CN1283793C - Chemokine-like factor superfamily having skeletal muscle stimulating activity and immunoregulation function - Google Patents

Abstract

The present invention relates to a new nucleotide sequence and an amino acid sequence of the chemotactic factor superfamily having the stimulation activity of skeletal muscles and the action of immunological regulation, a method for producing the chemotactic factor superfamily, and a carrier and a host cell containing the chemotactic factor superfamily, which also relates to an antibody as a member of the chemotactic factor superfamily, and a medicine compound, an antagonist and an activating agent containing the chemotactic factor superfamily. The polyribonucleotide and the polypeptide of the chemotactic factor superfamily has important significance on aspects of treating muscular dystrophy or other retrograde affections, diseases of a hematopoietic system and/or diseases of an immune system.

Description

Chemokine-like factor superfamily with skeletal muscle stimulating activity and immunoregulation effect

Technical field

The present invention relates to cytokine.More particularly, the present invention relates to the nucleotide sequence and the aminoacid sequence of new chemokine-like factor superfamily, produce the method for chemokine-like factor superfamily and contain the carrier and the host cell of chemokine-like factor superfamily with skeletal muscle stimulating activity and immunoregulation effect.The invention still further relates to the antibody of chemokine-like factor superfamily, contain the pharmaceutical composition of chemokine-like factor superfamily, and antagonist and activator.The polynucleotide and the polypeptide that the invention further relates to chemokine-like factor superfamily are preparing amyotrophy or other degeneration, the application in the pharmaceutical composition of disease of hematopoietic system and/or disease of immune system.

Background of invention

Cytokine is the general name by the micromolecule polypeptide that plays a significant role in the physiology of body and pathologic process of the synthetic justacrine of cell.Cytokine comprises interleukin-, G CFS, Interferon, rabbit, tumour necrosis factor, somatomedin and chemokine etc.

Cytokine has different physiological roles: they can regulate body's immunological function, participate in the propagation and the differentiation of cell, promote vascular endothelial cell proliferation and new vessel to generate, and are also significant aspect antitumor and anti-microbial infection.In recent years, continuous development along with Protocols in Molecular Biology, the structure function of people's pair cell factor and acceptor thereof has had more deep understanding, and it is clinical to utilize the recombinant cytokine of genetic engineering technique production and antagonist thereof to be used for as medicine, and obtains good efficacy.Be used for as the hematopoiesis protective material as marrow sample hemopoietic progenitor cell supressor 1 (MPIF-1) that the tumour heavy dose is put, chemotherapy; be expected to become biotechnology medicine (the Marshall A of a new generation; HGS Launches " first " genomics product in clinic.Nat Biotechnol 1998,16:129).This prompting cytokine has broad application prospects in the treatment of difficult and complicated illness such as tumour, hematopoietic disorder disease, autoimmune disorder.

Chemokine (chemokine, claim the chemotactic element again), be that a class formation is similar, molecular weight 8-12KD, micromolecule polypeptide with 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/CH00/00026), and CKLF2 is the full gene product of CKLF, 152 amino acid of encoding.

The invention summary

In the present invention, the inventor further discloses the function of CKLF2, and on the albumen and nucleotide sequence basis of CKLF2, utilize blastp, blastn and the tblast database of NCBI, successfully obtained other 8 chemokine-like factor superfamily members' new gene, by retrieval, the protein of these 8 new genes encodings is new sequence.Hereinafter referred to as chemokine-like factors associated protein 1-8 (chemokine-like factor related protein 1-8, CKLF-RP1-8).New CKLF-RP1-8 and CKLF2 have constituted the present invention's said " chemokine-like factor superfamily " jointly, are called for short the CKLF superfamily, and it has skeletal muscle stimulating activity and immunoregulation effect.

According to an aspect of the present invention, the invention provides a kind of isolating nucleotide sequence, it comprises nucleotide sequence or its fragment of the new chemokine-like factor superfamily with skeletal muscle stimulating activity and immunoregulation effect, described nucleotide sequence can be DNA or RNA, and wherein DNA comprises cDNA, genomic dna and synthetic DNA.

The preferred a kind of isolating nucleotide sequence of the present invention, it comprises polynucleotide or its fragment shown in SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17.

The present invention also provides the multi-nucleotide hybrid with chemokine-like factor superfamily, and has the polynucleotide of at least 70% homology with it.

The invention provides the carrier of the polynucleotide sequence that contains chemokine-like factor superfamily.

The invention provides the host cell of the carrier that contains chemokine-like factor superfamily, it can obtain through described carrier conversion, transfection or transduction.

The invention provides method with the mature polypeptide of genetic engineering means production chemokine-like factor superfamily of the present invention.

According to another aspect of the present invention, the invention provides the aminoacid sequence that contains chemokine-like factor superfamily.

The present invention also provide chemokine-like factor superfamily varient, analogue, derivative or active fragments.

The present invention comprises that also chemokine-like factor superfamily member's of the present invention polypeptide or its fragment are had specific polyclone and monoclonal antibody.

The present invention also provides a kind of pharmaceutical composition, and it contains chemokine-like factor superfamily member of the present invention or its active fragments, and one or more pharmaceutically acceptable carrier or vehicle.

The invention provides a kind of antagonist, it can suppress or seal chemokine-like factor superfamily of the present invention or its segmental biological activity.

The invention provides a kind of activator, it can activate chemokine-like factor superfamily of the present invention or its segmental biological activity.

In addition, polynucleotide and the polypeptide that the present invention further provides chemokine-like factor superfamily of the present invention treated amyotrophy or other degeneration, the application in the pharmaceutical composition of hemopoietic system and/or disease of immune system in preparation.

The accompanying drawing summary

Fig. 1 shows the conserved amino acid sequence between the chemokine-like factor superfamily member.

Fig. 2 shows close source property synoptic diagram between the chemokine-like factor superfamily member.

Fig. 3 shows chemokine-like factor superfamily member's transmembrane domains structural representation.

Fig. 4 shows chemokine-like factor superfamily member's chromosomal localization synoptic diagram.

Fig. 5 shows chemokine-like factor superfamily member's vector construction synoptic diagram.

Fig. 6 A and Fig. 6 B show that CKLF2 causes that the mouse myoblast is the variation of C2C12 cellular form, wherein Fig. 6 A:C2C12/pcDB; Fig. 6 B:C2C12/CKLF2, as seen from the figure, CKLF2 promotes C2C12 cell myotube to form.

Fig. 7 A-Fig. 7 D shows the result of four kinds of different methods research CKLF2 to the short proliferation function of C2C12 cell, wherein Fig. 7 A. cell counting; Fig. 7 B.MTT method; Fig. 7 C. cell cycle is studied; Fig. 7 D.H3 mixes experiment.

Fig. 8 shows that CKLF2 promotes the activity of C2C12 cell-specific molecule MCK.

Fig. 9 shows that CKLF2 promotes C2C12 cytodifferentiation specific transcription factor Myogenin to express.Wherein

1:C2C12/pcDB induces differentiation 1 day; 2:C2C12/pcDB induces differentiation 2 days;

3:C2C12/pcDB induces differentiation 3 days; 4:C2C12/pcDB induces differentiation 4 days;

5:C2C12/CKLF2 induces differentiation 1 day; 6:C2C12/CKLF2 induces differentiation 2 days;

7:C2C12/CKLF2 induces differentiation 3 days; 8:C2C12/CKLF2 induces differentiation 4 days.

Figure 10 A-Figure 10 C shows the distribution expression pattern of CKLF-RP2, wherein Figure 10 A:1. molecular weight standard, 2. skeletal muscle, 3. brain, 4. kidney, 5. marrow, 6. liver, 7. heart; Figure 10 B:1. molecular weight standard, 2. prostate cancer, 3. intestinal cancer, 4. adenocarcinoma of lung, 5. mammary cancer, 6. ovary; Figure 10 C:1. molecular weight standard, 2. fetal thymus, 3. fetal skeletal muscle, 4. tire spleen, 5. tire liver, 6. fetal rhythm.

Figure 11 shows the short proliferation function of CKLF-RP2 to the Hela cell.

Figure 12 A and Figure 12 B show the short proliferation function of CKLF-RP2 to splenocyte, wherein Figure 12 A:pcDB empty carrier injection mouse; Figure 12 B:pcDB-CKLF-RP2 injects mouse.

Detailed Description Of The Invention

The present invention is achieved through the following technical solutions: the inventor utilizes bioinformatics method successfully to clone rat and mouse CKLF2 sequence on the albumen and nucleotide sequence basis of people CKLF2, and they have similar 26S Proteasome Structure and Function to people CKLF2. On the albumen and nucleotide sequence basis of people and mouse CKLF2, utilize Protein Data Bank and people's EST (Expression Sequence Tag, abbreviation EST) carry out data retrieval and homology relatively, (network address is by EST Assembly machineHttp: ∥ www.tigem.it) carry out the EST splicing, according to the special primer of full length cDNA sequence design, and select the strand cDNA library of high expressed corresponding gene to increase, successfully separate and cloned other 8 chemokine-like factor superfamily members' new gene. Through retrieval, these 8 chemokine-like factor superfamily members' protein is new sequence. According to their found sequencings and different from the pro-borne of CKLF2, be named as respectively CKLF-RP1-8 (CKLF-RP1-8).

The previously disclosed CKLF2 of new CKLF-RP1-8 and the inventor has consisted of the present invention's said " chemokine-like factor superfamily " jointly. Chemokine-like factor superfamily of the present invention has skeletal muscle stimulating activity and immunoregulation effect.

CKLF-RP1-8 and CKLF2 have obvious homology at the protein primary sequence, and the similar membrane structure of wearing is arranged, and on chromosome close linkage (referring to embodiment one), this meets the feature of gene superfamily. Present research finds, a lot of gene families as: chemotactic factor (CF), the gene family members such as defensin become the gene cluster form to occur at chromosome, in primary structure homology is arranged, and similar function is arranged. Gene family member homology on primary structure is not high, namely can have identical space structure but the minority key amino acid is identical, and the space structure of protein is even more important for the function of Protein requirement.

CKLF2 and CKLF-RP1-8 and A4 differential protein have homology (the A4 differential protein is high expressed in the atomization of colon epithelial cell, has the effect of regulating cell propagation and differentiation). This homology analysis is to find that with the BlastP search function of NCBI the result of this sequence homology comparison is international endorsement. The homology of CKLF2 and CKLF-RP1-8 and A4 differential protein is between 22.4%-33%.

According to an aspect of the present invention, the invention provides polynucleotide sequence or its fragment of the separation that contains the chemokine-like factor superfamily of encoding, described polynucleotide sequence comprises cDNA, genomic DNA and RNA.

The polynucleotide sequence of chemokine-like factor superfamily of the present invention comprises the polynucleotide sequence of CKLF-RP1-8 and the polynucleotide sequence of CKLF2.

The polynucleotide sequence of CKLF-RP1-8 of the present invention and CKLF2 can be encoded respectively such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, mature polypeptide shown in SEQ ID NO:16 or the SEQ ID NO:18, coded sequence and the additional code sequence that also can comprise above-mentioned mature polypeptide, coded sequence and the non-coding sequence that can also comprise above-mentioned mature polypeptide, such as introne, coded sequence 5 ' or the 3 ' non-coding sequence of holding etc. The fragment of above-mentioned polynucleotide sequence also should comprise within the scope of the present invention. These fragments can be used as primer or hybridization probe, for detection of the polynucleotide sequence of coding chemokine-like factor superfamily of the present invention.

The invention still further relates to the variant of above-mentioned polynucleotides. The variant of polynucleotides can be the different variants of shearing variant or non-natural existence of naturally occurring allelic variation body, mRNA. These variants can be identical on function, similar or different from chemokine-like factor superfamily member of the present invention, but preferably encode and the essentially identical polypeptide of its BA. For example, the inventor has found that at present CKLF-RP1 has 4 kinds of different variants at least, encode respectively 286,242,240 and 170 amino acid, other member's comparativity of 170 amino acid whose sequences and CKLF superfamily of wherein encoding is best, so this patent is as representative (the signal peptide analysis discovery of CKLF-RP1, the signal peptide cutting site of the albumen of four kinds of different sizes is identical namely to produce identical maturation protein, therefore optional one of them as representative). 149 amino acid of another form A KLF-H1-A coding of CKLF-RP1, its eukaryotic cell transfection supernatant can obvious stimulation bone marrow cell and Proliferation of lymphocytes (see patent publication No. for details: CN 1283693A). Each given gene can not have, and has 1, perhaps a plurality of allelic variation bodies, and it can replace, insert or add with nucleotides, but does not change in fact the alternative form of polynucleotides of the function of coded polypeptide.

The polynucleotide sequence of described chemokine-like factor superfamily preferably provides with unpack format. " separation " of the present invention refers to that the sequence that this DNA or fragment have been positioned at its both sides under the native state separates, refer to that also this DNA or its fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

The polynucleotide sequence of chemokine-like factor superfamily of the present invention can be DNA or RNA, wherein DNA comprises cDNA, genomic DNA and synthetic DNA, DNA can be two strands or single stranded form, and single stranded DNA can be coding strand or noncoding strand (antisense strand). Those of ordinary skills are known, and antisense strand or its part (ASON) can be used for suppressing the expression of chemokine-like factor superfamily member of the present invention in the cell. The nucleotide sequence of chemokine-like factor superfamily of the present invention can comprise ox, sheep, pig, mouse, horse from any species, particularly mammal, and is preferred human.

The nucleotide sequence of the preferred a kind of separation of the present invention, it comprises polynucleotides or its fragment shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, SEQ ID NO:17. Polynucleotides shown in the SEQ ID NO:1,3,5,7,9,11,13,15 or 17 are respectively the cDNA sequence of code book invention chemokine-like factor superfamily member CKLF-RP1-8 and CKLF2, and the accession number in Genbank is respectively: CKLF-RP1:AF278577; CKLF-RP2:AF479260; CKLF-RP3:AF479813; CKLF-RP4:AF479814; CKLF-RP5:AF479262; CKLF-RP6:AF479261; CKLF-RP7:AF 479263; CKLF-RP8:AF474370; CKLF2:AF135380.

