CN1464057A - Chemokine-like factor superfamily having skeletal muscle stimulating activity and immunoregulation function - Google Patents

Abstract

The present invention relates to the nucleotide sequence and amino acid sequence of chemotactic factor super-family possessing skeletal muscle stimulating activity and immunoregulation function, the production process of chemotactic factor super-family, carrier and host cell containing chemotactic factor super-family. The present invention also relates to antibody of chemotactic factor super-family member, and medicine composition, agonist and activator containing chemotactic factor super-family. The polynucleotides and polypeptide of the chemotactic factor super-family is significant in treating muscular atrophy and other retrogressive diseases, hemopoietic system disease and/or immunological system disease.

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 the biotechnology medicine (MarshallA 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 protein1-8, 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, SEQID NO:7, SEQ ID NO:9, SEQID 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, SEQ ID 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: CN1283693A). 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:AF479263; 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 SEQID 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, SEQID NO:5, SEQ ID NO:7, SEQIDNO: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 Tm20-25 ℃ 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) carry 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, SEQID 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 SEQID 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 Chemical Company, Rockford, I11., (1984)) method described press the solid state chemistry technology with Appl ied 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, aminoacid sequence of the present invention has the polypeptide of 95% homology shown in the SEQID 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), (network address is http: ∥ www.tigem.it) carry out the EST splicing by EST Assembly machine, 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 the XL-1Blue bacterium, with blue hickie screening positive clone.The order-checking plasmid carries out purifying with Tip-20 Mini-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-RP1-8cDNA 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:AF479263; 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 4 CKLF 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 5 CKLF 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	?1260	?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 EcoR I 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 lMTT (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 0.5mlPBS, 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 6 CKLF2 promotes C2C12 to express myocyte special molecular MCK and MLC2

Molecule marker MCK MLC2	C2C12/CKLF2 cell multiple changed the 1st day the 2nd day the 3rd day the 4th day
		????+6??????+4??????+18??????+3528 ????+1.6????+1.2????+5.8?????+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 lMTT, 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〉have 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〉1cgtagagccg ggtgcacgcg ccggggaggt agccaacagc cgttgggacg cgacgctggt 60tcccatggtg gagagccagc cgctgccgcg tgcactggtt cagacggcca ggccctaggg 120acccacc 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???????????????????10tca?ggg?aac?ttg?aaa?caa?ccg?gag?act?gcc?gca?gcc?ctg?ctg?agt?ctt??????217Ser?Gly?Asn?Leu?Lys?Gln?Pro?Glu?Thr?Ala?Ala?Ala?Leu?Leu?Ser?Leu15??????????????????20??????????????????25????30atc?tta?gga?gca?tta?gct?tgt?ttc?atc?atc?acc?caa?gcc?aat?gag?tca??????265Ile?Leu?Gly?Ala?Leu?Ala?Cys?Phe?Ile?Ile?Thr?Gln?Ala?Asn?Glu?Ser

35??????????????????40??????????????????45ttt?ata?aca?atc?aca?agt?ctg?gaa?atc?tgc?att?gtc?gtt?ttt?ttt?att??????313Phe?Ile?Thr?Ile?Thr?Ser?Leu?Glu?Ile?Cys?Ile?Val?Val?Phe?Phe?Ile

50??????????????????55??????????????????60cta?ata?tat?gtg?cta?acc?ctt?cac?cac?ttg?ctg?acc?tat?tta?cat?tgg??????361Leu?Ile?Tyr?Val?Leu?Thr?Leu?His?His?Leu?Leu?Thr?Tyr?Leu?His?Trp

65??????????????????70??????????????????75ccc?tta?ctt?gat?ctt?acc?aac?agt?atc?att?aca?gct?gtg?ttc?ctt?tca??????409Pro?Leu?Leu?Asp?Leu?Thr?Asn?Ser?Ile?Ile?Thr?Ala?Val?Phe?Leu?Ser

80??????????????????85??????????????????90gta?gtt?gcc?atc?ttg?gcc?atg?caa?gaa?aag?aaa?aga?agg?cat?tta?ctc??????457Val?Val?Ala?Ile?Leu?Ala?Met?Gln?Glu?Lys?Lys?Arg?Arg?His?Leu?Leu95??????????????????100?????????????????105??????????????????????????110tat?gtc?ggg?ggg?tcc?ctg?tgt?ctc?aca?gcg?gta?atc?gtg?tgt?tgc?atc??????505Tyr?Val?Gly?Gly?Ser?Leu?Cys?Leu?Thr?Ala?Val?Ile?Val?Cys?Cys?Ile

115?????????????????120?????????????????125gat?gcg?ttt?gtg?gtc?acc?acg?aag?atg?agg?acc?aac?ttg?aaa?aga?ttc??????553Asp?Ala?Phe?Val?Val?Thr?Thr?Lys?Met?Arg?Thr?Asn?Leu?Lys?Arg?Phe

130?????????????????135?????????????????140ctg?gga?gtc?gaa?gtt?gaa?agg?aag?ctt?tcc?ccc?gcc?aag?gac?gcc?tac??????601Leu?Gly?Val?Glu?Val?Glu?Arg?Lys?Leu?Ser?Pro?Ala?Lys?Asp?Ala?Tyr

145?????????????????150?????????????????155ccc?gaa?acc?ggc?ccc?gac?gcc?ccg?cag?agg?ccc?gcc?tga?agccagcccg???????650Pro?Glu?Thr?Gly?Pro?Asp?Ala?Pro?Gln?Arg?Pro?Ala

160 165 170gcgccctagc agatgcacgt gtctgtcgaa tcgctgcctc cgagcccacc cccgagctcg 710catgctgtca cccattccag cctaaatgtg accataaaat tagggctgct gcttttatcg 770agaaaaaaaa aaaaaaaaaa aaa, 793＜210〉2＜211〉170＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉2Met Asp Pro Glu His Ala Lys Pro Glu Ser Ser Glu Ala Pro Ser Gly1,5 10 15Asn Leu Lys Gln Pro Glu Thr Ala Ala Ala Leu Leu Ser Leu Ile Leu

20??????????????????25??????????????????30Gly?Ala?Leu?Ala?Cys?Phe?Ile?Ile?Thr?Gln?Ala?Asn?Glu?Ser?Phe?Ile

35??????????????????40??????????????????45Thr?Ile?Thr?Ser?Leu?Glu?Ile?Cys?Ile?Val?Val?Phe?Phe?Ile?Leu?Ile

50??????????????????55??????????????????60Tyr?Val?Leu?Thr?Leu?His?His?Leu?Leu?Thr?Tyr?Leu?His?Trp?Pro?Leu65??????????????????70??????????????????75??????????????????80Leu?Asp?Leu?Thr?Asn?Ser?Ile?Ile?Thr?Ala?Val?Phe?Leu?Ser?Val?Val

85??????????????????90??????????????????95Ala?Ile?Leu?Ala?Met?Gln?Glu?Lys?Lys?Arg?Arg?His?Leu?Leu?Tyr?Val

100?????????????????105?????????????????110Gly?Gly?Ser?Leu?Cys?Leu?Thr?Ala?Val?Ile?Val?Cys?Cys?Ile?Asp?Ala

115??????????????????120?????????????????125Phe?Val?Val?Thr?Thr?Lys?Met?Arg?Thr?Asn?Leu?Lys?Arg?Phe?Leu?Gly

130????????????????135?????????????????140Val?Glu?Val?Glu?Arg?Lys?Leu?Ser?Pro?Ala?Lys?Asp?Ala?Tyr?Pro?Glu145?????????????????150?????????????????155?????????????????160Thr?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〉3ggttggcatt cggtggtcct ggcagttagc tgagcacgcc ctctgagccg ctcggtggac 60accaggcact ctagtaggcc tggcctaccc agaaacagca ggagagagaa gaaacaggcc 120agctgtgaga agccaaggac accgagtcag tc atg gca cct aag gcg gca aag 173

Met?Ala?Pro?Lys?Ala?Ala?Lys

1???????????????5ggg?gcc?aag?cca?gag?cca?gca?cca?gct?cca?cct?cca?ccc?ggg?gcc?aaa???????221Gly?Ala?Lys?Pro?Glu?Pro?Ala?Pro?Ala?Pro?Pro?Pro?Pro?Gly?Ala?Lys

