expression amount of CKLFSF2 is low, in various cells, increases for the first time and 30 take turns and there is no obvious band; After amplification for the second time, the expression of visible CKLFSF2 in Jurkat, HeLa S3 (human cervical carcinoma cell system), A204 (human rhabdomyosarcoma's clone) and human peripheral leucocytes that Jurkat (human leukemia cell line), PMA stimulate.

CKLFSF2 clone distributes similar with the distribution expression pattern result to the bioinformation prompting, and the clone of positive expression can be used for later gene function and study on regulation.

Embodiment 3:CKLFSF2 C holds the structure and the evaluation of the procaryotic cell expression plasmid of 51 amino acid polypeptides

In order further to understand the function of CKLFSF2, at first its aminoacid sequence is carried out bioinformatic analysis, and according to the information of pointing out, select stronger 51 amino acid of CKLFSF2 carboxyl terminal of antigenicity, be used for the proteic expression of protokaryon, CKLFSF2 C51 can be used for functional study and Antibody Preparation.Because CKLFSF2 C51 and the present invention have identical functions, so it can replace full-length polypeptide of the present invention, prepare pharmaceutical composition of the present invention or are used to prevent and/or treat immune deficiency, acquired immune deficiency syndrome (AIDS) or hematopoietic disorder.

1. bioinformatic analysis

ProtScale software analysis hydrophilic and hydrophobic, the CKLFSF2 hydrophobicity is strong as can be seen; The albumen that hydrophobicity is strong is difficult uses the procaryotic cell expression intact proteins, thus further carry out the antigenicity prediction according to aminoacid sequence, to select the strong part expressing protein fragment of antigenicity; The visible carboxyl terminal of DNAStar software analysis (198-248 amino acid) antigenicity is stronger, and therefore, the present invention has carried out the proteic preparation of CKLFSF2C51 protokaryon.

2.pGEM-Teasy-CKLFSF2 the structure of C51 and order-checking

1) the segmental amplification of CKLFSF2 C51: according to CKLFSF2 cDNA sequences Design the upstream and downstream primer of its C of amplification end 198-248 amino acid (being called for short CKLFSF2 C51), and primer sequence added EcoRI restriction enzyme site and protection base.Dash area is the EcoRI restriction enzyme site and the protection base of design.P5 5′ CCTTCAAAGAAACCACTTCAGAGGC-3′(SEQ ID NO：7)，P6 5′

TATTTCTTTCCCTTTGCTGGCC-3′(SEQ ID NO：8)。

With pcDNA3.1B-CKLFSF2 is template, carries out pcr amplification, and amplification condition is as follows: 94 ℃, and 5 minutes; 94 ℃, 15 seconds, 59 ℃, 15 seconds, 72 ℃, 30 seconds, 25 circulations; 72 ℃, 7 minutes.

2) purifying of PCR product: the PCR product is after the agarose electrophoresis of TAE preparation is separated, downcut the PCR band, the NaI (6M) that adds 3 times of volumes, 45-55 ℃ melts glue, add GLASSMILK suspension 5-10 μ l, on gyroscope, hatch 10min, of short duration centrifugation Glassmilk/DNA mixture, wash 3 times with the DNA washing soln, add H ₂O is in 45-55 ℃ of wash-out.

3) the PCR product cloning is to pGEM-T Easy carrier: 1 μ l, 10 * T4 dna ligase damping fluid, 1 μ l pGEM-TEasy carrier (promega) (50ng), PCR product 25ng, 1 μ l T4 dna ligase (3 Weiss units/ μ l) adds H ₂O to 10 μ l, 4 ℃ of connections are spent the night.Connect product and transform the XL-1Blue bacterium, screening and cloning can adopt blue hickie color reaction.

4) extraction of plasmid and purifying: be used to check order and the Qiagen plasmid purification test kit extraction of the plasmid of transfecting eukaryotic cells.With Qiagen-tip20 is example, operate as follows: choose single bacterium colony and contain in the LB substratum of 50-100 μ g/ml penbritin in 3ml, cultivated 12-16 hour, centrifugal, be resuspended among the 0.3ml damping fluid P1 that contains 100 μ g/ml RNase A, add 0.3ml damping fluid P2, put upside down 4-6 time, room temperature was placed 5 minutes, added the 0.3ml damping fluid P3 of 4 ℃ of precoolings, put upside down ice bath 5 minutes 4-6 time.High speed centrifugation, supernatant adds in the good Qiagen-tip20 post of 1ml damping fluid QBT balance, and QC washes post with the 1ml damping fluid, repeats 4 times, adds 0.8ml damping fluid QF wash-out plasmid, adds the Virahol of 0.7 times of volume, room temperature high speed centrifugation 30 minutes.Abandon supernatant, wash two times with 70% ethanol.Dry air 5 minutes is dissolved in H ₂Among the O.According to the difference of selected column volume, the volume of corresponding expansion damping fluid.

5) order-checking: use ABI PRISM 3100 Sequencer genetic analyzers to carry out determined dna sequence.In the sequencing kit that sequencing reaction provides by company method.

3.pGEX-4T-3-CKLFSF2 the structure of C51 prokaryotic expression plasmid and evaluation

The pGEM-Teasy-CKLFSF2 C51 plasmid that order-checking is correct, cut with the EcoRI enzyme, discharge CKLFSF2 C51 fragment, be connected on the pGEX-4T-3 carrier (Amersham-Pharmacia Biotech company) of EcoRI processing, and guarantee that frameshit does not take place ORF, correctly express CKLFSF2 C51 fusion rotein.The plasmid construction schema as shown in Figure 3.

