WO1999028472A1 - A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN - Google Patents
A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN Download PDFInfo
- Publication number
- WO1999028472A1 WO1999028472A1 PCT/KR1998/000145 KR9800145W WO9928472A1 WO 1999028472 A1 WO1999028472 A1 WO 1999028472A1 KR 9800145 W KR9800145 W KR 9800145W WO 9928472 A1 WO9928472 A1 WO 9928472A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lkn
- recombinant
- ser
- leukotactin
- cdna
- Prior art date
Links
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 title claims abstract description 156
- 102100023703 C-C motif chemokine 15 Human genes 0.000 title claims abstract description 148
- 239000002299 complementary DNA Substances 0.000 title claims abstract description 53
- 210000004027 cell Anatomy 0.000 claims abstract description 64
- 150000001413 amino acids Chemical class 0.000 claims abstract description 51
- 210000000440 neutrophil Anatomy 0.000 claims abstract description 28
- 210000001616 monocyte Anatomy 0.000 claims abstract description 27
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 26
- 230000035605 chemotaxis Effects 0.000 claims abstract description 17
- 239000013604 expression vector Substances 0.000 claims abstract description 13
- 230000005757 colony formation Effects 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims abstract description 10
- 241000238631 Hexapoda Species 0.000 claims abstract description 9
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 8
- 239000011886 peripheral blood Substances 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 108010017319 CCR1 Receptors Proteins 0.000 claims abstract description 5
- 102000004499 CCR3 Receptors Human genes 0.000 claims abstract description 5
- 108010017316 CCR3 Receptors Proteins 0.000 claims abstract description 5
- 102000004500 CCR1 Receptors Human genes 0.000 claims abstract description 4
- 102000005962 receptors Human genes 0.000 claims description 19
- 108020003175 receptors Proteins 0.000 claims description 19
- 102000004890 Interleukin-8 Human genes 0.000 claims description 17
- 108090001007 Interleukin-8 Proteins 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- CWSIBTLMMQLPPZ-FXQIFTODSA-N Val-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N CWSIBTLMMQLPPZ-FXQIFTODSA-N 0.000 claims description 8
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 claims description 6
- WXASLRQUSYWVNE-FXQIFTODSA-N Asp-Cys-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N WXASLRQUSYWVNE-FXQIFTODSA-N 0.000 claims description 4
- YTJFXEDRUOQGSP-DCAQKATOSA-N Lys-Pro-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YTJFXEDRUOQGSP-DCAQKATOSA-N 0.000 claims description 4
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 claims description 4
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 claims description 4
- AAIUGNSRQDGCDC-ZLUOBGJFSA-N Asp-Cys-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N)C(=O)O AAIUGNSRQDGCDC-ZLUOBGJFSA-N 0.000 claims description 3
- WZJLBUPPZRZNTO-CIUDSAMLSA-N Cys-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N WZJLBUPPZRZNTO-CIUDSAMLSA-N 0.000 claims description 3
- 108010065920 Insulin Lispro Proteins 0.000 claims description 3
- DAYQSYGBCUKVKT-VOAKCMCISA-N Leu-Thr-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O DAYQSYGBCUKVKT-VOAKCMCISA-N 0.000 claims description 3
- HVAUKHLDSDDROB-KKUMJFAQSA-N Lys-Lys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HVAUKHLDSDDROB-KKUMJFAQSA-N 0.000 claims description 3
- MIROMRNASYKZNL-ULQDDVLXSA-N Lys-Pro-Tyr Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MIROMRNASYKZNL-ULQDDVLXSA-N 0.000 claims description 3
- XOFDBXYPKZUAAM-GUBZILKMSA-N Met-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)O)N XOFDBXYPKZUAAM-GUBZILKMSA-N 0.000 claims description 3
- BFYHIHGIHGROAT-HTUGSXCWSA-N Phe-Glu-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFYHIHGIHGROAT-HTUGSXCWSA-N 0.000 claims description 3
- WOJYIMBIKTWKJO-KKUMJFAQSA-N Ser-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CO)N WOJYIMBIKTWKJO-KKUMJFAQSA-N 0.000 claims description 3
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 3
- LHEZGZQRLDBSRR-WDCWCFNPSA-N Thr-Glu-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LHEZGZQRLDBSRR-WDCWCFNPSA-N 0.000 claims description 3
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 claims description 3
- 108010090333 leucyl-lysyl-proline Proteins 0.000 claims description 3
- 108010091871 leucylmethionine Proteins 0.000 claims description 3
- 108010054155 lysyllysine Proteins 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- GJLXVWOMRRWCIB-MERZOTPQSA-N (2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-5-(diaminomethylideneamino)pentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanamide Chemical compound C([C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=C(O)C=C1 GJLXVWOMRRWCIB-MERZOTPQSA-N 0.000 claims description 2
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 claims description 2
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 claims description 2
- VTBGVPWSWJBERH-DCAQKATOSA-N Cys-Leu-Met Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CS)N VTBGVPWSWJBERH-DCAQKATOSA-N 0.000 claims description 2
- YVKSMSDXKMSIRX-GUBZILKMSA-N Leu-Glu-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O YVKSMSDXKMSIRX-GUBZILKMSA-N 0.000 claims description 2
- IBSGMIPRBMPMHE-IHRRRGAJSA-N Leu-Met-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O IBSGMIPRBMPMHE-IHRRRGAJSA-N 0.000 claims description 2
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 claims description 2
- YPLVCBKEPJPBDQ-MELADBBJSA-N Lys-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N YPLVCBKEPJPBDQ-MELADBBJSA-N 0.000 claims description 2
- WTHGNAAQXISJHP-AVGNSLFASA-N Met-Lys-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O WTHGNAAQXISJHP-AVGNSLFASA-N 0.000 claims description 2
- BJEYSVHMGIJORT-NHCYSSNCSA-N Phe-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BJEYSVHMGIJORT-NHCYSSNCSA-N 0.000 claims description 2
- JHSRGEODDALISP-XVSYOHENSA-N Phe-Thr-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O JHSRGEODDALISP-XVSYOHENSA-N 0.000 claims description 2
- HQVPQXMCQKXARZ-FXQIFTODSA-N Pro-Cys-Ser Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O HQVPQXMCQKXARZ-FXQIFTODSA-N 0.000 claims description 2
- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 claims description 2
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 claims description 2
- 108010077245 asparaginyl-proline Proteins 0.000 claims description 2
- 108010090894 prolylleucine Proteins 0.000 claims description 2
- 108010048397 seryl-lysyl-leucine Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 4
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 claims 4
- 229940096397 interleukin-8 Drugs 0.000 claims 4
- 238000000034 method Methods 0.000 claims 4
- 230000001131 transforming effect Effects 0.000 claims 4
- 101100496569 Homo sapiens C6 gene Proteins 0.000 claims 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 claims 1
- 108010040471 CC Chemokines Proteins 0.000 abstract description 19
- 102000001902 CC Chemokines Human genes 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 108010076504 Protein Sorting Signals Proteins 0.000 abstract description 8
- 210000000265 leukocyte Anatomy 0.000 abstract description 7
- 208000031886 HIV Infections Diseases 0.000 abstract description 5
- 108700026244 Open Reading Frames Proteins 0.000 abstract description 5
- 210000001185 bone marrow Anatomy 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 210000000130 stem cell Anatomy 0.000 abstract description 5
- 238000002512 chemotherapy Methods 0.000 abstract description 4
- 238000001959 radiotherapy Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 230000016784 immunoglobulin production Effects 0.000 abstract description 3
- 208000032839 leukemia Diseases 0.000 abstract description 3
- 235000001014 amino acid Nutrition 0.000 description 31
- 108020004414 DNA Proteins 0.000 description 30
- 239000012634 fragment Substances 0.000 description 22
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 102100031172 C-C chemokine receptor type 1 Human genes 0.000 description 12
- 101710149814 C-C chemokine receptor type 1 Proteins 0.000 description 12
- 230000000638 stimulation Effects 0.000 description 12
- 238000010240 RT-PCR analysis Methods 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 102100024167 C-C chemokine receptor type 3 Human genes 0.000 description 9
- 101710149862 C-C chemokine receptor type 3 Proteins 0.000 description 9
- 102000019034 Chemokines Human genes 0.000 description 9
- 108010012236 Chemokines Proteins 0.000 description 9
- 101710139422 Eotaxin Proteins 0.000 description 9
- 102100023688 Eotaxin Human genes 0.000 description 9
- 230000003185 calcium uptake Effects 0.000 description 9
- 239000000523 sample Substances 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 239000000556 agonist Substances 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 241001529936 Murinae Species 0.000 description 6
- 239000011543 agarose gel Substances 0.000 description 6
- 210000002798 bone marrow cell Anatomy 0.000 description 6
- 210000002360 granulocyte-macrophage progenitor cell Anatomy 0.000 description 6
- 210000005260 human cell Anatomy 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 5
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 102000004388 Interleukin-4 Human genes 0.000 description 5
- 108090000978 Interleukin-4 Proteins 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- 229940028885 interleukin-4 Drugs 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 108010051242 phenylalanylserine Proteins 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 102100037853 C-C chemokine receptor type 4 Human genes 0.000 description 3
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 210000002960 bfu-e Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010081551 glycylphenylalanine Proteins 0.000 description 3
- 108010037850 glycylvaline Proteins 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- ZAHQPTJLOCWVPG-UHFFFAOYSA-N mitoxantrone dihydrochloride Chemical compound Cl.Cl.O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO ZAHQPTJLOCWVPG-UHFFFAOYSA-N 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- WZFUQSJFWNHZHM-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 WZFUQSJFWNHZHM-UHFFFAOYSA-N 0.000 description 2