10??????????????????15??????????????????20ccc?gag?gaa?gac?aag?aag?gac?ggt?aag?gag?cca?tcg?gac?aaa?cct?caa??????269Pro?Glu?Glu?Asp?Lys?Lys?Asp?Gly?Lys?Glu?Pro?Ser?Asp?Lys?Pro?Gln

25??????????????????30??????????????????35aag?gcg?gtg?cag?gac?cat?aag?gag?cca?tcg?gac?aaa?cct?caa?aag?gcg??????317Lys?Ala?Val?Gln?Asp?His?Lys?Glu?Pro?Ser?Asp?Lys?Pro?Gln?Lys?Ala40??????????????????45??????????????????50??????????????????55gtg?cag?ccc?aag?cac?gaa?gtg?ggc?acg?agg?agg?ggg?tgt?cgc?cgc?tac??????365Val?Gln?Pro?Lys?His?Glu?Val?Gly?Thr?Arg?Arg?Gly?Cys?Arg?Arg?Tyr

60??????????????????65??????????????????70cgg?tgg?gaa?tta?aaa?gac?agc?aat?aaa?gag?ttc?tgg?ctc?ttg?ggg?cac??????413Arg?Trp?Glu?Leu?Lys?Asp?Ser?Asn?Lys?Glu?Phe?Trp?Leu?Leu?Gly?His

75??????????????????80??????????????????85gct?gag?atc?aag?att?cgg?agt?ttg?ggc?tgc?cta?ata?gct?gca?atg?ata??????461Ala?Glu?Ile?Lys?Ile?Arg?Ser?Leu?Gly?Cys?Leu?Ile?Ala?Ala?Met?Ile

90??????????????????95?????????????????100ctg?ttg?tcc?tca?ctc?acc?gtg?cac?ccc?atc?ttg?agg?ctt?atc?atc?acc??????509Leu?Leu?Ser?Ser?Leu?Thr?Val?His?Pro?Ile?Leu?Arg?Leu?Ile?Ile?Thr???105??????????????????110?????????????????115atg?gag?ata?tcc?ttc?ttc?agc?ttc?ttc?atc?tta?ctg?tac?agc?ttt?gcc??????557Met?Glu?Ile?Ser?Phe?Phe?Ser?Phe?Phe?Ile?Leu?Leu?Tyr?Ser?Phe?Ala120????????????????125??????????????????130??????????????????135att?cat?aga?tac?ata?ccc?ttc?atc?ctg?tgg?ccc?att?tct?gac?ctc?ttc??????605Ile?His?Arg?Tyr?Ile?Pro?Phe?Ile?Leu?Trp?Pro?Ile?Ser?Asp?Leu?Phe

140??????????????????145??????????????????150aac?gac?ctg?att?gct?tgt?gcg?ttc?ctt?gtg?gga?gcc?gtg?gtc?ttt?gct??????653Asn?Asp?Leu?Ile?Ala?Cys?Ala?Phe?Leu?Val?Gly?Ala?Val?Val?Phe?Ala

155?????????????????160?????????????????165gtg?aga?agt?cgg?cga?tcc?atg?aat?ctc?cac?tac?tta?ctt?gct?gtg?atc??????701Val?Arg?Ser?Arg?Arg?Ser?Met?Asn?Leu?His?Tyr?Leu?Leu?Ala?Val?Ile

170??????????????????175?????????????????180ctt?att?ggt?gcg?gct?gga?gtt?ttt?gct?ttt?atc?gat?gtg?tgt?ctt?caa??????749Leu?Ile?Gly?Ala?Ala?Gly?Val?Phe?Ala?Phe?Ile?Asp?Val?Cys?Leu?Gln???185??????????????????190?????????????????195aga?aac?cac?ttc?aga?ggc?aag?aag?gcc?aaa?aag?cat?atg?ctg?gtt?cct??????797Arg?Asn?His?Phe?Arg?Gly?Lys?Lys?Ala?Lys?Lys?His?Met?Leu?Val?Pro200?????????????????205?????????????????210??????????????????215cct?cca?gga?aag?gaa?aaa?gga?ccc?cag?cag?ggc?aag?gga?cca?gaa?ccc??????845Pro?Pro?Gly?Lys?Glu?Lys?Gly?Pro?Gln?Gln?Gly?Lys?Gly?Pro?Glu?Pro

220?????????????????225??????????????????230gcc?aag?cca?cca?gaa?cct?ggc?aag?cca?cca?ggg?cca?gca?aag?gga?aag??????893Ala?Lys?Pro?Pro?Glu?Pro?Gly?Lys?Pro?Pro?Gly?Pro?Ala?Lys?Gly?Lys

235 240 245aaa tga cttggaggag gctcctggtg tctgaaacgg cagtgtattt tacagcaata 949Lystgtttccact ctcttccttg tcttctttct ggaatggttt tcttttccat tttcattacc 1009acctttgctt ggaaaagaat ggattaatgg attctaaaag cctaaaaaaa aaaaaa, 1065＜210〉4＜211〉248＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉4Met Ala Pro Lys Ala Ala Lys Gly Ala Lys Pro Glu Pro Ala Pro Ala1,5 10 15Pro Pro Pro Pro Gly Ala Lys Pro Glu Glu Asp Lys Lys Asp Gly Lys

20??????????????????25??????????????????30Glu?Pro?Ser?Asp?Lys?Pro?Gln?Lys?Ala?Val?Gln?Asp?His?Lys?Glu?Pro

35?????????????????40???????????????????45Ser?Asp?Lys?Pro?Gln?Lys?Ala?Val?Gln?Pro?Lys?His?Glu?Val?Gly?Thr

50??????????????????55??????????????????60Arg?Arg?Gly?Cys?Arg?Arg?Tyr?Arg?Trp?Glu?Leu?Lys?Asp?Ser?Asn?Lys65??????????????????70???????????????????75?????????????????80Glu?Phe?Trp?Leu?Leu?Gly?His?Ala?Glu?Ile?Lys?Ile?Arg?Ser?Leu?Gly

85??????????????????????90??????????????????95Cys?Leu?Ile?Ala?Ala?Met?Ile?Leu?Leu?Ser?Ser?Leu?Thr?Val?His?Pro

100?????????????????105?????????????????110Ile?Leu?Arg?Leu?Ile?Ile?Thr?Met?Glu?Ile?Ser?Phe?Phe?Ser?Phe?Phe

115?????????????????120?????????????????125Ile?Leu?Leu?Tyr?Ser?Phe?Ala?Ile?His?Arg?Tyr?Ile?Pro?Phe?Ile?Leu

130?????????????????135?????????????????140Trp?Pro?Ile?Ser?Asp?Leu?Phe?Asn?Asp?Leu?Ile?Ala?Cys?Ala?Phe?Leu145?????????????????150?????????????????155?????????????????160Val?Gly?Ala?Val?Val?Phe?Ala?Val?Arg?Ser?Arg?Arg?Ser?Met?Asn?Leu

165?????????????????170?????????????????175His?Tyr?Leu?Leu?Ala?Val?Ile?Leu?Ile?Gly?Ala?Ala?Gly?Val?Phe?Ala

180?????????????????185?????????????????190Phe?Ile?Asp?Val?Cys?Leu?Gln?Arg?Asn?His?Phe?Arg?Gly?Lys?Lys?Ala

195?????????????????200?????????????????205Lys?Lys?His?Met?Leu?Val?Pro?Pro?Pro?Gly?Lys?Glu?Lys?Gly?Pro?Gln

210?????????????????215?????????????????220Gln?Gly?Lys?Gly?Pro?Glu?Pro?Ala?Lys?Pro?Pro?Glu?Pro?Gly?Lys?Pro225?????????????????230?????????????????235?????????????????240Pro?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〉5ccgggtttcc gcttccctcc gggcgcgaga agaggggagc caggccgagc cccggcccta 60ccgacgccgc 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???????????????????10gag?cct?gcc?ggc?ggc?tcc?cgt?ccc?ggc?ccc?gcg?gtc?ccc?ggg?ctc?cgc??????158Glu?Pro?Ala?Gly?Gly?Ser?Arg?Pro?Gly?Pro?Ala?Val?Pro?Gly?Leu?Arg