Connect product transformed into escherichia coli JM105, there is the possibility of two kinds of insertions in CKLFSF2 C51 cDNA, and promptly forward and reverse two kinds of insertions may.Carry out PCR with GST upstream primer and CKLFSF2 downstream primer and identify that agarose electrophoretic analysis obtains the recombinant plasmid that forward connects.After identifying correctly, extract plasmid in order to expressing former nucleoprotein, plasmid called after pGEX-4T-3-CKLFSF2 C51.This prokaryotic expression plasmid can be expressed GST-CKLFSF2 C51 fusion rotein, is used for immune animal, preparation antibody, and functional study.

4.CKLFSF2 proteic expression of C51 protokaryon and Immunological Identification

In order to obtain recombinant human CKLFSF2 C51 albumen, present embodiment is with pGEX-4T-3-CKLFSF2 C51 expression plasmid transformed into escherichia coli BL21, is that the IPTG of 10 μ mmol/L has carried out abduction delivering with final concentration.The result shows that referring to Fig. 4 SDS-PAGE analyzes short run abduction delivering product result, a tangible protein band occurs at estimating position, and relative molecular mass is about 31kD, size and desired value basically identical.

Monoclonal antibody with anti-GST detects Expression of Fusion Protein, and as seen from Figure 5, the fusion rotein of this prokaryotic expression can combine with the GST antibodies specific, is gst fusion protein.

Embodiment 4: reorganization GST-CKLFSF2 C51 Expression of Fusion Protein and purifying

1. the abduction delivering of reorganization GST-CKLFSF2 C51 fusion rotein

Transform pGEX-4T-3-CKLFSF2 C51 plasmid to expression strain E coli.BL21, with primer P5 and P6 the clone is carried out PCR and identify.Select positive colony, cultivate amplification for 37 ℃, to OD ₆₀₀1.5 the adding final concentration is that the IPTG of 100 μ mol/L induced 1 hour for 30 ℃.

2. the purifying of reorganization GST-CKLFSF2 C51 fusion rotein

Induce bacterium with the centrifugal collection of 6000r/min, every 1L induces bacterium to be resuspended in 50-100ml PBS (10mmol/L PB pH7.4,0.14mol/L NaCl), contains the DTT of 5mmol/L.Carrying out ultrasonic bacteria breaking under condition of ice bath is collected the liquid after ultrasonic, mixing, and centrifugal 20 minutes of 4 ℃ of 20000r/min (Beckman JA25.50) collect supernatant, are kept at 4 ℃.Carry out proteic purifying with Glutathione SepharoseTM 4B affinity column material (swelling) at 4 ℃.Carry out pre-equilibration with cold PBS, last column flow rate is 5ml/min, is washed till OD with cold PBS behind the upper prop ₂₈₀To baseline values.Use 50mM Tris HCl then, PH8.0 (reduced glutathion that contains 10mM) carries out wash-out.Eluted protein is carried out Bradford method protein quantification.To can obtain the 5mg fusion rotein behind the method purifying of the colibacillary expression product of 1L with Glutathione-Sepharose 4B affinity chromatography, the GST-CKLFSF2 C51 purity of protein behind this method purifying can reach more than 95%, sees Fig. 4.The albumen of this purity can be used for Antibody Preparation.Sample segment carries out SDS-PAGE and analyzes.

3. the zymoplasm cutting of reorganization GST-CKLFSF2 C51 fusion rotein

Estimate the adsorbed protein content (the roughly adsorbable 20mg albumen of every ml post material) of post material, needed zymoplasm (10U/mg albumen) be diluted among the PBS of proper volume that carry out zymoplasm and cut on pillar, the enzyme time of cutting was generally 1-2 hour.Collect the protein product after enzyme is cut, with the ultra-filtration equipment protein concentrate concentration of suitable big or small molecular weight cut-off to 2-3mg/ml, separate target protein with Sephacryl S-200 gel-filtration column again, purifying protein carries out quantitatively with BCA (Pierce company product) method.

4.GST-CKLFSF2 the Immunological Identification of C51 fusion rotein

Electrophoresis: adopt ordinary method, will contain the expression engineering bacteria abduction delivering of recombinant plasmid, the cracking thalline, supernatant liquor carries out the 15%SDS-PAGE electrophoresis;

Change film: behind the electrophoresis with the albumen electrotransfer to through on the methyl alcohol activatory Bio Trace pvdf membrane (Amersham-Pharmaciabiotech company), use 1 * CAPS electricity to change liquid, 90V, 120 minutes;

Sealing: behind the electrotransfer, film sealed 1 hour with 5% skim-milk (TBST preparation) room temperature;

Adding one resists: add mouse anti human GST monoclonal antibody, in 4 ℃ of reaction overnight;

Adding two resists: use TBST (TBS+0.05% Tween-20) thorough washing 4-6 time, each 10 minutes; Add then HRP (horseradish peroxidase) mark sheep anti-mouse igg (1: 5000, Promega), room temperature effect 1 hour;

Luminous: TBST thorough washing 4-6 time, each 10 minutes; After TBS washing once, use ECL luminescence reagent box, the X-film exposure of luminous back, developing fixing post analysis protein band.

5. recombinant human CKLFSF2 C51 protein sequencing is identified

1L induces the albumen behind the bacterium purifying to remove GST through the cutting of 10U zymoplasm, the CKLFSF2 C51 protein fragments that downcuts carries out the order-checking of N end in Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, it is GSPNSLQRNHFRG that the result records 13 amino acid of N end, consistent with expected sequence.

Recombinant C KLFSF2 C51 fragment is used to carry out functional study.

Embodiment 5: the preparation and the evaluation of anti-GST-CKLFSF2 C51 fusion rotein antibody

One, the preparation of anti-GST-CKLFSF2 C51 fusion rotein antibody

1. immunization protocol

Immunity for the first time: the part rabbit hair, the tincture of iodine and the alcohol disinfecting that cut off rabbit two hind paws.Draw Freund's complete adjuvant (FCA) emulsive 500ug GST-CKLFSF2C51 (1: 1) 1ml fully with the 2ml syringe, the subcutaneous 0.5ml that injects of every batter palm.

