Abstract:
An isolated human dendritic cell receptor comprising amino acid sequences selected from: TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQNCCYN FIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNFVLEQLLYFNYMA SWVMLGITYRNNSL amino acid at position 1208-1323 of SEQ ID NO:1 and SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML amino acid at position 71-106 of SEQ ID NO:1.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a 371 of WO 97/45449. 
     FIELD OF THE INVENTION 
     This invention relates to dendritic cell receptors. In particular, it relates to human DEC-205, to the production and use thereof, and to ligands which bind to it. Human DEC-205 and its ligands are useful in prophylaxis and therapy. 
     BACKGROUND OF THE INVENTION 
     Dendritic cells perform important immunoregulatory functions by presenting antigens in the form of peptides bound to cell-surface major histocompatibility complex (MHC) molecules to T cells. Identification of the mechanism by which this antigen presentation function is achieved therefore has important implications in manipulating immune response in prophylaxis and therapy, particularly in humans. 
     Jiang et al,  Nature  375: 151-155 (1995) disclose a murine dendritic cell receptor having a molecular weight of 205 kDa (murine DEC-205). However, they do not disclose a receptor on human dendritic cells. 
     The applicant has now identified a receptor on human dendritic cells. It is broadly to this receptor (likely to be the human homolog of murine DEC-205) that the present invention is directed. 
     SUMMARY OF THE INVENTION 
     The present invention has a number of aspects. In a first aspect, the invention provides isolated human DEC-205 which has an approximate molecular weight of 198-205 kDa and which includes the following amino acid sequences: 
     (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPF QNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKE NNFVLEQLLYFNYMASWVMLGITYRNNSL (amino acids at positions 1208-1323 of SEQ ID NO:1) and 
     (ii) SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML (amino acids at positions 71-106 of SEQ ID NO:1) 
     or a functionally equivalent fragment thereof. 
     In a further aspect, the invention provides isolated human DEC-205 which comprises the amino acid sequence shown in FIG. 11 or a functionally equivalent fragment thereof 
     In a still further aspect, the invention provides isolated mature human DEC-205, which comprises the amino acids 27 to 1722 shown for human DEC-205 in FIG.  11 . 
     In yet a further aspect, the invention provides an extracellular domain of human DEC-205 or a functionally-equivalent fragment thereof. 
     In a preferred embodiment, the extracellular domain fragment includes at least a portion of carbohydrate recognition domain (CRD7), spacer, and a portion of carbohydrate recognition domain (CRD8) of human DEC-205 (amino acids 1208 to 1323 of the amino acid sequence of FIG.  11 ). 
     In a still further aspect, the invention provides a polynucleotide encoding human DEC-205 or its extracellular domain as defined above. This polynucleotide is preferably DNA, more preferably cDNA, but can also be RNA. 
     In a specific embodiment, the polynucleotide coding for human DEC-205 includes the following nucleotide sequences (SEQ ID NOS:3&amp; 4, respectively, in order of appearance): 
     
       
         
               
               
             
           
               
                 (iii)  A ACA GTT GAT TGC AAT GAC AAT CAA CCA GGT GCT ATT TGC 
                   
               
               
                   
               
               
                      TAC TAT TCA GGA AAT GAG ACT GAA AAA GAG GTC AAA CCA GTT 
               
               
                   
               
               
                      GAC AGT GTT AAA TGT CCA TCT CCT GTT CTA AAT ACT CCG TGG 
               
               
                   
               
               
                      ATA CCA TTT CAG AAC TGT TGC TAC AAT TTC ATA ATA ACA AAG 
               
               
                   
               
               
                      AAT AGG CAT ATG GCA ACA ACA CAG GAT GAA GTT CAT ACT AAA 
               
               
                   
               
               
                      TGC CAG AAA CTG AAT CCA AAA TCA CAT ATT CTG AGT ATT CGA 
               
               
                   
               
               
                      GAT GAA AAG GAG AAT AAC TTT GTT CTT GAG CAA CTG CTG TAC 
               
               
                   
               
               
                      TTC AAT TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TAT 
               
               
                   
               
               
                      AGA AAT AAX TCT CTT;and 
               
               
                   
               
               
                  (iv)  ATT AAT ATG CTG TGG AAG TGG GTG TCC CAG CAT CGG CTC TTT 
               
               
                   
               
               
                        CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAA 
               
               
                   
               
               
                        TCG GTA AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC 
               
               
                   
               
               
                        ATG CTG TGG TGG AAA TGC GAG CAC CA 
               
             
          
         
       
     
     where X is T or G. 
     In a further embodiment, the polynucleotide comprises part or all of the nucleotide sequence of FIG.  10 . 
     In yet a further aspect, the invention provides a vector including a polynucleotide as defined above. 
     In still a flrter aspect, the invention provides a method of producing human DEC-205, the extracellular domain thereof or a fimctional fragment comprising the steps of: 
     (a) culturing a host cell which has been transformed or transfected with a vector as defined above to express the encoded human DEC-205, extracelhular domain or fragment; and 
     (b) recovering the expressed human DEC-205, extracellular domain or fragment. 
     As yet an additional aspect, the invention provides a ligand that binds to human DEC-205 or its extracellular domain as defined above. 
     Preferably, the ligand is an antibody or antibody binding fragment or carbohydrate bearing protein. 
     The antibody or antibody binding fragment can be used in methods for extracting or isolating activated dendritic cells. 
     In still a further aspect, the invention provides a construct for use in therapy or prophylaxis. The construct will usually be a ligand-antigen construct or a DEC-205-antigen construct although ligand-toxin and DEC-205-toxin constructs are also contemplated. The ligand-antigen construct preferably consists of an antibody or antibody binding fragment which binds to human DEC-205 and a host-protective antigen. The DEC-205-antigen construct preferably consists of at least the extra-cellular domain of human DEC-205 and a host-protective antigen. 
     In yet further aspects, the invention contemplates methods of therapy or prophylaxis which employ human DEC-205, ligands or constructs containing them. 
     In yet a further aspect, the invention provides a molecule (hapten) which may be used to generate antibodies for identifying or puring human dendritic cells, which includes a peptide based upon part or all of the sequence of FIG.  11 . 
    
    
     DESCRIPTION OF THE DRAWINGS 
     While the invention is broadly as defined above, it will be appreciated by those persons skilled in this art that it is not limited thereto and that it includes embodiments more particularly described below. It will also be better understood by reference to the accompanying drawings, in which 
     FIG. 1 shows the structure of human DEC-205; 
     FIG. 2 shows the strategy for isolation of human DEC-205 cDNA. A. A schematic presentation of human DEC-205 mRNA with the regions corresponding to DEC-205 domains. The positions of the primers used for the cDNA cloning and analysis are indicated with arrows. The positions of reverse transcriptase-polymerase chain reaction (RT-PCR) fragments 1 to 6 and the clone pBK14-1 are indicated with bars: B. RT-PCR amplification of fragment 1 and 2 from L428 and HEL cell line RNA. L428 and HEL cells were subjected to RT-PCR with two pairs of degenerate primers (DEC-a/-b, and DEC-d/-e), fractionated by electrophoresis through 2% agarose gel, and stained with ethidium bromide. C. RT-PCR and 3′-RACE amplification of fragment 3 and 4 from L428 cells using the primers 028/023 and 029/019, respectively. A cDNA pool of L428 cells was subjected to 3′-RACE and RT-PCR, electrophoresed through 0.8% agarose gel, and stained with ethidium bromide. The numbers on the top correspond to the name of fragment in FIG.  2 A. The positions of DNA molecular size standard are indicates to the right. The estimated molecular size of the RT-PCR products are indicated to the left; 
     FIG. 3 shows protein similarity between human and mouse DEC-205. 
     A. The predicted amino acid sequence of human DEC-205 (SEQ ID NO:1) is aligned with the mouse homolog (SEQ ID NO:31). The regions corresponding to DEC-205 domain structure are bracketed. The positions of amino acids are shaded where there are identical or conservatively replaced amino acids between the sequences, and the asterisks indicates conserved cysteines. The diamonds indicates potential N-glycosylation sites conserved between the sequences. The arrow indicates one amino acid deletion in CRD-5 of human DEC-205. The circles indicate conserved potential serine-phosphorylation sites by protein kinase C (open circle) or casein kinase (closed circle). B. The % identity between human and mouse DEC-205 is indicated above each domain (boxed, See FIG. 2A for key); 
     FIG. 4 shows that human DEC-205 is probably a one-copy gene. Genomic DNA isolated from the peripheral blood of four individuals was digested with the restriction enzymes BgIII, BamHI, HindIII or EcoRI and subjected to Southern blot analysis with the [ 32 P]cysteine-rich domain probe. The final wash was 0.3×SSC at 65° C. The positions of the DNA molecular size standards are indicated to the right; 
     FIG. 5 shows that human DEC-205 gene localizes on chromosome 2. A somatic cell hybrid panel blot (restriction-digested with PstI) was subjected to Southern blot analysis with the [ 32 P]cysteine-rich domain probe. The final wash was 0.3×SSC at 65° C. The positions of the DNA molecular size standards are indicated to the right. The estimated molecular size of the probe-specific bands are indicated to the left. The asterisk indicates weakly hybridized bands. M, male; F, female; 
     FIG. 6 shows that human DEC-205 gene maps to chromosome band 2q24. A. A metaphase spread of human chromosomes were subjected to fluorescent in situ hybridization (FISH) with 6.6 kb human DEC-205 cDNA probe. The final wash was 0.1×SSC at 60° C. The FISH image was overlaid with a DAPI-stained chromosome image. The DEC-205 specific signals are indicated by the arrowheads. B. An inverted image of chromosome 2 containing DEC-205-specific signal (see FIG. 6A) is aligned with an ideogram of chromosome 2. The chromosome band corresponding to DEC-205 gene is indicated to the right; 
     FIG. 7 shows that expression of DEC-205 transcripts within human hematopoetic cell lines. Total RNA prepared from the cell lines were subjected to Northern blot analysis with the [ 32 P]fragment 3 (A and B), or [ 32 P]-actin (C) probes. The final wash was 0.1×SSC at 65° C. The positions of the RNA molecular size standards are indicated to the right. The estimated molecular size of DEC-205 transcripts are indicated to the left. A, 24 h exposure; B, 72 h exposure; 
     FIG. 8 shows RT-PCR analysis of DEC-205 mRNA in human DC preparations. Specific product is seen using lineage negative; fresh DC (lane 2) and a stronger signal with CMRF44 +  low density cultured DC (lane 3). CD8 +  T lymphocytes (lane 1) contain no DEC-205 mRNA Ethidium stain. 
     FIG. 9 represents the result of an ELISA assay showing a monoclonal antibody binding specifically to DEC-205 peptide 1 and not peptide 3. Positive control binding of a hyperimmunized rabbit anti-DEC-205-peptide 1 serum and hyperimmunized rabbit anti-DEC-205-peptide 2 serum are shown; 
     FIG. 10 gives the DNA sequence (SEQ ID NO:1) for human DEC-205 (coding region only); 
     FIG. 11 gives the human DEC-205 amino acid sequence (SEQ ID NO:2). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A. Human DEC-205 
     The human DEC-205 of the invention is believed to be the human homolog of murine DEC-205 and has an approximate molecular weight of 198 to 205 kDa. It has the structure shown in FIGS. 1 and 2A. It also has the deduced amino acid sequence shown in FIG.  11 . 
     Human DEC-205 can usefully be provided in a number of different forms. These include human DEC-205 itself the “mature” form of human DEC-205, and the extracellular receptor domain of human DEC-205. 
     The “mature” form of human DEC-205 of the invention is human DEC-205 less its native amino-terminus leader or signal sequence, whereas the extracellular receptor domain is human DEC-205 lacking the signal sequence, the transmembrane region and cytoplasmic domain (where present). 
     The extracellular domain may be identified through commonly recognised criteria of extracellular amino acid sequences. The determination of appropriate criteria is known to those skilled in the at, and has been described, for example by Hopp et al.,  Proc. Natl. Acad. Sci. USA  78, 3824-3828 (1991); Kyte et al.,  J. Mol. Biol . 157 105-132 (1982); Emini,  J. Virol  55, 836-839 (1985); Jameson et al.  CA BIOS  4 181-186 (1988); and Karplus et al.  Naturwissenschaften  72, 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed. are characteristic of extracellular domains. 
     The amino acid sequences of the predicted regions for human DEC-205 are shown in FIG.  3 A. These include the amino acid sequences for the signal peptide, cysteine-rich domain, fibronectin type II domain, Carbohydrate Recognition Domain-1, (CRD-1), CRD-2, CRD-3, CRD-4, CRD-5. CRD-6, CRD-7, CRD-8, CRD-9, CRD-10, transmembrane domain and cytoplasmic domain. 
     Human DEC-205 of the invention or its extracellular receptor domain (or parts thereof) may be prepared by methods known in the art. Such methods include protein synthesis from individual amino acids as described by Stuart and Young in “Solid Phase Peptide Synthesis”, Second Edition, Pierce Chemical Company (1984). It is however preferred that human DEC-205 and/or its extracellular receptor domain or parts thereof be prepared by recombinant methods as will be detailed hereinafter. 
     Example 1 provides further details of human DEC-205. 
     EXAMPLE 1 
     Langerhans cells were prepared from human skin. Epidermal cell suspensions were prepared from split thickness normal human breast skin by 30 min dispase (Boehringer-Mannheir, Mannheim, Germany; 0.5% in PBS) treatment at 37° C., followed by 10 min disaggregation in the presence of trypsin (0.25% in PBS), DNase I (5U/ml in PBS) and 5 mM EDTA at room temperature. Langerhans cells were then enriched by Ficoll/Metrizoate gradient separation (d=1.077g/cm 3 ). Final cell suspensions contained 3-15% Langerhans cells as determined by HLA-DR positivity. Total RNA was extracted using Trizol reagent according to the manufacturer&#39;s instructions. 
     Degenerate primers were prepared on an Applied Biosystems DNA Synthesizer with the primer sequences (d) and (e) as set out below (SEQ ID NOS:5 &amp; 6, respectively, in order of appearance): 
     