In detail, the polynucleotide sequence that comprises the nucleotide sequence shown in SEQ ID NO:1 of the preferred a kind of separation of the present invention, wherein, nucleotide sequence shown in SEQ ID NO:1 derives from people's TESTIS cDNA library, 793 nucleotides of total length, nucleotides 128-640) and 5 ' noncoding region (nucleotides 1-127) and 3 ' noncoding region (nucleotides 441-793) and comprise the sequence of coding CKLF-RP1 albumen, (coded sequence (CDS) for example:. Perhaps, more preferably a kind of nucleotide sequence of separation, it comprises coding CKLF-RP1 protein sequence, for example coded sequence shown in the SEQ ID NO:1: nucleotides 128-640.

The polynucleotide sequence that comprises the nucleotide sequence shown in SEQ ID NO:3 of the preferred a kind of separation of the present invention, wherein the nucleotide sequence shown in SEQ ID NO:3 derives from people's TESTIS cDNA library, 1065 nucleotides of total length, the sequence that comprises coding CKLF-RP2 albumen, nucleotides 153-899) and 5 ' noncoding region (nucleotides 1-152) and 3 ' noncoding region (nucleotides 900-1065) (coded sequence (CDS) for example:. Perhaps, more preferably a kind of nucleotide sequence of separation, it comprises coding CKLF-RP2 protein sequence, for example coded sequence shown in the SEQ ID NO:3: nucleotides 153-899.

The polynucleotide sequence of the preferred a kind of separation of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:5. Wherein the nucleotide sequence shown in SEQ ID:5 derives from the human placenta cDNA library, 1514 nucleotides of total length, nucleotides 75-623) and 5 ' noncoding region (nucleotides 1-74) and 3 ' noncoding region (nucleotides 624-891) it comprises the sequence of coding CKLF-RP3 albumen, (coded sequence (CDS) for example:. Perhaps, more preferably a kind of nucleotide sequence of separation, it comprises coding CKLF-RP3 protein sequence, for example coded sequence shown in the SEQ ID NO:5: nucleotides 75-623.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:7.Nucleotide sequence shown in this SEQ ID:7 derives from people's spleen cDNA library, 1118 Nucleotide of total length, Nucleotide 183-809) and 5 ' non-coding region (Nucleotide 1-182) and 3 ' non-coding region (Nucleotide 810-1118) it comprises coding CKLF-RP4 proteic sequence, (encoding sequence (CDS) for example:.Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CKLF-RP4 protein sequence, for example encoding sequence shown in the SEQ ID NO:7: Nucleotide 183-809.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:9.Wherein the nucleotide sequence shown in the SEQ ID:9 derives from human prostata cancer cDNA library, 988 Nucleotide of total length, Nucleotide 191-661) and 5 ' non-coding region (Nucleotide 1-190) and 3 ' non-coding region (Nucleotide 662-988) it comprises coding CKLF-RP5 proteic sequence, (encoding sequence (CDS) for example:.Perhaps, individual more preferably a kind of isolating nucleotide sequence, it comprises coding CKLF-RP5 protein sequence, for example encoding sequence shown in the SEQ ID NO:9: Nucleotide 191-661.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:11.Wherein the nucleotide sequence shown in the SEQ ID:11 derives from human prostata cancer cDNA library, 1917 Nucleotide of total length, Nucleotide 123-674) and 5 ' non-coding region (Nucleotide 1-122) and 3 ' non-coding region (Nucleotide 675-1917) it comprises coding CKLF-RP6 proteic sequence, (encoding sequence (CDS) for example:.Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CKLF-RP6 protein sequence, for example encoding sequence shown in the SEQ ID NO:11: Nucleotide 123-674.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:13.Nucleotide sequence shown in this SEQ ID:13 derives from the human placenta cDNA library, 1177 Nucleotide of total length, Nucleotide 44-571) and 5 ' non-coding region (Nucleotide 1-143) and 3 ' non-coding region (Nucleotide 572-1177) it comprises coding CKLF-RP7 proteic sequence, (encoding sequence (CDS) for example:.Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CKLF-RP7 protein sequence, for example encoding sequence shown in the SEQ ID NO:13: Nucleotide 44-571.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:15.Nucleotide sequence shown in this SEQ ID:15 derives from human tonsil cDNA library, 1177 Nucleotide of total length, it comprises the proteic sequence of coding CKLF-RP8, (for example encoding sequence (CDS: Nucleotide 262-783) and 5 ' non-coding region (Nucleotide 1-261) and 3 ' non-coding region (Nucleotide 784-1177) wherein, Nucleotide 17 is n, and expression can not determine still at present which kind of Nucleotide this position is.Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CKLF-RP8 protein sequence, for example encoding sequence shown in the SEQ ID NO:15: Nucleotide 262-783.

The preferred a kind of isolating polynucleotide sequence of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:17.459 Nucleotide of nucleotide sequence total length shown in this SEQ ID:17, it comprises the proteic sequence of coding CKLF2, for example encoding sequence (CDS): Nucleotide 1-459.

Those of ordinary skills are known, chemokine-like factor superfamily member's of the present invention nucleotide sequence can be entirely identical to the NO:1 as SEQ ID, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, encoding sequence shown in the SEQ ID NO:17 also can not exclusively be equal to the encoding sequence of above-mentioned Nucleotide owing to the degeneracy of genetic code.For example,, can select corresponding codon, thereby improve described polynucleotide expression efficiency in corresponding host according to the frequency difference of each concrete prokaryotic hosts or the employed codon of eucaryon host.Also can be in order to obtain to have the polynucleotide of better performance than natural nucleotide sequence, as the longer transformation period, additive cipher.

The present invention also further relates to the polynucleotide with chemokine-like factor superfamily polynucleotide sequence of the present invention hybridization, and condition is to exist between sequence to have 70% at least, and more preferably 90%, the polynucleotide sequence of 95% homology most preferably.Be particularly related under stringent condition polynucleotide with chemokine-like factor superfamily gene recombination of the present invention, said " stringent condition " known hybridization occurs in and is lower than 5 ℃ of Tm (melting temperature, melting temp) to being lower than under Tm 20-25 ℃ the condition.Described polynucleotide can contain at least 20 bases, preferred at least 30 bases and more preferably at least 50 bases and chemokine-like factor superfamily member of the present invention multi-nucleotide hybrid and have homology as mentioned above with it, and can keep or retentive activity not.For example, such polynucleotide can be used as the present invention such as SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, the probe of the polynucleotide shown in the SEQ ID NO:17 is used to reclaim polynucleotide or is used as diagnostic probe or PCR primer.

Chemokine-like factor superfamily member polynucleotide sequence of the present invention can establishing criteria the pcr amplification technology with cDNA, mRNA or genomic dna be as template, and choose suitable Oligonucleolide primers amplification and obtain.Obtain Nucleotide like this and can clone in the suitable carriers, and carry out sequence description with the DNA analysis technology.Also can obtain by the standard DNA synthetic technology, for example, use can be synthetic on dna synthesizer by solid phase phosphorous acid acid amides triester method well known in the art.

(referring to embodiment one) in a preferred embodiment of the present invention, CKLF-RP1-5,7,8 full length cDNA sequence is based on the CKLF2 protein sequence, Protein Data Bank and people's expressed sequence tag (EST) is carried out data retrieval and homology comparison, and (network address is by EST Assembly machine Http:// www.tigem.it) carries out the EST splicingAfter, utilize complete single open reading frame design special primer, the sequences Design primer that discharges with reference to NCBI of the full length cDNA sequence of CKLF-RP6 wherein, and by selecting to express the strand cDNA library of corresponding gene, carry out pcr amplification and obtain.

The invention discloses the carrier that carries the invention described above chemokine-like factor superfamily polynucleotide sequence.Such carrier comprises that karyomit(e), non-chromosome are originated or the synthetic dna sequence dna, the for example derivative of SV40, bacterial plasmid, phage DNA, yeast plasmid, baculovirus and viral DNA (as vaccinia virus, adenovirus, domestic animal poxvirus), condition is that these carriers can duplicate in selected host cell and survive.

Available various known method is inserted into suitable dna sequence dna in the suitable restriction endonuclease site of carrier.Forward or oppositely be inserted into dna sequence dna in the expression vector to be operably connected to suitable expression control sequenc be promotor, synthetic to instruct mRNA.The example of such promotor comprises RSV, HIV, CMV or SV40 promotor, intestinal bacteria lac or trp promotor, phage PL promotor and other promotors that controlling gene is expressed in protokaryon or eukaryotic cell or its virus.In addition, expression vector can contain ribosome bind site and the transcription terminator that starts translation, can also comprise being used to the proper sequence that increases and express.In addition, carrier preferably contains one or more selected marker genes, providing, as be applicable to eukaryotic neomycin resistance or dihydrofolate reductase gene, or be applicable to penbritin or tetracycline resistance gene in the intestinal bacteria by the phenotypic characteristic selected of transformed host cells.In case of necessity, for the DNA that improves code book invention chemokine-like factor superfamily member transcribes efficient in higher eucaryotic cells, can in carrier, insert enhancer sequence.In addition, also can instruct translation product excretory leader sequence (secretion signal) in pericentral siphon or extracellular substratum in case of necessity in one of insertion between promotor and the downstream configurations sequence.Perhaps, can import the allogeneic dna sequence of encoding fusion protein matter as required, said fusion rotein can comprise that is given a required feature, as is used for stable not held identification polypeptide by the N product of expression or that simplify its purification step.

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 (ATCC37017).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.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 one) in a preferred embodiment of the invention, the polynucleotide sequence of chemokine-like factor superfamily of the present invention utilize PCR product cloning (Promega) to the pGEM-T Easy carrier is carried out the purifying order-checking; In another preferred embodiment of the present invention (referring to embodiment two), the carrier that carries chemokine-like factor superfamily member of the present invention is to adopt eukaryon expression plasmid pcDNA3-Myc-His (B) (Invitrogen) to make up.

The invention provides the cloning vector that carries chemotactic like factor superfamily gene of the present invention or the host cell of expression vector transduction, conversion or transfection.The method of transduction, conversion or transfection comprises virus infection known in the art, calcium chloride infection protocol, liposome transfection method, electroporation or microprojectile bombardment methods etc.In order to activate promotor, to select transformant or the required gene that increases, can in the conventional nutritional medium of suitably modifying, cultivate by transduction, transfection or transformed host cells.Culture condition such as employed temperature, pH generally all are by the decision of the host cell of selected expression specified protein in the cultivation, and these conditions all are well known to those skilled in the art.

Can use any appropriate host cell to express polynucleotide of the present invention, the suitable host's that can mention example has: prokaryotic cell prokaryocyte such as intestinal bacteria, genus bacillus, streptomycete etc.; Low eukaryotic cell such as the yeast cell of waiting; Insect cell such as fruit bat and fortunatus cell; Vegetable cell; Higher eucaryotic cells such as mammalian cell such as CHO, COS cell.The example of mammalian expression system comprises the human cell line, as Hela, 293 and U937 clone, and COS, CHO and bhk cell system.(referring to embodiment one) in a preferred embodiment of the invention, the prokaryotic host cell of employing is the XL-1Blue bacterium, eucaryon host is C2C12 clone (referring to embodiment three) for the mouse myoblast, and Hela clone (referring to embodiment five).

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

(1) under the condition that is suitable for expressing, cultivates the host cell of chemokine-like factor superfamily of the present invention;

(2) polypeptide of the polynucleotide encoding that the described carrier of acquisition carries from 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 purifying chemokine-like factor superfamily member of the present invention and varient polypeptide thereof 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.

According to another aspect of the present invention, the chemokine-like factor superfamily member's of the present invention that encodes nucleotide sequence or its fragment can be used as hybridization probe, are used for karyomit(e) and identify.Utilize known technology, this sequence is certain special karyomit(e) of target or chromosomal certain specific site specifically.These technology comprise fluorescence in situ hybridization (FISH), fluorescence amplifying cell separator (FACS) or structure artificial chromosome storehouse are for example, the yeast artificial chromosome storehouse, the bacterial artificial chromosome storehouse, bacterium P1 makes up storehouse or karyomit(e) cDNA library etc. (referring to Price, C.M. (1993) Blood Rev.7:127-134, and Trask, B.J. (1991) Trends Genet.7:149-154.).

In a preferred embodiment of the invention, utilize chemokine-like factor superfamily member's of the present invention full length cDNA sequence or its fragment in NCBI HGP database, to retrieve, analyze its genomic constitution and chromosomal localization.Shown in embodiment one, CKLF2 and CKLF-RP1-4 are positioned at karyomit(e) No. 16, and close linkage, do not find to have other known at present as yet therebetween.CKLF-RP5 is positioned at karyomit(e) No. 14, CKLF-RP6, and 7,8 become the gene cluster form (see figure 4) to occur on No. 3 karyomit(e)s.Genomic constitution analyze to be found, CKLF2 and CKLF-RP1, and 2,6,8 are made up of 4 exons, CKLF-RP3,4,5,7 form (seeing Table 5) by 5 exons.

According to the present invention, this bright DNA is carried out first important step that chromosome mapping connects these sequences and disease related gene.In case finished the accurate chromosomal localization mapping of sequence, the physical location and the genetic map data of this sequence can have been connected.Identify that by linkage analysis (the common heredity of the adjacent gene of physics) mapping is positioned gene on the same chromosomal region and the relation between the disease then.Then, can determine impaired or the difference of the cDNA between injured individual or genomic dna not.If in some or all injured individual, observe sudden change, and do not observe this sudden change in the normal individual, just then this sudden change is likely paathogenic factor.