Immunity for the second time: behind the At intervals of two to three weeks, subcutaneous 4-6 point injects Freund's incomplete adjuvant (FIA) emulsive antigen 1 ml fully in the both sides, back.At interval after 7-10 days, from ear edge vein exploitating blood 0.5ml, separation of serum, the ELISA method detects serum titer.

Booster immunization: every 2-3 week booster immunization, method is the same.5 immunity reach bloodletting immediately after the requirement to tiring.

2. bloodletting, separation of serum and purifying antibody

The heart depletion method: rabbit faces upward, and four limbs are tied up on the animal anchor.Cut off the left chest rabbit hair, sterilization skin.Left side thumb is touched the strongest position of heartbeat, use the 50ml syringe, tilts 45 °, and thrust the strength of aligning heartbeat, draws blood to rabbit death.

Separation of serum: blood is put into Erlenmeyer flask, and room temperature left standstill 1 hour, put 4 ℃ of refrigerator 3-4 hours again.After treating the blood coagulation blood clot retraction, sucking-off serum, centrifugal 20 minutes of 2000rpm gets supernatant, and 10000rpm is centrifugal 20 minutes again, gets supernatant, adds sanitas (final concentration 0.01% Thiomersalate or 0.02% sodium azide), preserves standby in the packing postposition-20 ℃ refrigerator.

Purifying antibody: part serum is after GST absorbs, and HiTrapTM Protein G affinity chromatography is operated and is stored among the 0.01M PBS by explanation.

Two, the ELISA method detects serum titer

1. method

The bag quilt: CKLFSF2 C51 is diluted to 1 μ g/ml with carbonate buffer solution, according to the amount bag in 100 μ l/ holes by NUNC 96 hole enzyme plates, 4 ℃ of bag quilts that spend the night;

Sealing: (0.1%Tween20 pH7.4) washs 3 times for 10mmol/L PB, 140mmol/L NaCl, and the amount according to 200 μ l/ holes adds confining liquid (3%BSA+PBS) then with PBS-T;

Adding one resists: with PBS-T washing 3 times, every hole adds the immune serum (0.1%BSA+PBS dilution) of 100 μ l multiple proportions, and normal rabbit serum is as negative control, and each sample is established 3 multiple holes, hatches 2 hours for 37 ℃;

Adding two resists: with PBS-T washing 3 times, every hole all adds anti-sheep monoclonal antibody (the Anti-Goat-monoantibody)-HRP (1: 4000 Sigma company) of 100 μ l with the PBS dilution that contains 0.1%BSA, hatches for 37 ℃;

Colour developing and color development stopping: with PBS-T washing 3 times, add 50 μ l OPD colour developing liquid, room temperature lucifuge colour developing 10-15 minute is with the sulfuric acid termination reaction of 25 μ l 2mol/L;

Reading: under the 490nm wavelength, use EL-311SX ELISA Reader reading.

2. result: with the immunizing rabbit with fusion protein behind the purifying, obtain antiserum(antisera), measure tiring of antibody with conventional indirect elisa method.This sero-fast tiring is 1: 10000.

Three, Western blot identifies the specificity of antibody

In order further to determine the specificity of prepared polyclonal antibody, be that antigen carries out Westernblot and detects with the 293T cell pyrolysis liquid of transfection pcDNA3.1B-CKLFSF2 and pcDNA3.1B-CKLFSF2/Myc-His6.

1. method

1) makes up recombinant mammalian expressing vector pCDNA3.1B-CKLFSF2: according to CKLFSF2 cDNA coding region nucleotide sequence, be designed for the complete open reading frame of amplification CKLFSF2 (open reading frame, ORF) primer: upstream primer is that P1 ': 5 '-AAGGACACCGAGTCAGTCATG-3 ' (SEQ ID NO:9) and downstream primer are P2 ': 5 '-TCATTTCTTTCCCTTTGCTGGCC-3 ' (SEQ ID NO:10), with people's TESTIS cDNA library is template, carries out pcr amplification with P1 and P2.Agarose electrophoresis reclaims the PCR product, is connected in the pGEM-Teasy carrier; Connect product transformed into escherichia coli DH5 α; The picking positive colony, order-checking, correct plasmid is cut through the EcoRI enzyme, discharges the CKLFSF2 fragment, and enzyme is cut product and is connected in the pcDNA3.1B carrier (Invitrogen) that EcoRI, Cip handle, and the screening forward inserts the clone, obtains pcDNA3.1B-CKLFSF2.

2) plasmid transfection eukaryotic cell: adopt the calcium phosphate transfection method:

Reagent configuration: 2 * HBS solution: HEPES 50mM; NaCl 280mM; NaHPO ₄3mM; PH7.0-7.1,25 ℃;

Operation steps is that example is summarized as follows with the 35mm plate:

Repopulating cell: 18-24h is with 4 * 10 before the transfection ⁵/ 2ml substratum repopulating cell, the about 50-70% of the cell density when making transfection;

The following solution of configuration before the transfection:

A liquid: 2.5M CaCl ₂10 μ l

DNA 5μg

Add H ₂O to 100 μ l

B liquid: 2 * HBS 100 μ l

Transfection: B liquid dropwise is added drop-wise in the A liquid, mixes, make it to form DNA-Ca-PO while dripping ₄Mixture.Mixture is added drop-wise in the 293T cell culture medium immediately, rocks plate gently, make it mixing;

Cultivate: cell is put into CO ₂Incubator, cell be in 37 ℃, 5%CO ₂Cultivated 5 hours, renew bright culture medium culturing after 48 hours respectively results go up cleer and peaceful cell and be used for next step experiment.