       
         
               
               
             
           
               
                   
                 (d) 5′-GAX ACY GAX GGY TTX TGG AA-3′ 
               
               
                   
                   
               
               
                   
                 (e) 3′-GCY GTX TTZ TCZ AAC CAC AT-5′ 
               
             
          
         
       
     
     wherein X is C or T, Y is A, C, G or T, and Z is G or A. 
     Single stranded cDNA was prepared using total RNA and reverse transcribed by AMV reverse transcriptase using the 3′ primer (e). Subsequently, the cDNA was amplified using the 5′(d) and 3′(e) primer using PCR amplification according to techniques known in the art. 
     The amplified products were run on a 2% agarose gel and visualized with ethidium bromide staining. 
     The DNA was purified and ligated into the T tailed pGEM vector (available from Promega) using standard techniques. The ligation mixture was transformed into competent  E. coli  JM 109 bacteria (available from Promega) which were grown on agar plates with appropriate antibiotic selection. Two colonies were isolated. DNA was prepared and digested with restriction enzymes. Two inserts of the same size as the PCR product were sequenced by double-stranded DNA sequencing techniques using a Sequence Kit (Sequence 2.0 USB Lab Supply, Pierce). The two clones corresponded to human DEC-205. 
     The amino acid sequence of human DEC-205 was determined to include the following amino acid sequences (portions of SEQ ID NO:1): 
     (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPF QNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKE NNFVLEQLLYFNYMASWVMLGITYRNNSL; and 
     (ii) SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML. 
     Determination of these sequences was fundamental to isolating the cDNA for human DEC-205 detailed below. 
     In the partial sequences given above, individual amino acids are represented by the single letter code as follows: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Three-letter 
                 One-letter 
               
               
                   
                 Amino Acid 
                 abbreviation 
                 symbol 
               
               
                   
                   
               
             
             
               
                   
                 Alanine 
                 Ala 
                 A 
               
               
                   
                 Arginine 
                 Arg 
                 R 
               
               
                   
                 Asparagine 
                 Asn 
                 N 
               
               
                   
                 Aspartic acid 
                 Asp 
                 D 
               
               
                   
                 Asparagine or 
                 Asx 
                 B 
               
               
                   
                 aspartic acid 
               
               
                   
                 Cysteine 
                 Cys 
                 C 
               
               
                   
                 Glutamine 
                 Gln 
                 Q 
               
               
                   
                 Glutamic Acid 
                 Glu 
                 E 
               
               
                   
                 Glutamine or 
                 Glx 
                 Z 
               
               
                   
                 glutamic acid 
               
               
                   
                 Glycine 
                 Gly 
                 G 
               
               
                   
                 Histidine 
                 His 
                 H 
               
               
                   
                 Isoleucine 
                 Ile 
                 I 
               
               
                   
                 Leucine 
                 Leu 
                 L 
               
               
                   
                 Lysine 
                 Lys 
                 K 
               
               
                   
                 Methionine 
                 Met 
                 M 
               
               
                   
                 Phenylalanine 
                 Phe 
                 F 
               
               
                   
                 Proline 
                 Pro 
                 P 
               
               
                   
                 Serine 
                 Ser 
                 S 
               
               
                   
                 Threonine 
                 Thr 
                 T 
               
               
                   
                 Tryptophan 
                 Trp 
                 W 
               
               
                   
                 Tyrosine 
                 Tyr 
                 Y 
               
               
                   
                 Valine 
                 Val 
                 V 
               
               
                   
                 Unidentified 
                   
                 X 
               
               
                   
                   
               
             
          
         
       
     
     This code also applies to the predicted full sequence of FIG. 11, deduced from the cDNA encoding human DEC-205 isolated as described below. 
     B. Polynucleotides Encoding Human DEC-205 
     In another aspect of this invention, the applicants provide polynucleotides encoding human DEC-205 or its extracellular domain. These polynucleotides may be DNA (isolated from nature, synthesised or cDNA) or RNA. Most often, the polynucleotides will be DNA. 
     The polynucleotides of the invention specifically include those which include the nucleotides (SEQ ID NOS 3 &amp; 4, respectively, in order of appearance) 
     
       
         
               
               
             
           
               
                 (iii)  A ACA GTT GAT TGC AAT GAC AAT CAA CCA GGT GCT ATT TGC 
                   
               
               
                   
               
               
                      TAC TAT TCA GGA AAT GAG ACT GAA AAA GAG GTC AAA CCA GTT 
               
               
                   
               
               
                      GAC AGT GTT AAA TGT CCA TCT CCT GTT CTA AAT ACT CCG TGG 
               
               
                   
               
               
                      ATA CCA TTT CAG AAC TGT TGC TAC AAT TTC ATA ATA ACA AAG 
               
               
                   
               
               
                      AAT AGG CAT ATG GCA ACA ACA CAG GAT GAA GTT CAT ACT AAA 
               
               
                   
               
               
                      TGC CAG AAA CTG AAT CCA AAA TCA CAT ATT CTG AGT ATT CGA 
               
               
                   
               
               
                      GAT GAA AAG GAG AAT AAC TTT GTT CTT GAG CAA CTG CTG TAC 
               
               
                   
               
               
                      TTC AAT TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TAT 
               
               
                   
               
               
                      AGA AAT AAX TCT CTT; and 
               
               
                   
               
               
                 (iv)  ATT AAT ATG CTG TGG AAG TGG GTG TCC CAG CAT CGG CTC TTT 
               
               
                   
               
               
                       CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAA 
               
               
                   
               
               
                       TCG GTA AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC 
               
               
                   
               
               
                       ATG CTG TGG TGG AAA TGC GAG CAC CA 
               
             
          
         
       
