WO1991004986A1 - Oligopeptides/expression products of the aujeszky's disease virus, and recombinant polynucleotides coding therefor - Google Patents
Oligopeptides/expression products of the aujeszky's disease virus, and recombinant polynucleotides coding therefor Download PDFInfo
- Publication number
- WO1991004986A1 WO1991004986A1 PCT/NL1990/000143 NL9000143W WO9104986A1 WO 1991004986 A1 WO1991004986 A1 WO 1991004986A1 NL 9000143 W NL9000143 W NL 9000143W WO 9104986 A1 WO9104986 A1 WO 9104986A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ala
- pro
- leu
- arg
- gly
- Prior art date
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- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 91
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 91
- 230000014509 gene expression Effects 0.000 title claims abstract description 53
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 title claims abstract description 46
- 108091033319 polynucleotide Proteins 0.000 title claims description 5
- 239000002157 polynucleotide Substances 0.000 title claims description 5
- 102000040430 polynucleotide Human genes 0.000 title claims description 5
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 7
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 7
- 230000002068 genetic effect Effects 0.000 claims abstract description 7
- 150000001413 amino acids Chemical group 0.000 claims description 61
- 239000012634 fragment Substances 0.000 claims description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 108020004511 Recombinant DNA Proteins 0.000 claims description 13
- 241000282887 Suidae Species 0.000 claims description 13
- 230000002163 immunogen Effects 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- KMGOBAQSCKTBGD-DLOVCJGASA-N Ala-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CN=CN1 KMGOBAQSCKTBGD-DLOVCJGASA-N 0.000 claims description 8
- OLVCTPPSXNRGKV-GUBZILKMSA-N Ala-Pro-Pro Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 OLVCTPPSXNRGKV-GUBZILKMSA-N 0.000 claims description 8
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 claims description 8
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 claims description 8
- 108010040443 aspartyl-aspartic acid Proteins 0.000 claims description 8
- 108010082286 glycyl-seryl-alanine Proteins 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 claims description 7
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 claims description 6
- JGDBHIVECJGXJA-FXQIFTODSA-N Asp-Asp-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JGDBHIVECJGXJA-FXQIFTODSA-N 0.000 claims description 6
- SBHUBSDEZQFJHJ-CIUDSAMLSA-N Asp-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O SBHUBSDEZQFJHJ-CIUDSAMLSA-N 0.000 claims description 6
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 claims description 6
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- HWPXGQCMZITGFN-XVYDVKMFSA-N Ala-Cys-His Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HWPXGQCMZITGFN-XVYDVKMFSA-N 0.000 claims description 4
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 claims description 4
- IORKCNUBHNIMKY-CIUDSAMLSA-N Ala-Pro-Glu Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O IORKCNUBHNIMKY-CIUDSAMLSA-N 0.000 claims description 4
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 claims description 4
- HULHGJZIZXCPLD-FXQIFTODSA-N Arg-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N HULHGJZIZXCPLD-FXQIFTODSA-N 0.000 claims description 4
- KWKQGHSSNHPGOW-BQBZGAKWSA-N Arg-Ala-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)NCC(O)=O KWKQGHSSNHPGOW-BQBZGAKWSA-N 0.000 claims description 4
- OTOXOKCIIQLMFH-KZVJFYERSA-N Arg-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N OTOXOKCIIQLMFH-KZVJFYERSA-N 0.000 claims description 4
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 claims description 4
- PPPXVIBMLFWNSK-BQBZGAKWSA-N Arg-Gly-Cys Chemical compound C(C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N PPPXVIBMLFWNSK-BQBZGAKWSA-N 0.000 claims description 4
- JTZUZBADHGISJD-SRVKXCTJSA-N Arg-His-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JTZUZBADHGISJD-SRVKXCTJSA-N 0.000 claims description 4
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 claims description 4
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 claims description 4
- SYDJILXOZNEEDK-XIRDDKMYSA-N Glu-Arg-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SYDJILXOZNEEDK-XIRDDKMYSA-N 0.000 claims description 4