- RCQRKPUXJAGEEC-ZLUOBGJFSA-N Ala-Cys-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O RCQRKPUXJAGEEC-ZLUOBGJFSA-N 0.000 description 2
- OSASDIVHOSJVII-WDSKDSINSA-N Arg-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCNC(N)=N OSASDIVHOSJVII-WDSKDSINSA-N 0.000 description 2
- FAUPLTGRUBTXNU-FXQIFTODSA-N Asp-Pro-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O FAUPLTGRUBTXNU-FXQIFTODSA-N 0.000 description 2
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 description 2
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 description 2
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 2
- 102100036305 C-C chemokine receptor type 8 Human genes 0.000 description 2
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 description 2
- 108091008927 CC chemokine receptors Proteins 0.000 description 2
- 102000005674 CCR Receptors Human genes 0.000 description 2
- QJUDRFBUWAGUSG-SRVKXCTJSA-N Cys-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N QJUDRFBUWAGUSG-SRVKXCTJSA-N 0.000 description 2
- 108010092408 Eosinophil Peroxidase Proteins 0.000 description 2
- 102100031939 Erythropoietin Human genes 0.000 description 2
- IDOGEHIWMJMAHT-BYPYZUCNSA-N Gly-Gly-Cys Chemical compound NCC(=O)NCC(=O)N[C@@H](CS)C(O)=O IDOGEHIWMJMAHT-BYPYZUCNSA-N 0.000 description 2
- JBCLFWXMTIKCCB-VIFPVBQESA-N Gly-Phe Chemical compound NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-VIFPVBQESA-N 0.000 description 2
- NSVOVKWEKGEOQB-LURJTMIESA-N Gly-Pro-Gly Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NCC(O)=O NSVOVKWEKGEOQB-LURJTMIESA-N 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 description 2
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 2
- 101000716063 Homo sapiens C-C chemokine receptor type 8 Proteins 0.000 description 2
- 101100402552 Homo sapiens MARCKSL1 gene Proteins 0.000 description 2
- YUAXTFMFMOIMAM-QWRGUYRKSA-N Lys-Lys-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O YUAXTFMFMOIMAM-QWRGUYRKSA-N 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- 230000004988 N-glycosylation Effects 0.000 description 2
- CMHTUJQZQXFNTQ-OEAJRASXSA-N Phe-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CC=CC=C1)N)O CMHTUJQZQXFNTQ-OEAJRASXSA-N 0.000 description 2
- RMJZWERKFFNNNS-XGEHTFHBSA-N Pro-Thr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O RMJZWERKFFNNNS-XGEHTFHBSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- NNFMANHDYSVNIO-DCAQKATOSA-N Ser-Lys-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NNFMANHDYSVNIO-DCAQKATOSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- TZJSEJOXAIWOST-RHYQMDGZSA-N Thr-Lys-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N TZJSEJOXAIWOST-RHYQMDGZSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000003399 chemotactic effect Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000012847 fine chemical Substances 0.000 description 2
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000003917 human chromosome Anatomy 0.000 description 2
- -1 i.e. Proteins 0.000 description 2
- 102000010681 interleukin-8 receptors Human genes 0.000 description 2
- 108010038415 interleukin-8 receptors Proteins 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000004897 n-terminal region Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WQVYAWIMAWTGMW-ZLUOBGJFSA-N Ala-Asp-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N WQVYAWIMAWTGMW-ZLUOBGJFSA-N 0.000 description 1
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 1
- KUDREHRZRIVKHS-UWJYBYFXSA-N Ala-Asp-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KUDREHRZRIVKHS-UWJYBYFXSA-N 0.000 description 1
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- BHTBAVZSZCQZPT-GUBZILKMSA-N Ala-Pro-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N BHTBAVZSZCQZPT-GUBZILKMSA-N 0.000 description 1
- CQJHFKKGZXKZBC-BPNCWPANSA-N Ala-Pro-Tyr Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CQJHFKKGZXKZBC-BPNCWPANSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- IGULQRCJLQQPSM-DCAQKATOSA-N Arg-Cys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IGULQRCJLQQPSM-DCAQKATOSA-N 0.000 description 1
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 1
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 1
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 1
- OMSMPWHEGLNQOD-UWVGGRQHSA-N Asn-Phe Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OMSMPWHEGLNQOD-UWVGGRQHSA-N 0.000 description 1
- GMUOCGCDOYYWPD-FXQIFTODSA-N Asn-Pro-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O GMUOCGCDOYYWPD-FXQIFTODSA-N 0.000 description 1
- SONUFGRSSMFHFN-IMJSIDKUSA-N Asn-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O SONUFGRSSMFHFN-IMJSIDKUSA-N 0.000 description 1
- GVPSCJQLUGIKAM-GUBZILKMSA-N Asp-Arg-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GVPSCJQLUGIKAM-GUBZILKMSA-N 0.000 description 1
- MGSVBZIBCCKGCY-ZLUOBGJFSA-N Asp-Ser-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MGSVBZIBCCKGCY-ZLUOBGJFSA-N 0.000 description 1
- GCACQYDBDHRVGE-LKXGYXEUSA-N Asp-Thr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC(O)=O GCACQYDBDHRVGE-LKXGYXEUSA-N 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 102000018211 CC chemokine receptor 1 Human genes 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- 108050006947 CXC Chemokine Proteins 0.000 description 1
- 108091008928 CXC chemokine receptors Proteins 0.000 description 1
- 102000054900 CXCR Receptors Human genes 0.000 description 1
- 101100512078 Caenorhabditis elegans lys-1 gene Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- QFMCHXSGIZPBKG-ZLUOBGJFSA-N Cys-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CS)N QFMCHXSGIZPBKG-ZLUOBGJFSA-N 0.000 description 1
- BCFXQBXXDSEHRS-FXQIFTODSA-N Cys-Ser-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BCFXQBXXDSEHRS-FXQIFTODSA-N 0.000 description 1
- GGRDJANMZPGMNS-CIUDSAMLSA-N Cys-Ser-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O GGRDJANMZPGMNS-CIUDSAMLSA-N 0.000 description 1
- WTXCNOPZMQRTNN-BWBBJGPYSA-N Cys-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CS)N)O WTXCNOPZMQRTNN-BWBBJGPYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- MPZWMIIOPAPAKE-BQBZGAKWSA-N Glu-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N MPZWMIIOPAPAKE-BQBZGAKWSA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- LXAUHIRMWXQRKI-XHNCKOQMSA-N Glu-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O LXAUHIRMWXQRKI-XHNCKOQMSA-N 0.000 description 1
- QPRZKNOOOBWXSU-CIUDSAMLSA-N Glu-Asp-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N QPRZKNOOOBWXSU-CIUDSAMLSA-N 0.000 description 1
- XEKAJTCACGEBOK-KKUMJFAQSA-N Glu-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N XEKAJTCACGEBOK-KKUMJFAQSA-N 0.000 description 1
- MXJYXYDREQWUMS-XKBZYTNZSA-N Glu-Thr-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O MXJYXYDREQWUMS-XKBZYTNZSA-N 0.000 description 1
- MIWJDJAMMKHUAR-ZVZYQTTQSA-N Glu-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N MIWJDJAMMKHUAR-ZVZYQTTQSA-N 0.000 description 1
- YSWHPLCDIMUKFE-QWRGUYRKSA-N Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 YSWHPLCDIMUKFE-QWRGUYRKSA-N 0.000 description 1
- QRWPTXLWHHTOCO-DZKIICNBSA-N Glu-Val-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QRWPTXLWHHTOCO-DZKIICNBSA-N 0.000 description 1
- RLFSBAPJTYKSLG-WHFBIAKZSA-N Gly-Ala-Asp Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O RLFSBAPJTYKSLG-WHFBIAKZSA-N 0.000 description 1
- DKEXFJVMVGETOO-LURJTMIESA-N Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CN DKEXFJVMVGETOO-LURJTMIESA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-N 0.000 description 1
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 1
- OCRQUYDOYKCOQG-IRXDYDNUSA-N Gly-Tyr-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 OCRQUYDOYKCOQG-IRXDYDNUSA-N 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 1
- 101000716729 Homo sapiens Kit ligand Proteins 0.000 description 1
- 101000668058 Infectious salmon anemia virus (isolate Atlantic salmon/Norway/810/9/99) RNA-directed RNA polymerase catalytic subunit Proteins 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- KWLWZYMNUZJKMZ-IHRRRGAJSA-N Leu-Pro-Leu Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O KWLWZYMNUZJKMZ-IHRRRGAJSA-N 0.000 description 1
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 1
- VVURYEVJJTXWNE-ULQDDVLXSA-N Lys-Tyr-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O VVURYEVJJTXWNE-ULQDDVLXSA-N 0.000 description 1
- VZBXCMCHIHEPBL-SRVKXCTJSA-N Met-Glu-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN VZBXCMCHIHEPBL-SRVKXCTJSA-N 0.000 description 1
- CGUYGMFQZCYJSG-DCAQKATOSA-N Met-Lys-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CGUYGMFQZCYJSG-DCAQKATOSA-N 0.000 description 1
- 102100035971 Molybdopterin molybdenumtransferase Human genes 0.000 description 1
- 101710119577 Molybdopterin molybdenumtransferase Proteins 0.000 description 1
- 101000946797 Mus musculus C-C motif chemokine 9 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- OOLOTUZJUBOMAX-GUBZILKMSA-N Pro-Ala-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O OOLOTUZJUBOMAX-GUBZILKMSA-N 0.000 description 1
- HMNSRTLZAJHSIK-YUMQZZPRSA-N Pro-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 HMNSRTLZAJHSIK-YUMQZZPRSA-N 0.000 description 1
- OCSACVPBMIYNJE-GUBZILKMSA-N Pro-Arg-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O OCSACVPBMIYNJE-GUBZILKMSA-N 0.000 description 1
- ZSKJPKFTPQCPIH-RCWTZXSCSA-N Pro-Arg-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSKJPKFTPQCPIH-RCWTZXSCSA-N 0.000 description 1
- OLTFZQIYCNOBLI-DCAQKATOSA-N Pro-Cys-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O OLTFZQIYCNOBLI-DCAQKATOSA-N 0.000 description 1
- RWCOTTLHDJWHRS-YUMQZZPRSA-N Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RWCOTTLHDJWHRS-YUMQZZPRSA-N 0.000 description 1
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- KIDXAAQVMNLJFQ-KZVJFYERSA-N Pro-Thr-Ala Chemical compound C[C@@H](O)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](C)C(O)=O KIDXAAQVMNLJFQ-KZVJFYERSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 1
- BGOWRLSWJCVYAQ-CIUDSAMLSA-N Ser-Asp-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BGOWRLSWJCVYAQ-CIUDSAMLSA-N 0.000 description 1
- BPMRXBZYPGYPJN-WHFBIAKZSA-N Ser-Gly-Asn Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O BPMRXBZYPGYPJN-WHFBIAKZSA-N 0.000 description 1