     
     where X is T or G, 
     as well as the full nucleotide sequence shown in FIG. 10, but are not limited thereto. 
     The invention also includes within its scope functional equivalents of these polynucleotides. 
     This aspect of the invention will now be illustrated by the following Examples. 
     EXAMPLE 2 
     EXPERIMENTAL PROCEDURES 
     Cell culture—The cell lines, HEL, K562, KG-1, THP-1, U937, Mann and Jurkat were obtained from the American Type Culture Collection (Rockville, Md.). L428 cells were provided by V. Diehl (Klinik for lnnere Medizin, Cologne, Germany). HDLM2 and KMH2 cells were obtained from the German Collection of Micro-organisms and Cell Culture (Braunscfweig, Germany). Mono Mac 6 cells (Bufler et al (1995)  Eur. J. Immunol . 25, 604-610) were provided by H. Engelmann (Institute for Immunology, Munchen, Germany). All cell lines were maintained in RPMI 1640, 10% fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin except that HDLM2 cells were with 20% fetal calf serum. 
     Isolation of Leukocytes—Leukocyte populations were isolated using standard laboratory procedures. 
     Isolation of cDNA encoding for human DEC-205—A set of degenerate oligonucleotide primers were designed based on the published amino acid sequence of mouse DEC-205 (Jiang etal (1995), above) and synthesized in house or by Life Technologies (Auckland, New Zealand) (see FIG.  2 A). These primers were (SEQ ID NOS:7-10 respectively, in order of appearance) 
     DEC-a (5′-AAYATGCTNTGGAARTGGGT-3′), 
     DEC-b (5′-TGRTGYTCRCAYTTCCACCA-3′), 
     DEC-d (5′-GAYACNGAYGGNTTYTGGAA-3′) and 
     DEC-e (5′-GCNGTYTTRTCRAACCACAT-3′), 
     where Y=C or T, R=A or G, N=A or C or G or T. Total RNA isolated from L428 or HEL cells was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.) at 55° C. for 1 h using the primers DEC-b or DEC-e. PCR was performed using the resultant cDNA and Taq polymerase (Boebrunger Mannheim, Auckland, New Zealand) with the primers DEC-a/-b for DEC-b-primed or DEC-d/-e for DEC-e-primed cDNAs. The PCR conditions used were the initial denaturation at 94° C. for 5 min, 35 cycles of denaturation at 94° C. for 1 min, annealing at 54° C. for 1 min, extension at 72° C. for 1 min, and the final extension at 72° C. for 5 min. The PCR reactions were fractionated with 2% agarose gel in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA (TAE) buffer, and stained with 0.5 ug/ml ethidium bromide. The PCR fragments (fragment 1 and 2, see FIG. 2A and 2B) were cloned into pGEM-T vector (Promega), and sequenced manually using Sequenase DNA sequencing kit (Amersham Life Science, Auckland, New Zealand). 
     A set of oligonucleotide primers nested within the DNA sequence of fragment 1 and 2 were synthesized (see FIG.  2 A). These primers were (SEQ ID NOS:11-13, respectively, in order of appearance): 
     023(5′-GCTCTAGAAACATGACCCATGAAGCC-3′ containing a XbaI site), 
     028(5′-GCTCTAGACATCGGCTCTTTCATTTGT-3′ containing a XbaI site) and. 
     029(5′-CGGGATTCACAGTTGATTGCAATGACA-3′ containing a EcoRI site) 
     where incorporated restriction sites are underlined. Two ug of poly(A) RNA from L428 cells was reverse transcribed with 200 U of SuperScriptII (Life Technologies) at 45° C. for 1 h using an oligo d(T) adaptor primer (SEQ ID NO:14) 
     018(5′-GACTAGTCTGCAGAATTCTTTTTTTTTTTTTTTTT-3′, containing a SpeI, PstI, and EcoRI sites). After heat-inactivation at 70° C. for 15 min, the reaction was incubated with 1 U RNaseH (Life Technologies) at 37° C. for 30 min, heat-inactivated at 70° C. for 15 min, and diluted to 1 ml with 10 mM Tris-HCI, pH 8.0,1 mM EDTA (L428 cDNA pool). In order to isolate the fragment 3 (connecting the fragment 1 and 2) (see FIG.  2 A), PCR was performed with 5 ul of L428 cDNA pool, the primers 028 and 023, and 2.5 U of Expand enzyme mix (Boehringer Mannheim). The PCR conditions were the initial denaturation at 94° C. for 2 min, 10 cycles of 10 cycles of denaturation at 94° C. for 15 sec, annealing at 53° C. for 30 sec, and extension at 68° C. for 4 min, followed by 20 cycles of denaturation at 94° C. for 15 sec. annealing at 53° C. for 30 sec, and extension at 68° C. for 4 min plus additional 20 sec for each cycle, and the final extension at 68° C. for 15 min. 3′-rapid amplification of cDNA ends (3′-RACE) (Frohman et al (1988)  Proc. Natl. Acad. Sci. USA  85, 8998-9002) was performed in order to isolate the fragment 4 (connecting the fragment 1 and the 3′-untranslated region of DEC-205) (see FIG.  2 A). PCR was performed with 5 ul of L428 cDNA pool and the primer 029 and an adaptor primer 019 (SEQ ID NO:15) (5′-GACTAGTCTGCAGAATTC, containing a SpeI, PstI and EcoRI site), in the same conditions for the fragment 3. The PCR reactions were fractionated with 0.8% agarose gel in TAE buffer, and stained with ethidium bromide. Both the fragment.3 and 4 were restriction digested with XbaI and EcoRI, respectively, and cloned into pBluescript 11 (Stratagene, La Jolla, Calif.). The representative clones from the fragment 3 (pB38fl) and 4 (pb30-3) were sequenced with a LI-COR automated sequencer (LI-COR, Lincoln, Neb.) using SequiTherm cycle sequencing kit (Epicentre Technologies, Madison, Wis.). If required, these plasmids were subjected to exonucleaseIII-nested deletion using Erase-A-Base system (Promega), and used for sequencing. 
     An oligo dT-primed L428 cDNA library was prepared using ZAP Express cDNA Gigapack Cloning kit (Stratagene) according to manufacturers instruction. The fragment 3 was labeled with [α-32P]dCTP (NEN) using Multiprime system (Amersham Life Science). The library was screened by plaque hybridization with the [ 32 P]fragment 3 using standard techniques (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)  Molecular Cloning. A Laboratory Manual , 2Ed., Cold Spring Harbour Laboratory, New York, USA). The specific activity of the probe was 0.8×10 9  cpm/ug DNA and used at 1×10 6  cpml/ml. The final wash was in 0.1×SSC, 0.5% SDS at 65° C. (1×SSC is 0.15 M NaCl, 15 mMM Na-citrate, pH7.0). Positive clones were converted to phagemid pBK-CMV (Stratagene) and sequenced using an automated sequencer. 
     In order to verify the DNA sequence obtained from the PCR clones, pB38f for fragment 3 and pB30-3 for fragment 4, the fragment 5 was PCR-amplified from a L428 cDNA pool using primers 058 (SEQ ID NO:16) (5′-CGGGATCCCTCTGGCCGCGCACTAATGA-3′ containing a BamHl site) and 050 (SEQ ID NO:17)(5′-CCGCTCGAGCTGTGGATACCAGCACATGCCT-3′ containing a XhoI site) (see FIG.  2 A). The PCR conditions were identical to that for the fragment 3 except using longer extension period (6 min) for cycling. The fragment 5 was sequenced directly using the IRD 40 -labeled custom primers (MWG-Biotech, Ebersberg, Germany) and a LI-COR automated sequencer without cloning. These primers were (SEQ ID NOS:18-25, respectively, in order of appearance): IRD001 (5′-GATGGGAACTCTTATGGGAGACCT-3′ at nucleotide 523-555), IRD002 (5′-TGATGCAGGCTGGCTGCCAAATAA-3′ at nucleotide 1134-1157), IRD003 (5′-AACTGGGCAACTGTTGGTGGAAGA-3′ at nucleotide 1759-1782), IRD004 (5′-ATGGCGAAGAGGCTGGCATTTCTA-3′ at nucleotide 2334-2357), IRD005 (5′-CTCAAGCAAGCGATACCTGTCACT-3′ at nucleotide 2972-2995), IRD006 (5′-TGGGCAACTCGAAGACTGTGTAGT-3′ at nucleotide 3624-3647), IRD007 (5′-CACCAGCACAGCATTCTTGCTTGT-3′ at nucleotide 4168-4191) and IRD008 (5′ATTTGTGAGCAGACTGATGAGGGA-3′ at nucleotide 4797-4820). The sequences of these primers were based on those of pb38fl and pb30-3, and they were positioned as 540-650 bp apart, ensuring the generation of contigs overlapping by at least 100 bp after automated sequencing. 
     Southern Blot Analysis—Genomic DNA was prepared from peripheral blood of patients with hematological disorders (each patient was karyotyped at Canterbury Health Laboratories, Christchurch, New Zealand). Approximately 8 ug of genomic DNA was digested with BgIII, BamHI, EcoRI, or HindIII, fractionated in 0.8% agarose gel in 89 mM Tris-borate, pH 8.3, 2 mM EDTA, and transferred to Hybond N+ by capillary reaction. A PCR-fragment corresponding to the cyteine-rich domain was PCR-amplified from pBK14-1 using the primers 058 and 059 (SEQ ID NO:26) (5′-CGGAATTCGATCTCATGATAAGGCTGGTCACA-3′ containing a EcoRI site) (see FIG.  2 A). Briefly, PCR was performed with 2 ng of pBK14-1, the primer 058 and 059, and Taq polymerase. The PCR conditions used were the initial denaturation at 94° C. for 2 min, 30 cycles of denaturation at 94° C. for 15 sec, annealing at 55° C. for 15 sec. extension at 72° C. for 30 sec, and the final extension at 72° C. for 5 min. The 450 bp PCR product was labeled with [α- 32 P]dCTP using Multiprime labeling system (Amersham Life Science). The blot was hybridized with the probe using standard technique (Sambrook et al, (1989), above). The specific activity of the probe was 0.8×10 9  cpm/ug DNA and used at 1×10 6  cpm/ml. The final wash was in 0.3×SSC, 0.5% SDS at 65° C., and exposed to X-OMAT AR film (Kodak) with an intensifying screen at −70° C. 
     A blot containing PstI-digested genomic DNA from a human-rodent somatic hybrid cell panel was obtained from Oncor (Gaithersburg, Md.), and probed with the [ 32 P]cysteine-rich domain fragment as described above. 
     Fluorescent in Situ Hybridization—Metaphase spreads were prepared from phytohaemagluttinin-stimulated peripheral blood lymphocytes of a 46,XY male donor using standard cytogenetic procedures. The fragment 6 was amplified by recombinant PCR with the fragment 3 and 4 (see FIG.  2 A). PCR was performed with each of the fragment 3 and 4 and the primers 028 and 019 in the same conditions for the fragment 3 except using longer extension period (7 min) for cycling. The fragment 6 was labelled with biotin-14-dCTP using a BioPrime random prime labelling kit (Bethesda Research Laboratories, Gathersberg, Md.), and hybridized to metaphase cells on slides. Conditions for hybridization and immununofluorescent detection were essentially as described (Morris et al, (1993)  Human Genetics , 91, 31-36), except that Cot 1 suppression was not required, slides were washed to a stringency of 0.1×SSC, 60° C. after hybridization, and an additional amplification step was needed because of the small size of the probe. For precise chromosome band localization, DAPI and FITC images were captured separately for each metaphase from the fluorescent microscope to computer using a Photometrics KAF1400 CCD camera and IPLAB Spectrum software (Signal Analytics, Va.), and colour-joined using Multiprobe extension software. 
     Northern Blot Analysis—Approximately 10 ug of total RNA from cultured cells were fractionated in formaldehyde-denatured 1% agarose gel and transferred to Hybond N+ (Amersham) using 3 M NaCl, 8 mM NaOH, 2 mM sarkosyl with Turboblotter (Schleicher &amp; Schuell, Keene, N.H.) for 3 h. The membrane was UV-crosslinked (Stratalinker, Stratagene), and hybridized with [ 32 P]fragment 3 or [ 32 P]human §-actin probe using standard techniques (Sambrook et al (1989), above). The specific activity of the probes were 0.9-1.1×10 9  cpm/ug DNA and used at 0.7-1.1×10 6  cpm/ml. The final wash was in 0.1×SSC, 0.5% SDS at 68° C., and exposed to X-OMAT AR film (Kodak) with intensifying screen at −70° C. 
     Reverse Transcription-PCR Analysis—Total RNA from isolated leukocytes was incubated with RNase-free DNasel (Life Technologies), and was reverse transcribed using Superscriptll with the oligo dT adaptor primer 018. PCR was performed using a pair of DEC-205 specific primers 060 (SEQ ID NO:27) (GTGGATCCAGTACAAGGGTCA at nucleotide 4655-4686) and 056 (SEQ ID NO:28) (ACCAAATCAGTCCGCCCATGA at nucleotide 5116-5096) with Taq polymerase in the presence of a PCR additive, Q buffer (Qiagen) by touch down PCR (Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S., (1991)  Nucleic Acid Res . 19, 4008). PCR conditions used were the initial denaturation at 92° C. for 2 min, 21 cycles of denaturation at 92 20   C. for 15 sec, annealing at 60° C. minus 0.5° C./cycle for 15 sec, extension at 68° C. for 30 sec, 15 cycles of denaturation at 92° C., annealing at 50° C., extension at 68° C. for 1 min and the final extension at 68° C. for 5 min. Human glycelaldehyde-3-phosphate dehydrogenase (GAPDH) (Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K., Ohkubo, M., Sawada, K., and Sakiyama, S. (1987)  Cancer Res . 47, 5616-5619) was used for normalization. The primers for GAPDH were 053 (SEQ ID NO:29) (ATGGGGAAGGTGAAGGTCGGA-3′ at nucleotide 61-81), and 055 (SEQ ID NO: 30) (AGGGGCCATCCACAGTCTTCT-3′ at nucleotide 634-614). The PCR reactions were fractionated with 1.5 % agarose gel in TAE buffer, and stained with 0.5 ug/ml ethidium bromide. 
     Sequence Data Analysis 
     The National Center of Biotechnology Information (NCBI) Center electronic mail server BLAST was used to search for homologous sequences. Sequence alignments and motif search were done using Bestfit and Motifs programs, respectively, of GCG computer package (Madison, Wis.). 
     RESULTS 
     Isolation of CDNA for human DEC-205. —Based on the amino acid sequence of mouse DEC-205, a set of degenerate primers were synthesized and used to perform RT-PCR using the Hodgkin&#39;s disease-derived L428 cell line and the myeloid HEL cell lines (FIG.  2 ). The two pair of primers (DEC-d/-e, and DEC-a/-b) gave rise to the specific RT-PCR products, fragment 1 (390 bp) and 2 (150 bp), respectively (FIG.  2 A and  2 B). These specific fragments were cloned and sequenced (data not shown). The deduced amino acid sequences of fragment 1 and 2 were ˜80% identical to that of mouse DEC-205, indicating that these fragments were derived from the cDNA of human DEC-205. 
     Primers nested within these fragments were synthesized and further RT-PCR and 3′-RACE performed using a L428 cDNA pool reverse transcribed with an oligo dT adapter primer 018. A 3.8 kb RT-PCR product (fragment 3) was obtained using primer 028 and 023 (FIG.  2 A and  2 C). A 3.2 kb 3′-RACE product (fragment 4) was obtained using primer 029 and an adaptor primer 019 (FIG.  2 A and  2 C). The fragment 3 was cloned and several identical clones were identified by restriction enzyme map analysis (data not shown), and one of which, pb38fl, was fully sequenced: The DNA sequence of the fragment 3 (pB38fl) extending from the middle of cysteine-rich domain to the middle of CRD-8 (FIG.  2 A), was 82% identical to the published mouse DEC-205 cDNA sequence. The fragment 4 was cloned and two distinct clones identified by restriction enzyme map analysis. Both clones were partially sequenced and the 3′ end DNA sequence of one clone (eg. pb30-3) was found to contain a poly A tail and with 72% identical to 3′-untranslated region of mouse DEC-205 (data not shown). Therefore, the pb30-3 was sequenced to obtain the DNA sequence of the coding region of DEC-205 plus partial 3′-untranslated region. The resulting DNA sequence for the coding region was ˜80% identical to that of mouse DEC-205 spanning from the middle of CRD-8 to the end of cytoplasmic domain (FIG.  2 A). The DNA sequences obtained from pb38fl and pb30-3 overlapped by 320 bp, covering 95% of human DEC-205 coding region. 
     In order to complete the 5′ end of the DEC-205 cDNA sequences a L428 cDNA library was screened by plaque hybridization using  32 P-labeled fragment 3 as a probe. A clone (pBKI4-1) was isolated, and the 1.5 kb insert of this clone was sequenced (FIG.  2 A). The sequence was  18  80% identical to the mouse sequence and corresponded to the signal peptide, cysteine-rich domain, fibronectin type II domain, CRD-1 and part of the CRD-2. The pBK14-1 contained 51 bp 5′-untranslated region, and overlapped with fragment 3 by ˜1.2 kb. 
     To validate the DNA sequence obtained from the PCR clones, a further RT-PCR fragment (fragment 5) amplified with primers 058 (nested in the cysteine-rich domain) and 050 (located ˜130 bp downstream of the stop codon) was prepared (FIG.  2 A). The fragment 5 PCR product was sequenced directly using MRD 41 -labeled custom primers without cloning. A total of 10 point mutations, presumably generated because of the low fidelity of thermostable polymerases were found and corrected in the PCR clone-derived DNA sequence. The complete cDNA sequence for human DEC-205 is 5166 bp in size, and encodes for a predicted 198 kDa type I transmembrane protein with 1722 amino acids before post translational modification. 
     The deduced amino acid sequence of human DEC-205 showed 77% overall identity with the homologous mouse protein (FIG.  3 A). All the cysteines, and putative N-glycosylation sites in the extracellular domain of mouse DEC-205, were conserved in the human sequence. In the cytoplasmic domain the putative serine phosphorylation sites by protein kinase C or casein kinase, and a tyrosine, which appears to be important for coated pit-mediated internalization (Ezekowitz, R. A. B., Sastry, K., Bailly, P., and Warner, A. (1990)  J. Exp. Med . 172, 1785-1794; and Zvaritch, E., Lambeau, G., and Lazdunski, M. (1996)  J. Biol. Chem . 271, 250-257), were also conserved. There was one amino acid deletion within the CRD-5 in human DEC-205. All the extracelluar domains, including the cysteine-rich domain, fibronectin type II domain, and CRD1-10 were 74-87% identical between human and mouse sequences (FIG.  3 B), suggesting the importance of these domains for the function of DEC-205. In contrast, the two hydrophobic domains, including the signal peptide and transmembrane domain, showed much lower identity (57% and 52%, respectively (FIG.  3 B)) with the mouse protein, confirming the observation that these hydrohobic domains are more variable, and rapidly evolved structures (Von Heijne, G. (1990)  J. Membrane Biol . 115, 195-201). 