- RMWAOBGCZZSJHE-UMNHJUIQSA-N Glu-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N RMWAOBGCZZSJHE-UMNHJUIQSA-N 0.000 claims description 4
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 claims description 4
- DHNXGWVNLFPOMQ-KBPBESRZSA-N Gly-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)CN DHNXGWVNLFPOMQ-KBPBESRZSA-N 0.000 claims description 4
- GGLIDLCEPDHEJO-BQBZGAKWSA-N Gly-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)CN GGLIDLCEPDHEJO-BQBZGAKWSA-N 0.000 claims description 4
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 claims description 4
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 claims description 4
- VSLXGYMEHVAJBH-DLOVCJGASA-N His-Ala-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O VSLXGYMEHVAJBH-DLOVCJGASA-N 0.000 claims description 4
- PTRKPHUGYULXPU-KKUMJFAQSA-N Leu-Phe-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O PTRKPHUGYULXPU-KKUMJFAQSA-N 0.000 claims description 4
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 claims description 4
- JDBQSGMJBMPNFT-AVGNSLFASA-N Leu-Pro-Val Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O JDBQSGMJBMPNFT-AVGNSLFASA-N 0.000 claims description 4
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 claims description 4
- MFDDVIJCQYOOES-GUBZILKMSA-N Met-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCSC)N MFDDVIJCQYOOES-GUBZILKMSA-N 0.000 claims description 4
- 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 claims description 4
- 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 4
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 claims description 4
- KDIIENQUNVNWHR-JYJNAYRXSA-N Pro-Arg-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KDIIENQUNVNWHR-JYJNAYRXSA-N 0.000 claims description 4
- SMCHPSMKAFIERP-FXQIFTODSA-N Pro-Asn-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 SMCHPSMKAFIERP-FXQIFTODSA-N 0.000 claims description 4
- KPDRZQUWJKTMBP-DCAQKATOSA-N Pro-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 KPDRZQUWJKTMBP-DCAQKATOSA-N 0.000 claims description 4
- QPPYAWVLAVXISR-DCAQKATOSA-N Ser-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QPPYAWVLAVXISR-DCAQKATOSA-N 0.000 claims description 4
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 claims description 4
- YXSSXUIBUJGHJY-SFJXLCSZSA-N Trp-Thr-Phe Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)[C@H](O)C)C(O)=O)C1=CC=CC=C1 YXSSXUIBUJGHJY-SFJXLCSZSA-N 0.000 claims description 4
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 claims description 4
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 claims description 4
- 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 description 4
- RYQUMYBMOJYYDK-NHCYSSNCSA-N Val-Pro-Glu Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RYQUMYBMOJYYDK-NHCYSSNCSA-N 0.000 claims description 4
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 claims description 4
- 108010086434 alanyl-seryl-glycine Proteins 0.000 claims description 4
- 108010029539 arginyl-prolyl-proline Proteins 0.000 claims description 4
- 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 claims description 4
- 108010081985 glycyl-cystinyl-aspartic acid Proteins 0.000 claims description 4
- 108010025306 histidylleucine Proteins 0.000 claims description 4
- 108010085325 histidylproline Proteins 0.000 claims description 4
- 108010018006 histidylserine Proteins 0.000 claims description 4
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 claims description 4
- 108010073472 leucyl-prolyl-proline Proteins 0.000 claims description 4
- 108010004914 prolylarginine Proteins 0.000 claims description 4
- 108010070643 prolylglutamic acid Proteins 0.000 claims description 4
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 claims description 3
- IJPNNYWHXGADJG-GUBZILKMSA-N Arg-Ala-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O IJPNNYWHXGADJG-GUBZILKMSA-N 0.000 claims description 3
- 108010010777 Arg-Gly-Asp-Gly Proteins 0.000 claims description 3
- DBWYWXNMZZYIRY-LPEHRKFASA-N Asp-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O DBWYWXNMZZYIRY-LPEHRKFASA-N 0.000 claims description 3
- DTNUIAJCPRMNBT-WHFBIAKZSA-N Asp-Gly-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O DTNUIAJCPRMNBT-WHFBIAKZSA-N 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 3
- VOEGKUNRHYKYSU-XVYDVKMFSA-N His-Asp-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O VOEGKUNRHYKYSU-XVYDVKMFSA-N 0.000 claims description 3