- IAORETPTUDBBGV-CIUDSAMLSA-N Ser-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N IAORETPTUDBBGV-CIUDSAMLSA-N 0.000 description 1
- GVMUJUPXFQFBBZ-GUBZILKMSA-N Ser-Lys-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GVMUJUPXFQFBBZ-GUBZILKMSA-N 0.000 description 1
- PTWIYDNFWPXQSD-GARJFASQSA-N Ser-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N)C(=O)O PTWIYDNFWPXQSD-GARJFASQSA-N 0.000 description 1
- XZKQVQKUZMAADP-IMJSIDKUSA-N Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(O)=O XZKQVQKUZMAADP-IMJSIDKUSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 1
- HAUVENOGHPECML-BPUTZDHNSA-N Ser-Trp-Val Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CO)=CNC2=C1 HAUVENOGHPECML-BPUTZDHNSA-N 0.000 description 1
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 1
- GZYNMZQXFRWDFH-YTWAJWBKSA-N Thr-Arg-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O GZYNMZQXFRWDFH-YTWAJWBKSA-N 0.000 description 1
- NLSNVZAREYQMGR-HJGDQZAQSA-N Thr-Asp-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NLSNVZAREYQMGR-HJGDQZAQSA-N 0.000 description 1
- XOTBWOCSLMBGMF-SUSMZKCASA-N Thr-Glu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOTBWOCSLMBGMF-SUSMZKCASA-N 0.000 description 1
- SIMKLINEDYOTKL-MBLNEYKQSA-N Thr-His-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C)C(=O)O)N)O SIMKLINEDYOTKL-MBLNEYKQSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- LWFWZRANSFAJDR-JSGCOSHPSA-N Trp-Val Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O)=CNC2=C1 LWFWZRANSFAJDR-JSGCOSHPSA-N 0.000 description 1
- RCLOWEZASFJFEX-KKUMJFAQSA-N Tyr-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RCLOWEZASFJFEX-KKUMJFAQSA-N 0.000 description 1
- KOVXHANYYYMBRF-IRIUXVKKSA-N Tyr-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KOVXHANYYYMBRF-IRIUXVKKSA-N 0.000 description 1
- LRHBBGDMBLFYGL-FHWLQOOXSA-N Tyr-Phe-Glu Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LRHBBGDMBLFYGL-FHWLQOOXSA-N 0.000 description 1
- RWOKVQUCENPXGE-IHRRRGAJSA-N Tyr-Ser-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RWOKVQUCENPXGE-IHRRRGAJSA-N 0.000 description 1
- XUIOBCQESNDTDE-FQPOAREZSA-N Tyr-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O XUIOBCQESNDTDE-FQPOAREZSA-N 0.000 description 1
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 1
- QVYFTFIBKCDHIE-ACRUOGEOSA-N Tyr-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCCN)C(=O)O)N)O QVYFTFIBKCDHIE-ACRUOGEOSA-N 0.000 description 1
- TYGHOWWWMTWVKM-HJOGWXRNSA-N Tyr-Tyr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 TYGHOWWWMTWVKM-HJOGWXRNSA-N 0.000 description 1
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 1
- ZLNYBMWGPOKSLW-LSJOCFKGSA-N Val-Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLNYBMWGPOKSLW-LSJOCFKGSA-N 0.000 description 1
- HEYSFDAMRDTCJM-UUOKFMHZSA-N [(2r,3s,4r,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methyl phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H]1O HEYSFDAMRDTCJM-UUOKFMHZSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 108010045023 alanyl-prolyl-tyrosine Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000009460 calcium influx Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010004073 cysteinylcysteine Proteins 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 108010073628 glutamyl-valyl-phenylalanine Proteins 0.000 description 1
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 102000044890 human EPO Human genes 0.000 description 1
- 102000055151 human KITLG Human genes 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- DZNKOAWEHDKBEP-UHFFFAOYSA-N methyl 2-[6-[bis(2-methoxy-2-oxoethyl)amino]-5-[2-[2-[bis(2-methoxy-2-oxoethyl)amino]-5-methylphenoxy]ethoxy]-1-benzofuran-2-yl]-1,3-oxazole-5-carboxylate Chemical compound COC(=O)CN(CC(=O)OC)C1=CC=C(C)C=C1OCCOC(C(=C1)N(CC(=O)OC)CC(=O)OC)=CC2=C1OC(C=1OC(=CN=1)C(=O)OC)=C2 DZNKOAWEHDKBEP-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 108010066052 multidrug resistance-associated protein 1 Proteins 0.000 description 1
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 1
- 230000003039 myelosuppressive effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010077037 tyrosyl-tyrosyl-phenylalanine Proteins 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
- C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
Definitions
- PCR reverse transcriptase-polymerase chain reaction
- Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of a Lkn-1 cDNA and an amino acid sequence deduced therefrom (SEQ ID NO: 4) of a Lkn-1 cDNA and an amino acid sequence deduced therefrom (SEQ ID NO: 4)
- Lkn-1 PCR primers permitting amplification of a lOObp-DNA fragment from the Lkn-1 exon sequence confirmed as above were prepared, and RT-PCR was carried out using total RNAs isolated from various human cell lines as a template, respectively, to select cell lines having Lkn-1 mRNA.
- a human monocytic THP-1 cell line activated by interleukin-4 (IL-4) was chosen and its cDNA library was prepared.
- a nucleotide sequence (SEQ ID N0:1) of the said DNA fragment of 202bp was determined to find intron and a part of exon translated into amino acids (see : Figure 1(A)) .
- the amino acid sequence (SEQ ID NO: 2) of the part of exon showed homology of 50% to that of mMRP-2 from the 87th to 110th amino acid and the second cysteine (indicated as "*") of two additional cysteines common inmMRP-1 andmMRP-2 was conserved inLkn-1 (see : Figure 1 (B) ) .
- boxes showed amino acids conserved in Lkn-1 and mMRP-2.
- lanes 1 to 4 show results of PCR using the 202bp-DNA fragment as a positive control, total RNA of the THP-1 cell line, total RNA of the U937 cell line and total RNA of the HL-60 cell line, respectively; and, lane M shows lOObp-ladder as a DNA size-marker.
- the THP-1 cell line activated by IL-4 produced Lkn-1 mRNA.
- Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of the cDNA clone obtained in Example 2 and an amino acid sequence deduced therefrom (SEQ ID NO: 5) .
- the underlined is a signal peptide; the sequence in a box is the amino acid sequence (SEQ ID N0:2) of Lkn-1 shown in Figure 1(B) which was used as a probe during the screening of THP-1 cDNA library; and, (***) indicates a translation stop codon.
- Figure 4 shows comparison of an amino acid sequence of mature Lkn-1 (SEQ ID NO: 7) with those of mMRP-1 (SEQ ID NO: 8) , mMRP-2 (SEQ ID N0:9) , hMIP-l ⁇ (SEQ ID NO : 10) (see : Youn, B.S. et al., J. Immunol., 155:2661(1995)), mMIP-l ⁇ (SEQ ID NO: 11) (see : Kwon, B.S. and Weissman, S.M., Proc . Natl . Acad.
- Lkn-2 belongs to C6 ⁇ -chemokine family having two cysteine residues further as in mMRP-1 andmMRP-2 besides four cysteine residues common in ⁇ -chemokine family. Moreover, it was found that the amino acid sequence (SEQ ID NO: 7) of mature Lkn-1 shows homology of only about 43% to that of mMRP-1 (SEQ ID NO: 8) or mMRP-2 (SEQ ID NO: 9) and homology of 40% to that of hMIP-l ⁇ (SEQ ID NO:10) although Lkn-1 was isolated as a human counterpart of mMRP-2.
- Example 4-1 Expression of a recombinant Lkn-1 in E. coli
- the DNA fragment thus amplified was digested with Ndel/Xhol and cloned into a plasmid pET21a (Novagen, USA).
- the recombinant plasmid thus constructed was designated as pET21a-Lkn-l and introduced into E. coli XL-1 Blue.
- the recombinant plasmid pET21a-Lkn-l permits expression of a recombinant Lkn-1 which has one additional methionine residue in N-terminus and six additional histidines at C-terminus of a mature Lkn-1.
- a Sf-21 insect cell was transfected with both the said recombinant plasmid and AcNPV (Autographa California nuclear polyhedrosis baculovirus) to transfer the Lkn-1 cDNA in the said recombinant plasmid to the AcNPV.
- AcNPV-Lkn-1 viral plaque was isolated based on the characteristics that the virion is occlusion-negative, and grown in the Sf-21 insect cell in a serum-free Ex-Cell 400 medium (JRH Biosciences, USA) for later use.
- peripheral blood mononuclear cells PBMC
- monocytes were isolated from the PBMC thus obtained based on their attached activity onto the surface of plastic, and the said isolation step was repeated twice.
- cells remained after the step for the monocyte isolation were obtained as lymphocytes. Purity of the monocytes and lymphocytes thus obtained was determined by microscopic examination of cytospin dyed with Diff- Quick (Baxter Scientific, USA). As a result, it was found that purity of the monocytes and lymphocytes was 90% and 88%, respectively .
- level of cell migration in a 48-well microchamber was determined as followings.
- Lower wells of the microchamber were filled with only a buffer solution (control) or a buffer containing the recombinant Lkn-1, hMIP-l ⁇ (PeproTech. , USA), RANTES (PeproTech. , USA), IL-8 (PeproTech. , USA) or eotaxin (PeproTech. , USA) , and upper wells were filled with 50 ⁇ l of the said cell suspensions.
- a well was partitioned by a suitable filter without polyvinylpyrolidine to give a lower well and an upper well.
- Lkn-1 may bind to a receptor for activation of lymphocytes, monocytes and neutrophils to induce calcium efflux. Its competitive relationships with other agonists against the receptor was also examined. Receptor activation was determined by measuring a change in [Ca 2+ ] in subsets (lymphocytes, monocytes and neutrophils) of peripheral blood leukocytes isolated using a MSIII fluorimeter (Photon Technology Internationl , USA) .
- TELEPHONE 02-584-0131
- TITLE OF INVENTION A PROCESS FOR PREPARING A cDNA ENCODINGAC6 ⁇ -CHEMOKINE LEUKOTACTIN-1 (Lkn-1) ISOLATED FROM HUMAN
- MOLECULE TYPE protein
Abstract
The present invention relates to a cDNA coding for a novel protein which belongs to C6 β-chemokines and attracts subsets of peripheral blood leukocytes, and a process for preparing the said protein by employing expression vector therefor. Open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and molecular weight of mature protein consisting of 92 amino acids among them is supposed to be 10,162 dalton. A recombinant Lkn-1 which was expressed in E.coli or insect cell employing the said Lkn-1 cDNA and purified, inhibited colony formation significantly, attracted neutrophils, monocytes and lymphocytes to cause chemotaxis, and bound to CCR1 and CCR3 receptors. Accordingly, it was determined that the recombinant Lkn-1 protein can be used as a potential drug for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.