According to another aspect of the present invention, the invention provides the aminoacid sequence that contains the chemokine-like factor superfamily member who infers, the chemokine-like factor superfamily member's who infers aminoacid sequence is respectively as SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14 is shown in SEQ ID NO:16 or the SEQ ID NO:18.The fragment of above-mentioned aminoacid sequence also should comprise within the scope of the present invention.Such polypeptide fragment comprises the NO:2 as SEQ ID, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, at least 15 successive aminoacid sequences shown in SEQ ID NO:16 or the SEQ ID NO:18.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP1 shown in SEQ ID NO:2,170 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 23-40,47-69,79-101,108-130.CKLF-RP-1 has typical C ys-Cys constitutional features (being called for short the CC structure), and promptly Bao Shou two halfcystines are adjacent, amino-acid residue 124-125 shown in SEQ ID NO:2, and it is the constructional feature of most of chemotactic prime factors.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP2 shown in SEQ ID NO:4,248 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.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP3 shown in SEQ ID NO:6,182 amino acid of total length.The prosite computer software analysis, it has 3 and strides the film district, lays respectively at amino-acid residue 61-83,103-122,132-151.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP4 shown in SEQ ID NO:8,208 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 59-77,87-109,121-143,148-170.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP5 shown in SEQ ID NO:10,156 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 35-56,60-82,95-114,119-136.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP6 shown in SEQ ID NO:12,183 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 43-64,73-94,107-126,135-156.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP7 shown in SEQ ID NO:14,175 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 40-59,69-91,103-125,145-167.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF-RP8 shown in SEQ ID NO:16,173 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 46-68,78-100,113-130,140-162.

In a preferred embodiment of the invention, the aminoacid sequence of CKLF2 shown in SEQ ID NO:18,152 amino acid of total length.The prosite computer software analysis, it has 4 and strides the film district, lays respectively at amino-acid residue 24-40,45-67,74-96,106-128.

As it be known to those skilled in the art that the above-mentioned preferred a series of hydrophobic amino acids in film district of striding, leucine for example, Xie Ansuan, L-Ala etc.

Chemokine-like factor superfamily albumen of the present invention is carried out sequential analysis, and showing has conservative aminoacid sequence (referring to Fig. 1) between CKLF2 and the CKLF-RP1-8; Homology is in various degree arranged each other, and CKLF2 and CKLF-RP1 have the typical C ys-Cys constitutional features of (being called for short the CC structure), and CKLF-RP2-8 does not then possess the CC structure; Evolutionary tree analysis finds that the close source relation of CKLF2 and CKLF-RP1 is nearest, and the close source of CKLF-RP3 and CKLF-RP5 concerns (referring to Fig. 2) recently.Transmembrane domains analysis discovery, except CKLF-RP3 has 3 potential transmembrane domains, CKLF2 and CKLF-RP1,2,4,5,6,7,8 are 4 transmembrane proteins (referring to table 4).Chromosomal localization discovers, CKLF2 and CKLF-RP1-4 be close linkage on No. 16 karyomit(e), and CKLF-RP5 is positioned karyomit(e) No. 14, CKLF-RP6, and 7,8 become the gene cluster form to occur on No. 3 karyomit(e)s.Therefore, chemokine-like factor superfamily of the present invention is represented a new gene superfamily, CKLF-RP1-8 and CKLF2 not only have homology on prlmary structure of protein, and similar transmembrane domains arranged, studies show that this superfamily member has skeletal muscle stimulating activity and immunoregulation effect in the external and body.

The polypeptide of chemokine-like factor superfamily of the present invention can be natural, and synthetic is semisynthetic, and perhaps reorganization produces.Chemokine-like factor superfamily member of the present invention can biological engineering method routinely produce polypeptide product by the coding of the recombinant DNA sequence in the host cell.Also can be according to Steward and Young (Steward, J.M.andYoung, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemicai 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 protein.

The present invention provides chemokine-like factor superfamily member's varient (variant), analogue (mimetic), derivative (derivative) or active fragments simultaneously.

" varient " described here is meant that aminoacid sequence has one or more change.Known as those of ordinary skills, varient can have " conservative property " to be changed, and promptly the amino acid of Ti Huaning has similar structure and chemical property, for example, replaces leucine with Isoleucine.Under a few cases, varient also can have " non-conservation " to be changed, and for example, replaces glycine with tryptophane.Varient can also comprise amino acid whose disappearance, perhaps inserts or both have concurrently.Do not eliminate bioactive amino acid and replace, insert or lack and to utilize the known computer software analysis in field, for example DNADNASTAR software.Varient described here, preferably the aminoacid sequence with chemokine-like factor superfamily member of the present invention has at least 80%, more preferably the polypeptide of at least 90% homology.Most preferably with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, the polypeptide that aminoacid sequence of the present invention has 95% homology shown in the SEQ ID NO:18." homology " is definite by the quantity of the comparison of aminoacid sequence between the polypeptide and conserved sequence amino-acid residue.

Therefore term described here " analogue " is meant that its structure is by chemokine-like factors of the present invention or its segmental structural development, can influence partly or completely the effect with chemokine-like factor superfamily of the present invention.

" derivative " described here can refer to the chemokine-like factor superfamily of the present invention of chemically modified; one or more residues in the amino acid sequence of polypeptide for example of the present invention have alkyl, acyl group; perhaps substituting group such as amino also comprises having chemokine-like factor superfamily of the present invention appended sequence or that merge with other compound.For example for the ease of purifying, merge other amino-acid residue at flank of the present invention, and for example merge to improve the half life of polypeptide with polyoxyethylene glycol or lipid.

The polypeptide fragment of so-called chemokine-like factors is meant the polypeptide with part or all of chemokine-like factors member of the present invention, it is made up of 20 amino acid at least, preferably form with upper amino acid by 40, polypeptide fragment preferably has basically and the same or analogous biological activity of polypeptide of the present invention.

The invention still further relates to and use polynucleotide of the present invention, detect the gene of mutant form and express deficiency or express excessive disease that is caused or pathological state because of chemokine-like factor superfamily member of the present invention in the body with diagnosis as diagnostic reagent.

Available various technology detects the individuality that carries chemokine-like factor superfamily member transgenation on dna level.Can be from patient's body fluid, as obtaining being used for the nucleic acid of diagnostic test in blood, urine, saliva or live body or the postmortem material.Can use gene DNA or RNA directly to detect, detect again after also can using PCR method (Saiki et al., Nature324:163-166,1986) DNA amplification.Available direct dna sequencing method is analyzed with reference to the sequence difference between gene and the mutator gene.In addition, the dna fragmentation that also can utilize the clone detects specific dna fragmentation as probe and in conjunction with PCR method with hypersensitivity and specificity, for example can unite the strand PCR product or the single-stranded template molecule that use sequencing primer and produced by amplification PCR products.

Can be at the gel that contains or do not contain denaturing agent, the electrophoretic mobility that particularly detects DNA in the high resolving power gel is finished the genetics test based on dna sequence dna difference.Can on sex change methane amide gradient gel, distinguish not homotactic dna fragmentation (as referring to Myers et al., Science 230:1242,1985).In addition, also available nucleic acid enzyme protection detection method or chemical cracking method (as referring to Cotton et al., PNAS, USA, 85:4397-4401,1985) disclose the sequence change of privileged site.

The invention still further relates to the diagnostic test method that chemokine-like factor superfamily member protein matter level changes in the various tissues that detects.The detection method that is used for detecting the chemokine-like factor superfamily member protein matter level of host-derived vivo sample is well known by persons skilled in the art, and comprises radioimmunology, competitive combined techniques, Western engram analysis method and enzyme-linked immunosorbent assay (ELISA).Wherein preferable methods is the ELISA method.

The present invention comprises that also chemokine-like factor superfamily member's of the present invention polypeptide or its fragment are had specific polyclone and monoclonal antibody, especially monoclonal antibody." specificity " described here is meant that antibody capable is incorporated into chemokine-like factor superfamily gene product of the present invention or fragment.Preferred those can combine with the gene product of chemokine-like factor superfamily of the present invention but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the antibody of chemokine-like factor superfamily member gene product of the present invention, comprise that also those do not influence the antibody of chemokine-like factor superfamily member function of the present invention.The present invention also comprises those energy chemokine-like factor superfamily member varient of the present invention, analogue, derivative or active fragments bonded antibody.

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 be prepared by the known the whole bag of tricks of those of ordinary skills.For example, the chemokine-like factors member 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.Similarly, express chemokine-like factor superfamily member of the present invention or have its antigenic segmental cell and can be used to immune animal and produce antibody.The preferred monoclonal antibody of antibody of the present invention, 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 chemokine-like factor superfamily 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.(for example, the gene product of producing in E.Coli) is come immune animal and is produced can to use prokaryotic cell prokaryocyte with the unmodified form bonded antibody of chemokine-like factors member's of the present invention gene product; With posttranslational modification form bonded antibody (as the albumen 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).

The present invention also provides a kind of pharmaceutical composition, and it contains chemokine-like factor superfamily member of the present invention or its active fragments, and the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle.

" the acceptable salt of medicine " refers to be suitable for contact with human or animal's tissue, and does not have too much toxicity, stimulation, transformation reactions etc.The acceptable salt of medicine is well known in the art.This salt can also can prepare free alkali or sour and suitable organic or inorganic acid or alkali reaction separately in the final separation of chemokine-like factor superfamily member polypeptide of the present invention and the process of preparing of purifying.Representative acid salt includes but not limited to acetate, two hexanoates, alginate, Citrate trianion, aspartate, benzoate, benzene sulfonate, hydrosulfate, butyrates, camphorate, camsilate, glycerophosphate, Hemisulphate, enanthate, hexanoate, fumarate, hydrochloride, hydrobromate, hydriodate, the 2-isethionate, lactic acid salt, maleate, mesylate, nicotinate, the 2-naphthalenesulfonate, oxalate, 3-phenylpropionic acid salt, propionic salt, succinate, tartrate, phosphoric acid salt, glutaminate, supercarbonate, tosilate and undecane hydrochlorate.The preferred acid that can be used to form drug acceptable salt is hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, oxalic acid, toxilic acid, succsinic acid and citric acid.Positively charged ion in the acceptable base addition salt of medicine includes but not limited to basic metal or alkaline-earth metal ions such as lithium, sodium, potassium, calcium, magnesium and aluminium etc., and non-toxicity quaternary ammonium cation such as ammonium, tetramethyl-ammonium, tetraethyl ammonium, methylamine, dimethyl amine, Trimethylamine, triethylamine, diethylamide, ethylamine, diethylamine, thanomin, diethanolamine, piperidines, piperazine etc.Preferred base addition salt comprises phosphoric acid salt, tris and acetate.

Chemokine-like factor superfamily member protein of the present invention can combine with pharmaceutically-acceptable excipients or carrier and form pharmaceutical composition.Medicine acceptable carrier or vehicle refer to nontoxic solid-state, semi-solid state or liquid weighting agent, thinner, coating material or other pharmaceutical adjuncts.Described pharmaceutical composition is suitable in parenteral, hypogloeeis, the brain pond, in the intravaginal, intraperitoneal, internal rectum, cheek, in the tumour or the epidermis administration.Parenteral admin comprises that intravenously, intramuscular, intraperitoneal, breastbone are interior, subcutaneous, intra-articular injection and infusion.The pharmaceutical composition that is suitable for parenteral admin comprises aseptic aqueous solution or non-aqueous solution, dispersion liquid, suspension or emulsion, and is used for before facing use in sterile injectable solution or dispersion liquid powder formulated.Suitable water-based or non-aqueous carrier, thinner, solvent or vehicle comprise water, ethanol, glycerine, propylene glycol, polyoxyethylene glycol, carboxymethyl cellulose, vegetables oil and injectable organic ester such as ethyl oleate.These compositions can also contain adjuvants such as sanitas, wetting agent, emulsifying agent and dispersion agent.Adding isotonic agent may be favourable as carbohydrate, sodium-chlor etc.The epidermis administration is included on skin, the mucous membrane and in the surperficial administration of lung and eye.Such pharmaceutical composition comprises pulvis, ointment, drops, transdermal patch, Iontophoretic device and inhalation etc.In the composition that is suitable for sucking, chemokine-like factor superfamily member of the present invention is compressed or is contained in pressurized gas such as nitrogen or the liquefied gas propellant.Preferably, chemokine-like factor superfamily member of the present invention is insoluble in the liquefaction propulsive medium.The composition of rectum or vagina administration is preferably suppository, it can be by being mixed with chemokine-like factor superfamily member of the present invention and suitable non-irritating excipient or carrier such as theobroma oil, polyoxyethylene glycol or suppository wax, described vehicle or carrier are solid-state in room temperature, be liquid under body temperature, therefore fusing and discharge active compound in rectum or vaginal canal.

When treating with above-mentioned or other modes, the acceptable salt form of respective pure form, medicine that the chemokine-like factor superfamily member of the present invention of treatment significant quantity can be a polypeptide of the present invention, or selectivity is used pharmaceutically-acceptable excipients.Concrete treatment effective dose to any particular patient depends on many factors, comprises gentle its severity of the disease of being treated; The activity of used particular compound; Used particular composition; Patient's age, body weight, sex, diet and general health situation; Administration time; Route of administration; The drainage rate of particular compound; The time length associating of treatment or the other drug of taking simultaneously etc.For example, when topical, the total per daily dose of chemokine-like factor superfamily member polypeptide of the present invention is the 0.05-20mg/kg body weight, and the per daily dose when being administered systemically is the 0.15-50mg/kg body weight.Therefore, the single dose form of composition can contain the whole of per daily dose or the amount that it is a part of of constituting.

The invention provides a kind of antagonist, it can suppress or seal chemokine-like factor superfamily member of the present invention or its segmental biological activity, and it can comprise albumen, nucleic acid, carbohydrate etc.Antagonist can combine with the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle and form pharmaceutical composition.