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 claims description 3
- GRVMHFCZUIYNKQ-UFYCRDLUSA-N Phe-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O GRVMHFCZUIYNKQ-UFYCRDLUSA-N 0.000 claims description 3
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 claims description 3
- KDGARKCAKHBEDB-NKWVEPMBSA-N Ser-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CO)N)C(=O)O KDGARKCAKHBEDB-NKWVEPMBSA-N 0.000 claims description 3
- PWKMJDQXKCENMF-MEYUZBJRSA-N Tyr-Thr-Leu 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](CC(C)C)C(O)=O PWKMJDQXKCENMF-MEYUZBJRSA-N 0.000 claims description 3
- 238000002405 diagnostic procedure Methods 0.000 claims description 3
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 108010077112 prolyl-proline Proteins 0.000 claims description 3
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 claims description 2
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- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 claims description 2
- IICZCLFBILYRCU-WHFBIAKZSA-N Asn-Gly-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IICZCLFBILYRCU-WHFBIAKZSA-N 0.000 claims description 2
- BKFXFUPYETWGGA-XVSYOHENSA-N Asn-Phe-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BKFXFUPYETWGGA-XVSYOHENSA-N 0.000 claims description 2
- PQKSVQSMTHPRIB-ZKWXMUAHSA-N Asn-Val-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O PQKSVQSMTHPRIB-ZKWXMUAHSA-N 0.000 claims description 2
- 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 claims description 2
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 claims description 2
- TWROVBNEHJSXDG-IHRRRGAJSA-N His-Leu-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O TWROVBNEHJSXDG-IHRRRGAJSA-N 0.000 claims description 2
- BPANDPNDMJHFEV-CIUDSAMLSA-N Leu-Asp-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O BPANDPNDMJHFEV-CIUDSAMLSA-N 0.000 claims description 2
- 108010079364 N-glycylalanine Proteins 0.000 claims description 2
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 claims description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/087—Herpes simplex virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention relates to novel oligopeptides/expression products which can be used for diagnostic purposes or for the production of polyclonal or monoclonal antibodies.
- the present invention relates to nove oligopeptides/expression products derived from glycoprotein I (referred to hereinafter by the conventional abbreviation gl) of the Aujeszky's disease virus (ADV, sometimes referred to a pseudorabies virus or PRV) , and suitable for use in serological diagnostics directed_to the differentiation of animals (specifically pigs) infected by the wild-type virus from animals not infected by the wild-type virus expressing the gl, more specifically animals vaccinated against ADV with a vaccin not containing or expressing gl (a gl-negative vaccin) .
- animals specifically pigs infected by the wild-type virus from animals not infected by the wild-type virus expressing the gl, more specifically animals vaccinated against ADV with a vaccin not containing or expressing gl (a gl-negative vaccin) .
- the present invention relates to these oligopeptides/expression products themselves, to immunogenic compositions containing one or more of these novel oligopeptides/expression products in a form suitable for immunisation purposes, to the use of the novel oligopeptides/expression products for diagnostic purposes or for producing polyclonal or monoclonal antibodies, and to recombinant genetic information (recombinant polynucleotides, i.e. recombinant DNA or recombinant RNA) coding for the novel oligopeptides/expression products.
- recombinant polynucleotides i.e. recombinant DNA or recombinant RNA
- the virus causes huge losses in pig farms, especially due to fertility problems in sows, high mortality among piglets, and growth retardation among fatteners . All infected pigs form antibodies against the gl of ADV. These antibodies continue to be demonstrable in the blood for many years. Pigs vaccinated with gl-negative vaccins, on the other hand, do not form antibodies against gl of ADV. To reduce economic losses, pigs are being vaccinated againt the disease on a large scale in many countries . However, vaccination alone is not sufficient to eradicate the ADV; the virus can continue to circulate in vaccinated pig populations.