3) total cell pyrolysis liquid preparation:

Collecting cell: calcium phosphate transfection renews bright substratum after 5 hours, continues to cultivate collecting cell 48 hours;

Lysing cell: following operation all on ice or 4 ℃ carry out, all liquid is refrigeration in advance.After the culturing cell results, wash 2 times, add lysis liquid (1 * 10 with PBS ⁷/ ml lysate) (1%Triton X-100 joins among the PBS, with before adding proteinase inhibitor); Soft fully suspension cell is placed in 4 ℃ and constantly shakes abundant cracking 30 minutes on the vertical turntable; Centrifugal then 12,000 * g, 4 ℃, 10 minutes;

Harvested cell lysate: centrifugal supernatant is transferred in the new pipe, is total cell pyrolysis liquid.

4)Western blot：

The cell pyrolysis liquid of transfection empty carrier is made negative control, and the reaction band of anti-Myc antibody and CKLFSF2/Myc-His6 fusion rotein is made positive control, is antigen with the cell pyrolysis liquid, and the Westernblot method is identified CKLFSF2 antibody.

2. result

The result sees also shown in Figure 6,293T cell pyrolysis liquid with transfection pcDNA3.1B-CKLFSF2 and pcDNA3.1B-CKLFSF2/Myc-His6 is that antigen carries out Western blot detection, can form the reaction band, the 293T cell pyrolysis liquid of transfection pcDNA3.1B carrier does not then react with the CKLFSF2 polyclonal antibody, the reaction band of anti-Myc antibody and CKLFSF2/Myc-His6 fusion rotein is as positive control, and the reaction band of CKLFSF2 polyclonal antibody and CKLFSF2/Myc-His6 fusion rotein and positive control are at same position.Point out prepared antibody can with the CKLFSF2 albumen and the CKLFSF2/Myc-His6 fusion rotein generation specific reaction of eukaryotic cell expression.

Embodiment 6: organization chip and testis tissue immunohistochemistry detect

One, method

Embodiment 5 confirms that anti-CKLFSF2 antibody titer height, specificity are good, can be used for detecting and functional study.So present embodiment has carried out the CKLFSF2 expression with this antibody to organization chip, normal testis tissue and the tumor of testis organization chip that comprises 6 kinds of tissues and has detected.Concrete grammar is as follows:

Dewaxing: place dimethylbenzene to soak dewaxing (repeating once) in 20 minutes organization chip, normal testis tissue or tumor of testis tissue paraffin section de;

Take off dimethylbenzene: take off dimethylbenzene respectively at immersion 5 seconds (twice), 90% ethanol (twice) in 100% ethanol again;

Remove peroxidase: after washing with PBS, tissue slice is with 3% H ₂O ₂Hatch 5-10min under the room temperature, to eliminate endogenous Peroxidase activity;

Antigen retrieval: after soaking 5min with the PBS of 0.01M again, carry out antigen retrieval, the beaker of the antigen retrieval liquid that 500ml is housed is put in section, microwave oven heating 15min (maintaining the temperature at 92 ℃-98 ℃) cools off 20-30min under the room temperature;

Sealing: after the PBS balance, the normal sheep serum room temperature sealing with 10% 20 minutes;

Add one anti-: discard sheep blood serum, do not wash, add 1: 100 anti-CKLFSF2 C51 IgG antibody, 37 ℃ 1 hour.Substitute an anti-negative control of doing with normal rabbit igg;

Adding two resists: the PBS (PH 7.4) with 0.01M cleans each 5 minutes 3 times.Two is anti-with goat anti-rabbit antibodies (dilution in 1: 200), 37 ℃ 1 hour;

Colour developing and mounting: with the PBS buffer solution for cleaning of 0.01M 3 times, each 5 minutes, add the DAB colour developing, again with behind the haematoxylin redyeing, mounting is observed and the record result in microscopically.

Two, result

1, the organization chip that comprises 6 kinds of tissues

Present embodiment adopts the antibody of embodiment 5, detected expression and the distribution of CKLFSF2 in 6 kinds of tissues on organization chip; Every kind of tissue includes healthy tissues (n=2) and tumor tissues (n=8).The result does not see positive expression at the healthy tissues and the tumor tissues of mammary gland, lung, stomach, colon and rectum as shown in Figure 7, only has than strong positive to express in the keratinocyte of normal esophagus epithelium, has illustrated that on protein level the express spectra of CKLFSF2 is narrower.

2, normal testis tissue

Information biology among the embodiment 2 and tissue expression spectrum analysis are all pointed out CKLFSF2 high expression level in testis tissue, therefore, present embodiment has detected the expression of CKLFSF2 in the normal testis tissue, the result sees also Fig. 8, mainly be present in the seminiferous tubule with secreted form in healthy tissues, spermatogonium only has a small amount of expression.

3, tumor of testis organization chip

In addition, present embodiment also detects the expression of CKLFSF2 in the tumor of testis organization chip with the antibody among the embodiment 5.This chip comprises tumor tissues (n=60), and spermocytoma (n=42), B cell lymphoma (n=15) and large B cell lymphoid tumor (n=3) are wherein arranged, and also has 2 routine moderate ischemic tissue and 1 routine healthy tissues.

See also shown in Figure 9ly, compare with other tissue, the positive expression that testis tissue is as seen stronger matches with the result of expression pattern analysis.The tumor tissues of testis is different with the expressive site of healthy tissues and existence form, mainly is present in the seminiferous tubule with secreted form at the tissue of healthy tissues and moderate atrophy, and spermatogonium only has a small amount of expression; And spermocytoma expression abundance is higher, mainly is present in endochylema and after birth.

Different tumor type expressions also there are differences.CKLFSF2 has positive expression at seminomatous endochylema, and wherein positive degree is +～++ 41 examples altogether account for 97.6% of spermocytoma sum; Most of spermocytoma after birth also has expression, and wherein positive degree is +～++ 32 examples altogether account for 76.2%.5 routine positive expressions being arranged in endochylema in B cell lymphoma, positive degree is+, account for 33.3%, then there is not positive expression on the after birth.Expression and B cell lymphoma at large B cell lymphoid tumor are similar, 1 routine positive expression in endochylema, positive degree is+, account for 33.3%, do not have positive expression on the after birth, the result is referring to table 1.