The invention provides a kind of activator, it can activate chemokine-like factor superfamily member of the present invention or its segmental biological activity.Activator can comprise albumen, nucleic acid, and carbohydrate, small molecules, perhaps other directly or indirectly strengthens chemokine-like factor superfamily member of the present invention or its segmental compound or composition.Activator can the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle in conjunction with forming pharmaceutical composition.

The invention still further relates to the polynucleotide and the application of polypeptide in the pharmaceutical composition of preparation treatment tumour, hemopoietic system and/or disease of immune system of chemokine-like factor superfamily of the present invention.

In one embodiment of the invention, (referring to embodiment 3) further studied the function of CKLF2, it is except having international patent application (referring to international application: the function of the promotion COS-7 cell bone cells propagation of mentioning PCT/CH00/00026), also has the function that promotes C2C12 cell proliferation and differentiation, Fig. 6 has shown that especially CKLF2 promotes C2C12 cell myotube to form (referring to embodiment three), shows that chemokine-like factor superfamily member of the present invention can be used for the treatment of amyotrophy or other degeneration.What should be mentioned in that is, bone cells in the propagation is easier to accept foreign DNA, thereby when using dna vaccination or injection DNA pharmacological agent disease, can inject chemokine-like factor superfamily member of the present invention simultaneously, to promote dna vaccination or drug absorption, improve immunity and result of treatment.

And in another preferred embodiment of the present invention (referring to embodiment four, five), the inventor is representative with CKLF-RP2, has studied the distribution expression pattern and the function of chemokine-like factor superfamily.Utilize the Auele Specific Primer of CKLF-RP2, with the strand cDNA library template that Clontech company produces, the result who carries out pcr amplification shows that the expression level of CKLF-RP2 in normal adult tissue is lower, and the expression level in embryo and tumor tissues is higher.(referring to patent publication No.: express spectra CN1283693A) is similar for the cytokine CKLF-H1-A of the express spectra of CKLF-RP2 and CKLF1 and patent applied for.The expression level of CKLF-RP2 in embryo and tumor tissues is higher, show that polynucleotide sequence of the present invention can be used as a diagnosis index, in conjunction with restriction fragment length polypeptide analysis (RFLP), fluorescence in situ hybridization method methods such as (FISH), detection bodies internal cause chemokine-like factor superfamily member of the present invention expresses the pathological state due to not enough or excessive.Equally, also can use the purified product of chemokine-like factor superfamily member protein of the present invention, reach identical purpose by radioimmunoassay, competitive combined techniques, Western engram analysis method or enzyme-linked immunosorbent assay (ELISA).

In vitro study is found, CKLF-RP2 has significantly short proliferation function to the Hela cell, (referring to embodiment five), expression pattern analysis shows that it is in tumor tissues simultaneously, prostate cancer for example, intestinal cancer, adenocarcinoma of lung, the expression level of mammary cancer etc. higher (referring to embodiment four), prompting CKLF-RP2 is in the generation of tumour, play an important role in development and the transfer, utilize CKLF-RP2 antisense oligonucleotide blocking-up CKLF-RP2 to express or utilize in the anti-CKLF-RP2 antibody and the effect of CKLF-RP2, to oncotherapy, prostate cancer especially, intestinal cancer, adenocarcinoma of lung, oncotherapies such as mammary cancer will play an important role.

Discover in the body, compare with injection empty carrier control mice, various cell densities obviously increase (referring to embodiment five) in the spleen of injection CKLF-RP2 eukaryon expression plasmid mouse, show that CKLF-RP2 has the hematopoietic cell hormesis, (referring to patent publication No.: hemopoinesis stimulating activity CN 1283693A) is similar for the cytokine CKLF-H1-A of this active and CKLF1, CKLF2 and patent applied for, prompting CKLF-RP2 has the effect that promotes proliferation of bone marrow cells, and the short proliferation of bone marrow cells effect of this and CKLF1, CKLF2 and CKLF-H1A is closely similar.Therefore chemokine-like factor superfamily member of the present invention can stimulate the hemopoietic function of body, can be used for the treatment of disease of hematopoietic system, comprises illnesss such as primary or Secondary cases hematopoietic disorder.Especially, red corpuscle and lymphocyte density obviously increase in the spleen of injection CKLF-RP2 eukaryon expression plasmid mouse, point out chemokine-like factor superfamily member of the present invention to have vital role in immunomodulatory and hematopoietic stimulation.

Preferred embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment one, CKLF-RP1-8 Full Length cDNA Cloningization and specificity analysis

1, CKLF-RP1-5, the EST splicing of 7,8 full-length cDNAs:

Utilize expressed sequence tag (the Expression Sequence Tag of CKLF2 protein sequence to Protein Data Bank (blastp of NCBI) and people, 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; The full length cDNA sequence of CKLF-RP6 increases with reference to the sequences Design primer that NCBI discharges.

Table 1 is used for CKLF-RP1-5, the est sequence of 7,8 full length cDNA sequences splicing

	The est sequence of full length cDNA sequence splicing
		CKLF-RP1	BI464188，AL038679，AI632227，AI242300，BG389971，AI540221， AI269738，AI217152，AI962574，AA921931，AA193255，AI825627， AI695900
CKLF-RP2	BG826826，AA778552，BG212551，AI201364，AW663435，AI223025
		CKLF-RP3	AL542357，AL551269，AL514858，AL526558，BG744169，AL544210， AL550478，BG758446，BG748053，AL527977，BI199176，BG697860， BG323663，BG323505
CKLF-RP4	AL530351，BF327262，AW297066，BF331942，and AI208468
		CKLF-RP5	BF530674，BF346190，AI147740，AA452430，AI553750，AI567497， AW296102，AA452257，AI471151
CKLF-RP7	BG182013，BG189368，BG189930，BG193535，AW080832，BE869501， BF793712，AI693734，AI564525，BG213702，BG216957，BG656425
		CKLF-RP8	AL528672，AL570224，AL528671，AL543749，BG761693，BE740519， BG761693，BE740519，BG477819，BG340845，BG282018，BG478235， BI459684

2, the cDNA cloning of CKLF-RP1-8:

The CKLF-RP1-5 that splicing obtains according to EST, 7, the special primer (seeing Table 2) of full length cDNA sequence design of the CKLF-RP6 of 8 full length cDNA sequences and NCBI prediction, different and the Unigene analytical results according to the EST source, select the strand cDNA library (available from Clontech) of high expression level corresponding gene, carry out pcr amplification, (94 ℃, 5 minutes; 94 ℃, 20 seconds, 58 ℃, 20 seconds, 72 ℃, 30 seconds, 35 circulations; 72 ℃, 7 minutes).Purified pcr product is cloned in the pGEM-T Easy carrier (Promega), connects product and transforms XL-1 Blue bacterium, with blue hickie screening positive clone.The order-checking plasmid carries out purifying with Tip-20Mini-preparation test kit (Qiagen), and 3100 sequenators carry out dna sequence analysis, proves that the EST splicing is correct.

Table 2 is used for the primer sequence and the cDNA library of CKLF-RP1-8 cDNA amplification

	Upstream primer	Downstream primer	The cDNA library
				CKLF-RP1	CACCATGGATCCTGAACACG C	AGCGATTCGACAGACACGTG C	Testis
CKLF-RP2	AAGGACACCGAGTCAGTCAT G	TCATTTCTTTCCCTTTGCTG GCC	Testis
				CKLF-RP3	CGCGAGAAGAGGGGAGCCAG	ATCTCGCAGTGCCCATAGCC	Placenta
CKLF-RP4	GGGCGGCAGCATGCGG	GGCAGGTCCTCACGTGTCCA G	Spleen
				CKLF-RP5	GCCTCCATCTCTGCCTACATG	GGCTCCACTGTCCTCTCTGC	Prostate cancer
CKLF-RP6	ACGATGGAGGAGCCGC	TCACTGTATGGTCCTGGATC TC	Prostate cancer
				CKLF-RP7	CTGGGGCCGCGCAATG	TAACAAAGGCAGAGATGGAG AGGAG	Placenta
CKLF-RP8	CACGATGGAGAACGGAGCGG	GGGCTCAGGCACCACAATG	Tonsilla

3, the homology between CKLF2 and the CKLF-RP1-8 relatively

After obtaining the cDNA sequence of CKLF-RP1-8, find that its encoded protein matter is new sequence, as SEQ ID NO:2,4,6,8,10,12,14, shown in 16.SEQ ID NO:1,3,5,7,9,11,13,15 is corresponding nucleotide sequence, and wherein, the accession number of CKLF2 in Genbank is AF135380, and the Accession numbers of CKLF-RP1-8 is respectively: CKLF-RP1:AF278577; CKLF-RP2:AF479260; CKLF-RP3:AF479813; CKLF-RP4:AF479814; CKLF-RP5:AF479262; CKLF-RP6:AF479261; CKLF-RP7:AF 479263; CKLF-RP8:AF474370.New patent searching finds that the CKLF-RP1-8 sequence is new sequence.Protein sequence analysis shows conservative aminoacid sequence (Fig. 1) between CKLF2 and the CKLF-RP1-8; Homology (table 3) is in various degree arranged each other, and CKLF2 and CKLF-RP1 have the typical C ys-Cys constitutional features of (being called for short the CC structure), and CKLF-RP2-8 does not then possess the CC structure; During evolution, CKLF2 and CKLF-RP1, the close source property of CKLF-RP3 and CKLF-RP5 is (Fig. 2) recently.

Conserved sequence analysis between the table 3 chemokine-like factor superfamily member

4, the transmembrane domains analysis of CKLF2 and CKLF-RP1-8:

Protein sequence with Prosite computer software analysis CKLF2 and CKLF-RP1-8.

The result:

Except CKLF-RP3 has 3 potential transmembrane domains, CKLF2 and CKLF-RP1,2,4-8 is 4 transmembrane proteins (as table 4 and shown in Figure 3).

Table 4CKLF superfamily member transmembrane domains is analyzed

	The 1st	The 2nd	The 3rd	The 4th
					CKLF2	24-40	45-67	74-96	106-128
CKLF-RP1	23-40	47-69	79-101	108-130
					CKLF-RP2	94-111	116-135	142-166	179-198
CKLF-RP3	61-83	103-122	132-151
					CKLF-RP4	59-77	87-109	121-143	148-170
CKLF-RP5	35-56	60-82	95-114	119-136
					CKLF-RP6	43-64	73-94	107-126	135-156
CKLF-RP7	40-59	69-91	103-125	145-167
					CKLF-RP8	46-68	78-100	113-130	140-162

5, chromosomal localization and genomic constitution analysis:

After obtaining the CKLF-RP1-8 sequence and carrying out corresponding analysis, find to have certain close source property between CKLF2 and the CKLF-RP1-8, for further studying the dependency between them, full length cDNA sequence with CKLF2 and CKLF-RP1-8 is retrieved in NCBI HGP database, and analyzes its genomic constitution and chromosomal localization.

The result:

CKLF2 and CKLF-RP1-4 are positioned at karyomit(e) No. 16, and close linkage, 325bp is only arranged between CKLF2 and the CKLF-RP1,322bp is only arranged between CKLF-RP1 and the CKLF-RP2, between CKLF-RP2 and the CKLF-RP3, be respectively 15kb and 6kb between CKLF-RP3 and the CKLF-RP4.Do not find to have other known at present as yet therebetween.CKLF-RP5 is positioned at karyomit(e) No. 14, CKLF-RP6, and 7,8 become the gene cluster form (see figure 4) to occur on No. 3 karyomit(e)s.Genomic constitution analyze to be found, CKLF2 and CKLF-RP1, and 2,6,8 are made up of 4 exons, CKLF-RP3,4,5,7 form (seeing Table 5) by 5 exons.

The genomic constitution of table 5CKLF superfamily member

	Exons

1	Exon 2	Exon 3	Exon 4	Exon 5
					CKLF2	1-226	225-385	386-482	483-663
CKLF-RP1	1-208	209-371	372-469	470-774
					CKLF-RP2	1-437	438-597	598-698	699-1055
CKLF-RP3	1-222	223-378	379-476	477-593							594-1508
					CKLF-RP4	1-368	369-547	548-645	646-914	922-1118
CKLF-RP5	1-317	318-470	471-564	565-648							649-726
					CKLF-RP6	1-260	261-438	439-538	539-1917
CKLF-RP7	1-188	189-361	362-459	460-541							542-1143
					CKLF-RP8	1-410	411-583	584-699	700-1133

The structure of embodiment two, eukaryon expression plasmid

Eukaryon expression plasmid pcDNA3-Myc-His (B) (being called for short pcDB) available from Invitrogen company.As shown in Figure 5, cut with the EcoRI enzyme and to discharge CKLF2 and CKLF-RP1-8 fragment, be connected to it after EcoR I enzyme is cut and handle with CIP after the pcDB carrier in, made up pcDB-CKLF2 and pcDB-CKLF-RP1-8 carrier.

Embodiment three, CKLF2 are to the short propagation and the short Differentiation of C2C12 cell

1, the foundation of C2C12/CKLF2 stable expression cell strain:

Utilize efficient transfection reagent SuperFect (Qiagen), pcDB-CKLF2 and pcDB difference transfection C2C12 cell (available from U.S. ATCC cell bank) with purifying, behind the plasmid transfection cell 72 hours, in substratum, add G418, G418 rises to high density gradually from lower concentration, to 500 μ g/ml, sift out high-expression clone after two weeks, carry out subsequent experimental.PcDB and pcDB-CKLF2 stable transfection clone be called after C2C12/pcDB and C2C12/CKLF2 respectively.

2, the variation of the C2C12 cellular form that causes of CKLF2:

(1) under similarity condition, cultivates C2C12/CKLF2 and C2C12/pcDB cell.

(2) cell dyeing and cellular form.

With FITC-Phalloidin the C2C12 cell is dyeed, method is as follows: the PBS (PH7.4) with precooling washes cell, 2% Paraformaldehyde 96 is fixed, 0.1%TrintonX100 handles, PBS (PH7.4) with precooling washes cell again one time, add the FITC-Phalloidin (Sigma) of 200ng/ml in cell, room temperature was placed 30 minutes.