- EP-A- 0 223 382 discloses recombinant DNA containing the genetic information coding for gl of PRV, which consists of 577 amino acids. Although it also refers to fragments of gl exhibiting "pseudorabies virus activity", it does not make clear which fragments of the gl have such "pseudorabies virus activity", or which fragments or parts of the gl are immunologically functional. Both for diagnostic purposes and for the production of polyclonal and monoclonal antibodies with a high specific affinity to gl, it may be useful to use a suitable fragment instead of the complete protein.
- oligopeptides/expression products of the gl which are-capable of generating antibodies in the pig, the natural host of ADV.
- Such oligopeptides/expression products could mean an important advance in the field of diagnostics.
- Such oligopeptides/expression products could be used for a., diagnostic test to differentiate infected pigs from pigs vaccinated with gl-negative vaccins .
- Such oligopeptides/expression products could also be used for the development of a so-called "pig-side" test in accordance with the principle described in an article by Kempt et al. , Scienc 241, 1988, 1352-1354.
- Such a test could be based upon a binding of the antigenic sequence of the oligopeptide/expression product by antibodies against the gl, present in infected animals.
- the oligopeptide/expression product would have to be coupled to a monoclonal antibody directed against pig erythrocytes.
- Another possibility would be for the oligopeptides/expression products to be used for generating an immune response within the framework of a process for producing polyclonal or monoclonal antibodies specific for the antigenic region(s) concerned, and, in turn, suitable for use in a diagnostic test.
- oligopeptide is used herein in a broad sense, and, specifically, should not be construed as being in any way limitative with regard to the way in which it has been obtained.
- the term includes both peptides prepared by chemical synthesis, and products obtained by the expression of relevant genetic information in suitable (micro)biological systems. Therefore, to leave no doubt, the phrase
- oligopeptides/expression products is used in many places in the present text .
- the invention is embodied in an oligopeptide having the following amino acid sequence (according to the three-letter code)
- oligopeptide in question is a relatively small one
- chemical synthesis by techniques known per se
- Production by expression of relevant genetic information in a (micro) biological host is also possible.
- One possible embodiment of the present invention relates to an oligopeptide having the following amino acid sequence (according to the three-letter code)
- Another possible embodiment of the present invention concerns an oligopeptide having the following amino acid sequence (according to the three-letter code) Asn Gly Asp Asp Arg Arg Ala Gly -Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 62-85 o gl of ADV, or an immunologically functional fragment or derivative thereof.
- a possibility within this embodiment is an oligopeptide having the following amino acid sequence (according tQ.the three-letter code) Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 68-85 o gl of ADV, or an immunologically functional fragment or derivative thereof.
- An important preferred embodiment of the present invention concerns an oligopeptide having the following amino acid sequence (according to the three-letter code) Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu Asp Ala Arg Gly ' Asp Gly Ala Val Val Ala Gly lie Trp Thr- Phe Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys Phe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu Ala Pro Glu Arg Gly lie Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gin Arg Glu Pro Pro lie Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu His Asp Ala Ser Gly Pro Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp Arg
- the oligopeptide can be produced by both chemical synthesis and expression. In the case of this oligopeptide, however, production by expression of relevant genetic information is much preferred.
- the phrase "immunologically functional fragment or derivative" is used herein to designate parts or modified forms of the oligopeptide/expres-sion product which are also reactive with antibodies against gl . Fragments of the oligopeptide will be at least eight to nine amino acids long for them to have a specific reactivity with antibodies against gl. In the case of derivatives of the oligopeptide, such modifications of the oligopeptide are permitted that the reactivity is not reduced below one-fifth of the reactivity of the oligopeptide.
- Modifications include, for example, replacing a limited number of amino acids by other amino acids, deleting or adding (e.g. inserting) a limited number of amino acids, and chemical modifications, such as the introduction of substituents at a limited number of amino acids and blocking the terminal amino and carboxyl groups .