15??????????????????20??????????????????25gcc?ctg?ctg?ccg?gcg?cgg?gct?ttc?ctc?tgc?tct?ctc?aaa?ggc?cgc?ctc??????206Ala?Leu?Leu?Pro?Ala?Arg?Ala?Phe?Leu?Cys?Ser?Leu?Lys?Gly?Arg?Leu

30??????????????????35??????????????????40ctg?ctg?gcc?gag?tcg?ggt?ctc?tca?ttc?atc?act?ttt?atc?tgc?tat?gtg??????254Leu?Leu?Ala?Glu?Ser?Gly?Leu?Ser?Phe?Ile?Thr?Phe?Ile?Cys?Tyr?Val45??????????????????50??????????????????55??????????????????60gcg?tcc?tca?gca?tct?gcc?ttc?ctc?aca?gcg?cct?ctg?ctg?gag?ttc?ctg??????302Ala?Ser?Ser?Ala?Ser?Ala?Phe?Leu?Thr?Ala?Pro?Leu?Leu?Glu?Phe?Leu

65??????????????????70??????????????????75ctg?gcc?ttg?tac?ttc?ctc?ttt?gct?gat?gcc?atg?cag?ctg?aat?gac?aag??????350Leu?Ala?Leu?Tyr?Phe?Leu?Phe?Ala?Asp?Ala?Met?Gln?Leu?Asn?Asp?Lys

80??????????????????85??????????????????????90tgg?cag?ggc?ttg?tgc?tgg?ccc?atg?atg?gac?ttc?ctg?cgc?tgt?gtc?acc??????398Trp?Gln?Gly?Leu?Cys?Trp?Pro?Met?Met?Asp?Phe?Leu?Arg?Cys?Val?Thr

95??????????????????100?????????????????105gcg?gcc?ctc?atc?tac?ttt?gct?atc?tcc?atc?acg?gcc?atc?gcc?aag?tac??????446Ala?Ala?Leu?Ile?Tyr?Phe?Ala?Ile?Ser?Ile?Thr?Ala?Ile?Ala?Lys?Tyr

110?????????????????115?????????????????120tcg?gat?ggg?gct?tcc?aaa?gcc?gct?ggg?gtg?ttt?ggc?ttc?ttt?gct?acc??????494Ser?Asp?Gly?Ala?Ser?Lys?Ala?Ala?Gly?Val?Phe?Gly?Phe?Phe?Ala?Thr125?????????????????130?????????????????135??????????????????140atc?gtg?ttt?gca?act?gat?ttc?tac?ctg?atc?ttt?aac?gac?gtg?gcc?aaa??????542Ile?Val?Phe?Ala?Thr?Asp?Phe?Tyr?Leu?Ile?Phe?Asn?Asp?Val?Ala?Lys

145?????????????????150?????????????????155ttc?ctc?aaa?caa?ggg?gac?tct?gca?gat?gag?acc?aca?gcc?cac?aag?aca??????590Phe?Leu?Lys?Gln?Gly?Asp?Ser?Ala?Asp?Glu?Thr?Thr?Ala?His?Lys?Thr

160?????????????????165?????????????????170gaa?gaa?gag?aat?tcc?gac?tcg?gac?tct?gac?tga?aggcctggcg?ggtgccttgg????643Glu?Glu?Glu?Asn?Ser?Asp?Ser?Asp?Ser?Asp

175 180caacctgagc cacacaggcc tccacccctg cgcctcacag gggtcgctgg cgttggagcg 703gaggcctgga cttctgagtt gcagaggggg ctgcggacac agcaggcccc ctacagcctc 763aggttctgcc tgagcccagc ctaccaggct tgcccctcag ctcagcactg ttgaccacgc 823tgcgtatgag ggcatcttgg gtatcccact ccttctcccc atttctgtcc cacagccttc 883agccctttaa cgtctctgcc aaaaaccagc acaaggagac aaagcagagc cttgtctgta 943tctgggcagc aggtgttcca tgctgctagg tggcgggggt cgggggtctt ctgtttcact 1003aacaggaaca aagacagaaa ccatgacagg gctgccccgc caggccccgg tgggtttgtc 1063tgcacttggt gctcctgccc acaccagcca ctttggtgac aatgaccctt ccaagaatct 1123ttggttcaag gagcaccagt tccctcttca ttcttgaagc agggagaaat tgacctttgc 1183cttgtcgccc aggaagtggg gctcggcacc cataactaac acctcccacc cttggaaacc 1243atgtcttctg ggggtgagat gaccattctg ggtctaagac tgtttcaaag aagagctcat 1303agactgactg gtccagaaga cagagggtac aacagtggca tcacagtgac agtgtcatgg 1363ggagctgggc gggcccagcc aaaccctcct tcttcctaga gcccagccag caggcaggag 1423ttcctggacc ctcaggacag gaaacttcag acttagggca ggctatgggc actgcgagat 1483gagaccgctt tgtgtcacct acgatatacg a 1514＜210〉6＜211〉182＜212〉PRT＜213〉 ( Homo sapiens )＜400〉6Met Trp Pro Pro Asp Pro Asp Pro Asp Pro Asp Pro Glu Pro Ala Gly1 5 10 15Gly Ser Arg Pro Gly Pro Ala Val Pro Gly Leu Arg Ala Leu Leu Pro

20??????????????????25??????????????????30Ala?Arg?Ala?Phe?Leu?Cys?Ser?Leu?Lys?Gly?Arg?Leu?Leu?Leu?Ala?Glu

35??????????????????40??????????????????45Ser?Gly?Leu?Ser?Phe?Ile?Thr?Phe?Ile?Cys?Tyr?Val?Ala?Ser?Ser?Ala

50??????????????????55??????????????????60Ser?Ala?Phe?Leu?Thr?Ala?Pro?Leu?Leu?Glu?Phe?Leu?Leu?Ala?Leu?Tyr65??????????????????70??????????????????75??????????????????80Phe?Leu?Phe?Ala?Asp?Ala?Met?Gln?Leu?Asn?Asp?Lys?Trp?Gln?Gly?Leu

85??????????????????90??????????????????95Cys?Trp?Pro?Met?Met?Asp?Phe?Leu?Arg?Cys?Val?Thr?Ala?Ala?Leu?Ile

100?????????????105?????????????110Tyr?Phe?Ala?Ile?Ser?Ile?Thr?Ala?Ile?Ala?Lys?Tyr?Ser?Asp?Gly?Ala

115?????????????????120?????????????????125Ser?Lys?Ala?Ala?Gly?Val?Phe?Gly?Phe?Phe?Ala?Thr?Ile?Val?Phe?Ala

130?????????????????135?????????????????140Thr?Asp?Phe?Tyr?Leu?Ile?Phe?Asn?Asp?Val?Ala?Lys?Phe?Leu?Lys?Gln145?????????????????150?????????????????155?????????????????160Gly?Asp?Ser?Ala?Asp?Glu?Thr?Thr?Ala?His?Lys?Thr?Glu?Glu?Glu?Asn

165?????????????????170?????????????175Ser?Asp?Ser?Asp?Ser?Asp

180＜210〉7＜211〉1118＜212〉DNA＜213〉homo sapiens (Homo sapiens)＜220〉＜221〉CDS＜222〉(183) .. (809)＜400〉7agcggaccga gccgcagccg cagccgggcg gcgggcgaga ggcggcggcg gcgggcgggc 60cgcgggcagt cagtcgggcg gcggcggcgg cggcggcggc ggcgatgcgg cggccccgct 120gagtccgccc gctcctggcg ccgggagcca gccgcgcgag gcggcccggg ccgggcggca 180gc 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???????????????????15agc?acc?tcc?atg?atc?tcg?ggc?gcc?agc?agc?ccg?tac?cag?ccc?acc?acc??????275Ser?Thr?Ser?Met?Ile?Ser?Gly?Ala?Ser?Ser?Pro?Tyr?Gln?Pro?Thr?Thr