The result:

As shown in Figure 6, compare with the C2C12/pcDB cell, the C2C12/CKLF2 cytogamy is quickened, and myotube increases and be more.

3, CKLF2 is to the short proliferation function of C2C12 cell:

CKLF2 promotes C2C12 cell myotube to form, and prompting CKLF2 can promote the C2C12 cytodifferentiation can promote that maybe its propagation or the two have both at the same time.For further studying its mechanism of action, at first observed the short proliferation function of CKLF2 to the C2C12 cell.The C2C12/CKLF2 and the C2C12/pcDB cell of same amount are spread to Tissue Culture Plate, cultivated after 2-3 days, mix experiment, mtt assay and cell cycle analysis with cell counting, H3 respectively, research CKLF2 is to the short proliferation function of C2C12 cell, and method is as follows:

(1) cell counting: C2C12/CKLF2 cell and C2C12/pcDB cell are seeded in 6 orifice plates with the density of 8000 cells/well respectively, cultured continuously 5 days, with 0.25%trypsin-EDTA cell dissociation is got off, be resuspended in the PBS damping fluid that contains 0.4% trypan blue, carry out viable cell dyeing, counting.

(2) mtt assay: C2C12/CKLF2 cell and C2C12/pcDB cell are seeded in 96 orifice plates with the density of 1000 cells/well respectively, cultured continuously 5 days, every hole add 10 μ l MTT (10mg/ml), continue to cultivate 6 hours, add 100 μ l lysates, the 570nm wavelength is surveyed the OD value.

(3) cell cycle analysis: when C2C12/CKLF2 and C2C12/pcDB cell grow to 70-80% when full, wash cell with PBS, the 0.25%trypsin-EDTA peptic cell is then with the 75% ethanol fixed cell that spends the night.Cell is washed and is resuspended in PBS among the 0.5ml PBS, and RnaseA handles 30 minutes with digestion RNA.At last, with propidium iodide (PI) transfect cell (final concentration is 20 μ g/ml), placed the dark place 30 minutes, the FACScan detection with Becton-Dickinson company utilizes CellFIT software analytical data.

(4) [ ³H] mix experiment: by detecting the synthetic of DNA, reflect the propagation situation of cell indirectly.C2C12/CKLF2 cell and C2C12/pcDB cell are seeded in 96 orifice plates with the density of 1000 cells/well respectively, after 24 hours, add in every hole 1 μ Ci/ml [ ³H], continue to cultivate 6 hours, collect cell with TamTec (Perkin Elmer) cell harvesting instrument, MicroBetaWindows Workstation (Wallac) detection mix [ ³H] value.

The result:

As shown in Figure 7, four kinds of different methods (A. cell countings; The B.MTT method; C. cell cycle research; D.H3 mixes experiment) obtain similar result, promptly compare with the empty carrier contrast, CKLF2 can obviously promote the propagation of C2C12 cell.

4, CKLF2 is to the short Differentiation of C2C12 cell:

Fig. 7 shows that CKLF2 can directly promote C2C12 cell proliferation, for whether clear and definite CKLF2 can directly promote the C2C12 cytodifferentiation, observed when C2C12/CKLF2 cell 80% is expired, the expression level that the expression of C2C12 cytodifferentiation specific molecular MCK (mouse creatine kinase), MLC (myosin light chain), the activity of MCK and myocyte break up specific transcription factor Myogenin, its method is as follows:

(1) RNA extraction and real-time quantitative RT-PCR detect the expression of MCK, MLC

Extract cell total rna with Trizol (available from U.S. life company), SUPERSCRIPTTM prepares strand cDNA library, (PE Applied Biosystems, Foster City CA) carry out the real-time quantitative PCR analysis to MLC2 and MCK molecule to use ABI PRISM 7700.

(2) creatine kinase activity detects

The creatine phosphokinase activation analysis test kit of using Sigma company detects the mouse creatine kinase activity.With deionized water dissolving NADH substrate, add cell lysate, mixing was placed 5 minutes, with POLARstar galaxy (BMGtechnologies, the absorption spectrum when Australia) spectrophotometer detects 340nm.In each cell cultures hole, add 300 μ l substrates and 10 μ l cell lysates.All experiments repeat twice at least.

(3) Western Blot analyzes the expression level of Myogenin

From myoblast and myotube, extract nucleoprotein, carry out quantitatively extracting albumen with Coomassie Plus Protein Assay Reagent Kit (Pierce).SDS-PAGE (concentration is 10%) isolated protein, and go on the Hybond-ECL film.After one anti-and the albumen test, add the anti-mouse IgG. antibody of horseradish peroxidase (HRP) mark, with ECL colour developing liquid colour developing (Pierce).

The result:

As shown in table 6, to compare with C2C12/pcDB, the MCK of C2C12/CKLF2 cell, the expression level of MLC increase, and the activity of MCK also obviously improves (Fig. 8); The expression level of Myogenin also obviously increases (Fig. 9), proves that CKLF2 can promote the C2C12 cytodifferentiation.

Table 6CKLF2 promotes C2C12 to express myocyte special molecular MCK and MLC2

Molecule marker	C2C12/CKLF2 cell multiple changes
					The 1st day	The 2nd day	The 3rd day	The 4th day
	MCK MLC2	+6 +1.6	+4 +1.2	+18 +5.8					+3528 +6.9

The distribution expression pattern research of embodiment four, CKLF-RP2

Utilize the Auele Specific Primer (seeing Table 2) of CKLF-RP2 and the strand cDNA library of Clontech company production to carry out pcr amplification.In 25 μ l reaction systems, add 1 μ l template, amplification condition is: 95 ℃, 5 minutes; 95 ℃, 20 seconds, 58 ℃, 20 seconds, 72 ℃, 30 seconds, 35cycles; 72 ℃, 7 minutes.Get 10 μ lPCR products electrophoresis in agarose gel.

The result:

As shown in figure 10, the expression level of CKLF-RP2 in normal adult tissue is very low, and the expression of higher level is arranged in embryo and tumor tissues, and this distribution expression pattern to CKLF1, CKLF2 and CKLF-H1A is similar.

Embodiment five, CKLF-RP2 are to the short proliferation function of Hela cell

Utilize electroporation with pcDB-CKLF-RP2 and pcDB difference transfection Hela cell (ATCC CCL 2), density with 2000 cells/well is spread cell to 96 orifice plates, after 72 hours, every hole adds 10 μ l MTT, and (MTT of concentration 500 μ g/ml continues to cultivate after 4-6 hour, adds 100 μ l cell pyrolysis liquid (20%SDS, 50%DMSO, PH4.5), after 37 ℃ of overnight incubation, on BioTek Elisa Reader, read OD _570nmAbsorption value.

The result:

As shown in figure 11, compare with the empty carrier control group, CKLF-RP2 can obviously promote the propagation of Hela cell.

Embodiment six, CKLF-RP2 are to the short proliferation function of hematopoietic cell

100 μ gCKLF-RP2 eukaryon expression plasmid pcDB-CKLF-RP2 and 100 μ g empty carrier control plasmid pcDB are injected 6 all Balb/c mice skeletal respectively.After 2 weeks, get each important organ of mouse, fixing, paraffin section is made in embedding, phenodin and eosin (HE) dyeing, and mirror is observed down.(magnification is 10 * 10)

The result:

As shown in figure 12, compare with the empty carrier control group, various cell densities obviously increase in the spleen of pcDB-CKLF-RP2 injection group mouse, show that CKLF-RP2 has the hematopoietic cell hormesis, and this is active similar to the hemopoinesis stimulating activity of CKLF1, CKLF2 and CKLF-H1A.

Embodiment seven preparation antibody

Recombinant protein or synthetic polypeptide with purifying come immune animal to produce antibody.Concrete grammar is as follows: recombinant molecule is standby after separating with chromatography.Also can separate with the SDS-PAGE gel electrophoresis, electrophoretic band is downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion, downcut from gel with the protein band of 50-100 μ g/0.2ml emulsification, mouse is carried out abdomen or subcutaneous injection.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.

The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation gene product of the present invention with it.

Sequence table

＜110〉Peking University

＜120〉has the chemokine-like factor superfamily of skeletal muscle stimulating activity and immunoregulation effect

<130>D0204068F

<160>18

<170>PatentIn version 3.1

<210>1

<211>793

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(128)..(640)

<400>1

cgtagagccg ggtgcacgcg ccggggaggt agccaacagc cgttgggacg cgacgctggt 60

tcccatggtg gagagccagc cgctgccgcg tgcactggtt cagacggcca ggccctaggg 120

acccacc atg gat cct gaa cac gcc aaa cct gag tca tcc gag gca cct 169

Met Asp Pro Glu His Ala Lys Pro Glu Ser Ser Glu Ala Pro

1 5 10

tca ggg aac ttg aaa caa ccg gag act gcc gca gcc ctg ctg agt ctt 217

Ser Gly Asn Leu Lys Gln Pro Glu Thr Ala Ala Ala Leu Leu Ser Leu

15 20 25 30

atc tta gga gca tta gct tgt ttc atc atc acc caa gcc aat gag tca 265

Ile Leu Gly Ala Leu Ala Cys Phe Ile Ile Thr Gln Ala Asn Glu Ser

35 40 45

ttt ata aca atc aca agt ctg gaa atc tgc att gtc gtt ttt ttt att 313

Phe Ile Thr Ile Thr Ser Leu Glu Ile Cys Ile Val Val Phe Phe Ile

50 55 60

cta ata tat gtg cta acc ctt cac cac ttg ctg acc tat tta cat tgg 361

Leu Ile Tyr Val Leu Thr Leu His His Leu Leu Thr Tyr Leu His Trp

65 70 75

ccc tta ctt gat ctt acc aac agt atc att aca gct gtg ttc ctt tca 409

Pro Leu Leu Asp Leu Thr Asn Ser Ile Ile Thr Ala Val Phe Leu Ser

80 85 90

gta gtt gcc atc ttg gcc atg caa gaa aag aaa aga agg cat tta ctc 457

Val Val Ala Ile Leu Ala Met Gln Glu Lys Lys Arg Arg His Leu Leu

95 100 105 110

tat gtc ggg ggg tcc ctg tgt ctc aca gcg gta atc gtg tgt tgc atc 505

Tyr Val Gly Gly Ser Leu Cys Leu Thr Ala Val Ile Val Cys Cys Ile

115 120 125

gat gcg ttt gtg gtc acc acg aag atg agg acc aac ttg aaa aga ttc 553

Asp Ala Phe Val Val Thr Thr Lys Met Arg Thr Asn Leu Lys Arg Phe

130 135 140

ctg gga gtc gaa gtt gaa agg aag ctt tcc ccc gcc aag gac gcc tac 601

Leu Gly Val Glu Val Glu Arg Lys Leu Ser Pro Ala Lys Asp Ala Tyr

145 150 155

ccc gaa acc ggc ccc gac gcc ccg cag agg ccc gcc tga agccagcccg 650

Pro Glu Thr Gly Pro Asp Ala Pro Gln Arg Pro Ala

160 165 170

gcgccctagc agatgcacgt gtctgtcgaa tcgctgcctc cgagcccacc cccgagctcg 710

catgctgtca cccattccag cctaaatgtg accataaaat tagggctgct gcttttatcg 770

agaaaaaaaa aaaaaaaaaa aaa 793

<210>2

<211>170

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Asp Pro Glu His Ala Lys Pro Glu Ser Ser Glu Ala Pro Ser Gly

1 5 10 15

Asn Leu Lys Gln Pro Glu Thr Ala Ala Ala Leu Leu Ser Leu Ile Leu

20 25 30

Gly Ala Leu Ala Cys Phe Ile Ile Thr Gln Ala Asn Glu Ser Phe Ile

35 40 45

Thr Ile Thr Ser Leu Glu Ile Cys Ile Val Val Phe Phe Ile Leu Ile

50 55 60

Tyr Val Leu Thr Leu His His Leu Leu Thr Tyr Leu His Trp Pro Leu

65 70 75 80

Leu Asp Leu Thr Asn Ser Ile Ile Thr Ala Val Phe Leu Ser Val Val

85 90 95

Ala Ile Leu Ala Met Gln Glu Lys Lys Arg Arg His Leu Leu Tyr Val

100 105 110

Gly Gly Ser Leu Cys Leu Thr Ala Val Ile Val Cys Cys Ile Asp Ala

115 120 125

Phe Val Val Thr Thr Lys Met Arg Thr Asn Leu Lys Arg Phe Leu Gly

130 135 140

Val Glu Val Glu Arg Lys Leu Ser Pro Ala Lys Asp Ala Tyr Pro Glu

145 150 155 160

Thr Gly Pro Asp Ala Pro Gln Arg Pro Ala

165 170

<210>3

<211>1065

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(153)..(899)

