Abstract:
The invention provides isolated human DEC-205, its extracellular domain and functionally equivalent fragments thereof. Also provided are polynucleotides encoding same and vectors which include such polynucleotides. Further provided are methods of recombinantly producing human DEC-205, an extracellular domain thereof or a functionally equivalent fragment, and ligands that bind to human DEC-205 or a fragment thereof. Also provided are constructs for use in prophylaxis or therapy comprising such a ligand, human DEC-205 or an extracellular domain thereof coupled to a toxin or to an antigen capable of inducing a protective immune response in a patient.

Description:
FIELD OF THE INVENTION  
         [0001]    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  
         [0002]    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.  
           [0003]    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.  
           [0004]    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  
         [0005]    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)   TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQ           NCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNF           VLEQLLYFNYMASWVMLGITYRNNSL; and               (ii)   SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML;          
 
           [0006]    or a functionally equivalent fragment thereof  
           [0007]    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.  
           [0008]    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.  
           [0009]    In yet a further aspect, the invention provides an extracellular domain of human DEC-205 or a functionally-equivalent fragment thereof.  
           [0010]    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).  
           [0011]    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.  
           [0012]    In a specific embodiment, the polynucleotide coding for human DEC-205 includes the following nucleotide sequences:  
                           (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          
 
           [0013]    wherein X is T or G.  
           [0014]    In a further embodiment, the polynucleotide comprises part or all of the nucleotide sequence of FIG. 10.  
           [0015]    In yet a further aspect, the invention provides a vector including a polynucleotide as defined above.  
           [0016]    In still a further aspect, the invention provides a method of producing human DEC-205, the extracellular domain thereof or a functional fragment comprising the steps of:  
           [0017]    (a) culturing a host cell which has been transformed or transfected with a vector as defined above to express the encoded human DEC-205, extracellular domain or fragment; and  
           [0018]    (b) recovering the expressed human DEC-205, extracellular domain or fragment.  
           [0019]    As yet an additional aspect, the invention provides a ligand that binds to human DEC-205 or its extracellular domain as defined above.  
           [0020]    Preferably, the ligand is an antibody or antibody binding fragment or carbohydrate bearing protein.  
           [0021]    The antibody or antibody binding fragment can be used in methods for extracting or isolating activated dendritic cells.  
           [0022]    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.  
           [0023]    In yet further aspects, the invention contemplates methods of therapy or prophylaxis which employ human DEC-205, ligands or constructs containing them.  
           [0024]    In yet a further aspect, the invention provides a molecule (hapten) which may be used to generate antibodies for identifying or purifying human dendritic cells, which includes a peptide based upon part or all of the sequence of FIG. 11. 
       
    
    
     DESCRIPTION OF THE DRAWINGS  
       [0025]    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  
         [0026]    [0026]FIG. 1 shows the structure of human DEC-205;  
         [0027]    [0027]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. 2A. The positions of DNA molecular size standard are indicateds to the right. The estimated molecular size of the RT-PCR products are indicated to the left;  
         [0028]    [0028]FIG. 3 shows protein similarity between human and mouse DEC-205. A. The predicted amino acid sequence of human DEC-205 is aligned with the mouse homolog. 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);  
         [0029]    [0029]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;  
         [0030]    [0030]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;  
         [0031]    [0031]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;  
         [0032]    [0032]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;  
         [0033]    [0033]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.  
         [0034]    [0034]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;  
         [0035]    [0035]FIG. 10 gives the DNA sequence for human DEC-205 (coding region only);  
         [0036]    [0036]FIG. 11 gives the human DEC-205 amino acid sequence. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0037]    A. Human DEC-205  
         [0038]    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.  
         [0039]    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.  
         [0040]    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).  
         [0041]    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 art, and has been described, for example by Hopp et al.,  Proc. Natl. Acad. Sci. U.S.A.  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.  
         [0042]    The amino acid sequences of the predicted regions for human DEC-205 are shown in FIG. 3A. 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.  
         [0043]    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.  
         [0044]    Example 1 provides further details of human DEC-205.  
       EXAMPLE 1  
       [0045]    Langerhans cells were prepared from human skin. Epidermal cell suspensions were prepared from split thickness normal human breast skin by 30 min dispase (Boehringer-Mannheim, 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=1.077 g/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.  
         [0046]    Degenerate primers were prepared on an Applied Biosystems DNA Synthesizer with the primer sequences (d) and (e) as set out below:  
                                       (d)   5′-GAX ACY GAX GGY TTX TGG AA-3′                       (e)   3′-GCY GTX TTZ TCZ AAC CAC AT-5′          
 