20??????????????????25???????????????????30gag?ccg?gtg?agc?cag?cgc?cgc?ggg?ctg?gcc?ggc?ctg?cgc?tgc?gac?ccc??????323Glu?Pro?Val?Ser?Gln?Arg?Arg?Gly?Leu?Ala?Gly?Leu?Arg?Cys?Asp?Pro

35??????????????????40??????????????????45gac?tac?ctg?cgc?ggc?gcg?ctc?ggc?cgc?ctc?aag?gtc?gcc?caa?gtg?atc??????371Asp?Tyr?Leu?Arg?Gly?Ala?Leu?Gly?Arg?Leu?Lys?Val?Ala?Gln?Val?Ile

50??????????????????55???????????????????60ttg?gcc?ctg?att?gca?ttc?atc?tgc?ata?gag?acc?atc?atg?gca?tgc?tcc??????419Leu?Ala?Leu?Ile?Ala?Phe?Ile?Cys?Ile?Glu?Thr?Ile?Met?Ala?Cys?Ser

65??????????????????70??????????????????75ccg?tgt?gaa?ggc?ctc?tac?ttt?ttt?gag?ttt?gtg?agc?tgc?agt?gcg?ttt??????467Pro?Cys?Glu?Gly?Leu?Tyr?Phe?Phe?Glu?Phe?Val?Ser?Cys?Ser?Ala?Phe80??????????????????85??????????????????90??????????????????95gtg?gtg?act?ggc?gtc?ttg?ctg?att?atg?ttc?agt?ctc?aac?ctg?cac?atg??????515Val?Val?Thr?Gly?Val?Leu?Leu?Ile?Met?Phe?Ser?Leu?Asn?Leu?His?Met

100??????????????????105?????????????????110agg?atc?ccc?cag?atc?aac?tgg?aat?ctg?aca?gat?ttg?gtc?aac?act?gga??????563Arg?Ile?Pro?Gln?Ile?Asn?Trp?Asn?Leu?Thr?Asp?Leu?Val?Asn?Thr?Gly

115?????????????????120?????????????????125ctc?agc?gct?ttc?ctt?ttc?ttt?att?gct?tca?atc?gta?ctg?gct?gct?tta??????611Leu?Ser?Ala?Phe?Leu?Phe?Phe?Ile?Ala?Ser?Ile?Val?Leu?Ala?Ala?Leu

130?????????????????135?????????????????140aac?cat?aga?gcc?gga?gca?gaa?att?gct?gcc?gtg?ata?ttt?ggc?ttc?ttg??????659Asn?His?Arg?Ala?Gly?Ala?Glu?Ile?Ala?Ala?Val?Ile?Phe?Gly?Phe?Leu

145?????????????????150?????????????????155gcg?act?gcg?gca?tat?gca?gtg?aac?aca?ttc?ctg?gca?gtg?cag?aaa?tgg??????707Ala?Thr?Ala?Ala?Tyr?Ala?Val?Asn?Thr?Phe?Leu?Ala?Val?Gln?Lys?Trp?160????????????????165?????????????????170?????????????????175aga?gtc?agc?gtc?cgc?cag?cag?agc?acc?aat?gac?tac?atc?cga?gcc?cgc??????755Arg?Val?Ser?Val?Arg?Gln?Gln?Ser?Thr?Asn?Asp?Tyr?Ile?Arg?Ala?Arg

180?????????????????185?????????????????190acg?gag?tcc?agg?gat?gtg?gac?agt?cgc?cct?gag?atc?cag?cgc?ctg?gac??????803Thr?Glu?Ser?Arg?Asp?Val?Asp?Ser?Arg?Pro?Glu?Ile?Gln?Arg?Leu?Asp

195 200 205acg tga ggacctgcct ggatcctcct ccctcccgtc ctcgtgcctg tctctcagtc 859Thraagttttctt tcccttgtcc tgtcagattt ccatgtgatt cagttgaatt ttgtcctctt 919tggtaaaagt tcattttggg aaagggtttc ccgagtggcc cagtgggcgg gtccgcggtg 979gagttgggag gcccacgttc ctcagcgttt ggggctgtgg agcagccggc gtcactgccc 1039aggtccactt gaggtcttac ctgccctgaa gcacaggctt cctttgactc tgagccacct 1099tgtacgtgtg tagaaaata, 1118＜210〉8＜211〉208＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉8Met Arg Ser Gly Glu Glu Leu Asp Gly Phe Glu Gly Glu Ala Ser Ser1,5 10 15Thr Ser Met Ile Ser Gly Ala Ser Ser Pro Tyr Gln Pro Thr Thr Glu

20??????????????????25??????????????????30Pro?Val?Ser?Gln?Arg?Arg?Gly?Leu?Ala?Gly?Leu?Arg?Cys?Asp?Pro?Asp

35??????????????????40??????????????????45Tyr?Leu?Arg?Gly?Ala?Leu?Gly?Arg?Leu?Lys?Val?Ala?Gln?Val?Ile?Leu

50??????????????????55??????????????????60Ala?Leu?Ile?Ala?Phe?Ile?Cys?Ile?Glu?Thr?Ile?Met?Ala?Cys?Ser?Pro65??????????????????70??????????????????75?????????????80Cys?Glu?Gly?Leu?Tyr?Phe?Phe?Glu?Phe?Val?Ser?Cys?Ser?Ala?Phe?Val

85??????????????????90??????????????????95Val?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?????????????????125Ser?Ala?Phe?Leu?Phe?Phe?Ile?Ala?Ser?Ile?Val?Leu?Ala?Ala?Leu?Asn

130?????????????????135?????????????????140His?Arg?Ala?Gly?Ala?Glu?Ile?Ala?Ala?Val?Ile?Phe?Gly?Phe?Leu?Ala145?????????????????150?????????????????155?????????????????160Thr?Ala?Ala?Tyr?Ala?Val?Asn?Thr?Phe?Leu?Ala?Val?Gln?Lys?Trp?Arg

165?????????????????170?????????????????175Val?Ser?Val?Arg?Gln?Gln?Ser?Thr?Asn?Asp?Tyr?Ile?Arg?Ala?Arg?Thr

180????????????????185??????????????????190Glu?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〉9ggagggccca tctgggcaag gcccccagcg cctgccttct ctcccggggc cctgtgggca 60agcctcctgc ttcactttca ggtttctcga agtgccttct tgctcctgtc tgtttcccca 120tcctgccaga tttctgtttc tcttgctggg cttttggcag tagggggctg tgttggtggg 180ccctacgaag 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???????????????????10gag?ggg?gta?gtt?gca?gag?ctc?cag?ggc?ttc?gcg?gtg?gac?aag?gcc?ttc??????277Glu?Gly?Val?Val?Ala?Glu?Leu?Gln?Gly?Phe?Ala?Val?Asp?Lys?Ala?Phe

15?????????????????20??????????????????25ctc?acc?tcc?cac?aag?ggc?atc?ctg?ctg?gaa?acc?gag?ctg?gcc?ctg?acc??????325Leu?Thr?Ser?His?Lys?Gly?Ile?Leu?Leu?Glu?Thr?Glu?Leu?Ala?Leu?Thr30??????????????????35??????????????????40??????????????????45ctc?atc?atc?ttc?atc?tgc?ttc?acg?gcc?tcc?atc?tct?gcc?tac?atg?gcc??????373Leu?Ile?Ile?Phe?Ile?Cys?Phe?Thr?Ala?Ser?Ile?Ser?Ala?Tyr?Met?Ala

50??????????????????55??????????????????60gcg?gcg?cta?ctg?gag?ttc?ttc?atc?aca?ctt?gcc?ttc?ctc?ttc?ctc?tat??????421Ala?Ala?Leu?Leu?Glu?Phe?Phe?Ile?Thr?Leu?Ala?Phe?Leu?Phe?Leu?Tyr

65??????????????????70??????????????????75gcc?acc?cag?tac?tac?cag?cgc?ttc?gac?cga?att?aac?tgg?ccc?tgt?ctg??????469Ala?Thr?Gln?Tyr?Tyr?Gln?Arg?Phe?Asp?Arg?Ile?Asn?Trp?Pro?Cys?Leu