     DEC-205 is a Single Copy Gene with Polymorphism—Peripheral blood-derived genomic DNA from 4 individuals was restriction enzyme-digested with BglII, BamHI, HindIII or EcoRI, and subjected to Southern blot analysis. The cysteine-rich domain of the macrophage mannose receptor (Kim, S. J., Ruiz, N., Bezouska, K, and Drickamer, K. (1992) Genomics 14, 721-727; and Harris, N., Peters, L. L., Eicher, E. M., Rits, M., Raspberry, D., Eichbaum, Q. G., Super, M., and Ezekowitz, R. A. B. (1994)  Biochem. Biophys. Res. Com . 198, 682-692) and phospholipase A2 receptor (Ancian, P., Lambeau, G., Mattei, M. G., and Lazdunski, M. (1995) 270, 8963-8970) is encoded by one exon. Therefore, we amplified the cysteine-rich domain of human DEC-205 using primers 058 and 059 as a potential single exon probe (450 bp), and used this to probe the Southern blot in high stringency. A single band appeared in BglII-, BamHI- or HindIII-digested genomic DNA from all individuals, indicating that DEC-205 is a single copy gene (FIG.  4 ). The EcoRI digests, however, produced a single band in two individuals and double bands in another, indicating that the DEC-205 gene is polymorphic. Further Southern blot analysis with larger panel of individuals showed identical results (data not shown). Therefore, DEC-205 is a single copy gene with at least one polymorphic site. 
     DEC-205 Gene Maps to Chromosome Band 2q24-In order to map the human DEC—205 gene, a somatic cell hybrid panel Southern blot (PstI-digested) was probed with the [ 32 P]cysteine-rich domain as described above (FIG.  5 ). A 3.0 kb band in human genomic DNA was found to hybridize strongly, and the identical band appeared in chromosome 2-containing somatic human-mouse hybrid cells, indicating that DEC-205 gene localizes on chromosome 2. The probe also hybridized weakly with hamster DNA, suggesting the presence of DEC-205 homolog in hamster as well as in the mouse (which also hybridized strongly). The origin of the weakly hybridized bands with apparent polymorphism in the human DNA-containing lanes is not known. The identical band appeared in chromosome 2, and may either be related to an alternative exon structure for this region of DEC-205 or result from weak cross hybridization to another gene on chromosome 2. 
     Fluorescent in situ hybridization then was used to map the DEC-205 gene in detail (FIGS.  6 A and  6 B). The 6.4 kb recombinant PCR fragment (fragment 6) (FIG. 2A) was prepared from fragment 3 and 4, labeled with biotinylated nucleotides, and used as a probe in a high stringency (FIG.  6 A). Ninety-one (80%) of a combined total 114 metaphase cells analysed from three experiments showed fluorescent signals on one (27) or both (64) chromosomes 2 in the middle of the long arm, specifically in band q24 (FIG.  6 B). High resolution banding analysis provided a more precise location of signals (not shown). No additional site-specific signals were detected on any other chromosome. 
     DEC-205 Exhibits Multiple Transcripts in Cell Lines—A panel of human cell lines, including myeloid, B lymphoid, T lymphoid and Hodgkin&#39;s desease-derived cell lines, were analyzed for the expression of DEC-205 transcripts by Northern blot analysis with the [ 32 P]fragment 3 as a probe (FIG.  7 A and  7 B). Two DEC-205 transcripts, 7.8 and 9.5 kb in size, were detected, and the 7.8 kb transcript was the most abundant. The expression level varied between cell lines, however the myeloid cell line THP-1, the B lymphoid cell line Mann and the Hodgkin&#39;s desease cell line KMH2 showed the highest level of expression. Even with longer exposure, DEC-205 transcripts were not detectable in K562, KG-1, Monomac and Jurkat cells, suggesting these cells are DEC-205 negative (FIG.  7 B). Interestingly all Hodgkin&#39;s disease-derived cell lines tested express the transcripts. Semiquantitative RT-PCR studies also support these results (data not shown). 
     C. Recombinant Expression of Human DEC-205 
     In yet another aspect, the present invention relates to the recombinant expression of human DEC-205 or of its extracellular domain. 
     The Polynucleotides that encode human DEC-205 or the extracellular domain of the invention may be inserted into known vectors for use in standard recombinant DNA techniques. Standard recombinant DNA techniques are those such as are described in Sambrook et al.; “Molecular Cloning” 2nd Edition Cold Spring Harbour Laboratory Press (1987) and by Ausubel et al., Eds, “Current Protocols in Molecular Biology” Greene Publishing Associates and Wiley-Interscience, New York (1987). 
     Vectors for expressing proteins in bacteria, especially  E. coli , are known. Such vectors include the PATH vectors described by Dieckmann and Tzagoloff in  J. Biol. Chem . 260, 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-galactosidase (pGEX); lambda P maltose binding protein (pMAL); and gluthathione S-transferase (pGST)—see  Gene  67, 31 (1988) and  Peptide Research  3, 167 (1990). 
     Vectors useful in yeast and insect cells are available and well known. A suitable example of a yeast vector is the 2μ plasmid. 
     Suitable vectors for use in mammlian cells are also known. Such vectors include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and vectors derived from combination of plasmids and phage DNA. 
     Further eucaryotic expression vectors are known in the art (e.g. P. J. Southern and P. Berg,  J. Mol. Appl. Genet . 1, 327-341 (1982); S. Subramani et al,  Mol. Cell. Biol . 1. 854-864 (1981); R. J. Kaufinann and P. A. Sharp, “Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene,”  J. Mol. Biol . 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp,  Mol. Cell. Biol . 159 601-664 (1982); S. I. Scahill et al, “Expression And Characterization Of The Product Of A Human Imnune Interferon DNA Gene In Chinese Hamster Ovary Cells,”  Proc. Natl. Acad. Sci. USA  80 4654-4659 (1983); G. Urlaub and L. A. Chasin,  Proc. Natl. Acad. Sci. USA  77, 4216-4220, (1980). 
     The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac stem, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g. the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g. the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic and eucaryotic cells and their viruses or combinations thereof. 
     Vectors containing the receptor-encoding DNA and control signals are inserted into a host cell for expression of the receptor. Some useful expression host cells include well-known prokaryotic and eucaryotic cells. Some suitable prokaryotic hosts include, for example,  E. coli . such as  E. coli  SG-936 , E. coli  HB 101 , E. coli  W3110 , E. coli  X1776 , E. coli  X2282 , E. coli  DHT, and  E. coli  MR01, Pseudomonas, Bacillus, such as  Bacillus subtilis , and Streptomyces. Suitable eucaryotic cells include yeast and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture. 
     D. Ligands 
     The invention also includes ligands that bind to human DEC-205 of the invention. 
     The ligand will usually be an antibody or an antibody binding fragment raised against human DEC-205 or its extracellular domain, or against fragments thereof. 
     Such antibodies may be polyclonal but are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in  Nature  256, 495-497 (1975) and Campbell in “Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas” in Burdon et al. Eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al. in  Science  246 1275-1281 (1989). 
     In yet another form, the ligand may also be a non-protein, probably carbohydrate containing, molecule that acts as a ligand when it binds to, or otherwise comes into contact with, human DEC-205. 
     In addition, ligands may be of two fimctional types. The first functional type of ligand is a molecule which binds to human DEC-205 and stimulates it in performing its normal function (a “stimulant ligand”). The second functional type of ligand is a molecule which binds to human DEC-205 and inhibits or prevents it performing its normal function (an “antagonistic ligand”). 
     Both types of ligand will find application in either therapeutic or prophylactic treatments as described below. 
     Example 3 describes the production of anti-DEC-205 antibodies. 
     EXAMPLE 3 
     Production of Anti-DEC-205 Antibodies 
     A BALB/c mouse was immunized ip/sc with L428 cells and boosted SC with two peptides derived from the DEC-205 cDNA sequence. DEC-205 peptide 1 ATTQDEVHTKC [amino acids at positions 267-277 of SEQ ID NO:1] and DEC-205-peptide 2 TEKEVKPVDSVKC [amino acids at positions 1227-1239 of SEQ ID NO:1] were synthesized by Chiron Mimotopes Pty Ltd (Clayton, Victoria, Australia). After a third immunization with the two DEC-205 peptides sc/ip/IV the mouse was sacrificed and a spleen cell suspension prepared. The spleen cells were fused with the NS-1 myeloma cell line using standard techniques (Hock et al, Immunology 1994;83:573). A hybridoma was subsequently isolated, 2F5, which produced monoclonal antibody binding to the DEC-205-peptide 1 but not the DEC-205-peptide 2 or a third control DEC-205-peptide 3 (KCLGLDITKSVNELR) [amino acids at positions 82-96 of SEQ ID NO:1]. This is shown by FIG.  9 . 
     E. Constructs 
     The invention also provides constructs. The constructs will generally include antigens against which an immune response is desired but can also include other products to be delivered specifically to dendritic cells. Toxins, such as the ricin A chain are not excluded. The other component of the construct will vary, being either a ligand as described above or at least the extracellular domain of human DEC-205. Both constructs will have the potential to manipulate the immune system of the host. 
     In the ligand-antigen constructs, ligands which bind to human DEC-205 (usually antibodies, antibody-binding fragments or carbohydrates expressing proteins) can be coupled or otherwise associated with the antigen against which an immune response is desired. An example of such antigens are sugar-coated antigens such as tumour-associated antigens In use, the ligand component binds to human DEC-205 and the dendritic cell is ‘primed’ with the associated antigen. This ‘priming’ action will assist in the induction of an immediate immune response against the antigen. 
     The ligand-antigen construct can take any appropriate form for administration to the dendritic cells. Such forms may differ depending upon whether the therapeutic protocol involves isolation of the patients dendritic cells (so that the priming action can take place in vitro) or whether the construct is to be administered to a patient in vivo. 
     The construct can be directly administered to a patient for in vivo treatment. It can also be administered in a form which allows the construct to be expressed within the patient. 
     One example of such a form for administration to a patient in vivo is a live recombinant viral vaccine. Such a vaccine includes a polynucleotide encoding the DEC-205 ligand (or a portion thereof) and the antigen. The vaccine is administered to the patient and, once within the patient, expresses the encoded ligand and antigen to bind to the patients dendritic cells (via human DEC-205). 
     A number of such live recombinant viral vaccine systems are known. An example of such a system is the  Vaccinia  virus system (U.S. Pat. No. 4,603,112; Brochier el al.,  Nature  354:520 (1991)). 
     Administration can be via intravenous, intramuscular, subcutaneous, topical, oral, intra nasal, rectal or intracerebroventricular routes, as appropriate. 
     F. Applications 
     Human DEC-205, its ligands and the constructs discussed above can be employed therapeutically or prophylactically in accordance with this invention to promote or inhibit any of the known actions of dendritic cells and/or to manipulate the immune system. 
     Thus, the antagonistic ligands per se have potential application  inter alia  blocking or inhibiting the immune response during transplantation procedures. 
     Ligands also have application in delivering other products with which they are associated directly to dendritic cells. This can be for therapeutic purposes (where the delivered product is an immunogenic antigen) as discussed above. It can also be to target a toxin (such as the ricin A-chain specifically to dendritic cells to selectively destroy them as part of an immunosuppressive process. 
     G. The Use of Human DEC-205 to Detect Dendritic Cells in Cell Suspensions on Tissues and to Purify Dendritic Cells 
     Monoclonal antibodies or other ligands binding to DEC-205 may be used to identify or isolate DC for scientific study or therapeutic application. For this application, the antibodies or ligands can be used in conjunction with conventional identification/separation systems. An example of such a system is the avidin-biotin immunoaffinity system available from Cell-Pro Inc, Washington, USA (see U.S. Pat. No. 5,215,927, U.S. Pat. No. 5,225,353, U.S. Pat. No. 5,262,334 and U.S. pat. No. 5,240,856). 
     This system employs directly or indirectly a biotinylated monoclonal antibody directed against a target cell and a column containing immunobilized avidin and can be readily adapted to extract activated human dendritic cells, in this case from human peripheral blood, using the anti-DEC-205 antibody as follows: 
     1. A sample of human peripheral blood containing the human dendritic cells is mixed with biotinylated anti-DEC-205 antibody and incubated to allow formation of antibody/human DC complexes. 
     2. Following incubation, the mixture is introduced into a CellPro continuous-flow imnuunoadsorption column filled with avidin-coated beads, the strong affinity between biotin and avidin causing the biotin-coated antibodies (together with the human DC to which they have bound) to adhere to the avidin-coated beads. 
     3. After unwanted cells present in the mixture are washed away, captured activated human DC are removed from the column by gentle agitation and are available for use. 
     Variations on this theme using the anti-DEC-205 antibody as primary antibody (to bind to activated DC) and a biotinylated secondary antibody (to bind to the anti-DEC-205 antibody) can also be employed. 
     It will be appreciated that before admixture with the anti-DEC-205 antibody in accordance with the above protocol, the human peripheral blood sample should be treated to ensure that the DC the sample contains are activated. This can easily be achieved by, for example, overnight incubation of the sample. 
     H. Functional Equivalents 
     The invention includes functional equivalents of human DEC-205, extracellular domains and nucleic acid molecules described above. 
     Human DEC-205 and its extracellular domain are or include proteins. A protein is considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has the same function as, the original protein. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein. 
     For example, it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids known normally to be equivalent are: 
     (a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G); 
     (b) Asn(N) Asp(D) Glu(E) Gln(Q); 
     (c) His(H) Arg(R) Lys(K); 
     (d) Met(M) Leu(L) Ile(I) Val(V); and 
     (e) Phe(F) Tyr(Y) Trp(W). 
     Substitutions, additions and/or deletions in human DEC-205 may be made as long as the resulting equivalent protein is immunologically cross-reactive with, and have the same function as, the native human DEC-205. 
     The equivalent human DEC-205 will normally have substantially the same amino acid sequence as the native human DEC-205. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions and/or deletions is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less tan 10%, and most preferably less than 5% of the number of amino acid residues in the amino acid sequence of the native human DEC-205 are substituted for, added to, or deleted from. 
     Equivalent nucleic acid molecules include nucleic acid sequences that encode proteins equivalent to human DEC-205 as defined above. Equivalent nucleic acid molecules also include nucleic acid sequences that, due to the degeneracy of the nucleic acid code, differ from native nucleic acid sequences in ways that do not affect the corresponding amino acid sequences. 
     Those persons skilled in the art will of course appreciate that the above description is provided by way of example only and that the invention is limited only by the lawful scope of the appended claims. 
     