<400>3

ggttggcatt cggtggtcct ggcagttagc tgagcacgcc ctctgagccg ctcggtggac 60

accaggcact ctagtaggcc tggcctaccc agaaacagca ggagagagaa gaaacaggcc 120

agctgtgaga agccaaggac accgagtcag tc atg gca cct aag gcg gca aag 173

Met Ala Pro Lys Ala Ala Lys

1 5

ggg gcc aag cca gag cca gca cca gct cca cct cca ccc ggg gcc aaa 221

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

10 15 20

ccc gag gaa gac aag aag gac ggt aag gag cca tcg gac aaa cct caa 269

Pro Glu Glu Asp Lys Lys Asp Gly Lys Glu Pro Ser Asp Lys Pro Gln

25 30 35

aag gcg gtg cag gac cat aag gag cca tcg gac aaa cct caa aag gcg 317

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

40 45 50 55

gtg cag ccc aag cac gaa gtg ggc acg agg agg ggg tgt cgc cgc tac 365

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

60 65 70

cgg tgg gaa tta aaa gac agc aat aaa gag ttc tgg ctc ttg ggg cac 413

Arg Trp Glu Leu Lys Asp Ser Asn Lys Glu Phe Trp Leu Leu Gly His

75 80 85

gct gag atc aag att cgg agt ttg ggc tgc cta ata gct gca atg ata 461

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

90 95 100

ctg ttg tcc tca ctc acc gtg cac ccc atc ttg agg ctt atc atc acc 509

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

105 110 115

atg gag ata tcc ttc ttc agc ttc ttc atc tta ctg tac agc ttt gcc 557

Met Glu Ile Ser Phe Phe Ser Phe Phe Ile Leu Leu Tvr Ser Phe Ala

120 125 130 135

att cat aga tac ata ccc ttc atc ctg tgg ccc att tct gac ctc ttc 605

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

140 145 150

aac gac ctg att gct tgt gcg ttc ctt gtg gga gcc gtg gtc ttt gct 653

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

155 160 165

gtg aga agt cgg cga tcc atg aat ctc cac tac tta ctt gct gtg atc 701

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

170 175 180

ctt att ggt gcg gct gga gtt ttt gct ttt atc gat gtg tgt ctt caa 749

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

185 190 195

aga aac cac ttc aga ggc aag aag gcc aaa aag cat atg ctg gtt cct 797

Arg Asn His Phe Arg Gly Lys Lys Ala Lys Lys His Met Leu Val Pro

200 205 210 215

cct cca gga aag gaa aaa gga ccc cag cag ggc aag gga cca gaa ccc 845

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

220 225 230

gcc aag cca cca gaa cct ggc aag cca cca ggg cca gca aag gga aag 893

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

235 240 245

aaa tga cttggaggag gctcctggtg tctgaaacgg cagtgtattt tacagcaata 949

Lys

tgtttccact ctcttccttg tcttctttct ggaatggttt tcttttccat tttcattacc 1009

acctttgctt ggaaaagaat ggattaatgg attctaaaag cctaaaaaaa aaaaaa 1065

<210>4

<211>248

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

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 Asn 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 Ile 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>5

<211>1514

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(75)..(623)

<400>5

ccgggtttcc gcttccctcc gggcgcgaga agaggggagc caggccgagc cccggcccta 60

ccgacgccgc cgcc atg tgg ccc cca gac ccc gac ccc gac ccg gac ccc 110

Met Trp Pro Pro Asp Pro Asp Pro Asp Pro Asp Pro

1 5 10

gag cct gcc ggc ggc tcc cgt ccc ggc ccc gcg gtc ccc ggg ctc cgc 158

Glu Pro Ala Gly Gly Ser Arg Pro Gly Pro Ala Val Pro Gly Leu Arg

15 20 25

gcc ctg ctg ccg gcg cgg gct ttc ctc tgc tct ctc aaa ggc cgc ctc 206

Ala Leu Leu Pro Ala Arg Ala Phe Leu Cys Ser Leu Lys Gly Arg Leu

30 35 40

ctg ctg gcc gag tcg ggt ctc tca ttc atc act ttt atc tgc tat gtg 254

Leu Leu Ala Glu Ser Gly Leu Ser Phe Ile Thr Phe Ile Cys Tyr Val

45 50 55 60

gcg tcc tca gca tct gcc ttc ctc aca gcg cct ctg ctg gag ttc ctg 302

Ala Ser Ser Ala Ser Ala Phe Leu Thr Ala Pro Leu Leu Glu Phe Leu

65 70 75

ctg gcc ttg tac ttc ctc ttt gct gat gcc atg cag ctg aat gac aag 350

Leu Ala Leu Tyr Phe Leu Phe Ala Asp Ala Met Gln Leu Asn Asp Lys

80 85 90

tgg cag ggc ttg tgc tgg ccc atg atg gac ttc ctg cgc tgt gtc acc 398

Trp Gln Gly Leu Cys Trp Pro Met Met Asp Phe Leu Arg Cys Val Thr

95 100 105

gcg gcc ctc atc tac ttt gct atc tcc atc acg gcc atc gcc aag tac 446

Ala Ala Leu Ile Tyr Phe Ala Ile Ser Ile Thr Ala Ile Ala Lys Tyr

110 115 120

tcg gat ggg gct tcc aaa gcc gct ggg gtg ttt ggc ttc ttt gct acc 494

Ser Asp Gly Ala Ser Lys Ala Ala Gly Val Phe Gly Phe Phe Ala Thr

125 130 135 140

atc gtg ttt gca act gat ttc tac ctg atc ttt aac gac gtg gcc aaa 542

Ile Val Phe Ala Thr Asp Phe Tyr Leu Ile Phe Asn Asp Val Ala Lys

145 150 155

ttc ctc aaa caa ggg gac tct gca gat gag acc aca gcc cac aag aca 590

Phe Leu Lys Gln Gly Asp Ser Ala Asp Glu Thr Thr Ala His Lys Thr

160 165 170

gaa gaa gag aat tcc gac tcg gac tct gac tga aggcctggcg ggtgccttgg 643

Glu Glu Glu Asn Ser Asp Ser Asp Ser Asp

175 180

caacctgagc cacacaggcc tccacccctg cgcctcacag gggtcgctgg cgttggagcg 703

gaggcctgga cttctgagtt gcagaggggg ctgcggacac agcaggcccc ctacagcctc 763

aggttctgcc tgagcccagc ctaccaggct tgcccctcag ctcagcactg ttgaccacgc 823

tgcgtatgag ggcatcttgg gtatcccact ccttctcccc atttctgtcc cacagccttc 883

agccctttaa cgtctctgcc aaaaaccagc acaaggagac aaagcagagc cttgtctgta 943

tctgggcagc aggtgttcca tgctgctagg tggcgggggt cgggggtctt ctgtttcact 1003

aacaggaaca aagacagaaa ccatgacagg gctgccccgc caggccccgg tgggtttgtc 1063

tgcacttggt gctcctgccc acaccagcca ctttggtgac aatgaccctt ccaagaatct 1123

ttggttcaag gagcaccagt tccctcttca ttcttgaagc agggagaaat tgacctttgc 1183

cttgtcgccc aggaagtggg gctcggcacc cataactaac acctcccacc cttggaaacc 1243

atgtcttctg ggggtgagat gaccattctg ggtctaagac tgtttcaaag aagagctcat 1303

agactgactg gtccagaaga cagagggtac aacagtggca tcacagtgac agtgtcatgg 1363

ggagctgggc gggcccagcc aaaccctcct tcttcctaga gcccagccag caggcaggag 1423

ttcctggacc ctcaggacag gaaacttcag acttagggca ggctatgggc actgcgagat 1483

gagaccgctt tgtgtcacct acgatatacg a 1514

<210>6

<211>182

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Met Trp Pro Pro Asp Pro Asp Pro Asp Pro Asp Pro Glu Pro Ala Gly

1 5 10 15

Gly Ser Arg Pro Gly Pro Ala Val Pro Gly Leu Arg Ala Leu Leu Pro

20 25 30

Ala Arg Ala Phe Leu Cys Ser Leu Lys Gly Arg Leu Leu Leu Ala Glu

35 40 45

Ser Gly Leu Ser Phe Ile Thr Phe Ile Cys Tyr Val Ala Ser Ser Ala

50 55 60

Ser Ala Phe Leu Thr Ala Pro Leu Leu Glu Phe Leu Leu Ala Leu Tyr

65 70 75 80

Phe Leu Phe Ala Asp Ala Met Gln Leu Asn Asp Lys Trp Gln Gly Leu

85 90 95

Cys Trp Pro Met Met Asp Phe Leu Arg Cys Val Thr Ala Ala Leu Ile

100 105 110

Tyr Phe Ala Ile Ser Ile Thr Ala Ile Ala Lys Tyr Ser Asp Gly Ala

115 120 125

Ser Lys Ala Ala Gly Val Phe Gly Phe Phe Ala Thr Ile Val Phe Ala

130 135 140

Thr Asp Phe Tyr Leu Ile Phe Asn Asp Val Ala Lys Phe Leu Lys Gln

145 150 155 160

Gly Asp Ser Ala Asp Glu Thr Thr Ala His Lys Thr Glu Glu Glu Asn

165 170 175

Ser Asp Ser Asp Ser Asp

180

<210>7

<211>1118

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(183)..(809)

<400>7

agcggaccga gccgcagccg cagccgggcg gcgggcgaga ggcggcggcg gcgggcgggc 60

cgcgggcagt cagtcgggcg gcggcggcgg cggcggcggc ggcgatgcgg cggccccgct 120

gagtccgccc gctcctggcg ccgggagcca gccgcgcgag gcggcccggg ccgggcggca 180

gc atg cgg agc ggc gag gag ctg gac ggc ttc gag ggc gag gcc tcg 227

Met Arg Ser Gly Glu Glu Leu Asp Gly Phe Glu Gly Glu Ala Ser

1 5 10 15

agc acc tcc atg atc tcg ggc gcc agc agc ccg tac cag ccc acc acc 275

Ser Thr Ser Met Ile Ser Glv Ala Ser Ser Pro Tvr Gln Pro Thr Thr

20 25 30

gag ccg gtg agc cag cgc cgc ggg ctg gcc ggc ctg cgc tgc gac ccc 323

Glu Pro Val Ser Gln Arg Arg Gly Leu Ala Gly Leu Arg Cys Asp Pro

35 40 45

gac tac ctg cgc ggc gcg ctc ggc cgc ctc aag gtc gcc caa gtg atc 371

Asp Tyr Leu Arg Gly Ala Leu Gly Arg Leu Lys Val Ala Gln Val Ile

50 55 60

ttg gcc ctg att gca ttc atc tgc ata gag acc atc atg gca tgc tcc 419

Leu Ala Leu Ile Ala Phe Ile Cys Ile Glu Thr Ile Met Ala Cys Ser

65 70 75

ccg tgt gaa ggc ctc tac ttt ttt gag ttt gtg agc tgc agt gcg ttt 467

Pro Cys Glu Gly Leu Tyr Phe Phe Glu Phe Val Ser Cys Ser Ala Phe

80 85 90 95

gtg gtg act ggc gtc ttg ctg att atg ttc agt ctc aac ctg cac atg 515

Val Val Thr Gly Val Leu Leu Ile Met Phe Ser Leu Asn Leu His Met

100 105 110

agg atc ccc cag atc aac tgg aat ctg aca gat ttg gtc aac act gga 563

Arg Ile Pro Gln Ile Asn Trp Asn Leu Thr Asp Leu Val Asn Thr Gly

115 120 125

ctc agc gct ttc ctt ttc ttt att gct tca atc gta ctg gct gct tta 611

Leu Ser Ala Phe Leu Phe Phe Ile Ala Ser Ile Val Leu Ala Ala Leu

130 135 140

aac cat aga gcc gga gca gaa att gct gcc gtg ata ttt ggc ttc ttg 659

Asn His Arg Ala Gly Ala Glu Ile Ala Ala Val Ile Phe Gly Phe Leu

145 150 155

gcg act gcg gca tat gca gtg aac aca ttc ctg gca gtg cag aaa tgg 707

Ala Thr Ala Ala Tyr Ala Val Asn Thr Phe Leu Ala Val Gln Lys Trp

160 165 170 175

aga gtc agc gtc cgc cag cag agc acc aat gac tac atc cga gcc cgc 755

Arg Val Ser Val Arg Gln Gln Ser Thr Asn Asp Tyr Ile Arg Ala Arg

180 185 190

acg gag tcc agg gat gtg gac agt cgc cct gag atc cag cgc ctg gac 803

Thr Glu Ser Arg Asp Val Asp Ser Arg Pro Glu Ile Gln Arg Leu Asp

195 200 205

acg tga ggacctgcct ggatcctcct ccctcccgtc ctcgtgcctg tctctcagtc 859

Thr

aagttttctt tcccttgtcc tgtcagattt ccatgtgatt cagttgaatt ttgtcctctt 919

tggtaaaagt tcattttggg aaagggtttc ccgagtggcc cagtgggcgg gtccgcggtg 979

gagttgggag gcccacgttc ctcagcgttt ggggctgtgg agcagccggc gtcactgccc 1039

aggtccactt gaggtcttac ctgccctgaa gcacaggctt cctttgactc tgagccacct 1099

tgtacgtgtg tagaaaata 1118

<210>8

<211>208

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Met Arg Ser Gly Glu Glu Leu Asp Gly Phe Glu Gly Glu Ala Ser Ser

1 5 10 15

Thr Ser Met Ile Ser Gly Ala Ser Ser Pro Tyr Gln Pro Thr Thr Glu

20 25 30

Pro Val Ser Gln Arg Arg Gly Leu Ala Gly Leu Arg Cys Asp Pro Asp

35 40 45

Tyr Leu Arg Gly Ala Leu Gly Arg Leu Lys Val Ala Gln Val Ile Leu

50 55 60

Ala Leu Ile Ala Phe Ile Cys Ile Glu Thr Ile Met Ala Cys Ser Pro

65 70 75 80

Cys Glu Gly Leu Tyr Phe Phe Glu Phe Val Ser Cys Ser Ala Phe Val

85 90 95

Val Thr Gly Val Leu Leu Ile Met Phe Ser Leu Asn Leu His Met Arg

100 105 110

Ile Pro Gln Ile Asn Trp Asn Leu Thr Asp Leu Val Asn Thr Gly Leu

115 120 125

Ser Ala Phe Leu Phe Phe Ile Ala Ser Ile Val Leu Ala Ala Leu Asn

130 135 140

His Arg Ala Gly Ala Glu Ile Ala Ala Val Ile Phe Gly Phe Leu Ala

145 150 155 160

Thr Ala Ala Tyr Ala Val Asn Thr Phe Leu Ala Val Gln Lys Trp Arg

165 170 175

Val Ser Val Arg Gln Gln Ser Thr Asn Asp Tyr Ile Arg Ala Arg Thr

180 185 190

Glu Ser Arg Asp Val Asp Ser Arg Pro Glu Ile Gln Arg Leu Asp Thr

195 200 205

<210>9

<211>988

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(191)..(661)