Description
A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1 (Lkn-1)
ISOLATED FROM HUMAN
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a cDNA encoding a novel C6 β-chemokine isolated from human body and a process for preparing the C6 β-chemokine, more specifically, to a cDNA coding for a novel protein which belongs to C6 β-chemokines and attracts subsets of peripheral blood leukocytes, and a process for preparing the said protein by employing expression vector therefor.
Description of the Prior Art
Chemokines are a family of small cytokines consisting of basic proteins of a low molecular weight, have four cysteines commonly, which are classified into four subfamilies of CXC(α) , CC(β) , C(γ) and CX3C depending on the position of the first and the second cysteines, i.e., whether they lie adjacent or an amino acid intervenes between the two cysteines (see : Baggiolini, M. and Dahinden, C.A., Immunol. Today, 15:127(1994); Kelner, S.G. et al . , Science, 266:1395 (1994) ; Bazan, J.F. et al . , Nature, 385:640 (1997) ) . Genes of chemokine subfamilies locate on a same chromosome in a cluster, for example, α -chemokine genes locate on the human chromosome 4ql2-21 and β-chemokine genes exist on the human chromosome 17qll-32 and the mouse chromosome 11.
Chemokines have biological activities such as HIV- inhibitory action, immunoregulatory action, leukocyte migration and inhibitory action against division of hematopoietic stem cells (see : Cocchi, F. et al . , Science,
270:1811(1995); olpe, S.D. et al . , J. Exp . Med . , 167:570(1988); Graham, G.J. et al . , Nature, 344:442(1990); Broxmeyer, H.E. et al., Blood, 76:1110(1990); Youn, B.-S. et al . , J. Immunol., 155:2661-2667(1995)). Also, chemokines bind to transmembrane domain G protein-coupled receptors to activate leukocytes and some of the receptors are also used as coreceptors during HIV-1 infection (see : Oh, K.-O. et al . , J. Immunol., 147:2978(1991); Alkabatih, G. et al . , Science, 272:1955(1996)). For example, among 8 subtypes of β- chemokine(CC chemokine) receptor, i.e., CCR1, CCR2 , CCR3 , CCR4 , CCR5 , CCR6 , CCR7 and CCR8 , four subtypes of CCR4 , CCR6 , CCR7 and CCR8 show a high affinity to one substance while CCR1, CCR2 , CCR3 and CCR5 have a binding affinity to various chemokines .
So far, nine chemokines belonging to β-chemokine subfamily have been found (see : Wilson, S.D. et al . , J. Exp. Med., 171:1301(1990); Modi, W.S. et al . , Hum. Genet., 84:185(1990)). Among them, a murine MRP-1 ( "mMRP-1" , MIP (macrophage inflammatory protein) -related protein-1 or CIO) (see: Orlofsky, A. et al . , Cell Regul . , 2:403 (1991) ) and a murine MRP-2 ("mMRP-2" ) (see: Youn, B.-S. et al . , J. Immunol. ,
155:2661(1995)) are distinguished from the rest of β- chemokines in that they have two extra cysteine residues, thereby forming the third disulfide bond and their N-terminal regions are very long. Based on the findings, they are classified into C6 β -chemokines .
Under the circumstances, there are strong reasons for exploring and developing novel human MRPs, since human MRPs may be employed as a potential drug for the treatment of HIV-1 infection or for the protection of bone marrow stem cells in the course of chemotherapy or radiotherapy.
SUMMARY OF THE INVENTION
The present inventors have made an effort to isolate MRP genes from various human cell lines. As a result, the inventors isolated a novel cDNA of MRP belonging to C6 β -chemokines from a human monocytic cell line and determined its nucleotide sequence and amino acid sequence deduced therefrom. Further, they successfully expressed the said MRP cDNA in E. coli or insect cell and discovered that the expressed recombinant protein attracts subsets of peripheral blood leukocytes (lymphocyte, monocyte and neutrophil) in chemotactic fashion. Hereinafter, the said MRP cDNA isolated from human body and the recombinant MRP are referred to as "Lkn-1 (leukotactin-1) cDNA" and "recombinant Lkn-1", respectively.
A primary object of the invention is, therefore, to provide a novel cDNA encoding C6 β-chemokine (Lkn-1) and an amino acid sequence deduced therefrom. The other object of the invention is to provide an expression vector comprising the said Lkn-1 cDNA and a recombinant microorganism transformed with the vector.
Another object of the invention is to provide a process for preparing a recombinant Lkn-1 from the said microorganism.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:
Figure 1(A) shows a nucleotide sequence(SEQ ID N0:1) of a DNA fragment of 202bp and an amino acid sequence deduced therefrom (SEQ
ID NO : 2 ) .
Figure 1 (B) shows a comparison of the amino acid sequence (SEQ ID NO: 2) of Lkn-1 with the 87th to 110th amino acid sequence (SEQ ID NO: 3) of mMRP-2.
Figure 2 is a photograph showing a result of RT-
PCR (reverse transcriptase-polymerase chain reaction) of total RNA isolated from various human cell lines.
Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of a Lkn-1 cDNA and an amino acid sequence deduced therefrom (SEQ ID
NO : 5 ) .
Figure 4 shows a comparison of an amino acid sequence of mature Lkn-1(SEQ ID NO: 7) with those of mMRP-l(SEQ ID NO: 8), mMRP-2 (SEQ ID
NO:9), hMIP-lα(SEQ ID NO: 10), mMIP- lα (SEQ ID NO:ll) and hMIP-lβ (SEQ ID NO: 12) .
Figure 5 (A) is a graph which shows a chemotaxis attracting lymphocytes by a recombinant Lkn-1 and RANTES (regulated on activation, normal T cell expressed and secreted) .
Figure 5 (B) is a graph which shows a chemotaxis attracting monocytes by a recombinant Lkn-1 and hMIP-l .
Figure 5 (C) is a graph which shows a chemotaxis attracting neutrophils by a
recombinant Lkn-1 and IL-8.
Figure 6 (A) is a graph showing relative fluorescence measured in lymphocytes which are stimulated by a series of addition of
RANTES and a recombinant Lkn-1.
Figure 6 (B) is a graph showing relative fluorescence measured in lymphocytes which are stimulated by a series of addition of a recombinant kn-1 and RANTES.
Figure 6 (C) is a graph showing relative fluorescence measured in monocytes which are stimulated by a series of addition of hMIP-lα and a recombinant Lkn-1.
Figure 6 (D) is a graph showing relative fluorescence measured in monocytes which are stimulated by a series of addition of a recombinant Lkn-1 and hMIP-l .
Figure 6 (E) is a graph showing relative fluorescence measured in neutrophils which are stimulated by a series of addition of
IL-8 and a recombinant Lkn-1.
Figure 6(F) is a graph showing relative fluorescence measured in neutrophils which are stimulated by a series of addition of a recombinant Lkn-1 and IL-8.
Figure 7 (A) is a graph showing relative fluorescence measured in a HOS cell line expressing CCR1 which are stimulated by a series of addition of various agents.
Figure 7 (B) is a graph showing relative fluorescence measured in a HOS cell line expressing CCR3 which are stimulated by a series of addition of various agents.
Figure 7 (C) is a graph showing peak amplitude of calcium responses depending on concentrations of Lkn-1 and hMIP-lα.
Figure 7 (D) is a graph showing peak amplitude of calcium responses depending on concentrations of Lkn-1, eotaxin and RANTES.
DETAILED DESCRIPTION OF THE INVENTION
To isolate the Lkn-1 gene from a human cell line, the present inventors first cloned an exon of the Lkn-1 gene, which may be used for the preparation of a probe. That is, human genomic DNA was digested with Hind III, separated on an agarose gel, and analyzed with southern blot by employing the 32P-labelled mMRP-2 cDNA(see: Youn, B.S. et al . , J. Immunol., 155:2661(1995)) as a probe to obtain a DNA fragment of 7. Okb which can be hybridized with mMRP-2. Then, to isolate an exon sequence of Lkn-1 gDNA from the Hindlll- digested DNA fragment of 7. Okb, human genomic DNAwas digested with HindiII completely, and separated on an agarose gel to isolate a DNA fragment of 7. Okb. The fragment thus obtained was inserted into a vector, and the vector thus obtained was introduced into a host cell. After transformation, a colony showing hybridization with the said mMRP-2 cDNA probe was selected. After a DNA fragment of 7. Okb was isolated from the colony and digested with Alul , a DNA fragment of 202bp which can be hybridized with mMRP-2 cDNA was cloned. And then, the nucleotide sequence of the fragment was determined, which differentiated parts of intron and exon from the DNA fragment .
Then, in order to clone a novel Lkn-1 cDNA from a human cell line, Lkn-1 PCR primers permitting amplification of a
lOObp-DNA fragment from the Lkn-1 exon sequence confirmed as above were prepared, and RT-PCR was carried out using total RNAs isolated from various human cell lines as a template, respectively, to select cell lines having Lkn-1 mRNA. One of the cell lines thus selected, a human monocytic THP-1 cell line activated by interleukin-4 (IL-4) was chosen and its cDNA library was prepared. On the other hand, RT-PCR was carried out using total RNA of the said THP-1 cell line as a template and the said Lkn-1 PCR primer to prepare a lOObp-DNA fragment of Lkn-1 exon. The cDNA library of a THP-1 cell line constructed as above was hybridized with a probe of the lOObp-DNA fragment of Lkn-1 exon. As a result, a Lkn-1 cDNA showing a positive reaction was obtained.
Determination of the nucleotide sequence of the said Lkn-1 cDNA and the deduced amino acid sequence revealed that the Lkn-1 cDNA is a novel one, open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and the predicted molecular weight of mature Lkn-1 protein consisting of 92 amino acids is 10,162 dalton. Also, it was found that the Lkn-1 has no potential
N-glycosylation site and belongs to C6 β-chemokine family having two cysteine residues as in mMRP-1 and mMRP-2, in addition to four cysteine residues commonly appeared in β-chemokine family. The novel Lkn-1 cDNA thus cloned was inserted into an expression vector and introduced into a host cell such as E. coli or insect cell for expression, to express the recombinant Lkn-1. The purified recombinant Lkn-1 inhibited colony formation by granulocyte-macrophage progenitors , etc . in a concentration-dependent manner, attracted human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis, and induced calcium flux in the said cells. Particularly, it was found that the recombinant Lkn-1 binds to the RANTES and hMIP-lα receptors and does not bind to a IL-8 receptor (although it attracts neutrophils strongly to cause chemotaxis, like IL-8) . Also,
it was revealed that receptors of the recombinant Lkn-1 are CC chemokine receptor 1(CCR1) and CCR3. The recombinant
Lkn-1 was a stronger agonist at CCR1 than hMIP-lα or RANTES which had been already known to bind to CCR1 , and it was a stronger agonist at CCR3 receptor than RANTES although it was a weaker agonist the receptor than eotaxin.