       
         
           
             31 
           
           
             1 
             1722 
             PRT 
             Homo sapiens 
           
            1
Met Arg Thr Gly Trp Ala His Pro Ser Pro Pro Gly Gly Ala Pro His
  1               5                  10                  15
Ala Ala Leu Leu Val Leu Arg Ser Arg Gly Ala Leu Trp Pro Arg Thr
             20                  25                  30
Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys
         35                  40                  45
Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr Glu Asp
     50                  55                  60
Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu His Ser
 65                  70                  75                  80
Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu Leu Arg
                 85                  90                  95
Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys Cys Glu His
            100                 105                 110
His Ser Leu Tyr Gly Ala Ala Arg Tyr Trp Leu Ala Leu Lys Asp Gly
        115                 120                 125
His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly
    130                 135                 140
Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg
145                 150                 155                 160
Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp
                165                 170                 175
Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro
            180                 185                 190
Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile
        195                 200                 205
Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn Glu
    210                 215                 220
Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu Ser Trp
225                 230                 235                 240
Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser
                245                 250                 255
Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys Glu Gly Ile
            260                 265                 270
Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly
        275                 280                 285
Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro
    290                 295                 300
Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala Arg Met
305                 310                 315                 320
Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys Glu Ala Gln Leu
                325                 330                 335
Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val Glu Leu Thr Asp
            340                 345                 350
Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly Trp Leu Pro Asn
        355                 360                 365
Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn Ser Trp Asp Lys
    370                 375                 380
Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu Ile Ser Ile His
385                 390                 395                 400
Ser Leu Ala Asp Val Glu Val Val Val Thr Lys Leu His Asn Glu Asp
                405                 410                 415
Ile Lys Glu Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr
            420                 425                 430
Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp
        435                 440                 445
Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser
    450                 455                 460
Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Glu Lys
465                 470                 475                 480
Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys Leu Asn Asp Ala Ser
                485                 490                 495
Ser Asp Lys Met Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu
            500                 505                 510
Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe Gly Thr Asn Cys
        515                 520                 525
Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp Leu
    530                 535                 540
Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu
545                 550                 555                 560
Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn Trp Ala Thr Val Gly Gly
                565                 570                 575
Arg Arg Arg Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala
            580                 585                 590
Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser Val Gly Lys
        595                 600                 605
Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala Leu Ser Ile Cys Lys
    610                 615                 620
Lys Met Ser Gly Pro Leu Gly Pro Glu Glu Ala Ser Pro Lys Pro Asp
625                 630                 635                 640
Asp Pro Cys Pro Glu Gly Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys
                645                 650                 655
Tyr Lys Val Phe His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu
            660                 665                 670
Glu Ala Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe
        675                 680                 685
Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln
    690                 695                 700
Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro
705                 710                 715                 720
Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser Thr
                725                 730                 735
Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile Arg Asp Cys
            740                 745                 750
Ala Ala Val Lys Val Phe His Arg Pro Trp Arg Arg Gly Trp His Phe
        755                 760                 765
Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro Phe Ala Cys Asp Thr
    770                 775                 780
Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Arg Thr Pro Lys Thr
785                 790                 795                 800
Pro Asp Trp Tyr Asn Pro Asp Arg Ala Gly Ile His Gly Pro Pro Leu
                805                 810                 815
Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn
            820                 825                 830
Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala
        835                 840                 845
Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys Asn Lys Ile Ala
    850                 855                 860
Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp Ile Arg Ile Ser Glu Trp
865                 870                 875                 880
Pro Ile Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe
                885                 890                 895
Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp
            900                 905                 910
Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe
        915                 920                 925
Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp
    930                 935                 940
Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn
945                 950                 955                 960
Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe Ser Gln Ala
                965                 970                 975
Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser
            980                 985                 990
Gln Ile Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala
        995                1000                1005
Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Lys Ile Asn Lys
   1010                1015                1020
Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu
1025                1030                1035               1040
Val Ser Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu Ser
               1045                1050                1055
Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys Ser Pro Phe Thr
           1060                1065                1070
Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu
       1075                1080                1085
Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg Gln Thr Leu Gln Asn Ala
   1090                1095                1100
Ser Glu Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Pro Lys
1105                1110                1115               1120
Thr Leu Thr Trp His Ser Ala Lys Arg Glu Cys Leu Lys Ser Asn Met
               1125                1130                1135
Gln Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala Phe Leu Ser Val
           1140                1145                1150
Gln Ala Leu Leu His Asn Ser Ser Leu Trp Ile Gly Leu Phe Ser Gln
       1155                1160                1165
Asp Asp Glu Leu Asn Phe Gly Trp Ser Asp Gly Lys Arg Leu His Phe
   1170                1175                1180
Ser Arg Trp Ala Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu
1185                1190                1195               1200
Asp Thr Asp Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro
               1205                1210                1215
Gly Ala Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys
           1220                1225                1230
Pro Val Asp Ser Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp
       1235                1240                1245
Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn Arg
   1250                1255                1260
His Met Ala Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln Lys Leu
1265                1270                1275               1280
Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys Glu Asn Asn
               1285                1290                1295
Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met Ala Ser Trp Val
           1300                1305                1310
Met Leu Gly Ile Thr Tyr Arg Asn Asn Ser Leu Met Trp Phe Asp Lys
       1315                1320                1325
Thr Pro Leu Ser Tyr Thr His Trp Arg Ala Gly Arg Pro Thr Ile Lys
   1330                1335                1340
Asn Glu Lys Phe Leu Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp Ile
1345                1350                1355               1360
Gln Thr Phe Lys Val Ile Glu Glu Ala Val Tyr Phe His Gln His Ser
               1365                1370                1375
Ile Leu Ala Cys Lys Ile Glu Met Val Asp Tyr Lys Glu Glu His Asn
           1380                1385                1390
Thr Thr Leu Pro Gln Phe Met Pro Tyr Glu Asp Gly Ile Tyr Ser Val
       1395                1400                1405
Ile Gln Lys Lys Val Thr Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln
   1410                1415                1420
Ser Gly Gly His Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe
1425                1430                1435               1440
Leu Glu Asp Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu
               1445                1450                1455
Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser
           1460                1465                1470
Thr Phe Asp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys
       1475                1480                1485
Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn Ser
   1490                1495                1500
Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys Lys Leu
1505                1510                1515               1520
Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys Glu Asn Gly
               1525                1530                1535
Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys Ser Asp Gln Ala
           1540                1545                1550
Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys Ser Lys His Asp His
       1555                1560                1565
Ser Ala Thr Ile Val Ser Ile Lys Asp Glu Asp Glu Asn Lys Phe Val
   1570                1575                1580
Ser Arg Leu Met Arg Glu Asn Asn Asn Ile Thr Met Arg Val Trp Leu
1585                1590                1595               1600
Gly Leu Ser Gln His Ser Val Asp Gln Ser Trp Ser Trp Leu Asp Gly
               1605                1610                1615
Ser Glu Val Thr Phe Val Lys Trp Glu Asn Lys Ser Lys Ser Gly Val
           1620                1625                1630
Gly Arg Cys Ser Met Leu Ile Ala Ser Asn Glu Thr Trp Lys Lys Val
       1635                1640                1645
Glu Cys Glu His Gly Phe Gly Arg Val Val Cys Lys Val Pro Leu Gly
   1650                1655                1660
Pro Asp Tyr Thr Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu
1665                1670                1675               1680
Val Leu Met Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu
               1685                1690                1695
His Leu Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu
           1700                1705                1710
Asp Glu Ile Met Leu Pro Ser Phe His Asp
       1715                1720
 