         [0047]    wherein X is C or T, Y is A, C, G or T, and Z is G or A.  
         [0048]    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.  
         [0049]    The amplified products were run on a 2% agarose gel and visualized with ethidium bromide staining.  
         [0050]    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.  
         [0051]    The amino acid sequence of human DEC-205 was determined to include the following amino acid sequences:  
                           (i)   TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQ           NCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNF           VLEQLLYFNYMASWVMLGITYRNNSL; and               (ii)   SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML.          
 
         [0052]    Determination of these sequences was fundamental to isolating the cDNA for human DEC-205 detailed below.  
         [0053]    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 aspartic acid   Asx   B           Cysteine   Cys   C           Glutamine   Gln   Q           Glutamic Acid   Glu   E           Glutamine or glutamic acid   Glx   Z           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           Typtophan   Trp   W           Tyrosine   Tyr   Y           Valine   Val   V           Unidentified       X                      
 
         [0054]    This code also applies to the predicted full sequence of FIG. 11, deduced from the cDNA encoding human DEC-205 isolated as described below.  
         [0055]    B. Polynucleotides Encoding Human DEC-205  
         [0056]    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.  
         [0057]    The polynucleotides of the invention specifically include those which include the nucleotides  
                           (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          
 
         [0058]    wherein X is T or G, as well as the full nucleotide sequence shown in FIG. 10, but are not limited thereto.  
         [0059]    The invention also includes within its scope functional equivalents of these polynucleotides.  
         [0060]    This aspect of the invention will now be illustrated by the following Examples.  
       EXAMPLE 2  
     Experimental Procedures  
       [0061]    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 Innere 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 i640, 10% fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin except that HDLM2 cells were with 20% fetal calf serum.  
         [0062]    Isolation of leukocytes—Leukocyte populations were isolated using standard laboratory procedures.  
         [0063]    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 et al (1995), above) and synthesized in house or by Life Technologies (Auckland, New Zealand) (see FIG. 2A). These primers were:  
         [0064]    DEC-a (5′-AAYATGCTNTGGAARTGGGT-3′),  
         [0065]    DEC-b (5′-TGRTGYTCRCAYTTCCACCA-3′),  
         [0066]    DEC-d (5′-GAYACNGAYGGNTTYTGGAA-3′) and  
         [0067]    DEC-e (5′-GCNGTYTTRTCRAACCACAT-3′),  
         [0068]    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 (Boehringer 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 FIGS. 2A and 2B) were cloned into pGEM-T vector (Promega), and sequenced manually using Sequenase DNA sequencing kit (Amersham Life Science, Auckland, New Zealand).  
         [0069]    A set of oligonucleotide primers nested within the DNA sequence of fragment 1 and 2 were synthesized (see FIG. 2A). These primers were:  
         [0070]    023 (5′-GCTCTAGAAACATGACCCATGAAGCC-3′ containing a XbaI site),  
         [0071]    028 (5′-GCTCTAGACATCGGCTCTTTCATITGT-3′ containing a XbaI site) and  
         [0072]    029 (5′-CGGGATTCACAGTTGATTGCAATGACA-3′ containing a EcoRI site)  
         [0073]    where incorporated restriction sites are underlined. Two ug of poly(A) RNA from L428 cells was reverse transcribed with 200 U of SuperScriptII (LifeTechnoloies) at 45° C. for 1 h using an oligo d(T) adaptor primer 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-HCl, pH 8.0, 1 mM EDTA (L428 cDNA pool). In order to isolate the fragment 3 (connecting the fragment 1 and 2) (see FIG. 2A), 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. U.S.A.  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. 2A). PCR was performed with 5 ul of L428 cDNA pool and the primer 029 and an adaptor primer 019 (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 II (Stratagene, La Jolla, Calif.). The representative clones from the fragment 3 (pB38f1) and 4 (pb30-3) were sequenced with a LI-COR automated sequencer (LI-COR, Lincoln, Nebr.) using SequiTherm cycle sequecing 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.  
         [0074]    An oligo dT-primed L428 cDNA library was prepared using ZAP Express cDNA Gigapack Cloning kit (Stratagene) according to manufacturer&#39;s 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, U.S.A.). 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 0.1×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.  