80??????????????????85??????????????????90gac?ttc?ctg?cgc?tgt?gtc?agt?gcc?atc?atc?atc?ttc?ctg?gtg?gtc?tcc??????517Asp?Phe?Leu?Arg?Cys?Val?Ser?Ala?Ile?Ile?Ile?Phe?Leu?Val?Val?Ser

95??????????????????100?????????????????105ttt?gca?gct?gtg?acc?tcc?cgg?gac?gga?gct?gcc?att?gct?gct?ttt?gtt??????565Phe?Ala?Ala?Val?Thr?Ser?Arg?Asp?Gly?Ala?Ala?Ile?Ala?Ala?Phe?Val110?????????????????115?????????????????120?????????????????125ttt?ggc?atc?atc?ctg?gtt?tcc?atc?ttt?gcc?tat?gat?gcc?ttc?aag?atc??????613Phe?Gly?Ile?Ile?Leu?Val?Ser?Ile?Phe?Ala?Tyr?Asp?Ala?Phe?Lys?Ile

130?????????????????135?????????????????140tac?cgg?act?gag?atg?gca?ccc?ggg?gcc?agc?cag?ggg?gac?cag?cag?tga??????661Tyr?Arg?Thr?Glu?Met?Ala?Pro?Gly?Ala?Ser?Gln?Gly?Asp?Gln?Gln

145 150 155ctctggggct acctggctcc taggcccagc cagccagaga ggacagtgga gcccagacac 721gtctcttgat tattatagcc cagcgctaaa cccaaccaag cctaaagagt tggctggagt 781actgggaatt atcttggagc tgtgtcagga ttggtatcat gaatttgcaa gagggacatg 841ggggggggga gggagaaatt gacctagctc ccataattat taaaaaaagg ggcctaatct 901caggccatac cacttttaag gctaataggt taataaggga aaggaaaagt tgactgttag 961acaaccagat ttttgttata tatccgc, 988＜210〉10＜211〉156＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉10Met Leu Ser Ala Arg Asp Arg Arg Asp Arg His Pro Glu Glu Gly Val1,5 10 15Val Ala Glu Leu Gln Gly Phe Ala Val Asp Lys Ala Phe Leu Thr Ser

20??????????????????25??????????????????30His?Lys?Gly?Ile?Leu?Leu?Glu?Thr?Glu?Leu?Ala?Leu?Thr?Leu?Ile?Ile

35??????????????????40??????????????????45Phe?Ile?Cys?Phe?Thr?Ala?Ser?Ile?Ser?Ala?Tyr?Met?Ala?Ala?Ala?Leu

50?????????????????55??????????????????60Leu?Glu?Phe?Phe?Ile?Thr?Leu?Ala?Phe?Leu?Phe?Leu?Tyr?Ala?Thr?Gln65??????????????????70??????????????????75??????????????????80Tyr?Tyr?Gln?Arg?Phe?Asp?Arg?Ile?Asn?Trp?Pro?Cys?Leu?Asp?Phe?Leu

85??????????????????90??????????????????95Arg?Cys?Val?Ser?Ala?Ile?Ile?Ile?Phe?Leu?Val?Val?Ser?Phe?Ala?Ala

100?????????????????105?????????????????110Val?Thr?Ser?Arg?Asp?Gly?Ala?Ala?Ile?Ala?Ala?Phe?Val?Phe?Gly?Ile

115?????????????????120?????????????????125Ile?Leu?Val?Ser?Ile?Phe?Ala?Tyr?Asp?Ala?Phe?Lys?Ile?Tyr?Arg?Thr

130 135 140Glu Met Ala Pro Gly Ala Ser Gln Gly Asp Gln Gln145,150 155＜210〉11＜211〉1917＜212〉DNA＜213〉homo sapiens (Homo sapiens)＜220〉＜221〉CDS＜222〉(123) .. (674)＜400〉11caggcggaag ccggggagaa ggcccaggaa gtgacggccg cctcccggct accggggact 60tctggagtcc gagaagtcaa cggcgcggtt gctgcggccg ccgcgctccc cggcccgagg 120cg 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 15 10 15ggc ccc gcc aga ggc ccc cgg agc ggc ctc gct gcc tac ttt ttc atg 215Gly Pro Ala Arg Gly Pro Arg Ser Gly Leu Ala Ala Tyr Phe Phe Met

20??????????????????25??????????????????30ggc?cgg?ctc?cca?ttg?ctc?cgg?cgc?gtt?ctc?aag?ggc?ttg?cag?ctg?ttg??????263Gly?Arg?Leu?Pro?Leu?Leu?Arg?Arg?Val?Leu?Lys?Gly?Leu?Gln?Leu?Leu

35??????????????????40??????????????????45ctg?tct?ctg?ctg?gcc?ttc?atc?tgt?gaa?gaa?gtt?gta?tca?caa?tgt?act??????311Leu?Ser?Leu?Leu?Ala?Phe?Ile?Cys?Glu?Glu?Val?Val?Ser?Gln?Cys?Thr

50??????????????????55??????????????????60tta?tgt?gga?gga?ctt?tat?ttt?ttt?gag?ttt?gta?agc?tgc?agt?gcc?ttt??????359Leu?Cys?Gly?Gly?Leu?Tyr?Phe?Phe?Glu?Phe?Val?Ser?Cys?Ser?Ala?Phe

65??????????????????70??????????????????75ctt?ctg?agt?ctc?ctt?ata?ctg?att?gtg?tat?tgc?act?cca?ttt?tat?gag??????407Leu?Leu?Ser?Leu?Leu?Ile?Leu?Ile?Val?Tyr?Cys?Thr?Pro?Phe?Tyr?Glu80??????????????????85??????????????????90??????????????????95aga?gtt?gat?acc?aca?aaa?gta?aaa?tca?tcg?gat?ttt?tat?att?act?ttg??????455Arg?Val?Asp?Thr?Thr?Lys?Val?Lys?Ser?Ser?Asp?Phe?Tyr?Ile?Thr?Leu

100????????????????105??????????????????110gga?aca?gga?tgt?gtg?ttt?ttg?ttg?gca?tcc?atc?att?ttt?gtt?tcc?aca??????503Gly?Thr?Gly?Cys?Val?Phe?Leu?Leu?Ala?Ser?Ile?Ile?Phe?Val?Ser?Thr

115?????????????????120?????????????????125cat?gac?agg?act?tca?gct?gag?att?gct?gca?att?gtg?ttt?gga?ttt?ata??????551His?Asp?Arg?Thr?Ser?Ala?Glu?Ile?Ala?Ala?Ile?Val?Phe?Gly?Phe?Ile

130?????????????????135?????????????????140gca?agt?ttt?atg?ttc?cta?ctt?gac?ttt?atc?act?atg?ctg?tat?gaa?aaa??????599Ala?Ser?Phe?Met?Phe?Leu?Leu?Asp?Phe?Ile?Thr?Met?Leu?Tyr?Glu?Lys

145?????????????150?????????????????????155cga?cag?gag?tcc?cag?ctg?aga?aaa?cct?gaa?aat?acc?act?agg?gct?gaa??????647Arg?Gln?Glu?Ser?Gln?Leu?Arg?Lys?Pro?Glu?Asn?Thr?Thr?Arg?Ala?Glu160?????????????????165?????????????????170?????????????????175gcc?ctc?act?gag?cca?ctt?aat?gcc?taa?agactctggg?gagcagatgt????????????694Ala?Leu?Thr?Glu?Pro?Leu?Asn?Ala