<400>9

ggagggccca tctgggcaag gcccccagcg cctgccttct ctcccggggc cctgtgggca 60

agcctcctgc ttcactttca ggtttctcga agtgccttct tgctcctgtc tgtttcccca 120

tcctgccaga tttctgtttc tcttgctggg cttttggcag tagggggctg tgttggtggg 180

ccctacgaag atg ctc agt gct cga gat cgc cgg gac cgg cac cct gag 229

Met Leu Ser Ala Arg Asp Arg Arg Asp Arg His Pro Glu

1 5 10

gag ggg gta gtt gca gag ctc cag ggc ttc gcg gtg gac aag gcc ttc 277

Glu Gly Val Val Ala Glu Leu Gln Gly Phe Ala Val Asp Lys Ala Phe

15 20 25

ctc acc tcc cac aag ggc atc ctg ctg gaa acc gag ctg gcc ctg acc 325

Leu Thr Ser His Lys Gly Ile Leu Leu Glu Thr Glu Leu Ala Leu Thr

30 35 40 45

ctc atc atc ttc atc tgc ttc acg gcc tcc atc tct gcc tac atg gcc 373

Leu Ile Ile Phe Ile Cys Phe Thr Ala Ser Ile Ser Ala Tyr Met Ala

50 55 60

gcg gcg cta ctg gag ttc ttc atc aca ctt gcc ttc ctc ttc ctc tat 421

Ala Ala Leu Leu Glu Phe Phe Ile Thr Leu Ala Phe Leu Phe Leu Tyr

65 70 75

gcc acc cag tac tac cag cgc ttc gac cga att aac tgg ccc tgt ctg 469

Ala Thr Gln Tyr Tyr Gln Arg Phe Asp Arg Ile Asn Trp Pro Cys Leu

80 85 90

gac ttc ctg cgc tgt gtc agt gcc atc atc atc ttc ctg gtg gtc tcc 517

Asp Phe Leu Arg Cys Val Ser Ala Ile Ile Ile Phe Leu Val Val Ser

95 100 105

ttt gca gct gtg acc tcc cgg gac gga gct gcc att gct gct ttt gtt 565

Phe Ala Ala Val Thr Ser Arg Asp Gly Ala Ala Ile Ala Ala Phe Val

110 115 120 125

ttt ggc atc atc ctg gtt tcc atc ttt gcc tat gat gcc ttc aag atc 613

Phe Gly Ile Ile Leu Val Ser Ile Phe Ala Tyr Asp Ala Phe Lys Ile

130 135 140

tac cgg act gag atg gca ccc ggg gcc agc cag ggg gac cag cag tga 661

Tyr Arg Thr Glu Met Ala Pro Gly Ala Ser Gln Gly Asp Gln Gln

145 150 155

ctctggggct acctggctcc taggcccagc cagccagaga ggacagtgga gcccagacac 721

gtctcttgat tattatagcc cagcgctaaa cccaaccaag cctaaagagt tggctggagt 781

actgggaatt atcttggagc tgtgtcagga ttggtatcat gaatttgcaa gagggacatg 841

ggggggggga gggagaaatt gacctagctc ccataattat taaaaaaagg ggcctaatct 901

caggccatac cacttttaag gctaataggt taataaggga aaggaaaagt tgactgttag 961

acaaccagat ttttgttata tatccgc 988

<210>10

<211>156

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>10

Met Leu Ser Ala Arg Asp Arg Arg Asp Arg His Pro Glu Glu Gly Val

1 5 10 15

Val Ala Glu Leu Gln Gly Phe Ala Val Asp Lys Ala Phe Leu Thr Ser

20 25 30

His Lys Gly Ile Leu Leu Glu Thr Glu Leu Ala Leu Thr Leu Ile Ile

35 40 45

Phe Ile Cys Phe Thr Ala Ser Ile Ser Ala Tyr Met Ala Ala Ala Leu

50 55 60

Leu Glu Phe Phe Ile Thr Leu Ala Phe Leu Phe Leu Tyr Ala Thr Gln

65 70 75 80

Tyr Tyr Gln Arg Phe Asp Arg Ile Asn Trp Pro Cys Leu Asp Phe Leu

85 90 95

Arg Cys Val Ser Ala Ile Ile Ile Phe Leu Val Val Ser Phe Ala Ala

100 105 110

Val Thr Ser Arg Asp Gly Ala Ala Ile Ala Ala Phe Val Phe Gly Ile

115 120 125

Ile Leu Val Ser Ile Phe Ala Tyr Asp Ala Phe Lys Ile Tyr Arg Thr

130 135 140

Glu Met Ala Pro Gly Ala Ser Gln Gly Asp Gln Gln

145 150 155

<210>11

<211>1917

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(123)..(674)

<400>11

caggcggaag ccggggagaa ggcccaggaa gtgacggccg cctcccggct accggggact 60

tctggagtcc gagaagtcaa cggcgcggtt gctgcggccg ccgcgctccc cggcccgagg 120

cg atg gag aac gga gcg gtg tac agc ccc act acg gag gag gac ccg 167

Met Glu Asn Gly Ala Val Tyr Ser Pro Thr Thr Glu Glu Asp Pro

1 5 10 15

ggc ccc gcc aga ggc ccc cgg agc ggc ctc gct gcc tac ttt ttc atg 215

Gly Pro Ala Arg Gly Pro Arg Ser Gly Leu Ala Ala Tyr Phe Phe Met

20 25 30

ggc cgg ctc cca ttg ctc cgg cgc gtt ctc aag ggc ttg cag ctg ttg 263

Gly Arg Leu Pro Leu Leu Arg Arg Val Leu Lys Gly Leu Gln Leu Leu

35 40 45

ctg tct ctg ctg gcc ttc atc tgt gaa gaa gtt gta tca caa tgt act 311

Leu Ser Leu Leu Ala Phe Ile Cvs Glu Glu Val Val Ser Gln Cvs Thr

50 55 60

tta tgt gga gga ctt tat ttt ttt gag ttt gta agc tgc agt gcc ttt 359

Leu Cys Gly Gly Leu Tyr Phe Phe Glu Phe Val Ser Cys Ser Ala Phe

65 70 75

ctt ctg agt ctc ctt ata ctg att gtg tat tgc act cca ttt tat gag 407

Leu Leu Ser Leu Leu Ile Leu Ile Val Tyr Cys Thr Pro Phe Tyr Glu

80 85 90 95

aga gtt gat acc aca aaa gta aaa tca tcg gat ttt tat att act ttg 455

Arg Val Asp Thr Thr Lys Val Lys Ser Ser Asp Phe Tyr Ile Thr Leu

100 105 110

gga aca gga tgt gtg ttt ttg ttg gca tcc atc att ttt gtt tcc aca 503

Gly Thr Gly Cys Val Phe Leu Leu Ala Ser Ile Ile Phe Val Ser Thr

115 120 125

cat gac agg act tca gct gag att gct gca att gtg ttt gga ttt ata 551

His Asp Arg Thr Ser Ala Glu Ile Ala Ala Ile Val Phe Gly Phe Ile

130 135 140

gca agt ttt atg ttc cta ctt gac ttt atc act atg ctg tat gaa aaa 599

Ala Ser Phe Met Phe Leu Leu Asp Phe Ile Thr Met Leu Tyr Glu Lys

145 150 155

cga cag gag tcc cag ctg aga aaa cct gaa aat acc act agg gct gaa 647

Arg Gln Glu Ser Gln Leu Arg Lys Pro Glu Asn Thr Thr Arg Ala Glu

160 165 170 175

gcc ctc act gag cca ctt aat gcc taa agactctggg gagcagatgt 694

Ala Leu Thr Glu Pro Leu Asn Ala

180

tacctaaggt agtgaccctg cattgtggtg cctgagccct ggcagaagct cttgtaaaat 754

ttgttaattg tttaaaccac ttcttttgga gagcaagggg aaggtcaaga aggcagtttt 814

atcaatattg tgtcagtcac cacaaagtag gccagataag ttaaaaaaaa ttttttttta 874

aataataatt gaaacttatc tcaaatggag attttggtgg gaggaggaga aaacaattgt 934

ttttaaatca cacagctcaa cggttgataa atgattctgt cattctgtta caggtcattc 994

ttttactagg cttagcttcc aaattatgct ttatagctgt ataaacatcg tgattatatt 1054

catctactta gaaattgttt tatttttaaa ttaatttgct tagctgtttg ttttgatgct 1114

tagattatgt tctgttaatg ggaatttaac atatttaaga aaccaatatt taaaatgttg 1174

gtctaggttt ttttccttaa catatattac caggctttac tgtatttcac tcagccttaa 1234

atgttataat atttttggat aacggttatt aattctttga gaccttcgta tagcctataa 1294

aatgtatggg agatgttggt attttatgtg tataaaagca acaatatcag caacttcgtg 1354

tttatactgc accttggttg ttgatgtcaa gtaaaaaaaa gattgttttg taacacataa 1414

aaaaatggaa gaaactgata ccacacctaa ggaccaaaga taagaaagac tttttgccca 1474

agacagtgaa agtaattata aaaacaagct ttgaccactt accaagtatc tgaagagatg 1534

agttcatact atgatttaga aagtggttca attcccctgt tggcatatga ttatttttac 1594

taaaattaat acagctctgt gggtcttcct tagtgttttc tttgaagcca atctgttttt 1654

tttaggacac cagcctttgg ttttteatct gttcgagatg cctcttctct gtctccttat 1714

cagatagaaa tggagtcatg tgctgctgct tcatctagca gaggttggcc tctggctctg 1774

acactttttg tcagttgtct ttaggtggtc ctgaatcttg ggcccttttg attgtgaata 1834

ctgtgtagca ggatcttgag agtccttgtt cttacatagg cattgctcta gtttgtcttt 1894

ggcaaaaaaa aaaaaaaaaa aaa 1917

<210>12

<211>183

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>12

Met Glu Asn Gly Ala Val Tyr Ser Pro Thr Thr Glu Glu Asp Pro Gly

1 5 10 15

Pro Ala Arg Gly Pro Arg Ser Gly Leu Ala Ala Tyr Phe Phe Met Gly

20 25 30

Arg Leu Pro Leu Leu Arg Arg Val Leu Lys Gly Leu Gln Leu Leu Leu

35 40 45

Ser Leu Leu Ala Phe Ile Cys Glu Glu Val Val Ser Gln Cys Thr Leu

50 55 60

Cys Gly Gly Leu Tyr Phe Phe Glu Phe Val Ser Cys Ser Ala Phe Leu

65 70 75 80

Leu Ser Leu Leu Ile Leu Ile Val Tyr Cys Thr Pro Phe Tyr Glu Arg

85 90 95

Val Asp Thr Thr Lys Val Lys Ser Ser Asp Phe Tyr Ile Thr Leu Gly

100 105 110

Thr Gly Cys Val Phe Leu Leu Ala Ser Ile Ile Phe Val Ser Thr His

115 120 125

Asp Arg Thr Ser Ala Glu Ile Ala Ala Ile Val Phe Gly Phe Ile Ala

130 135 140

Ser Phe Met Phe Leu Leu Asp Phe Ile Thr Met Leu Tyr Glu Lys Arg

145 150 155 160

Gln Glu Ser Gln Leu Arg Lys Pro Glu Asn Thr Thr Arg Ala Glu Ala

165 170 175

Leu Thr Glu Pro Leu Asn Ala

180

<210>13

<211>1177

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(44)..(571)