         [0075]    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 (5′-CGGGATCCCTCTGGCCGCGCACTAATGA-3′ containing a BamHI site) and 050 (5′-CCGCTCGAGCTGTGGATACCAGCACATGCCT-3′ containing a XhoI site) (see FIG. 2A). 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:  
         [0076]    IRD001 (5′-GATGGGAACTCTTATGGGAGACCT-3′ at nucleotide 523-555),  
         [0077]    IRD002 (5′-TGATGCAGGCTGGCTGCCAAATAA-3′ at nucleotide 1134-1157),  
         [0078]    IRD003 (5′-AACTGGGCAACTGTTGGTGGAAGA-3′ at nucleotide 1759-1782),  
         [0079]    IRD004 (5′-ATGGCGAAGAGGCTGGCATTTCTA-3′ at nucleotide 2334-2357),  
         [0080]    IRD005 (5′-CTCAAGCAAGCGATACCTGTCACT-3′ at nucleotide 2972-2995),  
         [0081]    IRD006 (5′-TGGGCAACTCGAAGACTGTGTAGT-3′ at nucleotide 3624-3647),  
         [0082]    IRD007 (5′-CACCAGCACAGCATTCTTGCTTGT-3′ at nucleotide 4168-4191) and  
         [0083]    IRD008 (5′-ATTTGTGAGCAGACTGATGAGGGA-3′ at nucleotide 4797-4820).  
         [0084]    The sequences of these primers were based on those of pb38f1 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.  
         [0085]    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 transfered 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 (5′-CGGAATTCGATCTCATGATAAGGCTGGTCACA-3′ containing a EcoRI site) (see FIG. 2A). 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.  
         [0086]    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.  
         [0087]    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. 2A). 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 immunofluorescent 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.  
         [0088]    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.  
         [0089]    Reverse transcription-PCR analysis—Total RNA from isolated leukocytes was incubated with RNase-free DNasel (Life Technologies), and was reverse transcribed using SuperscriptII with the oligo dT adaptor primer 018. PCR was performed using a pair of DEC-205 specific primers 060 (GTGGATCCAGTACAAGGGTCA at nucleotide 4655-4686) and 056 (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° 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., extention 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 (ATGGGGAAGGTGAAGGTCGGA-3′ at nucleotide 61-81), and 055 (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.  
         [0090]    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  
       [0091]    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. 2A and 2B). 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.  
         [0092]    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 (FIGS. 2A and 2C). A 3.2 kb 3′-RACE product (fragment 4) was obtained using primer 029 and an adaptor primer 019 (FIGS. 2A and 2C). The fragment 3 was cloned and several identical clones were identified by restriction enzyme map analysis (data not shown), and one of which, pb38f1, was fully sequenced: The DNA sequence of the fragment 3 (pB38f1) extending from the middle of cysteine-rich domain to the middle of CRD-8 (FIG. 2A), 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. 2A). The DNA sequences obtained from pb38f1 and pb30-3 overlapped by 320 bp, covering 95% of human DEC-205 coding region.  
         [0093]    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 (pBK14-1) was isolated, and the 1.5 kb insert of this clone was sequenced (FIG. 2A). The sequence was ˜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.  
         [0094]    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. 2A). The fragment 5 PCR product was sequenced directly using IRD 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.  
         [0095]    The deduced amino acid sequence of human DEC-205 showed 77% overall identity with the homologous mouse protein (FIG. 3A). 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. 3B), 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. 3B)) 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 BgIII, 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 BgIII-, 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.  
         [0096]    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.  
         [0097]    Fluorescent in situ hybridization then was used to map the DEC-205 gene in detail (FIGS. 6A and 6B). 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. 6A). 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. 6B). High resolution banding analysis provided a more precise location of signals (not shown). No additional site-specific signals were detected on any other chromosome.  
         [0098]    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 (FIGS. 7A and 7B). 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. 7B). Interestingly all Hodgkin&#39;s disease-derived cell lines tested express the transcripts. Semiquantitative RT-PCR studies also support these results (data not shown).  
         [0099]    C. Recombinant Expression of Human DEC-205  
         [0100]    In yet another aspect, the present invention relates to the recombinant expression of human DEC-205 or of its extracellular domain.  