           
             2 
             5169 
             DNA 
             Homo sapiens 
             
               CDS 
               (1)..(5166) 
             
           
            2
atg agg aca ggc tgg gcg cac ccc tcg ccg ccc ggc ggg gct cct cat       48
Met Arg Thr Gly Trp Ala His Pro Ser Pro Pro Gly Gly Ala Pro His
  1               5                  10                  15
gct gct ctt ctg gtt ctt cga tct cgc gga gcc ctc tgg ccg cgc act       96
Ala Ala Leu Leu Val Leu Arg Ser Arg Gly Ala Leu Trp Pro Arg Thr
             20                  25                  30
aat gac ccc ttc acc atc gtc cat gga aat acg ggc aag tgc atc aag      144
Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys
         35                  40                  45
cca gtg tat ggc tgg ata gta gca gac gac tgt gat gaa act gag gac      192
Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr Glu Asp
     50                  55                  60
aag tta tgg aag tgg gtg tcc cag cat cgg ctc ttt cat ttg cac tcc      240
Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu His Ser
 65                  70                  75                  80
caa aag tgc ctt ggc ctc gat att acc aaa tcg gta aat gag ctg aga      288
Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu Leu Arg
                 85                  90                  95
atg ttc agc tgt gac tcc agt gcc atg ctg tgg tgg aaa tgt gag cac      336
Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys Cys Glu His
            100                 105                 110
cac tct ctg tac gga gct gcc cgg tac tgg ctg gct ctg aag gat gga      384
His Ser Leu Tyr Gly Ala Ala Arg Tyr Trp Leu Ala Leu Lys Asp Gly
        115                 120                 125
cat ggc aca gca atc tca aat gca tct gat gtc tgg aag aaa gga ggc      432
His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly
    130                 135                 140
tca gag gaa agc ctt tgt gac cag cct tat cat gag atc tat acc aga      480
Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg
145                 150                 155                 160
gat ggg aac tct tat ggg aga cct tgt gaa ttt cca ttc tta att gat      528
Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp
                165                 170                 175
ggg acc tgg cat cat gat tgc att ctt gat gaa gat cat agt ggg cca      576
Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro
            180                 185                 190
tgg tgt gcc acc acc tta aat tat gaa tat gac cga aag tgg ggc atc      624
Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile
        195                 200                 205
tgc tta aag cct gaa aac ggt tgt gaa gat aat tgg gaa aag aac gag      672
Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn Glu
    210                 215                 220
cag ttt gga agt tgc tac caa ttt aat act cag acg gct ctt tct tgg      720
Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu Ser Trp
225                 230                 235                 240
aaa gaa gct tat gtt tca tgt cag aat caa gga gct gat tta ctg agc      768
Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser
                245                 250                 255
atc aac agt gct gct gaa tta act tac ctt aaa gaa aaa gaa ggc att      816
Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys Glu Gly Ile
            260                 265                 270
gct aag att ttc tgg att ggt tta aat cag cta tac tct gct aga ggc      864
Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly
        275                 280                 285
tgg gaa tgg tca gac cac aaa cca tta aac ttt ctc aac tgg gat cca      912
Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro
    290                 295                 300
gac agg ccc agt gca cct act ata ggt ggc tcc agc tgt gca aga atg      960
Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala Arg Met
305                 310                 315                 320
gat gct gag tct ggt ctg tgg cag agc ttt tcc tgt gaa gct caa ctg     1008
Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys Glu Ala Gln Leu
                325                 330                 335
ccc tat gtc tgc agg aaa cca tta aat aat aca gtg gag tta aca gat     1056
Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val Glu Leu Thr Asp
            340                 345                 350
gtc tgg aca tac tca gat acc cgc tgt gat gca ggc tgg ctg cca aat     1104
Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly Trp Leu Pro Asn
        355                 360                 365
aat gga ttt tgc tat ctg ctg gta aat gaa agt aat tcc tgg gat aag     1152
Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn Ser Trp Asp Lys
    370                 375                 380
gca cat gcg aaa tgc aaa gcc ttc agt agt gac cta atc agc att cat     1200
Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu Ile Ser Ile His
385                 390                 395                 400
tct cta gca gat gtg gag gtg gtt gtc aca aaa ctc cat aat gag gat     1248
Ser Leu Ala Asp Val Glu Val Val Val Thr Lys Leu His Asn Glu Asp
                405                 410                 415
atc aaa gaa gaa gtg tgg ata ggc ctt aag aac ata aac ata cca act     1296
Ile Lys Glu Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr
            420                 425                 430
tta ttt cag tgg tca gat ggt act gaa gtt act cta aca tat tgg gat     1344
Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp
        435                 440                 445
gag aat gag cca aat gtt ccc tac aat aag acg ccc aac tgt gtt tcc     1392
Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser
    450                 455                 460
tac tta gga gag cta ggt cag tgg aaa gtc caa tca tgt gag gag aaa     1440
Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Glu Lys
465                 470                 475                 480
cta aaa tat gta tgc aag aga aag gga gaa aaa ctg aat gac gca agt     1488
Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys Leu Asn Asp Ala Ser
                485                 490                 495
tct gat aag atg tgt cct cca gat gag ggc tgg aag aga cat gga gaa     1536
Ser Asp Lys Met Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu
            500                 505                 510
acc tgt tac aag att tat gag gat gag gtc cct ttt gga aca aac tgc     1584
Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe Gly Thr Asn Cys
        515                 520                 525
aat ctg act atc act agc aga ttt gag caa gaa tac cta aat gat ttg     1632
Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp Leu
    530                 535                 540
atg aaa aag tat gat aaa tct cta aga aaa tac ttc tgg act ggc ctg     1680
Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu
545                 550                 555                 560
aga gat gta gat tct tgt gga gag tat aac tgg gca act gtt ggt gga     1728
Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn Trp Ala Thr Val Gly Gly
                565                 570                 575
aga agg cgg gct gta acc ttt tcc aac tgg aat ttt ctt gag cca gct     1776
Arg Arg Arg Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala
            580                 585                 590
tcc ccg ggc ggc tgc gtg gct atg tct act gga aag tct gtt gga aag     1824
Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser Val Gly Lys
        595                 600                 605
tgg gag gtg aag gac tgc aga agc ttc aaa gca ctt tca att tgc aag     1872
Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala Leu Ser Ile Cys Lys
    610                 615                 620
aaa atg agt gga ccc ctt ggg cct gaa gaa gca tcc cct aag cct gat     1920
Lys Met Ser Gly Pro Leu Gly Pro Glu Glu Ala Ser Pro Lys Pro Asp
625                 630                 635                 640
gac ccc tgt cct gaa ggc tgg cag agt ttc ccc gca agt ctt tct tgt     1968
Asp Pro Cys Pro Glu Gly Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys
                645                 650                 655
tat aag gta ttc cat gca gaa aga att gta aga aag agg aac tgg gaa     2016
Tyr Lys Val Phe His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu
            660                 665                 670
gaa gct gaa cga ttc tgc caa gcc ctt gga gca cac ctt tct agc ttc     2064
Glu Ala Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe
        675                 680                 685
agc cat gtg gat gaa ata aag gaa ttt ctt cac ttt tta acg gac cag     2112
Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln
    690                 695                 700
ttc agt ggc cag cat tgg ctg tgg att ggt ttg aat aaa agg agc cca     2160
Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro
705                 710                 715                 720
gat tta caa gga tcc tgg caa tgg agt gat cgt aca cca gtg tct act     2208
Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser Thr
                725                 730                 735
att atc atg cca aat gag ttt cag cag gat tat gac atc aga gac tgt     2256
Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile Arg Asp Cys
            740                 745                 750
gct gct gtc aag gta ttt cat agg cca tgg cga aga ggc tgg cat ttc     2304
Ala Ala Val Lys Val Phe His Arg Pro Trp Arg Arg Gly Trp His Phe
        755                 760                 765
tat gat gat aga gaa ttt att tat ttg agg cct ttt gct tgt gat aca     2352
Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro Phe Ala Cys Asp Thr
    770                 775                 780
aaa ctt gaa tgg gtg tgc caa att cca aaa ggc cgt act cca aaa aca     2400
Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Arg Thr Pro Lys Thr
785                 790                 795                 800
cca gac tgg tac aat cca gac cgt gct gga att cat gga cct cca ctt     2448
Pro Asp Trp Tyr Asn Pro Asp Arg Ala Gly Ile His Gly Pro Pro Leu
                805                 810                 815
ata att gaa gga agt gaa tat tgg ttt gtt gct gat ctt cac cta aac     2496
Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn
            820                 825                 830
tat gaa gaa gcc gtc ctg tac tgt gcc agc aat cac agc ttt ctt gcg     2544
Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala
        835                 840                 845
act ata aca tct ttt gtg gga cta aaa gcc atc aaa aac aaa ata gca     2592
Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys Asn Lys Ile Ala
    850                 855                 860
aat ata tct ggt gat gga cag aag tgg tgg ata aga att agc gag tgg     2640
Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp Ile Arg Ile Ser Glu Trp
865                 870                 875                 880
cca ata gat gat cat ttt aca tac tca cga tat cca tgg cac cgc ttt     2688
Pro Ile Asp Asp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe
                885                 890                 895
cct gtg aca ttt gga gag gaa tgc ttg tac atg tct gcc aag act tgg     2736
Pro Val Thr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp
            900                 905                 910
ctt atc gac tta ggt aaa cca aca gac tgt agt acc aag ttg ccc ttc     2784
Leu Ile Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe
        915                 920                 925
atc tgt gaa aaa tat aat gtt tct tcg tta gag aaa tac agc cca gat     2832
Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp
    930                 935                 940
tct gca gct aaa gtg caa tgt tct gag caa tgg att cct ttt cag aat     2880
Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn
945                 950                 955                 960
aag tgt ttt cta aag atc aaa ccc gtg tct ctc aca ttt tct caa gca     2928
Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe Ser Gln Ala
                965                 970                 975
agc gat acc tgt cac tcc tat ggt ggc acc ctt cct tca gtg ttg agc     2976
Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser
            980                 985                 990
cag att gaa caa gac ttt att aca tcc ttg ctt ccg gat atg gaa gct     3024
Gln Ile Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala
        995                1000                1005
act tta tgg att ggt ttg cgc tgg act gcc tat gaa aag ata aac aaa     3072
Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Lys Ile Asn Lys
   1010                1015                1020
tgg aca gat aac aga gag ctg acg tac agt aac ttt cac cca tta ttg     3120
Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu
1025               1030                1035                1040
gtt agt ggg agg ctg aga ata cca gaa aat ttt ttt gag gaa gag tct     3168
Val Ser Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu Ser
               1045                1050                1055
cgc tac cac tgt gcc cta ata ctc aac ctc caa aaa tca ccg ttt act     3216
Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys Ser Pro Phe Thr
           1060                1065                1070
ggg acg tgg aat ttt aca tcc tgc agt gaa cgc cac ttt gtg tct ctc     3264
Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu
       1075                1080                1085
tgt cag aaa tat tca gaa gtt aaa agc aga cag acg ttg cag aat gct     3312
Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg Gln Thr Leu Gln Asn Ala
   1090                1095                1100
tca gaa act gta aag tat cta aat aat ctg tac aaa ata atc cca aag     3360
Ser Glu Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Pro Lys
1105               1110                1115                1120
act ctg act tgg cac agt gct aaa agg gag tgt ctg aaa agt aac atg     3408
Thr Leu Thr Trp His Ser Ala Lys Arg Glu Cys Leu Lys Ser Asn Met
               1125                1130                1135
cag ctg gtg agc atc acg gac cct tac cag cag gca ttc ctc agt gtg     3456
Gln Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala Phe Leu Ser Val
           1140                1145                1150
cag gcg ctc ctt cac aac tct tcc tta tgg atc gga ctc ttc agt caa     3504
Gln Ala Leu Leu His Asn Ser Ser Leu Trp Ile Gly Leu Phe Ser Gln
       1155                1160                1165
gat gat gaa ctc aac ttt ggt tgg tca gat ggg aaa cgt ctt cat ttt     3552
Asp Asp Glu Leu Asn Phe Gly Trp Ser Asp Gly Lys Arg Leu His Phe
   1170                1175                1180
agt cgc tgg gct gaa act aat ggg caa ctc gaa gac tgt gta gta tta     3600
Ser Arg Trp Ala Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu
1185               1190                1195                1200
gac act gat gga ttc tgg aaa aca gtt gat tgc aat gac aat caa cca     3648
Asp Thr Asp Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro
               1205                1210                1215
ggt gct att tgc tac tat tca gga aat gag act gaa aaa gag gtc aaa     3696
Gly Ala Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys
           1220                1225                1230
cca gtt gac agt gtt aaa tgt cca tct cct gtt cta aat act ccg tgg     3744
Pro Val Asp Ser Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp
       1235                1240                1245
ata cca ttt cag aac tgt tgc tac aat ttc ata ata aca aag aat agg     3792
Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn Arg
   1250                1255                1260
cat atg gca aca aca cag gat gaa gtt cat act aaa tgc cag aaa ctg     3840
His Met Ala Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln Lys Leu
1265               1270                1275                1280
aat cca aaa tca cat att ctg agt att cga gat gaa aag gag aat aac     3888
Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys Glu Asn Asn
               1285                1290                1295
ttt gtt ctt gag caa ctg ctg tac ttc aat tat atg gct tca tgg gtc     3936
Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met Ala Ser Trp Val
           1300                1305                1310
atg tta gga ata act tat aga aat aat tct ctt atg tgg ttt gat aag     3984
Met Leu Gly Ile Thr Tyr Arg Asn Asn Ser Leu Met Trp Phe Asp Lys
       1315                1320                1325
acc cca ctg tca tat aca cat tgg aga gca gga aga cca act ata aaa     4032
Thr Pro Leu Ser Tyr Thr His Trp Arg Ala Gly Arg Pro Thr Ile Lys
   1330                1335                1340
aat gag aag ttt ttg gct ggt tta agt act gac ggc ttc tgg gat att     4080
Asn Glu Lys Phe Leu Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp Ile
1345               1350                1355                1360
caa acc ttt aaa gtt att gaa gaa gca gtt tat ttt cac cag cac agc     4128
Gln Thr Phe Lys Val Ile Glu Glu Ala Val Tyr Phe His Gln His Ser
               1365                1370                1375
att ctt gct tgt aaa att gaa atg gtt gac tac aaa gaa gaa cat aat     4176
Ile Leu Ala Cys Lys Ile Glu Met Val Asp Tyr Lys Glu Glu His Asn
           1380                1385                1390
act aca ctg cca cag ttt atg cca tat gaa gat ggt att tac agt gtt     4224
Thr Thr Leu Pro Gln Phe Met Pro Tyr Glu Asp Gly Ile Tyr Ser Val
       1395                1400                1405
att caa aaa aag gta aca tgg tat gaa gca tta aac atg tgt tct caa     4272
Ile Gln Lys Lys Val Thr Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln
   1410                1415                1420
agt gga ggt cac ttg gca agc gtt cac aac caa aat ggc cag ctc ttt     4320
Ser Gly Gly His Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe
1425               1430                1435                1440
ctg gaa gat att gta aaa cgt gat gga ttt cca cta tgg gtt ggg ctc     4368
Leu Glu Asp Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu
               1445                1450                1455
tca agt cat gat gga agt gaa tca agt ttt gaa tgg tct gat ggt agt     4416
Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser
           1460                1465                1470
aca ttt gac tat atc cca tgg aaa ggc caa aca tct cct gga aat tgt     4464
Thr Phe Asp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys
       1475                1480                1485
gtt ctc ttg gat cca aaa gga act tgg aaa cat gaa aaa tgc aac tct     4512
Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn Ser
   1490                1495                1500
gtt aag gat ggt gct att tgt tat aaa cct aca aaa tct aaa aag ctg     4560
Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys Lys Leu
1505               1510                1515                1520
tcc cgt ctt aca tat tca tca aga tgt cca gca gca aaa gag aat ggg     4608
Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys Glu Asn Gly
               1525                1530                1535
tca cgg tgg atc cag tac aag ggt cac tgt tac aag tct gat cag gca     4656
Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys Ser Asp Gln Ala
           1540                1545                1550
ttg cac agt ttt tca gag gcc aaa aaa ttg tgt tca aaa cat gat cac     4704
Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys Ser Lys His Asp His
       1555                1560                1565
tct gca act atc gtt tcc ata aaa gat gaa gat gag aat aaa ttt gtg     4752
Ser Ala Thr Ile Val Ser Ile Lys Asp Glu Asp Glu Asn Lys Phe Val
   1570                1575                1580
agc aga ctg atg agg gaa aat aat aac att acc atg aga gtt tgg ctt     4800
Ser Arg Leu Met Arg Glu Asn Asn Asn Ile Thr Met Arg Val Trp Leu
1585               1590                1595                1600
gga tta tct caa cat tct gtt gac cag tct tgg agt tgg tta gat gga     4848
Gly Leu Ser Gln His Ser Val Asp Gln Ser Trp Ser Trp Leu Asp Gly
               1605                1610                1615
tca gaa gtg aca ttt gtc aaa tgg gaa aat aaa agt aag agt ggt gtt     4896
Ser Glu Val Thr Phe Val Lys Trp Glu Asn Lys Ser Lys Ser Gly Val
           1620                1625                1630
gga aga tgt agc atg ttg ata gct tca aat gaa act tgg aaa aaa gtt     4944
Gly Arg Cys Ser Met Leu Ile Ala Ser Asn Glu Thr Trp Lys Lys Val
       1635                1640                1645
gaa tgt gaa cat ggt ttt gga aga gtt gtc tgc aaa gtg cct ctg ggc     4992
Glu Cys Glu His Gly Phe Gly Arg Val Val Cys Lys Val Pro Leu Gly
   1650                1655                1660
cct gat tac aca gca ata gct atc ata gtt gcc aca cta agt atc tta     5040
Pro Asp Tyr Thr Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu
1665               1670                1675                1680
gtt ctc atg ggc gga ctg att tgg ttc ctc ttc caa agg cac cgt ttg     5088
Val Leu Met Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu
               1685                1690                1695
cac ctg gcg ggt ttc tca tca gtt cga tat gca caa gga gtg aat gaa     5136
His Leu Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu
           1700                1705                1710
gat gag att atg ctt cct tct ttc cat gac taa                         5169
Asp Glu Ile Met Leu Pro Ser Phe His Asp
       1715                1720
 