The expression of table 1:CKLFSF2 in the tumor of testis tissue

	Cytoplasm	Cytolemma and nuclear membrane
			Spermocytoma (42 example)	±1/42(2.4％) +12/42(28.6％) ++～+++29/42(69％)	-3/42(7.1％) ±7/42(16.7％) +14/42(33.3％) ++～+++18/42(42.9％)
B cell lymphoma (15 example)	-7/15(46.7％) ±3/15(20％) +5/15(33.3％)	-15/15(100％)
			Large B cell lymphoid tumor (3 example)	-2/3(66.7％) +1/3(33.3％)	-3/300％)
Moderate atrophy testis tissue (2 example)	+ seminiferous tubule liquid part spermatogonium
			Normal testis tissue (1 example)	+ seminiferous tubule liquid part spermatogonium

Embodiment 7: the chemotactic experiment

One, method

With pCDNA3.1B and pCDNA3.1B-CKLFSF2 plasmid difference transfection 293T cell, the collecting cell culture supernatant, the supernatant with original content, 5 times of dilutions and 25 times of dilutions carries out the chemotactic experiment to mouse boosting cell respectively.Chemotaxis represents that with chemotactic index so-called chemotactic index is meant: the ratio of the cell count of experimental group migration and the cell count of the non-special migration of control group, in general, chemotactic index is meaningful greater than 2.

48 hole Boyden cell methods are adopted in concrete experiment:

37 ℃ of pre-temperature of transfection supernatant with doubling dilution, the dissolved air is removed in concussion, the bottom plate is discharged water on the flat surface, the NP mark is positioned at the lower right, sample is added in the aperture, and the suitableeest amount of liquid is: after liquid injects aperture, can form a meniscus of protuberance a little, when covering filter membrane, can avoid bubble formation;

At one jiao of little angle that cuts about 1mm of filter membrane, clamp the filter membrane two ends with tweezers respectively, level descends, and the middle part contacts cell at first, lies on the bottom plate that adds sample, and unfilled corner is facing to the NP mark.Adjust the position of filter membrane a little;

Spread silicagel pad successively, load onto top plate, the NP mark of the unfilled corner of silicagel pad and top plate is positioned at the lower right, tightens whole device;

The various cell concns that will be used to do the chemotactic experimental analysis are adjusted to 1 * 10 ⁶/ ml adds an amount of cell suspension, and the amount of liquid of adding can make aperture form a meniscus that swells slightly;

The chemotactic cell that adds sample is placed on 37 ℃, 5%CO ₂Incubator in.Neutrophilic granulocyte was hatched 30 minutes, and lymphocyte and peritoneal macrophage were all hatched 2 hours;

Back out nut, whole cell is inverted, four jiaos (below now being positioned at) of holding in the palm top plate slowly level are placed down on the paper handkerchief, unload lower plywood;

Migrating cell now is positioned at filter membrane one side down, is called the cell face, picks up a jiao of filter membrane with tweezers, clamp the width of this end distance edge 1mm then with big plastic clip, carried filter membrane, clamped another edge with pincet rapidly, adhesional wetting acellular face in filling the plate of PBS;

Cell on flush away acellular face on the cleaning sheet allows cleaning sheet at first contact filter membrane near big clip place, is becoming 30 ° of angular direction to draw on gently with vertical direction.Repeat this process twice;

Carefully filter membrane is immersed in the methyl alcohol, 2 minutes, fixed cell; Cell after fixing is dyeed with Giemsa;

Select the numeration of 5 visuals field at random, average, the cell count of the supernatant group chemotactic of transfection CKLFSF2 is compared with the cell count of transfection pcDNA3.1-Myc-His B (-) vehicle group supernatant group chemotactic, promptly obtain chemotactic index.

Two, result

The result as shown in figure 10, the supernatant chemotactic index that the supernatant chemotactic index that undiluted pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant chemotactic index is 4.6,5 times of dilutions reaches 8.7,25 times of dilutions is 1.2.As can be seen, pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant has obvious chemotactic activity to the cell in the spleen of mouse.

Simultaneously, present embodiment has detected protokaryon PROTEIN C KLFSF2 C51 the splenocyte of mouse has also been had obvious chemotactic activity, and the result sees also Figure 11.

Embodiment 8:CKLFSF2 is to the effect of Turnover of Mouse Peritoneal Macrophages

One, method

1, the observation of situation is obtained and adhered to Turnover of Mouse Peritoneal Macrophages

Mouse is taken off cervical vertebra put to death, portable mouse tail immerses 3-5 second in 70% alcohol with full mouse.Then, put mouse on autopsy table, the syringe needle fixing limbs exposes the abdominal cavity, injects 5ml pre-warm PBS by the upper part with syringe in the abdominal cavity, does not extract syringe needle, gently syringe needle is provoked, and rubs repeatedly the about 1-2 in abdominal cavity minute.Use syringe pumpback peritoneal fluid then, or pick up peritonaeum (syringe needle inserting needle place) gently with tweezers and extract syringe needle, draw peritoneal fluid with sharp suction pipe and put in the clean tube.With peritoneal fluid piping and druming evenly, (avoiding producing bubble) is put in 24 orifice plates as far as possible.37 ℃, hatch 30min.Take out, with RPMI1640 flushing 3 times.The CKLFSF2 C51 that adds eukaryotic cell transfection supernatant or procaryotic cell expression, microscopically is observed the adhesion situation of scavenger cell.

2, the expression of Flow cytometry CD11b

The Xylotox peptic cell, digestion is 3-5 minute in 37 ℃ of incubators, PBS washing 2 times, the anti-CD11b antibody (BD Biosciences Clontech company) that adds the FITC mark, placed 30 minutes in 4 ℃ of lucifuges, the expression of Flow cytometry CD11b is carried out in PBS washing 2 times.