180tacctaaggt agtgaccctg cattgtggtg cctgagccct ggcagaagct cttgtaaaat 754ttgttaattg tttaaaccac ttcttttgga gagcaagggg aaggtcaaga aggcagtttt 814atcaatattg tgtcagtcac cacaaagtag gccagataag ttaaaaaaaa ttttttttta 874aataataatt gaaacttatc tcaaatggag attttggtgg gaggaggaga aaacaattgt 934ttttaaatca cacagctcaa cggttgataa atgattctgt cattctgtta caggtcattc 994ttttactagg cttagcttcc aaattatgct ttatagctgt ataaacatcg tgattatatt 1054catctactta gaaattgttt tatttttaaa ttaatttgct tagctgtttg ttttgatgct 1114tagattatgt tctgttaatg ggaatttaac atatttaaga aaccaatatt taaaatgttg 1174gtctaggttt ttttccttaa catatattac caggctttac tgtatttcac tcagccttaa 1234atgttataat atttttggat aacggttatt aattctttga gaccttcgta tagcctataa 1294aatgtatggg agatgttggt attttatgtg tataaaagca acaatatcag caacttcgtg 1354tttatactgc accttggttg ttgatgtcaa gtaaaaaaaa gattgttttg taacacataa 1414aaaaatggaa gaaactgata ccacacctaa ggaccaaaga taagaaagac tttttgccca 1474agacagtgaa agtaattata aaaacaagct ttgaccactt accaagtatc tgaagagatg 1534agttcatact atgatttaga aagtggttca attcccctgt tggcatatga ttatttttac 1594taaaattaat acagctctgt gggtcttcct tagtgttttc tttgaagcca atctgttttt 1654tttaggacac cagcctttgg tttttcatct gttcgagatg cctcttctct gtctccttat 1714cagatagaaa tggagtcatg tgctgctgct tcatctagca gaggttggcc tctggctctg 1774acactttttg tcagttgtct ttaggtggtc ctgaatcttg ggcccttttg attgtgaata 1834ctgtgtagca ggatcttgag agtccttgtt cttacatagg cattgctcta gtttgtcttt 1894ggcaaaaaaa aaaaaaaaaa aaa 1917＜210〉12＜211〉183＜212〉PRT＜213〉 ( Homo sapiens )＜400〉12Met Glu Asn Gly Ala Val Tyr Ser Pro Thr Thr Glu Glu Asp Pro Gly1 5 10 15Pro Ala Arg Gly Pro Arg Ser Gly Leu Ala Ala Tyr Phe Phe Met Gly

20??????????????????25??????????????????30Arg?Leu?Pro?Leu?Leu?Arg?Arg?Val?Leu?Lys?Gly?Leu?Gln?Leu?Leu?Leu

35??????????????????40??????????????????45Ser?Leu?Leu?Ala?Phe?Ile?Cys?Glu?Glu?Val?Val?Ser?Gln?Cys?Thr?Leu

50??????????????????55??????????????????60Cys?Gly?Gly?Leu?Tyr?Phe?Phe?Glu?Phe?Val?Ser?Cys?Ser?Ala?Phe?Leu65??????????????????70??????????????????75??????????????????80Leu?Ser?Leu?Leu?Ile?Leu?Ile?Val?Tyr?Cys?Thr?Pro?Phe?Tyr?Glu?Arg

85??????????????????90??????????????????95Val?Asp?Thr?Thr?Lys?Val?Lys?Ser?Ser?Asp?Phe?Tyr?Ile?Thr?Leu?Gly

100?????????????????105?????????????????110Thr?Gly?Cys?Val?Phe?Leu?Leu?Ala?Ser?Ile?Ile?Phe?Val?Ser?Thr?His

115?????????????????120?????????????????125Asp?Arg?Thr?Ser?Ala?Glu?Ile?Ala?Ala?Ile?Val?Phe?Gly?Phe?Ile?Ala

130?????????????????135?????????????????140Ser?Phe?Met?Phe?Leu?Leu?Asp?Phe?Ile?Thr?Met?Leu?Tyr?Glu?Lys?Arg145?????????????????150?????????????????155?????????????????160Gln?Glu?Ser?Gln?Leu?Arg?Lys?Pro?Glu?Asn?Thr?Thr?Arg?Ala?Glu?Ala

165?????????????????170?????????????????175Leu?Thr?Glu?Pro?Leu?Asn?Ala

180＜210〉13＜211〉1177＜212〉DNA＜213〉homo sapiens (Homo sa piens)＜220〉＜221〉CDS＜222〉(44) .. (571)＜400〉13ggcagctgcc cggggaggcg gtcaggcgag ctggggccgc gca atg tcg cac gga 55

Met?Ser?His?Gly

1gcc?ggg?ctc?gtc?cgc?acc?acg?tgc?agc?agc?ggc?agc?gcg?ctc?gga?ccc??????103Ala?Gly?Leu?Val?Arg?Thr?Thr?Cys?Ser?Ser?Gly?Ser?Ala?Leu?Gly?Pro5???????????????????10??????????????????15??????????????????20ggg?gcc?ggc?gcg?gcc?cag?ccc?agc?gcg?agc?ccc?ttg?gag?ggg?ctg?ctg??????151Gly?Ala?Gly?Ala?Ala?Gln?Pro?Ser?Ala?Ser?Pro?Leu?Glu?Gly?Leu?Leu

25??????????????????30??????????????????35gac?ctc?agc?tac?ccc?cgc?acc?cac?gcg?gcc?ctg?ctg?aaa?gtg?gcg?caa??????199Asp?Leu?Ser?Tyr?Pro?Arg?Thr?His?Ala?Ala?Leu?Leu?Lys?Val?Ala?Gln

40??????????????????45??????????????????50atg?gtc?acc?ctg?ctg?att?gcc?ttc?atc?tgt?gtg?cgg?agc?tcc?ctg?tgg??????247Met?Val?Thr?Leu?Leu?Ile?Ala?Phe?Ile?Cys?Val?Arg?Ser?Ser?Leu?Trp

55??????????????????60??????????????????65acc?aac?tac?agc?gcc?tac?agc?tac?ttt?gaa?gtg?gtc?acc?att?tgc?gac??????295Thr?Asn?Tyr?Ser?Ala?Tyr?Ser?Tyr?Phe?Glu?Val?Val?Thr?Ile?Cys?Asp

70??????????????????75??????????????????80ttg?ata?atg?atc?ctc?gcc?ttt?tac?ctg?gtc?cac?ctc?ttc?cgc?ttc?tac??????343Leu?Ile?Met?Ile?Leu?Ala?Phe?Tyr?Leu?Val?His?Leu?Phe?Arg?Phe?Tyr85??????????????????90??????????????????95??????????????????100cgc?gtg?ctc?acc?tgt?atc?agc?tgg?ccc?ctg?tcg?gaa?ctt?ctg?cac?tat??????391Arg?Val?Leu?Thr?Cys?Ile?Ser?Trp?Pro?Leu?Ser?Glu?Leu?Leu?His?Tyr

105?????????????????110?????????????????115tta?atc?ggt?acc?ctg?ctc?ctc?ctc?atc?gcc?tcc?att?gtg?gca?gct?tcc??????439Leu?Ile?Gly?Thr?Leu?Leu?Leu?Leu?Ile?Ala?Ser?Ile?Val?Ala?Ala?Ser

120?????????????????125?????????????????130aag?agt?tac?aac?cag?agc?gga?ctg?gta?gcc?gga?gcg?atc?ttt?ggt?ttc??????487Lys?Ser?Tyr?Asn?Gln?Ser?Gly?Leu?Val?Ala?Gly?Ala?Ile?Phe?Gly?Phe

135?????????????????140?????????????????145atg?gcc?acc?ttc?ctc?tgc?atg?gca?agc?ata?tgg?ctg?tcc?tat?aag?atc??????535Met?Ala?Thr?Phe?Leu?Cys?Met?Ala?Ser?Ile?Trp?Leu?Ser?Tyr?Lys?Ile

150 155 160tcg tgt gta acc cag tcc aca gat gca gcc gtc tga tgaggccaca 581Ser Cys Val Thr Gln Ser Thr Asp Ala Ala Val165 170 175acccctaggc ccctcaggag ctttgcagag aggaggacgt gtactccagg cgaggcctct 641ggacctgtgt tcctgtgcca aagtcctgtc aggctggtgg gcaccaggaa aggcctgcac 701cctcttcctg ctctcccagg aagccagctc cctgagctcc tgagccagcc ggaaactctt 761cctccagcct tccggggaga acatccctcc cattctggga aaggaaagca gcctccaggg 821aaatgttttc tgccttcctg cttctagaac cacctcaggt actgatgaac cccacttagc 881acagctgaag gggtttgtga atactcccgc ctaaatccct tctacttcac tcctcagggg 941agtgaagtgc cttaagaaac aaagccctgt cctaatttat ctagcttgtc agtccggtct 1001tagagatacc ctctttcctg aagtgaggcg tgcctgtaga aacactatgt ggtcagcctg 1061tccccaagga gatcttgtgt ctcctctcca tctctgcctt tgttaccagt gtgcatgtgt 1121ttgtgtgttt ttttaataaa atattgactc ggccagttaa aaaaaaaaaa aaaaaa 1177＜210〉14＜211〉175＜212〉PRT＜213〉 ( Homo sapiens )＜400〉14Met Ser His Gly Ala Gly Leu Val Arg Thr Thr Cys Ser Ser Gly Ser1 5 10 15Ala Leu Gly Pro Gly Ala Gly Ala Ala Gln Pro Ser Ala Ser Pro Leu