           
             3 
             349 
             DNA 
             Homo sapiens 
           
            3
aacagttgat tgcaatgaca atcaaccagg tgctatttgc tactattcag gaaatgagac     60
tgaaaaagag gtcaaaccag ttgacagtgt taaatgtcca tctcctgttc taaatactcc    120
gtggatacca tttcagaact gttgctacaa tttcataata acaaagaata ggcatatggc    180
aacaacacag gatgaagttc atactaaatg ccagaaactg aatccaaaat cacatattct    240
gagtattcga gatgaaaagg agaataactt tgttcttgag caactgctgt acttcaatta    300
tatggcttca tgggtcatgt taggaataac ttatagaaat aaktctctt                349
 
           
             4 
             152 
             DNA 
             Homo sapiens 
           
            4
attaatatgc tgtggaagtg ggtgtcccag catcggctct ttcatttgca ctcccaaaag     60
tgccttggcc tcgatattac caaatcggta aatgagctga gaatgttcag ctgtgactcc    120
gtgccatgc tgtggtggaa atgcgagcac ca                                   152
 
           
             5 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            5
gaycangayg gnttytggaa                                                 20
 
           
             6 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            6
tacaccaarc trttytgncg                                                 20
 
           
             7 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            7
aayatgctnt ggaartgggt                                                 20
 
           
             8 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            8
tgrtgytcrc ayttccacca                                                 20
 
           
             9 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            9
gayacngayg gnttytggaa                                                 20
 
           
             10 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            10
gcngtyttrt craaccacat                                                 20
 
           
             11 
             26 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            11
gctctagaaa catgacccat gaagcc                                          26
 
           
             12 
             27 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            12
gctctagaca tcggctcttt catttgt                                         27
 
           
             13 
             27 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            13
cgggattcac agttgattgc aatgaca                                         27
 
           
             14 
             35 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Oligo d(T)
      adaptor primer 
             
           
            14
gactagtctg cagaattctt tttttttttt ttttt                                35
 
           
             15 
             18 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Adaptor
      primer 
             
           
            15
gactagtctg cagaattc                                                   18
 
           
             16 
             28 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            16
cgggatccct ctggccgcgc actaatga                                        28
 
           
             17 
             31 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            17
ccgctcgagc tgtggatacc agcacatgcc t                                    31
 
           
             18 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            18
gatgggaact cttatgggag acct                                            24
 
           
             19 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            19
tgatgcaggc tggctgccaa ataa                                            24
 
           
             20 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            20
aactgggcaa ctgttggtgg aaga                                            24
 
           
             21 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            21
atggcgaaga ggctggcatt tcta                                            24
 
           
             22 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            22
ctcaagcaag cgatacctgt cact                                            24
 
           
             23 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            23
tgggcaactc gaagactgtg tagt                                            24
 
           
             24 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            24
caccagcaca gcattcttgc ttgt                                            24
 
           
             25 
             24 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            25
atttgtgagc agactgatga ggga                                            24
 
           
             26 
             32 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence
      PCR-fragment 
             
           
            26
cggaattcga tctcatgata aggctggtca ca                                   32
 
           
             27 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 060 
             
           
            27
gtggatccag tacaagggtc a                                               21
 
           
             28 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 056 
             
           
            28
accaaatcag tccgcccatg a                                               21
 
           
             29 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 053 
             
           
            29
atggggaagg tgaaggtcgg a                                               21
 
           
             30 
             21 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 053 
             
           
            30
aggggccatc cacagtcttc t                                               21
 
           
             31 
             1723 
             PRT 
             Murine sp. 
           