The recombinant Lkn-1 of the invention showing the characteristics mentioned above can be used for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.
The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.
Example 1 : Cloning of exon of Lkn-1 genomic DNA
In order to clone a genomic DNA homologous to mMRP-2 from human, total human genomic DNA was digested with BamHI , EcoRI, Hind III, PstI, Xbal and Xhol , fractionated on a 1.0% agarose gel , and analyzed by southern blot employing a 32P-labelled mMRP-2 cDNA as a probe to obtain a Hindlll fragment of 7. Okb showing a positive signal. In order to prepare sublibrary of the said Hindi11 fragment, lOOμg of human genomic DNA was digested with Hindi11 completely, and fractionated on a 1% agarose gel at a voltage of 20V for 16 hours. Then, a DNA fragment near 7. Okb was isolated employing Gene-clean kit (Bio 101, USA) and inserted into a HindiII -digested dephosphorylated pBluescript SK+ vector (Stratagene, USA) . Electro-max (GIBCO-BRL, USA) competent cell was transformed with the recombinant vector thus prepared by the aid of electroporation and cultured on a solid medium. About 2xl05 colonies formed was hybridized with the mMRP-2 probe. As a result, it was found that only one colony showed a positive signal of hybridization and
contained the insert DNA of 7. Okb.
In order to isolate an exon sequence from the said genomic DNA fragment of 7. Okb, the pBluescript SK+ vector containing the insert DNA of 7. Okb was isolated from the said colony showing a positive signal of hybridization, digested with Alul , fractionated on a 1.5% agarose gel, and analyzed by Southern blot to clone a DNA fragment of 202bp which was hybridized with a mMRP-2 cDNA.
A nucleotide sequence (SEQ ID N0:1) of the said DNA fragment of 202bp was determined to find intron and a part of exon translated into amino acids (see : Figure 1(A)) . As a result, it was found that the amino acid sequence (SEQ ID NO: 2) of the part of exon showed homology of 50% to that of mMRP-2 from the 87th to 110th amino acid and the second cysteine (indicated as "*") of two additional cysteines common inmMRP-1 andmMRP-2 was conserved inLkn-1 (see : Figure 1 (B) ) . In Figure 1 (B) , boxes showed amino acids conserved in Lkn-1 and mMRP-2.
Example 2 : Cloning of a Lkn-1 cDNA
In order to clone a Lkn-1 cDNA from a human cell line, PCR primers permitting amplification of the Lkn-1 exon of lOObp, i.e., a forward primer of 5 ' -TTCCTCACCAAGAAGGGG- 3 '(SEQ ID NO: 13) and a reverse primer of 5'- CTTTTTCATGCAATCCTG-3 ' (SEQ ID NO: 14) were first prepared based on the amino acid sequence (SEQ ID NO: 2) disclosed in Figure 1(B) . Then, PCR was carried out using the 202bp-DNA fragment prepared in Example 1 , total RNA of a human monocytic THP-1 cell line(ATCC TIB202) activated by lOO g/ml of IL-4 for 24 hours, total RNA of a macrophage cell line U937(ATCC CTL1593) and total RNA of a HL-60 cell line (ATCC CCL240) as a template, respectively (see : Figure 2) .
In Figure 2, lanes 1 to 4 show results of PCR using the 202bp-DNA fragment as a positive control, total RNA of the THP-1 cell line, total RNA of the U937 cell line and total RNA of the HL-60 cell line, respectively; and, lane M shows
lOObp-ladder as a DNA size-marker. As shown in Figure 2, the THP-1 cell line activated by IL-4 produced Lkn-1 mRNA.
In order to prepare THP-1 cDNA library, human mRNA was isolated from the THP-1 cell line activated by IL-4, reverse-transcribed employing Time Saver cDNA kit (Pharmacia Biotech, USA) to give double stranded cDNA, and attached with BstXI adapter (Invitrogen, USA) . Then, fractionation on an agarose gel was performed to isolate a cDNA of 0.5kb or more, which was inserted into a PRc/CMV (Invitrogen, USA) vector digested with BstXI to prepare cDNA library. The cDNA library was hybridized with the lOObp-DNA fragment of Lkn-1 exon amplified by PCR above as a probe. As a result, one cDNA clone showing a positive signal was isolated.
Example 3 : Nucleotide sequencing of the Lkn-1 cDNA
Figure 3 shows a nucleotide sequence (SEQ ID NO: 4) of the cDNA clone obtained in Example 2 and an amino acid sequence deduced therefrom (SEQ ID NO: 5) . In Figure 3, the underlined is a signal peptide; the sequence in a box is the amino acid sequence (SEQ ID N0:2) of Lkn-1 shown in Figure 1(B) which was used as a probe during the screening of THP-1 cDNA library; and, (***) indicates a translation stop codon.
As shown in Figure 3, open reading frame of the Lkn-1 cDNA(SEQ ID NO: 6) encodes 113 amino acids consisting of 21 amino acids forming a signal peptide and 92 amino acids forming a mature protein whose molecular weight is supposed to be 10,162 dalton. Also, it was found that there is no N-glycosylation site in the deduced amino acid sequence (SEQ ID NO: 5) of Lkn-1.
Figure 4 shows comparison of an amino acid sequence of mature Lkn-1 (SEQ ID NO: 7) with those of mMRP-1 (SEQ ID NO: 8) , mMRP-2 (SEQ ID N0:9) , hMIP-lα (SEQ ID NO : 10) (see : Youn, B.S. et al., J. Immunol., 155:2661(1995)), mMIP-lα (SEQ ID NO: 11) (see : Kwon, B.S. and Weissman, S.M., Proc . Natl . Acad.
Sci., USA, 86:1963(1989)) and hMIP-lβ(SEQ ID NO: 12) (see:
Sherry, B. et al . , J. Exp. Med. , 168 :2251 (1988) ) . In Figure 4, boxes and (■) indicate four conserved cysteine residues and two additional cysteine residues, respectively. As shown in Figure 4, it was revealed that: (l)Lkn-l has a long N-terminal region before the first two cysteine residues of four cysteine residues common in β-chemokine family; and,
(2) Lkn-2 belongs to C6 β-chemokine family having two cysteine residues further as in mMRP-1 andmMRP-2 besides four cysteine residues common in β-chemokine family. Moreover, it was found that the amino acid sequence (SEQ ID NO: 7) of mature Lkn-1 shows homology of only about 43% to that of mMRP-1 (SEQ ID NO: 8) or mMRP-2 (SEQ ID NO: 9) and homology of 40% to that of hMIP-lα(SEQ ID NO:10) although Lkn-1 was isolated as a human counterpart of mMRP-2.
Example 4 : Expression of a recombinant Lkn-1
Example 4-1: Expression of a recombinant Lkn-1 in E. coli
In order to express only a mature Lkn-1 protein without the putative signal peptide as a recombinant protein, PCR amplification of the open reading frame of the mature Lkn-1 was carried out using the Lkn-1 cDNA cloned in Example 2 as a template, the following PCR primers and Pfu polymerase (Stratagene, USA):
forward primer:
5 ' -CGAATTCCATATGCAGTTCACAAATGATGCAGAG-3 ' (SEQ ID NO:15)
reverse primer:
5 ' -CGCCGCTCGAGTTGAGTAGGGCTTCAGC-3 ' (SEQ ID NO:16)
The DNA fragment thus amplified was digested with Ndel/Xhol and cloned into a plasmid pET21a (Novagen, USA). The
recombinant plasmid thus constructed was designated as pET21a-Lkn-l and introduced into E. coli XL-1 Blue. The recombinant plasmid pET21a-Lkn-l permits expression of a recombinant Lkn-1 which has one additional methionine residue in N-terminus and six additional histidines at C-terminus of a mature Lkn-1.
Transformant thus prepared was designated as
'Escherichia coli (XL-1 Blue) hMRP-2 ' , and deposited with
American Type Culture Collection (ATCC) , an international depositary authority as deposition No. ATCC 98166 on September 10, 1996.
The said transformant was cultured to express Lkn-1, and inclusion bodies were obtained, dissolved in 20ml of denaturation buffer (6M guanidine-HCl , 20mM Tris-HCl , pH 7.9 , 500mM NaCl , 4mM n-octylglucopyranoside) and centrifuged to obtain a supernatant. Then, chromatography using an activated Ni-column (Novagen, USA) and a heparin-agarose column (Pharmacia Fine Chemicals, USA) was carried out to purify a His-tagged recombinant Lkn-1. Electrophoresis revealed that a molecular weight of a recombinant Lkn-1 is about 12kDa.
Example 4-2 : Expression of a recombinant Lkn-1 in an insect cell
In order to express a recombinant Lkn-1 containing a signal peptide, a Lkn-1 cDNA containing a signal peptide sequence was inserted with a PstI restriction site at N- terminus and a Xbal restriction site at C-terminus, and amplified by PCR. Then, the PCR product thus amplified was isolated and inserted into a PVL 1392 vector (Invitrogen, USA) digested with Pstl/Xbal to construct a recombinant plasmid PVL 1392-Lkn-l. And then, a Sf-21 insect cell was transfected with both the said recombinant plasmid and AcNPV (Autographa California nuclear polyhedrosis baculovirus) to transfer the Lkn-1 cDNA in the said recombinant plasmid to the AcNPV. AcNPV-Lkn-1 viral plaque
was isolated based on the characteristics that the virion is occlusion-negative, and grown in the Sf-21 insect cell in a serum-free Ex-Cell 400 medium (JRH Biosciences, USA) for later use. A recombinant Lkn-1 was expressed in a High five cell line (Invitrogen, USA) cultured in a Ex-cell 400 medium, and the Lkn-1 thus expressed was purified using a HiTrap-Heparin column (Pharmacia Biotech., USA) and a HiTrap-SP column (Pharmacia Biotech., USA). Western blot analysis revealed that a molecular weight of the recombinant Lkn-1 analyzed immediately after purification is about 12kDa.