20??????????????????25??????????????????30Glu?Gly?Leu?Leu?Asp?Leu?Ser?Tyr?Pro?Arg?Thr?His?Ala?Ala?Leu?Leu

35??????????????????40??????????????????45Lys?Val?Ala?Gln?Met?Val?Thr?Leu?Leu?Ile?Ala?Phe?Ile?Cys?Val?Arg

50??????????????????55??????????????????60Ser?Ser?Leu?Trp?Thr?Asn?Tyr?Ser?Ala?Tyr?Ser?Tyr?Phe?Glu?Val?Val65??????????????????70??????????????????75??????????????????80Thr?Ile?Cys?Asp?Leu?Ile?Met?Ile?Leu?Ala?Phe?Tyr?Leu?Val?His?Leu

85??????????????????90??????????????????95Phe?Arg?Phe?Tyr?Arg?Val?Leu?Thr?Cys?Ile?Ser?Trp?Pro?Leu?Ser?Glu

100?????????????????105?????????????????110Leu?Leu?His?Tyr?Leu?Ile?Gly?Thr?Leu?Leu?Leu?Leu?Ile?Ala?Ser?Ile

115?????????????????120?????????????????125Val?Ala?Ala?Ser?Lys?Ser?Tyr?Asn?Gln?Ser?Gly?Leu?Val?Ala?Gly?Ala

130?????????????????135?????????????????140Ile?Phe?Gly?Phe?Met?Ala?Thr?Phe?Leu?Cys?Met?Ala?Ser?Ile?Trp?Leu145?????????????????150?????????????????155?????????????????160Ser?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〉n=not yet is defined as which kind of nucleotides＜220 at present〉＜221〉CDS＜222〉(262) .. (783)＜400〉15ttttggcgac ccagcgnccg ctaactccag ggccccagcg cagctcggga gcccgcgcac 60cgaggcgcta ggggcaccgc gcactagagg gacacccgcc gcgcctggac agcccccggc 120gggcgccccc ctcgcacctc ctgccccgcg cgggccgcgc tcccctcccc cgcgcctgtg 180tccccagggc gcagggtcgc gcgtccagcc ccagacccgc cggggtccct ggggacgcgc 240cagcccggca 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???????????????????10aca?gtc?acc?acc?acc?gcc?agc?tcc?ttc?gca?gag?aac?ttc?ttc?acc?agc??????339Thr?Val?Thr?Thr?Thr?Ala?Ser?Ser?Phe?Ala?Glu?Asn?Phe?Phe?Thr?Ser

15??????????????????20??????????????????25agc?agc?agc?ttc?gcc?tac?gac?cgg?gag?ttc?ctc?cgc?acc?ctg?ccc?ggc??????387Ser?Ser?Ser?Phe?Ala?Tyr?Asp?Arg?Glu?Phe?Leu?Arg?Thr?Leu?Pro?Gly

30??????????????????35??????????????????40ttc?ctc?atc?gtg?gcc?gag?atc?gtt?ctg?ggg?ctg?ctg?gta?tgg?acg?ctt??????435Phe?Leu?Ile?Val?Ala?Glu?Ile?Val?Leu?Gly?Leu?Leu?Val?Trp?Thr?Leu

45??????????????????50??????????????????55att?gct?gga?act?gag?tac?ttc?cgg?gtc?ccc?gca?ttt?ggc?tgg?gtc?atg??????483Ile?Ala?Gly?Thr?Glu?Tyr?Phe?Arg?Val?Pro?Ala?Phe?Gly?Trp?Val?Met

60??????????????????65??????????????????70ttt?gta?gct?gta?ttt?tac?tgg?gtc?ctc?acc?gtc?ttc?ttc?ctc?att?atc??????531Phe?Val?Ala?Val?Phe?Tyr?Trp?Val?Leu?Thr?Val?Phe?Phe?Leu?Ile?Ile75??????????????????80???????????????????85?????????????????90?tac?ata?aca?atg?acc?tac?acc?agg?att?ccc?cag?gtg?ccc?tgg?aca?aca?????579Tyr?Ile?Thr?Met?Thr?Tyr?Thr?Arg?Ile?Pro?Gln?Val?Pro?Trp?Thr?Thr

95?????????????????100?????????????????105gtg?ggc?ctg?tgc?ttt?aac?ggc?agt?gcc?ttc?gtc?ttg?tac?ctc?tct?gcc??????627Val?Gly?Leu?Cys?Phe?Asn?Gly?Ser?Ala?Phe?Val?Leu?Tyr?Leu?Ser?Ala

110?????????????????115?????????????????120gct?gtt?gta?gat?gca?tct?tcc?gtc?tcc?cct?gag?agg?gac?agt?cac?aac??????675Ala?Val?Val?Asp?Ala?Ser?Ser?Val?Ser?Pro?Glu?Arg?Asp?Ser?His?Asn

125?????????????????130?????????????????135ttc?aac?agc?tgg?gcg?gcc?tca?tcg?ttc?ttt?gcc?ttc?ctg?gtc?acc?atc??????723Phe?Asn?Ser?Trp?Ala?Ala?Ser?Ser?Phe?Phe?Ala?Phe?Leu?Val?Thr?Ile

140 145 150tgc tac gct gga aat aca tat ttc agt ttt ata gca tgg aga tcc agg 771Cys Tyr Ala Gly Asn Thr Tyr Phe Ser Phe Ile Ala Trp Arg Ser Arg155 160 165 170acc ata cag tga tttaccattt tgataattaa aaggaaaaaa aaaggaagac 823Thr Ile Glntctcactgta aaaacagctg taggtataat gtatattccc agagaattgt atttaactaa 883ttaatgtttt ttatattctt aaatttgctc acaaattgtg gtttgttaca attaaactgg 943atacttattt gcaaagtgtt gtagcttata atggactctt aagtatctta ttaatgtatt 1003aatgtcttca tagatcatat tttcttagac aatgtttaaa tagataaatt gctaatattg 1063agaatgtgtc aagtttggaa acctaacttt taagatgcca gattcttttt tgattaaagt 1123tgcaaaatcc aaaaaaaaaa aaaaaatggc ctcgttggcc tcagcggtgg agct 1177＜210〉16＜211〉173＜212〉PRT＜213〉 ( Homo sapiens )＜400〉16Met Glu Glu Pro Gln Arg Ala Arg Ser His Thr Val Thr Thr Thr Ala1 5 10 15Ser Ser Phe Ala Glu Asn Phe Phe Thr Ser Ser Ser Ser Phe Ala Tyr

20??????????????????25??????????????????30Asp?Arg?Glu?Phe?Leu?Arg?Thr?Leu?Pro?Gly?Phe?Leu?Ile?Val?Ala?Glu

35??????????????????40??????????????????45Ile?Val?Leu?Gly?Leu?Leu?Val?Trp?Thr?Leu?Ile?Ala?Gly?Thr?Glu?Tyr

50??????????????????55??????????????????60Phe?Arg?Val?Pro?Ala?Phe?Gly?Trp?Val?Met?Phe?Val?Ala?Val?Phe?Tyr65??????????????????70??????????????????75??????????????????80Trp?Val?Leu?Thr?Val?Phe?Phe?Leu?Ile?Ile?Tyr?Ile?Thr?Met?Thr?Tyr

85??????????????????90??????????????????95Thr?Arg?Ile?Pro?Gln?Val?Pro?Trp?Thr?Thr?Val?Gly?Leu?Cys?Phe?Asn

100?????????????????105?????????????????110Gly?Ser?Ala?Phe?Val?Leu?Tyr?Leu?Ser?Ala?Ala?Val?Val?Asp?Ala?Ser