         [0101]    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).  
         [0102]    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).  
         [0103]    Vectors useful in yeast and insect cells are available and well known. A suitable example of a yeast vector is the 2 μ plasmid.  
         [0104]    Suitable vectors for use in mammalian 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.  
         [0105]    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. Kaufmann 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 Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA 80 46544659 (1983); G. Urlaub and L. A. Chasin,  Proc. Natl. Acad. Sci. USA  77, 4216-4220, (1980).  
         [0106]    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 system, 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.  
         [0107]    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  W3 110 , 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.  
         [0108]    D. Ligands  
         [0109]    The invention also includes ligands that bind to human DEC-205 of the invention.  
         [0110]    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.  
         [0111]    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 495497 (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).  
         [0112]    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.  
         [0113]    In addition, ligands may be of two functional 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”).  
         [0114]    Both types of ligand will find application in either therapeutic or prophylactic treatments as described below.  
         [0115]    Example 3 describes the production of anti-DEC-205 antibodies.  
       EXAMPLE 3  
       [0116]    Production of Anti-DEC-205 Antibodies  
         [0117]    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 (aa1267-aa1277) and DEC-205-peptide 2 TEKEVKPVDSVKC (aa1227-aa1239) 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) (aa82-aa96). This is shown by FIG. 9.  
         [0118]    E. Constructs  
         [0119]    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.  
         [0120]    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.  
         [0121]    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.  
         [0122]    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.  
         [0123]    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).  
         [0124]    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 et al.,  Nature  354:520 (1991)).  
         [0125]    Administration can be via intravenous, intramuscular, subcutaneous, topical, oral intra nasal, rectal or intracerebroventricular routes, as appropriate.  
         [0126]    F. Applications  
         [0127]    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.  
         [0128]    Thus, the antagonistic ligands per se have potential application inter alia blocking or inhibiting the immune response during transplantation procedures.  
         [0129]    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.  
         [0130]    G. The Use of Human DEC-205 to Detect Dendritic Cells in Cell Suspensions on Tissues and to Purify Dendritic Cells  
         [0131]    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, Wash., U.S.A. (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).  
         [0132]    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:  
         [0133]    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.  
         [0134]    2. Following incubation, the mixture is introduced into a CellPro continuous-flow immunoadsorption 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.  
         [0135]    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.  
         [0136]    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.  
         [0137]    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.  
         [0138]    H. Functional Equivalents  
         [0139]    The invention includes functional equivalents of human DEC-205, extracellular domains and nucleic acid molecules described above.  
         [0140]    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.  
         [0141]    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:  
         [0142]    (a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);  
         [0143]    (b) Asn(N) Asp(D) Glu(E) Gln(Q);  
         [0144]    (c) His(H) Arg(R) Lys(K);  
         [0145]    (d) Met(M) Leu(L) Ile(I) Val(V); and  
         [0146]    (e) Phe(F) Tyr(Y) Trp(W).  
         [0147]    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.  
         [0148]    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 than 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  
         [0149]    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.  
         [0150]    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.  
     
       
       
         1 
         
           
             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 

agtgccatgc 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