Example 5 : Inhibition of colony formation of bone marrow cells by the recombinant Lkri-1
Since some of the β -chemokines have a myelosuppressive activity in vitro, an effect of the recombinant Lkn-1 purified in Example 4-1 on the colony formation by myeloid progenitor cell present in human bone marrow was investigated. That is, in order to form colony of bone marrow cells by CFU-GM (colony forming unit-granulocyte-macrophage) , human bone marrow cells of low density obtained by centrifugation employing Ficoll-Hypaque gradient (1.070gm/cm3, Sigma Chemical Co., USA) were plated on a 0.3% agar culture medium containing 10% FBS in a concentration of 5xl04cells/ml , and stimulated by rhGM-CSF (recombinant human granulocyte-macrophage- colony stimulating factor, lOOU/ml, Immunex Corporation, USA) + rhSLF (recombinant human steel factor, 50ng/ml, Immunex Corporation, USA) . On the other hand, in order to form colony of bone marrow cells by CFU-GM, BFU- E (burstforming unit-erythroid) and CFU-GEMM (colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte) , the said bone marrow cells were plated on a 1% methylcellulose culture medium containing 30% FBS in a concentration of 5xl04 cells/ml, and stimulated by rhEPO (recombinant human erythropoietin, lU/ml , Amgen Corporation, USA), rhlL- 3 (recombinant human interleukin-3 , lOOU/ml, Immunex
Corporation, USA) or rhSLF.
After the stimulation, the cells were cultured in a BNP-210 incubator (Tabai ESPEC Corp., USA) under an environment of 5% C02 and 5% 02, and number of colonies was counted after 14 days (see : Table 1) . In this connection, the recombinant Lkn-1 was added in a concentration of 3-50ng/ml to a plate.
Table 1: Effect of the recombinant Lkn-1 on the colony formation of human bone marrow cells of low density
Experimental group without addition of the recombinant L n-1 to the reaction solution
Growth factor used for the stimulation of colony formation
Level of change compared with the control (%)
Level of change compared with the control is signi icant (p<0.001 ) .
As can be seen in Table 1 above, the recombinant Lkn-1 inhibited colony formation by CFU-GM, BFU-E and CFU-GEMM significantly in a concentration-dependent manner . Level of the inhibition of colony formation was 22-63% compared with the control. On the other hand, it was found that the recombinant Lkn-1 cannot inhibit colony formation by CFU-GM
stimulated by GM-CSF alone or by BFU-E stimulated by EPO alone, which demonstrates that the recombinant Lkn-1 has an inhibitory effect on immature progenitor cells which can be stimulated by various growth factors.
Example 6 : Investigation of the recombinant Lkn-1 as a chemokine
In order to investigate whether the recombinant Lkn-1 may cause chemotaxis, peripheral blood mononuclear cells (PBMC) of a healthy man were obtained by centrifugation employing Ficoll-Hypaque gradient (1.077gm/cm3) . And then, monocytes were isolated from the PBMC thus obtained based on their attached activity onto the surface of plastic, and the said isolation step was repeated twice. In this connection, cells remained after the step for the monocyte isolation were obtained as lymphocytes. Purity of the monocytes and lymphocytes thus obtained was determined by microscopic examination of cytospin dyed with Diff- Quick (Baxter Scientific, USA). As a result, it was found that purity of the monocytes and lymphocytes was 90% and 88%, respectively .
Also, red blood cells were precipitated by using 3% Dextran T 500 (Pharmacia Fine Chemicals, USA) and obtained by centrifugation employing Ficoll-Hypaque gradient. The red blood cells thus obtained were dissolved in a hypotonic solution to obtain human neutrophils. Purity of the neutrophils thus obtained was determined by morphology. As a result, it was found that the purity was 95%. The monocytes and lymphocytes isolated above were suspended in RPMI 1640 (Gibco, USA) containing 0.5% low endotoxin BSA (Sigma Chemical Co., USA) and 20mM Hepes in concentrations of 2xl06 cells/ml and 8xl06 cells/ml, respectively. The neutrophils were suspended in HBSS in a concentration of lxlO6 cells/ml.
Then, level of cell migration in a 48-well microchamber (Neuroprobe, USA) was determined as followings.
Lower wells of the microchamber were filled with only a buffer solution (control) or a buffer containing the recombinant Lkn-1, hMIP-lα (PeproTech. , USA), RANTES (PeproTech. , USA), IL-8 (PeproTech. , USA) or eotaxin (PeproTech. , USA) , and upper wells were filled with 50μl of the said cell suspensions. A well was partitioned by a suitable filter without polyvinylpyrolidine to give a lower well and an upper well. When neutrophils and lymphocytes were used, a diameter of the filter pore was 3μm. When monocytes were used, it was 5μm. After the said microchamber was incubated at 37°C for 1 hour (for neutrophils), 2 hours (for monocytes) or 4 hours (for lymphocytes), the filter was separated from the chamber and cashed. The cells on the filter were fixed and dyed with Diff-Quick. Thus, number of the cells was counted (see: Figures 5(A) to 5(C)).
In Figures 5 (A) to 5 (C) , a value obtained by dividing number of migrating cells in an experimental group treated with a chemokine by number of migrating cells in a control was represented as a chemotactic index. Figure 5(A) is a graph which shows chemotaxis attracting lymphocytes by the recombinant Lkn-1 and RANTES. Figure 5(B) is a graph which shows chemotaxis attracting monocytes by the recombinant Lkn-1 and hMIP-lα. Figure 5(C) is a graph which shows chemotaxis attracting neutrophils by the recombinant Lkn-1 and IL-8.
As shown in Figures 5 (A) to 5 (C) , it was revealed that the recombinant Lkn-1 is a strong chemokine attracting human peripheral blood neutrophils, monocytes and lymphocytes. Also, the recombinant Lkn-1 showed chemotaxis attracting neutrophils in a similar manner to IL-8 (see : Figure 5(C)) and chemotaxis attracting monocytes in a similar manner to hMIP-lα (see : Figure 5(B)); and, it showed an improved chemotaxis attracting lymphocytes in a concentration- dependent manner up to a concentration of lOμg/ml of Lkn-1 although it showed lower level of chemotaxis than RANTES (see : Figure 5 (A) ) .
Example 7 : Analysis of calcium flux by the recombinant Lkn-1
Example 7-1: Analysis of calcium flux in lymphocytes, monocytes and neutrophils
It was investigated whether the recombinant Lkn-1 may bind to a receptor for activation of lymphocytes, monocytes and neutrophils to induce calcium efflux. Its competitive relationships with other agonists against the receptor was also examined. Receptor activation was determined by measuring a change in [Ca2+] in subsets (lymphocytes, monocytes and neutrophils) of peripheral blood leukocytes isolated using a MSIII fluorimeter (Photon Technology Internationl , USA) . That is, cells were reacted with 2μM fura-2AM(Sigma Chemical Co., USA) at 37°C for 45 minutes, washed twice, and resuspended in HBSS (pH 7.4) containing 0.05% BSA in a concentration of lxlO7 cells/ml. 2ml of the cell suspension was added into a stirred, water-jacketed cuvette, and activated continuously at 340nm and 380nm at 37°C. Emitted fluorescence was measured at 510nm before and after addition of 25nM agonist (the recombinant Lkn-1, hMIP-lα , RANTES, IL-8 or eotaxin) (see: Figures 6 (A) to 6 (F) ) .
In Figures 6 (A) to 6 (F) , relative fluorescence was expressed as a relative ratio of fluorescence activated at 340nm and 380nm. Figure 6(A) shows relative fluorescence measured in lymphocytes which are stimulated by a series of addition of RANTES and the recombinant Lkn-1; Figure 6(B) shows relative fluorescence measured in lymphocytes which are stimulated by a series of addition of the recombinant Lkn-1 and RANTES; Figure 6(C) shows relative fluorescence measured in monocytes which are stimulated by a series of addition of hMIP-lα and the recombinant Lkn-1; Figure 6(D) shows relative fluorescence measured in monocytes which are stimulated by a series of addition of the recombinant Lkn-1 and hMIP-lα; Figure 6(E) shows relative fluorescence measured in neutrophils which are stimulated by a series of
addition of IL-8 and the recombinant Lkn-1; Figure 6 (F) shows relative fluorescence measured in neutrophils which are stimulated by a series of addition of the recombinant Lkn-1 and IL-8. As shown in Figures 6 (A) to 6 (F) , it was found that the recombinant Lkn-1 induced calcium influx in lymphocytes, monocytes and neutrophils.
On the other hand, when G protein-coupled receptors were continuously exposed to ligands having the same binding site as the receptor signal within a short time, the said receptors were desensitized. Such a phenomenon occurred when the said cells expressing receptors were stimulated by the recombinant Lkn-1.
As shown in Figures 6(A) to 6(F) , it was found that the recombinant Lkn-1 desensitized lymphocytes and monocytes completely when stimulation by RANTES and hMIP-lα followed (see: Figures 6(B) and 6(D)). Also, RANTES or hMIP-lα did not desensitize lymphocytes and monocytes completely when stimulation by the recombinant Lkn-1 followed (see : Figures 6(A) and 6(C)), which demonstrated that the recombinant Lkn-1 shares receptors of RANTES and hMIP-lα, and induces more calcium flux than RANTES and hMIP-lα . In addition, neutrophils were not desensitized by the recombinant Lkn-1 during a further stimulation by IL-8 although the recombinant Lkn-1 induced calcium flux in neutrophils (see : Figure 6(F)) . Moreover, IL-8 did not desensitize neutrophils during a further stimulation by the recombinant Lkn-1 (see : Figure 6(E)). Therefore, it was clearly demonstrated that the recombinant Lkn-1 does not share IL-8 receptors although it is a strong factor to induce calcium flux in neutrophils like IL-8.