            31
Met Arg Thr Gly Arg Val Thr Pro Gly Leu Ala Ala Gly Leu Leu Leu
  1               5                  10                  15
Leu Leu Leu Arg Ser Phe Gly Leu Val Glu Pro Ser Glu Ser Ser Gly
             20                  25                  30
Asn Asp Pro Phe Thr Ile Val His Glu Asn Thr Gly Lys Cys Ile Gln
         35                  40                  45
Pro Leu Ser Asp Trp Val Val Ala Gln Asp Cys Ser Gly Thr Asn Asn
     50                  55                  60
Met Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu Glu Ser
 65                  70                  75                  80
Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ala Thr Asp Asn Leu Arg
                 85                  90                  95
Met Phe Ser Cys Asp Ser Thr Val Met Leu Trp Trp Lys Cys Glu His
            100                 105                 110
His Ser Leu Tyr Thr Ala Ala Gln Tyr Arg Leu Ala Leu Lys Asp Gly
        115                 120                 125
Tyr Ala Val Ala Asn Thr Asn Thr Ser Asp Val Trp Lys Lys Gly Gly
    130                 135                 140
Ser Glu Glu Asn Leu Cys Ala Gln Pro Tyr His Glu Ile Tyr Thr Arg
145                 150                 155                 160
Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Gly
                165                 170                 175
Glu Thr Trp Tyr His Asp Cys Ile His Asp Glu Asp His Ser Gly Pro
            180                 185                 190
Trp Cys Ala Thr Thr Leu Ser Tyr Glu Tyr Asp Gln Lys Trp Gly Ile
        195                 200                 205
Cys Leu Leu Pro Glu Ser Gly Cys Glu Gly Asn Trp Glu Lys Asn Glu
    210                 215                 220
Gln Ile Gly Ser Cys Tyr Gln Phe Asn Asn Gln Glu Ile Leu Ser Trp
225                 230                 235                 240
Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser
                245                 250                 255
Ile His Ser Ala Ala Glu Leu Ala Tyr Ile Thr Gly Lys Glu Asp Ile
            260                 265                 270
Ala Arg Leu Val Trp Leu Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly
        275                 280                 285
Trp Glu Trp Ser Asp Phe Arg Pro Leu Lys Phe Leu Asn Trp Asp Pro
    290                 295                 300
Gly Thr Pro Val Ala Pro Val Ile Gly Gly Ser Ser Cys Ala Arg Met
305                 310                 315                 320
Asp Thr Glu Ser Gly Leu Trp Gln Ser Val Ser Cys Glu Ser Gln Gln
                325                 330                 335
Pro Tyr Val Cys Lys Lys Pro Leu Asn Asn Thr Leu Glu Leu Pro Asp
            340                 345                 350
Val Trp Thr Tyr Thr Asp Thr His Cys His Val Gly Trp Leu Pro Asn
        355                 360                 365
Asn Gly Phe Cys Tyr Leu Leu Ala Asn Glu Ser Ser Ser Trp Asp Ala
    370                 375                 380
Ala His Leu Lys Cys Lys Ala Phe Gly Ala Asp Leu Ile Ser Met His
385                 390                 395                 400
Ser Leu Ala Asp Val Glu Val Val Val Thr Lys Leu His Asn Gly Asp
                405                 410                 415
Val Lys Lys Glu Ile Trp Thr Gly Leu Lys Asn Thr Asn Ser Pro Ala
            420                 425                 430
Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asn
        435                 440                 445
Glu Asn Glu Pro Ser Val Pro Phe Asn Lys Thr Pro Asn Cys Val Ser
    450                 455                 460
Tyr Leu Gly Lys Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Lys Lys
465                 470                 475                 480
Leu Arg Tyr Val Cys Lys Lys Lys Gly Glu Ile Thr Lys Asp Ala Glu
                485                 490                 495
Ser Asp Lys Leu Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu
            500                 505                 510
Thr Cys Tyr Lys Ile Tyr Glu Lys Glu Ala Pro Phe Gly Thr Asn Cys
        515                 520                 525
Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Phe Leu Asn Tyr Met
    530                 535                 540
Met Lys Asn Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu
545                 550                 555                 560
Arg Asp Pro Asp Ser Arg Gly Glu Tyr Ser Trp Ala Val Ala Gln Gly
                565                 570                 575
Val Lys Gln Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala
            580                 585                 590
Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Thr Leu Gly Lys
        595                 600                 605
Trp Glu Val Lys Asn Cys Arg Ser Phe Arg Ala Leu Ser Ile Cys Lys
    610                 615                 620
Lys Val Ser Glu Pro Gln Glu Pro Glu Glu Ala Ala Pro Lys Pro Asp
625                 630                 635                 640
Asp Pro Cys Pro Glu Gly Trp His Thr Phe Pro Ser Ser Leu Ser Cys
                645                 650                 655
Tyr Lys Val Phe His Ile Glu Arg Ile Val Arg Lys Arg Asn Trp Glu
            660                 665                 670
Glu Ala Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Pro Ser Phe
        675                 680                 685
Ser Arg Arg Glu Glu Ile Lys Asp Phe Val His Leu Leu Lys Asp Gln
    690                 695                 700
Phe Ser Gly Gln Arg Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro
705                 710                 715                 720
Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser Ala
                725                 730                 735
Val Met Met Glu Pro Glu Phe Gln Gln Asp Phe Asp Ile Arg Asp Cys
            740                 745                 750
Ala Ala Ile Lys Val Leu Asp Val Pro Trp Arg Arg Val Trp His Leu
        755                 760                 765
Tyr Glu Asp Lys Asp Tyr Ala Tyr Trp Lys Pro Phe Ala Cys Asp Ala
    770                 775                 780
Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Ser Thr Pro Gln Met
785                 790                 795                 800
Pro Asp Trp Tyr Asn Pro Glu Arg Thr Gly Ile His Gly Pro Pro Val
                805                 810                 815
Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Pro His Leu Asn
            820                 825                 830
Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala
        835                 840                 845
Thr Ile Thr Ser Phe Thr Gly Leu Lys Ala Ile Lys Asn Lys Leu Ala
    850                 855                 860
Asn Ile Ser Gly Glu Glu Gln Lys Trp Trp Val Lys Thr Ser Glu Asn
865                 870                 875                 880
Pro Ile Asp Arg Tyr Phe Leu Gly Ser Arg Arg Arg Leu Trp His His
                885                 890                 895
Phe Pro Met Thr Phe Gly Asp Glu Cys Leu His Met Ser Ala Lys Thr
            900                 905                 910
Trp Leu Val Asp Leu Ser Lys Arg Ala Asp Cys Asn Ala Lys Leu Pro
        915                 920                 925
Phe Ile Cys Glu Arg Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro
    930                 935                 940
Asp Pro Ala Ala Lys Val Gln Cys Thr Glu Lys Trp Ile Pro Phe Gln
945                 950                 955                 960
Asn Lys Cys Phe Leu Lys Val Asn Ser Gly Pro Val Thr Phe Ser Gln
                965                 970                 975
Ala Ser Gly Ile Cys His Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu
            980                 985                 990
Ser Arg Gly Glu Gln Asp Phe Ile Ile Ser Leu Leu Pro Glu Met Glu
        995                1000                1005
Ala Ser Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Arg Ile Asn
   1010                1015                1020
Arg Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu
1025               1030                1035                1040
Leu Val Gly Arg Arg Leu Ser Ile Pro Thr Asn Phe Phe Asp Asp Glu
               1045                1050                1055
Ser His Phe His Cys Ala Leu Ile Leu Asn Leu Lys Lys Ser Pro Leu
           1060                1065                1070
Thr Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg His Ser Leu Ser
       1075                1080                1085
Leu Cys Gln Lys Tyr Ser Glu Thr Glu Asp Gly Gln Pro Trp Glu Asn
   1090                1095                1100
Thr Ser Lys Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Ser
1105               1110                1115                1120
Lys Pro Leu Thr Trp His Gly Ala Leu Lys Glu Cys Met Lys Glu Lys
               1125                1130                1135
Met Arg Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala Phe Leu Ala
           1140                1145                1150
Val Gln Ala Thr Leu Arg Asn Ser Ser Phe Trp Ile Gly Leu Ser Ser
       1155                1160                1165
Gln Asp Asp Glu Leu Asn Phe Gly Trp Ser Asp Gly Lys Arg Leu Gln
   1170                1175                1180
Phe Ser Asn Trp Ala Gly Ser Asn Glu Gln Leu Asp Asp Cys Val Ile
1185               1190                1195                1200
Leu Asp Thr Asp Gly Phe Trp Lys Thr Ala Asp Cys Asp Asp Asn Gln
               1205                1210                1215
Pro Gly Ala Ile Cys Tyr Tyr Pro Gly Asn Glu Thr Glu Glu Glu Val
           1220                1225                1230
Arg Ala Leu Asp Thr Ala Lys Cys Pro Ser Pro Val Gln Ser Thr Pro
       1235                1240                1245
Trp Ile Pro Phe Gln Asn Ser Cys Tyr Phe Asn Met Ile Thr Asn Asn
   1250                1255                1260
Arg His Lys Thr Val Thr Pro Glu Glu Val Gln Ser Thr Cys Glu Lys
1265               1270                1275                1280
Leu His Pro Lys Ala His Ser Leu Ser Ile Arg Asn Glu Glu Glu Asn
               1285                1290                1295
Thr Phe Val Val Glu Gln Leu Leu Tyr Phe Asn Tyr Ile Ala Ser Trp
           1300                1305                1310
Val Met Leu Gly Ile Thr Tyr Glu Asn Asn Ser Leu Met Trp Phe Asp
       1315                1320                1325
Lys Thr Ala Leu Ser Tyr Thr His Trp Arg Thr Gly Arg Pro Thr Val
   1330                1335                1340
Lys Asn Gly Lys Phe Leu Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp
1345               1350                1355                1360
Ile Gln Ser Phe Asn Val Ile Glu Glu Thr Leu His Phe Tyr Gln His
               1365                1370                1375
Ser Ile Ser Ala Cys Lys Ile Lys Met Val Asp Tyr Glu Asp Lys His
           1380                1385                1390
Asn Gly Thr Leu Pro Gln Phe Ile Pro Tyr Lys Asp Gly Val Tyr Ser
       1395                1400                1405
Val Ile Gln Lys Lys Val Thr Trp Tyr Glu Ala Leu Asn Ala Cys Ser
   1410                1415                1420
Gln Ser Gly Gly Glu Leu Ala Ser Val His Asn Pro Asn Gly Lys Leu
1425               1430                1435                1440
Phe Leu Glu Asp Ile Val Asn Arg Asp Gly Phe Pro Leu Asn Val Gly
               1445                1450                1455
Leu Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly
           1460                1465                1470
Arg Ala Phe Asp Tyr Val Pro Trp Gln Ser Leu Gln Ser Pro Gly Asp
       1475                1480                1485
Cys Val Val Leu Tyr Pro Lys Gly Ile Trp Arg Arg Glu Lys Cys Leu
   1490                1495                1500
Ser Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Asp Lys Lys
1505               1510                1515                1520
Leu Ile Phe His Val Lys Ser Ser Lys Cys Pro Val Ala Lys Arg Asp
               1525                1530                1535
Gly Pro Gln Trp Val Gln Tyr Gly Gly His Cys Tyr Ala Ser Asp Gln
           1540                1545                1550
Val Leu His Ser Phe Ser Glu Ala Lys Gln Val Cys Gln Glu Leu Asp
       1555                1560                1565
His Ser Ala Thr Val Val Thr Ile Ala Asp Glu Asn Glu Asn Lys Phe
   1570                1575                1580
Val Ser Arg Leu Met Arg Glu Asn Tyr Asn Ile Thr Met Arg Val Trp
1585               1590                1595                1600
Leu Gly Leu Ser Gln His Ser Leu Asp Gln Ser Trp Ser Trp Leu Asp
               1605                1610                1615
Gly Leu Asp Val Thr Phe Val Lys Trp Glu Asn Lys Thr Lys Asp Gly
           1620                1625                1630
Asp Gly Lys Cys Ser Ile Leu Ile Ala Ser Asn Glu Thr Trp Arg Lys
       1635                1640                1645
Val His Cys Ser Arg Gly Tyr Ala Arg Ala Val Cys Lys Ile Pro Leu
   1650                1655                1660
Ser Pro Asp Tyr Thr Gly Ile Ala Ile Leu Phe Ala Val Leu Cys Leu
1665               1670                1675                1680
Leu Gly Leu Ile Ser Leu Ala Ile Trp Phe Leu Leu Gln Arg Ser His
               1685                1690                1695
Ile Arg Trp Thr Gly Phe Ser Ser Val Arg Tyr Glu His Gly Thr Asn
           1700                1705                1710
Glu Asp Glu Val Met Leu Pro Ser Phe His Asp
       1715                1720