Two, result

1, see also shown in Figure 12ly, the Turnover of Mouse Peritoneal Macrophages that the former nucleoprotein of CKLFSF2 C51 is handled still has good adherent ability under serum-free condition, and cellular control unit then becomes round owing to removing serum, loses adherent ability.

2, detect the expression of peritoneal macrophage CD11b with fluorescently-labeled anti-mouse CD11b monoclonal antibody, the result sees also Figure 13, can find that the expression of the Turnover of Mouse Peritoneal Macrophages CD11b that the former nucleoprotein of cleer and peaceful CKLFSF2 C51 is handled on the CKLFSF2 eukaryotic cell transfection obviously strengthens.

Embodiment 9:CKLFSF2 is to the effect of bone marrow cells in mice

One, method

Collect medullary cell: mouse is put to death in the cervical vertebra dislocation, the bifilar medullary cell of mouse is got in aseptic technique, transfer cell to 5 * 106/ml, get 90 μ l and add in the 96 porocyte culture plates, the transfection supernatant of finite concentration dilution or the protein 10 μ l of procaryotic cell expression are pressed in adding in advance again.If 5 multiple holes are put 37 ℃, 5%CO ₂Incubator was cultivated 72 hours.

Mtt assay detects: stop preceding 5 hours h, mix MTT (5mg/ml) 10 μ l/ holes, when stopping cultivating, the centrifugal supernatant of abandoning, every hole add 100 μ l DMSO, 37 ℃, the 5%CO2 incubator spends the night, and measures its OD value in full-automatic microplate reader wavelength 560nm place, represents the medullary cell activity with the OD value.

Medullary cell gets rid of sheet: the cell of getting 1 hole gets rid of sheet, centrifugal 2000rpm, 2min; Gimsa dyeing.Microscopically is observed.

Two, result

See also shown in Figure 14 A and Figure 14 B, CKLFSF2 is high expression level in marrow, points out it to play a significant role in hematopoiesis, and in experiment in vitro, MTT result shows that CKLFSF2 promotes the propagation of bone marrow cells in mice.

See also Figure 15 simultaneously, medullary cell gets rid of the bone marrow cells in mice that the former nucleoprotein of sheet cleer and peaceful CKLFSF2 C51 on the visible CKLFSF2 eukaryotic cell transfection of microscopically handles and obviously bigger cell occurs, cleer and peaceful PBS no abnormality seen then in the control group pCDNA3.1B transfection.

Embodiment 10: the functional receptor of calcium current experimental identification CKLFSF2

One, method

Existing document shows: CCR5 belongs to sharing Chemokine Receptors (a kind of be subjected to physical efficiency in conjunction with a plurality of members in the family), and its known ligand comprises MIP-1 α, MIP-1 β, CCL5/RANTES, MCP-2 etc.Now clear and definite CCR5 is a co-receptor of having a liking for scavenger cell (M) HIV-1 virus infection human body cell, and therefore, CCR5 is the desired target of antiviral therapy.

Chemokine Receptors CCR4 is one of functional part of CKLF1, and CKLFSF2 and CKLF close linkage on karyomit(e), which kind of Chemokine Receptors to come functionating in order further to inquire into CKLFSF2 by, present embodiment adopts pCDI, pCDI-CCR4 and pCDI-CCR5 transient transfection HEK293 cell respectively, express CCR4 and CCR5, use the calcium current experiment and analyze.

Applying flow cytometry detects CCR4 and the CCR5 expression at the HEK293 cell respectively with fluorescently-labeled anti-CCR4 and anti-CCR5 monoclonal antibody, and the result shows the transfection efficiency height, can be used for the calcium current experiment.

CCL17/TARC is the part of CCR4, and CCL5/RANTES is the part of CCR5, and the two can distinguish abduction delivering CCR4 under proper concn, Ca takes place the CCR5 cell ²⁺Discharge, as positive control and seal research.Monitor Ca in the cell with fluorescence intensity ²⁺Changing conditions.

Concrete grammar is as follows:

1, the construction process of pCDI-CCR4 and pCDI-CCR5 recombinant mammalian expressing vector is as follows: according to cDNA sequence (CCR4, the NM_005508.2 of human chemokine receptor CCR4 that discharges in the database and CCR5; CCR5, NM_000579), the design primer, clonal expansion comprises the cDNA fragment of its complete open reading frame from the cDNA library of K562 cell, and is cloned in the pcDI carrier, makes it at HEK293 cell effective expression.

2, electroporation transfection Chemokine Receptors

The cultivation of going down to posterity of HEK293 cell routine, trypsin digestion cell is adjusted cell density, electroporation transfection pCDI, pCDI-CCR4 and pCDI-CCR5, transfection method such as preceding.Be inoculated in the centre hole of 35mm petridish/10mm microwell ware, density is 1 * 105/ml, 37 ℃ of overnight incubation.

3, the expression of Flow cytometry Chemokine Receptors

Carry out flow cytometry, detect the expression of CCR4 and CCR5.Flow cytometer is FACScan, power 1200w, and 2W argon ion excitor, excitation spectrum 488nm, after the signal logarithm of photomultiplier amplified, input CellQuest (Becton Dickison) computer software was analyzed each sample analysis 1 * 10 ⁴Individual cell, (histogram) quantitatively shows the FITC positive cell number with the one-parameter histogram.

4, laser scanning confocal microscope detects intracellular calcium current

Cell Hank ' s balanced salt to be checked (PH7.4) solution is washed secondary, load Fluo-3/AM lucifuge, 37 ℃, 60 minutes.After using Hank ' s balanced salt solution to wash the cell secondary, (for example: the TCS NT of Leica company) go up application of sample, detect, and add reagent to be detected, observe the variation of fluorescence power in the specific moment at laser scanning confocal microscope.

Two, result

Experimental result sees also Figure 16-Figure 22 (display part result).