<400>13

ggcagctgcc cggggaggcg gtcaggcgag ctggggccgc gca atg tcg cac gga 55

Met Ser His Gly

1

gcc ggg ctc gtc cgc acc acg tgc agc agc ggc agc gcg ctc gga ccc 103

Ala Gly Leu Val Arg Thr Thr Cys Ser Ser Gly Ser Ala Leu Gly Pro

5 10 15 20

ggg gcc ggc gcg gcc cag ccc agc gcg agc ccc ttg gag ggg ctg ctg 151

Gly Ala Gly Ala Ala Gln Pro Ser Ala Ser Pro Leu Glu Gly Leu Leu

25 30 35

gac ctc agc tac ccc cgc acc cac gcg gcc ctg ctg aaa gtg gcg caa 199

Asp Leu Ser Tyr Pro Arg Thr His Ala Ala Leu Leu Lys Val Ala Gln

40 45 50

atg gtc acc ctg ctg att gcc ttc atc tgt gtg cgg agc tcc ctg tgg 247

Met Val Thr Leu Leu Ile Ala Phe Ile Cys Val Arg Ser Ser Leu Trp

55 60 65

acc aac tac agc gcc tac agc tac ttt gaa gtg gtc acc att tgc gac 295

Thr Asn Tyr Ser Ala Tyr Ser Tyr Phe Glu Val Val Thr Ile Cys Asp

70 75 80

ttg ata atg atc ctc gcc ttt tac ctg gtc cac ctc ttc cgc ttc tac 343

Leu Ile Met Ile Leu Ala Phe Tyr Leu Val His Leu Phe Arg Phe Tyr

85 90 95 100

cgc gtg ctc acc tgt atc agc tgg ccc ctg tcg gaa ctt ctg cac tat 391

Arg Val Leu Thr Cys Ile Ser Trp Pro Leu Ser Glu Leu Leu His Tyr

105 110 115

tta atc ggt acc ctg ctc ctc ctc atc gcc tcc att gtg gca gct tcc 439

Leu Ile Gly Thr Leu Leu Leu Leu Ile Ala Ser Ile Val Ala Ala Ser

120 125 130

aag agt tac aac cag agc gga ctg gta gcc gga gcg atc ttt ggt ttc 487

Lys Ser Tyr Asn Gln Ser Gly Leu Val Ala Gly Ala Ile Phe Gly Phe

135 140 145

atg gcc acc ttc ctc tgc atg gca agc ata tgg ctg tcc tat aag atc 535

Met Ala Thr Phe Leu Cys Met Ala Ser Ile Trp Leu Ser Tyr Lys Ile

150 155 160

tcg tgt gta acc cag tcc aca gat gca gcc gtc tga tgaggccaca 581

Ser Cys Val Thr Gln Ser Thr Asp Ala Ala Val

165 170 175

acccctaggc ccctcaggag ctttgcagag aggaggacgt gtactccagg cgaggcctct 641

ggacctgtgt tcctgtgcca aagtcctgtc aggctggtgg gcaccaggaa aggcctgcac 701

cctcttcctg ctctcccagg aagccagctc cctgagctcc tgagccagcc ggaaactctt 761

cctccagcct tccggggaga acatccctcc cattctggga aaggaaagca gcctccaggg 821

aaatgttttc tgccttcctg cttctagaac cacctcaggt actgatgaac cccacttagc 881

acagctgaag gggtttgtga atactcccgc ctaaatccct tctacttcac tcctcagggg 941

agtgaagtgc cttaagaaac aaagccctgt cctaatttat ctagcttgtc agtccggtct 1001

tagagatacc ctctttcctg aagtgaggcg tgcctgtaga aacactatgt ggtcagcctg 1061

tccccaagga gatcttgtgt ctcctctcca tctctgcctt tgttaccagt gtgcatgtgt 1121

ttgtgtgttt ttttaataaa atattgactc ggccagttaa aaaaaaaaaa aaaaaa 1177

<210>14

<211>175

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>14

Met Ser His Gly Ala Gly Leu Val Arg Thr Thr Cys Ser Ser Gly Ser

1 5 10 15

Ala Leu Gly Pro Gly Ala Gly Ala Ala Gln Pro Ser Ala Ser Pro Leu

20 25 30

Glu Gly Leu Leu Asp Leu Ser Tyr Pro Arg Thr His Ala Ala Leu Leu

35 40 45

Lys Val Ala Gln Met Val Thr Leu Leu Ile Ala Phe Ile Cys Val Arg

50 55 60

Ser Ser Leu Trp Thr Asn Tyr Ser Ala Tyr Ser Tyr Phe Glu Val Val

65 70 75 80

Thr Ile Cys Asp Leu Ile Met Ile Leu Ala Phe Tyr Leu Val His Leu

85 90 95

Phe Arg Phe Tyr Arg Val Leu Thr Cys Ile Ser Trp Pro Leu Ser Glu

100 105 110

Leu Leu His Tyr Leu Ile Gly Thr Leu Leu Leu Leu Ile Ala Ser Ile

115 120 125

Val Ala Ala Ser Lys Ser Tyr Asn Gln Ser Gly Leu Val Ala Gly Ala

130 135 140

Ile Phe Gly Phe Met Ala Thr Phe Leu Cys Met Ala Ser Ile Trp Leu

145 150 155 160

Ser Tyr Lys Ile Ser Cys Val Thr Gln Ser Thr Asp Ala Ala Val

165 170 175

<210>15

<211>1177

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>misc_feature

<222>(17)..(17)

＜223〉which kind of Nucleotide n=is not defined as at present as yet

<220>

<221>CDS

<222>(262)..(783)

<400>15

ttttggcgac ccagcgnccg ctaactccag ggccccagcg cagctcggga gcccgcgcac 60

cgaggcgcta ggggcaccgc gcactagagg gacacccgcc gcgcctggac agcccccggc 120

gggcgccccc ctcgcacctc ctgccccgcg cgggccgcgc tcccctcccc cgcgcctgtg 180

tccccagggc gcagggtcgc gcgtccagcc ccagacccgc cggggtccct ggggacgcgc 240

cagcccggca gtggctcgac g atg gag gag ccg cag cgc gcc cgc tcg cac 291

Met Glu Glu Pro Gln Arg Ala Arg Ser His

1 5 10

aca gtc acc acc acc gcc agc tcc ttc gca gag aac ttc ttc acc agc 339

Thr Val Thr Thr Thr Ala Ser Ser Phe Ala Glu Asn Phe Phe Thr Ser

15 20 25

agc agc agc ttc gcc tac gac cgg gag ttc ctc cgc acc ctg ccc ggc 387

Ser Ser Ser Phe Ala Tyr Asp Arg Glu Phe Leu Arg Thr Leu Pro Gly

30 35 40

ttc ctc atc gtg gcc gag atc gtt ctg ggg ctg ctg gta tgg acg ctt 435

Phe Leu Ile Val Ala Glu Ile Val Leu Gly Leu Leu Val Trp Thr Leu

45 50 55

att gct gga act gag tac ttc cgg gtc ccc gca ttt ggc tgg gtc atg 483

Ile Ala Gly Thr Glu Tyr Phe Arg Val Pro Ala Phe Gly Trp Val Met

60 65 70

ttt gta gct gta ttt tac tgg gtc ctc acc gtc ttc ttc ctc att atc 531

Phe Val Ala Val Phe Tyr Trp Val Leu Thr Val Phe Phe Leu Ile Ile

75 80 85 90

tac ata aca atg acc tac acc agg att ccc cag gtg ccc tgg aca aca 579

Tyr Ile Thr Met Thr Tyr Thr Arg Ile Pro Gln Val Pro Trp Thr Thr

95 100 105

gtg ggc ctg tgc ttt aac ggc agt gcc ttc gtc ttg tac ctc tct gcc 627

Val Gly Leu Cys Phe Asn Gly Ser Ala Phe Val Leu Tyr Leu Ser Ala

110 115 120

gct gtt gta gat gca tct tcc gtc tcc cct gag agg gac agt cac aac 675

Ala Val Val Asp Ala Ser Ser Val Ser Pro Glu Arg Asp Ser His Asn

125 130 135

ttc aac agc tgg gcg gcc tca tcg ttc ttt gcc ttc ctg gtc acc atc 723

Phe Asn Ser Trp Ala Ala Ser Ser Phe Phe Ala Phe Leu Val Thr Ile

140 145 150

tgc tac gct gga aat aca tat ttc agt ttt ata gca tgg aga tcc agg 771

Cys Tyr Ala Gly Asn Thr Tyr Phe Ser Phe Ile Ala Trp Arg Ser Arg

155 160 165 170

acc ata cag tga tttaccattt tgataattaa aaggaaaaaa aaaggaagac 823

Thr Ile Gln

tctcactgta aaaacagctg taggtataat gtatattccc agagaattgt atttaactaa 883

ttaatgtttt ttatattctt aaatttgctc acaaattgtg gtttgttaca attaaactgg 943

atacttattt gcaaagtgtt gtagcttata atggactctt aagtatctta ttaatgtatt 1003

aatgtcttca tagatcatat tttcttagac aatgtttaaa tagataaatt gctaatattg 1063

agaatgtgtc aagtttggaa acctaacttt taagatgcca gattcttttt tgattaaagt 1123

tgcaaaatcc aaaaaaaaaa aaaaaatggc ctcgttggcc tcagcggtgg agct 1177

<210>16

<211>173

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>16

Met Glu Glu Pro Gln Arg Ala Arg Ser His Thr Val Thr Thr Thr Ala

1 5 10 15

Ser Ser Phe Ala Glu Asn Phe Phe Thr Ser Ser Ser Ser Phe Ala Tyr

20 25 30

Asp Arg Glu Phe Leu Arg Thr Leu Pro Gly Phe Leu Ile Val Ala Glu

35 40 45

Ile Val Leu Gly Leu Leu Val Trp Thr Leu Ile Ala Gly Thr Glu Tyr

50 55 60

Phe Arg Val Pro Ala Phe Gly Trp Val Met Phe Val Ala Val Phe Tyr

65 70 75 80

Trp Val Leu Thr Val Phe Phe Leu Ile Ile Tyr Ile Thr Met Thr Tyr

85 90 95

Thr Arg Ile Pro Gln Val Pro Trp Thr Thr Val Gly Leu Cys Phe Asn

100 105 110

Gly Ser Ala Phe Val Leu Tyr Leu Ser Ala Ala Val Val Asp Ala Ser

115 120 125

Ser Val Ser Pro Glu Arg Asp Ser His Asn Phe Asn Ser Trp Ala Ala

130 135 140

Ser Ser Phe Phe Ala Phe Leu Val Thr Ile Cys Tyr Ala Gly Asn Thr

145 150 155 160

Tyr Phe Ser Phe Ile Ala Trp Arg Ser Arg Thr Ile Gln

165 170

<210>17

<211>459

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(1)..(459)

<400>17

atg gat aac gtg cag ccg aaa ata aaa cat cgc ccc ttc tgc ttc agt 48

Met Asp Asn Val Gln Pro Lys Ile Lys His Arg Pro Phe Cys Phe Ser

1 5 10 15

gtg aaa ggc cac gtg aag atg ctg cgg ctg gca cta act gtg aca tct 96

Val Lys Gly His Val Lys Met Leu Arg Leu Ala Leu Thr Val Thr Ser

20 25 30

atg acc ttt ttt atc atc gca caa gcc cct gaa cca tat att gtt atc 144

Met Thr Phe Phe Ile Ile Ala Gln Ala Pro Glu Pro Tyr Ile Val Ile

35 40 45

act gga ttt gaa gtc acc gtt atc tta ttt ttc ata ctt tta tat gta 192

Thr Gly Phe Glu Val Thr Val Ile Leu Phe Phe Ile Leu Leu Tyr Val

50 55 60

ctc aga ctt gat cga tta atg aag tgg tta ttt tgg cct ttg ctt gat 240

Leu Arg Leu Asp Arg Leu Met Lys Trp Leu Phe Trp Pro Leu Leu Asp

65 70 75 80

att atc aac tca ctg gta aca aca gta ttc atg ctc atc gta tct gtg 288

Ile Ile Asn Ser Leu Val Thr Thr Val Phe Met Leu Ile Val Ser Val

85 90 95

ttg gca ctg ata cca gaa acc aca aca ttg aca gtt ggt gga ggg gtg 336

Leu Ala Leu Ile Pro Glu Thr Thr Thr Leu Thr Val Gly Gly Gly Val

100 105 110

ttt gca ctt gtg aca gca gta tgc tgt ctt gcc gac ggg gcc ctt att 384

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

115 120 125

tac cgg aag ctt ctg ttc aat ccc agc ggt cct tac cag aaa aag cct 432

Tyr Arg Lys Leu Leu Phe Asn Pro Ser Gly Pro Tyr Gln Lys Lys Pro

130 135 140

gtg cat gaa aaa aaa gaa gtt ttg taa 459

Val His Glu Lys Lys Glu Val Leu

145 150

<210>18

<211>152

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>18

Met Asp Asn Val Gln Pro Lys Ile Lys His Arg Pro Phe Cys Phe Ser

1 5 10 15

Val Lys Gly His Val Lys Met Leu Arg Leu Ala Leu Thr Val Thr Ser

20 25 30

Met Thr Phe Phe Ile Ile Ala Gln Ala Pro Glu Pro Tyr Ile Val Ile

35 40 45

Thr Gly Phe Glu Val Thr Val Ile Leu Phe Phe Ile Leu Leu Tyr Val

50 55 60

Leu Arg Leu Asp Arg Leu Met Lys Trp Leu Phe Trp Pro Leu Leu Asp

65 70 75 80

Ile Ile Asn Ser Leu Val Thr Thr Val Phe Met Leu Ile Val Ser Val

85 90 95

Leu Ala Leu Ile Pro Glu Thr Thr Thr Leu Thr Val Gly Gly Gly Val

100 105 110

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

115 120 125

Tyr Arg Lys Leu Leu Phe Asn Pro Ser Gly Pro Tyr Gln Lys Lys Pro

130 135 140

Val His Glu Lys Lys Glu Val Leu

145 150

Claims (15)

1, a kind of isolating polynucleotide, these polynucleotide comprise the polynucleotide of the aminoacid sequence of coding shown in SEQ ID NO:4.

2, polynucleotide according to claim 1, wherein said polynucleotide are cDNA.

3, polynucleotide according to claim 2, it comprises the polynucleotide shown in SEQ ID NO:3.

4, polynucleotide according to claim 1, wherein said polynucleotide are genomic dnas.

5, polynucleotide according to claim 1, wherein said polynucleotide are RNA.

6, a kind of isolating polynucleotide, the polypeptide of this polynucleotide encoding is that one or more amino acid are replaced, lack or insert resulting sequence in the polypeptide of the described polynucleotide encoding of arbitrary claim among the claim 1-5, and the polypeptide of the polypeptide of this polynucleotide encoding and the described polynucleotide encoding of the arbitrary claim of claim 1-5 has identical functions.

7, a kind of carrier that contains the described polynucleotide of arbitrary claim among the claim 1-5.

8, a kind of host cell that contains the carrier of claim 7, it can obtain through carrier conversion, transfection or the transduction of claim 7.

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

(1) under the condition that is suitable for expressing, cultivates the described host cell of claim 8;

(2) polypeptide of the polynucleotide encoding that the described carrier of acquisition claim 7 carries from cell culture.

10, a peptide species, it comprises and has the aminoacid sequence shown in SEQ ID NO:4 of inferring.

11, a peptide species, this polypeptide are that one or more amino acid are replaced, lack or insert resulting sequence in the described polypeptide of claim 10, and have identical functions with the described polypeptide of claim 10.

12, the described polypeptid specificity bonded of a kind of and claim 10 antibody.

13, a kind of pharmaceutical composition, it contains the described polypeptide of claim 10, and the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle.

14, the described polynucleotide of claim 1 are in preparation amyotrophy or other degeneration, the application in the pharmaceutical composition of disease of hematopoietic system and/or disease of immune system.

15, claim 10 or 11 described polypeptide are in preparation treatment amyotrophy or other degeneration, the application in the pharmaceutical composition of hemopoietic system and/or disease of immune system.

Images