115?????????????????120?????????????????125Ser?Val?Ser?Pro?Glu?Arg?Asp?Ser?His?Asn?Phe?Asn?Ser?Trp?Ala?Ala

130?????????????????135?????????????????140Ser?Ser?Phe?Phe?Ala?Phe?Leu?Val?Thr?Ile?Cys?Tyr?Ala?Gly?Asn?Thr145?????????????????150?????????????????155?????????????????160Tyr?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〉17atg gat aac gtg cag ccg aaa ata aaa cat cgc ccc ttc tgc ttc agt 48Met Asp Asn Val Gln Pro Lys Ile Lys His Arg Pro Phe Cys Phe Ser1 5 10 15gtg aaa ggc cac gtg aag atg ctg cgg ctg gca cta act gtg aca tct 96Val Lys Gly His Val Lys Met Leu Arg Leu Ala Leu Thr Val Thr Ser

20??????????????????25??????????????????30atg?acc?ttt?ttt?atc?atc?gca?caa?gcc?cct?gaa?cca?tat?att?gtt?atc??????144Met?Thr?Phe?Phe?Ile?Ile?Ala?Gln?Ala?Pro?Glu?Pro?Tyr?Ile?Val?Ile

35??????????????????40??????????????????45act?gga?ttt?gaa?gtc?acc?gtt?atc?tta?ttt?ttc?ata?ctt?tta?tat?gta??????192Thr?Gly?Phe?Glu?Val?Thr?Val?Ile?Leu?Phe?Phe?Ile?Leu?Leu?Tyr?Val

50??????????????????55??????????????????60ctc?aga?ctt?gat?cga?tta?atg?aag?tgg?tta?ttt?tgg?cct?ttg?ctt?gat??????240Leu?Arg?Leu?Asp?Arg?Leu?Met?Lys?Trp?Leu?Phe?Trp?Pro?Leu?Leu?Asp65??????????????????70??????????????????75??????????????????80att?atc?aac?tca?ctg?gta?aca?aca?gta?ttc?atg?ctc?atc?gta?tct?gtg??????288Ile?Ile?Asn?Ser?Leu?Val?Thr?Thr?Val?Phe?Met?Leu?Ile?Val?Ser?Val

85??????????????????90??????????????????95ttg?gca?ctg?ata?cca?gaa?acc?aca?aca?ttg?aca?gtt?ggt?gga?ggg?gtg??????336Leu?Ala?Leu?Ile?Pro?Glu?Thr?Thr?Thr?Leu?Thr?Val?Gly?Gly?Gly?Val

100??????????????????105??????????????????110ttt?gca?ctt?gtg?aca?gca?gta?tgc?tgt?ctt?gcc?gac?ggg?gcc?ctt?att??????384Phe?Ala?Leu?Val?Thr?Ala?Val?Cys?Cys?Leu?Ala?Asp?Gly?Ala?Leu?Ile

115?????????????????120?????????????????125tac?cgg?aag?ctt?ctg?ttc?aat?ccc?agc?ggt?cct?tac?cag?aaa?aag?cct??????432Tyr?Arg?Lys?Leu?Leu?Phe?Asn?Pro?Ser?Gly?Pro?Tyr?Gln?Lys?Lys?Pro

130 135 140gtg cat gaa aaa aaa gaa gtt ttg taa 459Val His Glu Lys Lys Glu Val Leu145,150＜210〉18＜211〉152＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉18Met Asp Asn Val Gln Pro Lys Ile Lys His Arg Pro Phe Cys Phe Ser

5???????????????????10??????????????????15Val?Lys?Gly?His?Val?Lys?Met?Leu?Arg?Leu?Ala?Leu?Thr?Val?Thr?Ser

20??????????????????25??????????????????30Met?Thr?Phe?Phe?Ile?Ile?Ala?Gln?Ala?Pro?Glu?Pro?Tyr?Ile?Val?Ile

35??????????????????40??????????????????45Thr?Gly?Phe?Glu?Val?Thr?Val?Ile?Leu?Phe?Phe?Ile?Leu?Leu?Tyr?Val

50??????????????????55??????????????????60Leu?Arg?Leu?Asp?Arg?Leu?Met?Lys?Trp?Leu?Phe?Trp?Pro?Leu?Leu?Asp65??????????????????70??????????????????75??????????????????80Ile?Ile?Asn?Ser?Leu?Val?Thr?Thr?Val?Phe?Met?Leu?Ile?Val?Ser?Val

85??????????????????90??????????????????95Leu?Ala?Leu?Ile?Pro?Glu?Thr?Thr?Thr?Leu?Thr?Val?Gly?Gly?Gly?Val

100????????????????105?????????????????110Phe?Ala?Leu?Val?Thr?Ala?Val?Cys?Cys?Leu?Ala?Asp?Gly?Ala?Leu?Ile

115??????????????????120?????????????????125Tyr?Arg?Lys?Leu?Leu?Phe?Asn?Pro?Ser?Gly?Pro?Tyr?Gln?Lys?Lys?Pro???130??????????????????135?????????????????140Val?His?Glu?Lys?Lys?Glu?Val?Leu145?????????????????150

Claims (17)

1, a kind of isolating polynucleotide sequence, described sequence comprises the member who is selected from following sequence:

(1) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:2;

(2) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:4;

(3) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:6;

(4) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:8;

(5) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:10;

(6) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:12;

(7) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:14;

(8) polynucleotide or its fragment of the aminoacid sequence of a kind of coding shown in SEQ ID NO:16.

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

3, polynucleotide according to claim 2, it comprises the member who is selected from following sequence:

(1) a kind of polynucleotide or its fragment shown in SEQ ID NO:1;

(2) a kind of polynucleotide or its fragment shown in SEQ ID NO:3;

(3) a kind of polynucleotide or its fragment shown in SEQ ID NO:5;

(4) a kind of polynucleotide or its fragment shown in SEQ ID NO:7;

(5) a kind of polynucleotide or its fragment shown in SEQ ID NO:9;

(6) a kind of polynucleotide or its fragment shown in SEQ ID NO:11;

(7) a kind of polynucleotide or its fragment shown in SEQ ID NO:13;

(8) a kind of polynucleotide or its fragment shown in SEQ ID NO:15.

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, it comprise can with the described multi-nucleotide hybrid of arbitrary claim among the claim 1-5, and and they have the polynucleotide sequence of at least 70% homology.

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 the member who is selected from following sequence:

(1) a kind of have the aminoacid sequence shown in SEQ ID NO:2 or its fragment of inferring;

(2) a kind of have the aminoacid sequence shown in SEQ ID NO:4 or its fragment of inferring;

(3) a kind of have the aminoacid sequence shown in SEQ ID NO:6 or its fragment of inferring;

(4) a kind of have the aminoacid sequence shown in SEQ ID NO:8 or its fragment of inferring;

(5) a kind of have the aminoacid sequence shown in SEQ ID NO:10 or its fragment of inferring;

(6) a kind of have the aminoacid sequence shown in SEQ ID NO:12 or its fragment of inferring;

(7) a kind of have the aminoacid sequence shown in SEQ ID NO:14 or its fragment of inferring;

(8) a kind of have the aminoacid sequence shown in SEQ ID NO:16 or its fragment of inferring.

11, the varient of the described polypeptide of claim 10, analogue, derivative or active fragments.

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

13, a kind of pharmaceutical composition, its contain the described polypeptide of claim 10 or shown in SEQ ID NO:18 polypeptide, and the acceptable salt of one or more medicines or pharmaceutically acceptable carrier or vehicle.

14, a kind of antagonist, it can suppress or seal the biological activity of the described polypeptide of claim 10.

15, a kind of activator, it can activate the biological activity of the described polypeptide of claim 10.

16, the described polynucleotide of claim 1 or shown in SEQ ID NO:17 polynucleotide in preparation amyotrophy or other degeneration, the application in the pharmaceutical composition of disease of hematopoietic system and/or disease of immune system.

17, the described polypeptide of claim 11 or shown in SEQ ID NO:18 polypeptide in preparation treatment amyotrophy or other degeneration, the application in the pharmaceutical composition of hemopoietic system and/or disease of immune system.

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