Example 7-2 : Analysis of calcium flux in a cell line expressing CC chemokine receptors
It was investigated whether the recombinant Lkn-1 can activate a cell line expressing CC or CXC chemokine receptors
because it was found in Example 7-1 that the recombinant Lkn-1 activated receptors which can be activated by RANTES and hMIP-l . Analysis was carried out in the same manner as in Example 7-1 except for the use of a HOS cell line expressing recombinant CCR1 , CCR2B, CCR3 , CCR4 , CCR5 or CXCR4 (see: AIDS Research and Reference Reagent Program of NIH, USA) instead of the use of subsets of leukocytes isolated. It has been known that hMIP-lα binds to both CCR1 and CCR5 , and RANTES binds to CCR1 , CCR3 and CCR5. Figure 7 (A) shows relative fluorescence measured in a HOS cell line expressing CCR1 which are stimulated by a series of addition of various agents, and Figure 7 (B) shows relative fluorescence measured in a HOS cell line expressing CCR3 which are stimulated by a series of addition of various agents. As shown in Figures 7 (A) and 7 (B) , it was revealed that the recombinant Lkn-1 strongly induced calcium flux in a HOS cell line expressing CCR1 or CCR3. However, calcium flux by the recombinant Lkn-1 in HOS cell lines expressing other receptors was not observed. Also, as shown in Figure 7(A) , CCR1-HOS cells were not stimulated further by hMIP-lα or RANTES after stimulation by the recombinant Lkn-1 while further stimulation of CCR1-H0S cells by the recombinant Lkn-1 was not affected after stimulation by hMIP-lα or RANTES. Figure 7(A) revealed that the recombinant Lkn-1 is a stronger agonist against CCR1 than hMIP-lα or RANTES, which was clearly confirmed from calcium responses depending on concentrations of the recombinant Lkn-1 and hMIP-lα as shown in Figure 7 (C) . Moreover, as shown in Figure 7(B) , CCR3-HOS cells were desensitized almost completely during further stimulation by eotaxin or the recombinant Lkn-1 after the cells were stimulated by eotaxin; CCR3-HOS cells were desensitized almost completely during further stimulation by RANTES after the cells were stimulated by the recombinant Lkn-1; and, further stimulation of CCR3-HOS cells by eotaxin was, however,
not affected after the cells were stimulated by the recombinant Lkn-1 or RANTES. Also, calcium responses depending on concentrations of eotaxin, the recombinant Lkn-1 and RANTES showed that the recombinant Lkn-1 is a stronger agonist against CCR3 than RANTES although it is not a stronger one than eotaxin (see : Figure 7(D)) .
As clearly illustrated and demonstrated as aboves, the present invention provides a cDNA coding for a novel Lkn-1 protein which belongs to C6 β -chemokines, and an amino acid sequence deduced therefrom. Open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and molecular weight of mature protein consisting of 92 amino acids among them is supposed to be 10,162 dalton. A recombinant Lkn-1 which was expressed in E. coli or insect cell employing the said Lkn-1 cDNA and purified, inhibited colony formation significantly, attracted neutrophils, monocytes and lymphocytes to cause chemotaxis, and bound to CCR1 and CCR3 receptors. Accordingly, it was determined that the recombinant Lkn-1 protein can be used as a potential drug for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT:
(A) NAME: KOREA GREEN CROSS CORPORATION
(B) STREET: 227, Gugal-Ri, Kiheung-Eup
(C) CITY: Yongin, Kyonggi-Do
(D) STATE: not applicable (E) COUNTRY: Korea
(F) POSTAL CODE (ZIP) : 449-900
(G) TELEPHONE: 02-584-0131 (H) TELEFAX: 02-582-6331 (ii) TITLE OF INVENTION: A PROCESS FOR PREPARING A cDNA ENCODINGAC6 β -CHEMOKINE LEUKOTACTIN-1 (Lkn-1) ISOLATED FROM HUMAN
(iii) NUMBER OF SEQUENCES: 16
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(EPO)
(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic DNA library from human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
CTGCCATCAG CAGAGAAAGG AAAAAACAGG CTGTGTTGAC TGGGAAATCT 50 GAGGAGCAGG GAGGATGGGG CCCCCTGTCT CCATCTGCCC ACACCTCAGT 100 TTGTAATCTT TCTCTCCCTT GTTCCCCAGA TTCCTCACCA AGAAGGGGCG 150 GCAAGTCTGT GCCAAACCCA GTGGTCCGGG AGTTCAGGAT TGCATGAAAA 200 AG 202
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : Phe Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser 1 5 10
Gly Pro Gly Val Gin Asp Cys Met Lys Lys 15 20
(2) INFORMATION FOR SEQ ID NO : 3
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: Murine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : Phe lie Ser Lys Arg Gly Phe Gin Val Cys Ala Asn Pro Ser 1 5 10
Asp Arg Arg Val Gin Arg Cys lie Glu Arg 15 20
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 582 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Human
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: DNA library from human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
CGGAGCCAGG AAGCAGTGAG CCCAGGAGTC CTCGGCCAGC CCTGCCTGCC 50 CACCAGGAGG ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA 100 TTGCTGTCCT TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG 150 TTAATGATGT CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT 200 TCACTTTGCT GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT 250 GTTCACTCAT GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA 300 GGTGTCATAT TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG 350 TGGTCCGGGA GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT 400 AATAATAAAC AGACAAAAGA GGCCAGCCAC CCACCTCCAA CACCTCCTGT 450 GAGTTTCTTG GTCTGAAATA CTTAAAAAAT ATATATATTG TTGTGTCTGG 500 TAATGAAAGT AATGCATCTA ATAAAGGTAT TCAATTTTTT AACTTTGCTT 550 GAGTTTTAAG AGGAAATAAA CTAATATAAA AC 582
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu lie 1 5 10
Ala Val Leu Gly Ser Gin Ala Gin Phe Thr Asn Asp Ala Glu 15 20 25
Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val 30 35 40
Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser 45 50 55 Tyr lie Ser Gin Ser lie Pro Cys Ser Leu Met Lys Ser Tyr
60 65 70
Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val lie Phe
75 80
Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser Gly 85 90 95
Pro Gly Val Gin Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser 100 105 110
He
(2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Human (vii) IMMEDIATE SOURCE:
(A) LIBRARY: DNA library from human (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA TTGCTGTCCT 50 TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG TTAATGATGT 100 CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT TCACTTTGCT 150 GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT GTTCACTCAT 200 GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA GGTGTCATAT 250 TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG TGGTCCGGGA 300 GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT AA 342 (2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
Gin Phe Thr Asn Asp Ala Glu Thr Glu Leu Met Met Ser Lys 1 5 10
Leu Pro Leu Glu Asn Pro Val Val Leu Asn Ser Phe His Phe 15 20 25 Ala Ala Asp Cys Cys Thr Ser Tyr He Ser Gin Ser He Pro 30 35 40
Cys Ser Leu Met Lys Ser Tyr Phe Glu Thr Ser Ser Glu Cys 45 50 55
Ser Lys Pro Gly Val He Phe Leu Thr Lys Lys Gly Arg Gin 60 65 70
Val Cys Ala Lys Pro Ser Gly Pro Gly Val Gin Asp Cys Met 75 80
Lys Lys Leu Lys Pro Tyr Ser He 85 90 (2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: Murine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Gly Leu He Gin He Met Glu Lys Glu Asp Arg Arg Tyr Asn 1 5 10
Pro Pro He He His Gin Gly Phe Gin Asp Thr Ser Ser Asp 15 20 25 Cys Cys Phe Ser Tyr Ala Thr Gin He Pro Cys Lys Arg Phe 30 35 40
He Tyr Tyr Phe Pro Thr Ser Gly Gly Cys He Lys Pro Gly 45 50 55
He He Phe He Ser Arg Arg Gly Thr Gin Val Cys Ala Asp 60 65 70
Pro Ser Asp Arg Arg Val Gin Arg Cys Leu Ser Thr Leu Lys 75 80
Gin Gly Pro Arg Ser Gly Asn Lys Val He Ala 85 90 95 (2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 101 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: Murine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
Gin He Thr His Ala Thr Glu Thr Lys Glu Val Gin Ser Ser 1 5 10
Leu Lys Ala Gin Gin Gly Leu Glu He Glu Met Phe His Met 15 20 25
Gly Phe Gin Asp Ser Ser Asp Cys Cys Leu Ser Tyr Asn Ser 30 35 40
Arg He Gin Cys Ser Arg Phe He Gly Tyr Phe Pro Thr Ser 45 50 55 Gly Gly Cys Thr Arg Pro Gly He He Phe He Ser Lys Arg
60 65 70
Gly Phe Gin Val Cys Ala Asn Pro Ser Asp Arg Arg Val Gin
75 80 Arg Cys He Glu Arg Leu Glu Gin Asn Ser Gin Pro Arg Thr 85 90 95
Tyr Tyr Lys 100
(2) INFORMATION FOR SEQ ID NO: 10
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser 1 5 10
Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He Ala Asp Tyr 15 20 25
Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Gly Val He Phe 30 35 40
Leu Thr Lys Arg Ser Arg Gin Val Cys Ala Asp Pro Ser Glu 45 50 55
Glu Trp Val Gin Lys Tyr Val Ser Asp Leu Glu 60 65
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: Murine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Ala Pro Tyr Gly Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser 1 5 10
Tyr Ser Arg Lys He Pro Arg Gin Phe He Val Glu Val Phe 15 20 25
Glu Thr Ser Ser Leu Cys Ser Gin Pro Gly Val He Phe Leu 30 35 40
Thr Lys Arg Asn Arg Gin He Cys Ala Asp Ser Lys Glu Thr 45 50 55
Trp Val Gin Glu Tyr He Thr Asp Leu Glu 60 65 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM: Human
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser 1 5 10
Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr 15 20 25 Tyr Glu Thr Ser Ser Leu Cys Ser Gin Pro Ala Val Val Phe 30 35 40
Gin Thr Lys Arg Ser Lys Gin Val Cys Ala Asp Pro Ser Glu 45 50 55
Ser Trp Val Gin Glu Val Tyr Tyr Asp Leu Glu 60 65
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vii) IMMEDIATE SOURCE: (B) CLONE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
TTCCTCACCA AGAAGGGG 18 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vii) IMMEDIATE SOURCE: (B) CLONE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CTTTTTCATG CAATCCTG 18
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (vii) IMMEDIATE SOURCE: (B) CLONE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGAATTCCAT ATGCAGTTCA CAAATGATGC AGAG 34
(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vii) IMMEDIATE SOURCE: (B) CLONE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CGCCGCTCGA GTTGAGTAGG GCTTCAGC 28
Claims
1. A cDNA of human C6 β-chemokine Lkn-1 (leukotactin- 1) whose nucleotide sequence is represented as following (SEQ ID NO: 6) :
ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTC ATG CTT 39 ATT GCT GTC CTT GGA TCC CAG GCC CAG TTC ACA AAT GAT 78 GCA GAG ACA GAG TTA ATG ATG TCA AAG CTT CCA CTG GAA 117 AAT CCA GTA GTT CTG AAC AGC TTT CAC TTT GCT GCT GAC 156 TGC TGC ACC TCC TAC ATC TCA CAA AGC ATC CCG TGT TCA 195 CTC ATG AAA AGT TAT TTT GAA ACG AGC AGC GAG TGC TCC 234 AAG CCA GGT GTC ATA TTC CTC ACC AAG AAG GGG CGG CAA 273 GTC TGT GCC AAA CCC AGT GGT CCG GGA GTT CAG GAT TGC 312 ATG AAA AAG CTG AAG CCC TAC TCA ATA TAA 342
2. Human C6 β-chemokine Lkn-1 (leukotactin-1) whose amino acid sequence is represented as following (SEQ ID NO: 5) :
Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu He 1 5 10
Ala Val Leu Gly Ser Gin Ala Gin Phe Thr Asn Asp Ala Glu 15 20 25
Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val 30 35 40
Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser 45 50 55
Tyr He Ser Gin Ser He Pro Cys Ser Leu Met Lys Ser Tyr 60 65 70 Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val He Phe
75 80
Leu Thr Lys Lys Gly Arg Gin Val Cys Ala Lys Pro Ser Gly 85 90 95
Pro Gly Val Gin Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser 100 105 110
He
3. A cDNA of human C6 β-chemokine Lkn-1 (leukotactin- 1) from which the 1st to 63th nucleotide sequences are deleted.
4. An expression vector which comprises the cDNA of claim
1.