Respectively add sample between second one time at 10-15 second, 135-140 respectively.At first, add pCDNA3.1B-CKLFSF2 eukaryotic cell transfection supernatant in second, HEK293/CCR5 cell fluorescence intensity in moment is obviously strengthened, show Ca in the cell at 10-15 ²⁺Significantly raise, Ca has taken place ²⁺Discharge; Then, add 100nmol/LCCL5/RANTES between 135s-140s, cell fluorescence intensity strengthens not obvious, i.e. CCL5/RANTES inductive HEK293/CCR5 cell Ca ²⁺The release major part is suppressed.

And add 100nmol/L recombinant C KLFSF2 C51 albumen earlier, the HEK293/CCR5 cell fluorescence intensity is obviously strengthened, i.e. inducing cell Ca ²⁺Discharge; But after adding 100nmol/L CCL5/RANTES, still can strengthen cell fluorescence intensity once more, promptly CKLFSF2 C51 can not suppress CCL5/RANTES inductive Ca ²⁺Discharge.

And cleer and peaceful recombinant C KLFSF2 C51 albumen on the pCDNA3.1B-CKLFSF2 eukaryotic cell transfection all can not induce HEK293/pCDI and HEK293/CCR4 cell that obvious Ca takes place ²⁺Discharge, also can not seal CCL17/TARC inductive HEK293/CCR4 cell Ca ²⁺Release action.

Otherwise adding CCL5/RANTES earlier also can the most of CKLFSF2 of inhibition inductive HEK293/CCR5 cell Ca ²⁺Discharge.This prompting CCR5 is one of functional receptor of CKLFSF2, and CCR4 is not the functional receptor of CKLFSF2.

Above-mentioned explanation, CKLFSF2 polypeptide or its function fragment are as one of part of CCR5, and it plays a significant role in anti-HIV-1 infects by combining with CCR5.

Sequence table

＜110〉Peking University

＜120〉has the cytokine of multiple function

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aacaggccag ctgtgagaag ccaaggacac cgagtcagtc atg gca cct aag gcg 175

Met Ala Pro Lys Ala

1 5

gca aag ggg gcc aag cca gag cca gca cca gct cca cct cca ccc ggg 223

Ala Lys Gly Ala Lys Pro Glu Pro Ala Pro Ala Pro Pro Pro Pro Gly

10 15 20

gcc aaa ccc gag gaa gac aag aag gac ggt aag gag cca tcg gac aaa 271

Ala Lys Pro Glu Glu Asp Lys Lys Asp Gly Lys Glu Pro Ser Asp Lys

25 30 35

cct caa aag gcg gtg cag gac cat aag gag cca tcg gac aaa cct caa 319

Pro Gln Lys Ala Val Gln Asp His Lys Glu Pro Ser Asp Lys Pro Gln

40 45 50

aag gcg gtg cag ccc aag cac gaa gtg ggc acg agg agg ggg tgt cgc 367

Lys Ala Val Gln Pro Lys His Glu Val Gly Thr Arg Arg Gly Cys Arg

55 60 65

cgc tac cgg tgg gaa tta aaa gac agc aat aaa gag ttc tgg ctc ttg 415

Arg Tyr Arg Trp Glu Leu Lys Asp Ser Asn Lys Glu Phe Trp Leu Leu

70 75 80 85

ggg cac gct gag atc aag att cgg agt ttg ggc tgc cta ata gct gca 463

Gly His Ala Glu Ile Lys Ile Arg Ser Leu Gly Cys Leu Ile Ala Ala

90 95 100

atg ata ctg ttg tcc tca ctc acc gtg cac ccc atc ttg agg ctt atc 511

Met Ile Leu Leu Ser Ser Leu Thr Val His Pro Ile Leu Arg Leu Ile

105 110 115

atc acc atg gag ata tcc ttc ttc agc ttc ttc atc tta ctg tac agc 559

Ile Thr Met Glu Ile Ser Phe Phe Ser Phe Phe Ile Leu Leu Tyr Ser

120 125 130

ttt gcc att cat aga tac ata ccc ttc atc ctg tgg ccc att tct gac 607

Phe Ala Ile His Arg Tyr Ile Pro Phe Ile Leu Trp Pro Ile Ser Asp

135 140 145

ctc ttc aac gac ctg att gct tgt gcg ttc ctt gtg gga gcc gtg gtc 655

Leu Phe Asn Asp Leu Ile Ala Cys Ala Phe Leu Val Gly Ala Val Val

150 155 160 165

ttt gct gtg aga agt cgg cga tcc atg aat ctc cac tac tta ctt gct 703

Phe Ala Val Arg Ser Arg Arg Ser Met Asn Leu His Tyr Leu Leu Ala

170 175 180

gtg atc ctt att ggt gcg gct gga gtt ttt gct ttt atc gat gtg tgt 751

Val Ile Leu Ile Gly Ala Ala Gly Val Phe Ala Phe Ile Asp Val Cys

185 190 195

ctt caa aga aac cac ttc aga ggc aag aag gcc aaa aag cat atg ctg 799

Leu Gln Arg Asn His Phe Arg Gly Lys Lys Ala Lys Lys His Met Leu

200 205 210

gtt cct cct cca gga aag gaa aaa gga ccc cag cag ggc aag gga cca 847

Val Pro Pro Pro Gly Lys Glu Lys Gly Pro Gln Gln Gly Lys Gly Pro

215 220 225

gaa ccc gcc aag cca cca gaa cct ggc aag cca cca ggg cca gca aag 895

Glu Pro Ala Lys Pro Pro Glu Pro Gly Lys Pro Pro Gly Pro Ala Lys

230 235 240 245

gga aag aaa tga cttggaggag gctcctggtg tctgaaacgg cagtgtattt 947

Gly Lys Lys

tacagcaata tgtttccact ctcttccttg tcttctttct ggaatggttt tcttttccat 1007

tttcattacc acctttgctt ggaaaagaat ggattaatgg attctaaaag cctaaaaaaa 1067

aaaaaa 1073

<210>2

<211>248

<212>PRT

＜213〉homo sapiens

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Met Ala Pro Lys Ala Ala Lys Gly Ala Lys Pro Glu Pro Ala Pro Ala