5. An expression vector PVL 1392-Lkn-l which comprises the cDNA of claim 1.
6. A process for preparing a recombinant leukotactin-1 which comprises the steps of transforming a host cell with the expression vector of claim 4 , culturing the transformant , and obtaining the recombinant Lkn-1 therefrom.
7. The process of claim 6 wherein the host cell is E. coli or insect cell.
8. A recombinant leukotactin-1 prepared by a process which comprises the steps of transforming a host cell with the expression vector of claim 4 , culturing the transformant , and obtaining the recombinant Lkn-1 from the culture.
9. The recombinant leukotactin-1 of claim 8 which inhibits colony formation.
10. The recombinant leukotactin-1 of claim 8 which attracts human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis .
11. The recombinant leukotactin-1 of claim 8 which binds to CCR1 and CCR3 receptors.
12. The recombinant leukotactin-1 of claim 11 which shares receptors with RANTES (regulated activated normal T cell expressed sequence) and hMIP-lα (human macrophage
inflammatory protein- lα) and does not share receptors with IL-8 (interleukin-8) .
13. An expression vector which comprises the cDNA of claim 2.
14. An expression vector pET21a-Lkn-l which comprises the cDNA of claim 2.
15. Escherichia coli (XL-1 Blue) hMRP-2 transformed with the expression vector of claim 14 (ATCC 98166) .
16. A process for preparing a recombinant leukotactin-1 which comprises the steps of transforming a host cell with the expression vector of claim 13 , culturing the transformant , and obtaining the recombinant Lkn-1 therefrom.
17. The process of claim 16 wherein the host cell is E. coli or insect cell.
18. A recombinant leukotactin-1 prepared by a process which comprises the steps of transforming a host cell with the expression vector of claim 13 , culturing the transformant , and obtaining the recombinant Lkn-1 therefrom.
19. The recombinant leukotactin-1 of claim 18 which inhibits colony formation.
20. The recombinant leukotactin-1 of claim 18 which attracts human peripheral blood neutrophils, monocytes and lymphocytes strongly to cause chemotaxis.
21. The recombinant leukotactin-1 of claim 18 which binds to CCR1 and CCR3 receptors .
22. The recombinant leukotactin-1 of claim 21 which shares receptors with RANTES (regulated activated normal T
cell expressed sequence) and hMIP-lα (human macrophage inflammatory protein- lα) and does not share receptors with IL-8 (interleukin-8) .
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019970063610A KR19990042713A (en) | 1997-11-27 | 1997-11-27 | Method for preparing CDNA and recombinant LKN-1 of C 6 beta-chemokine LKN-1 isolated from human |
KR1997/63610 | 1997-11-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999028472A1 true WO1999028472A1 (en) | 1999-06-10 |
Family
ID=19525811
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR1998/000145 WO1999028472A1 (en) | 1997-11-27 | 1998-06-05 | A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN |
PCT/KR1998/000381 WO1999028473A1 (en) | 1997-11-27 | 1998-11-27 | A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR1998/000381 WO1999028473A1 (en) | 1997-11-27 | 1998-11-27 | A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1034272A1 (en) |
JP (1) | JP2002505076A (en) |
KR (2) | KR19990042713A (en) |
CN (1) | CN1282371A (en) |
WO (2) | WO1999028472A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001036459A2 (en) * | 1999-11-15 | 2001-05-25 | Forssmann Wolf Georg | Novel use of hcc-2 |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1176200A3 (en) | 2000-06-20 | 2005-01-12 | Switch Biotech Aktiengesellschaft | Use of polyeptides or their encoding nucleic acids for the diagnosis or treatment of skin diseases or wound healing and their use in indentifying pharmacologically acitve substances |
CN104977414B (en) * | 2014-04-09 | 2016-09-28 | 上海交通大学医学院 | Identify test kit and the application thereof that whether there is protection leukemic stem cells microenvironment in bone marrow |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996034891A1 (en) * | 1995-05-05 | 1996-11-07 | Human Genome Sciences, Inc. | Human chemokine beta-8, chemokine beta-1 and macrophage inflammatory protein-4 |
WO1997019696A1 (en) * | 1995-11-30 | 1997-06-05 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, National Institutes Of Health, Office Of Technology Transfer | C-c chemokines that inhibit retrovirus infection |
WO1997025427A1 (en) * | 1996-01-12 | 1997-07-17 | Genetics Institute, Inc. | Beta-chemokine, h1305 (mcp-2) |
WO1997039126A1 (en) * | 1996-04-17 | 1997-10-23 | Incyte Pharmaceuticals, Inc. | Novel rantes homolog from prostate |
EP0807439A2 (en) * | 1996-05-14 | 1997-11-19 | Smithkline Beecham Corporation | Method of treating sepsis and adult respiratory distress syndrome |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1190991A (en) * | 1995-06-05 | 1998-08-19 | 人类基因组科学公司 | Human chemokine beta-11 and human chemokine alpha-1 |
WO1996039521A1 (en) * | 1995-06-06 | 1996-12-12 | Human Genome Sciences, Inc. | Human chemokine beta-13 |
AU2698795A (en) * | 1995-06-06 | 1996-12-24 | Human Genome Sciences, Inc. | Human chemokine beta-12 |
HUP9802699A3 (en) * | 1995-09-29 | 2000-09-28 | Smithkline Beecham Corp | Short forms of chemokine beta-8 |
-
1997
- 1997-11-27 KR KR1019970063610A patent/KR19990042713A/en active IP Right Grant
-
1998
- 1998-06-05 WO PCT/KR1998/000145 patent/WO1999028472A1/en active Application Filing
- 1998-11-27 EP EP98959235A patent/EP1034272A1/en not_active Withdrawn
- 1998-11-27 KR KR10-2000-7000931A patent/KR100367213B1/en not_active IP Right Cessation
- 1998-11-27 CN CN98811574A patent/CN1282371A/en active Pending
- 1998-11-27 WO PCT/KR1998/000381 patent/WO1999028473A1/en not_active Application Discontinuation
- 1998-11-27 JP JP2000523349A patent/JP2002505076A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996034891A1 (en) * | 1995-05-05 | 1996-11-07 | Human Genome Sciences, Inc. | Human chemokine beta-8, chemokine beta-1 and macrophage inflammatory protein-4 |
WO1997019696A1 (en) * | 1995-11-30 | 1997-06-05 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, National Institutes Of Health, Office Of Technology Transfer | C-c chemokines that inhibit retrovirus infection |
WO1997025427A1 (en) * | 1996-01-12 | 1997-07-17 | Genetics Institute, Inc. | Beta-chemokine, h1305 (mcp-2) |
WO1997039126A1 (en) * | 1996-04-17 | 1997-10-23 | Incyte Pharmaceuticals, Inc. | Novel rantes homolog from prostate |
EP0807439A2 (en) * | 1996-05-14 | 1997-11-19 | Smithkline Beecham Corporation | Method of treating sepsis and adult respiratory distress syndrome |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001036459A2 (en) * | 1999-11-15 | 2001-05-25 | Forssmann Wolf Georg | Novel use of hcc-2 |
WO2001036459A3 (en) * | 1999-11-15 | 2001-11-15 | Forssmann Wolf Georg | Novel use of hcc-2 |
Also Published As
Publication number | Publication date |
---|---|
KR20010022352A (en) | 2001-03-15 |
JP2002505076A (en) | 2002-02-19 |
WO1999028473A1 (en) | 1999-06-10 |
EP1034272A1 (en) | 2000-09-13 |
CN1282371A (en) | 2001-01-31 |
KR19990042713A (en) | 1999-06-15 |
KR100367213B1 (en) | 2003-01-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Patel et al. | Molecular and functional characterization of two novel human CC chemokines as inhibitors of two distinct classes of myeloid progenitors | |
Hromas et al. | Cloning and characterization of exodus, a novel β-chemokine | |
Youn et al. | Molecular cloning of leukotactin-1: a novel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3. | |
Youn et al. | Characterization of CKβ8 and CKβ8-1: two alternatively spliced forms of human β-chemokine, chemoattractants for neutrophils, monocytes, and lymphocytes, and potent agonists at CC chemokine receptor 1 | |
AU680851B2 (en) | Progenitor B cell stimulating factor | |
AU708743B2 (en) | Macrophage derived chemokine and chemokine analogs | |
CA2202889A1 (en) | Chemokine-like proteins and methods of use | |
US6096300A (en) | Treatment of myeloproliferative disease with exodus chemokine | |
AU711573B2 (en) | Short forms of chemokine beta-8 | |
AU733925C (en) | Monocyte chemotactic protein-5 materials and methods | |
WO1999028472A1 (en) | A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(Lkn-1) ISOLATED FROM HUMAN | |
EP1182211A1 (en) | Chemokine-like factors having cell chemokinesis function and proliferation-stimulating activity | |
KR100250091B1 (en) | Cdna encoding a human c6 beta-chemokine, leukotactin-3, and a process for preparing a recombinant leukotactin-3 | |
KR100250090B1 (en) | Cdna encoding a human c6 beta-chemokine, leukotactin-2, and a process for preparing a recombinant leukotactin-2 | |
US6320023B1 (en) | Macrophage derived chemokine | |
US20030138400A1 (en) | Short forms of chemokine beta-8 | |
Kwon et al. | Characterization of CK | |
AU1552602A (en) | Exodus chemokine materials and methods | |
MXPA98002380A (en) | Short shapes of bet chemiscino | |
CA2233367A1 (en) | Short forms of chemokine .beta.-8 | |
MXPA98005598A (en) | Exodus chemokine materials and methods |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA CN JP PL RU US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
122 | Ep: pct application non-entry in european phase |