1 5 10 15

Pro Pro Pro Pro Gly Ala Lys Pro Glu Glu Asp Lys Lys Asp Gly Lys

20 25 30

Glu Pro Ser Asp Lys Pro Gln Lys Ala Val Gln Asp His Lys Glu Pro

35 40 45

Ser Asp Lys Pro Gln Lys Ala Val Gln Pro Lys His Glu Val Gly Thr

50 55 60

Arg Arg Gly Cys Arg Arg Tyr Arg Trp Glu Leu Lys Asp Ser Asn Lys

65 70 75 80

Glu Phe Trp Leu Leu Gly His Ala Glu Ile Lys Ile Arg Ser Leu Gly

85 90 95

Cys Leu Ile Ala Ala Met Ile Leu Leu Ser Ser Leu Thr Val His Pro

100 105 110

Ile Leu Arg Leu Ile Ile Thr Met Glu Ile Ser Phe Phe Ser Phe Phe

115 120 125

Ile Leu Leu Tyr Ser Phe Ala Ile His Arg Tyr Ile Pro Phe Ile Leu

130 135 140

Trp Pro Ile Ser Asp Leu Phe Ash Asp Leu Ile Ala Cys Ala Phe Leu

145 150 155 160

Val Gly Ala Val Val Phe Ala Val Arg Ser Arg Arg Ser Met Asn Leu

165 170 175

His Tyr Leu Leu Ala Val Iie Leu Ile Gly Ala Ala Gly Val Phe Ala

180 185 190

Phe Ile Asp Val Cys Leu Gln Arg Asn His Phe Arg Gly Lys Lys Ala

195 200 205

Lys Lys His Met Leu Val Pro Pro Pro Gly Lys Glu Lys Gly Pro Gln

210 215 220

Gln Gly Lys Gly Pro Glu Pro Ala Lys Pro Pro Glu Pro Gly Lys Pro

225 230 235 240

Pro Gly Pro Ala Lys Gly Lys Lys

245

<210>3

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＜213〉artificial sequence

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＜223〉primer

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tcggtggtcc tggcagttag 20

<210>4

<211>24

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＜223〉primer

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caagcaaagg tggtaatgaa aatg 24

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

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agtcatggca cctaaggcgg 20

<210>6

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

cctccaagtc atttctttcc c 21

<210>7

<211>32

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<210>8

<211>30

<212>DNA

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<220>

＜223〉primer

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gcgaattcta tttctttccc tttgctggcc 30

<210>9

<211>21

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＜213〉artificial sequence

<220>

＜223〉primer

<400>9

aaggacaccg agtcagtcat g 21

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Claims (13)

1, a kind of polynucleotide, these polynucleotide comprise:

The polynucleotide of the 198-248 amino acids sequence of (1) polynucleotide of coding aminoacid sequence shown in SEQ ID NO:2, or coding shown in SEQ ID NO:2; Or

(2) polynucleotide that have at least 80% homology with (1), the polypeptide of this polynucleotide encoding has identical functions with (1) encoded polypeptides.

2, polynucleotide as claimed in claim 1, these polynucleotide comprise polynucleotide or its complementary sequence shown in SEQ ID NO:1, or the polynucleotide shown in the 752-904 position of SEQ ID NO:1.

3, a kind of engineering carrier, this engineering carrier contain claim 1 or 2 described polynucleotide.

4, a kind of host cell, this host cell obtains through the described engineering carrier conversion of claim 3, transfection or transduction.

5, a kind of method of producing polypeptide, this method may further comprise the steps:

(1) under the condition that is suitable for expressing, cultivates the described host cell of claim 4;

(2) polypeptide of the polynucleotide encoding that the described engineering carrier of acquisition claim 3 carries from described host cell culture.

6, a peptide species, this polypeptide comprises:

(1) aminoacid sequence shown in SEQ ID NO:2, or the polypeptide shown in the 198-248 amino acids of SEQ ID NO:2;

(2) polypeptide that has at least 80% homology with (1), the function of this polypeptide is identical with the function of (1).

7, the fragment of a kind of claim 1 or 2 described polynucleotide, this fragment are at least 10 continuous nucleotide sequences of described polynucleotide, are used as the primer or the probe that detect described polynucleotide, or suppress the expression of described polynucleotide.

8, a kind of pharmaceutical composition, this pharmaceutical composition contains claim 1 or 2 described polynucleotide or the acceptable salt of its medicine, the described carrier of claim 3, the described host cell of claim 4 or the described polypeptide of claim 6 or the acceptable salt of its medicine, and the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle.

9, polynucleotide as claimed in claim 1 or 2 or polypeptide as claimed in claim 6 prevent and/or treat application in the medicine of immune deficiency, acquired immune deficiency syndrome (AIDS) or hematopoietic disorder in preparation.

10, the method that whether changes from claim 1 in the person's to be measured sample or 2 described polynucleotide or polypeptide expression level as claimed in claim 6 of a kind of vitro detection, this method comprises:

(1) detects polynucleotide or polypeptide expression level described in the testing sample;

(2) polynucleotide or the polypeptide expression level with polynucleotide described in the testing sample or polypeptide expression level and normal specimens compares;

(3) determine in the testing sample whether polynucleotide or polypeptide expression level change.

11, method as claimed in claim 10, wherein said testing sample are person's to be measured testis tissue sample or seminal fluid sample.

12, a kind of mono-clonal or polyclonal antibody, this antibody combines with the described polypeptid specificity of claim 6.

13, mono-clonal as claimed in claim 12 or polyclonal antibody, the sequence of wherein said polypeptide is shown in the 198-248 amino acids of SEQ ID NO:2.

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