- Expression products which in addition to a sequence according to the present invention comprise a foreign protein fragment (i.e., an amino acid sequence not occurring in gl, such as an amino acid sequence of a protein different from gl) , in particular fusion proteins, are "also covered by this terminology.
- the oligopeptide according to the present invention can be synthesized by known per se methods. They can also be obtained, however, by expression (e.g. in bacteria or in eukaryotic cells) of recombinant DNA comprising nucleotide sequences coding for one or more-oligopeptides according to the present invention. Accordingly, the present invention also provides a recombinant polynucleotide comprising a nucleotide sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to the present invention, as defined hereinbefore.
- the invention provides recombinant DN comprising a DNA sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to the present invention.
- a recombinant DNA consisting of a vector portion and an insertion portion containing a DNA sequence coding for an oligopeptide or immunologically-functional fragment or derivative thereof in accordance with this invention is preferred.
- the present invention is further embodied in a host cell genetically engineered with the help of such a recombinant DN to be given the capacity of expressing an oligopeptide or immunologically functional fragment or derivative thereof in accordance with this invention.
- the nature of the host cell i relatively uncritical, and is mainly determined by considerations of production engineering and the available technical possibilities. Generally speaking, microorganisms, in particular bacteria (e.g. E . coli) will be selected, but eukaryotic cells, such as yeast cells, fungi, mammalian cells and plant cells can also be used.
- the present invention also relates to expression products that can be obtained by using such transformed host cells.
- oligopeptides/expression products in question require the oligopeptides/expression products in question to be used in a form in which they are capable of bringing about an immune reaction which includes a production of antibodies against the gl .
- the present invention is therefore further embodied in novel compositions comprising one or more oligopeptides/expression products according to this invention brought into an immunogenic form.
- novel compositions comprising one or more oligopeptides/expression products according to this invention brought into an immunogenic form.
- a first possibility is for the oligopeptides/expression products according to this invention to be coupled to a suitable carrier protein.
- the C-terminus or N-terminus can be used for this purpose. It is well known to those skilled in the art what coupling methods and what carrier proteins are eligible. By way of example, we mention here the possibility of coupling the oligopeptide by means of a suitable coupling agent to KLH (keyhole limpet hemocyanin) or to BSA (bovine serum albumin) . Toxoids and liposomes are also suitable carriers . If desired, the oligopeptide is provided at the N-terminus or at the
- compositions comprising an immunogenic conjugate of a protein and an oligopeptide according to the invention are primarily preferred. Another possibility is for the oligopeptide to be converted by cross-linking into some larger complex, or for the oligopeptide to be expressed by means of recombinant DNA engineering as a part of a (larger) protein.
- the invention is accordingly also embodied in compositions comprising an immunogenic complex or an immunogenic recombinant protein comprising an oligopeptide according to the invention.
- the oligopeptides/expression products according to the invention may be used in combination with one or more carriers and/or adjuvants suitable for immunization purposes to ensure a strong immune response.
- carriers and adjuvants are known per se.
- Carriers such as poly-L-lysine, poly-L-glutamic acid, muramyldipeptide and murabutidine can be included in the composition.
- the invention is naturally also embodied in novel compositions comprising one or more oligopeptides/expression products according to this invention in a form suitable for generating an immune response, whereby antibodies are formed, in combination with at least one immun adjuvant.
- Suitable immuno adjuvants are known to those skille in the art.
- suitable adjuvants are also, for example, aluminium hydroxide and other known adjuvants.
- Diluents such as distilled water, phosphate-buffered saline solutions, and buffer solutions (such as a citrate buffer) , suitable for the administration of the compositions, are also known per se.
- the invention also relates to the use of one or more oligopeptides/expression products according to the invention, or of one or more polyclonal or-monoclonal antibodies obtaine with the use of the oligopeptides/expression prod ⁇ cts for diagnostic purposes. More specifically, the invention include a test of pig sera by means of a set of oligopeptides/expression products according to this invention to determine whether the sera are from a pig infected with th ADV.
- Such tests can be instrumental to the control or eradication of the ADV in infected pig populations.
- Technique suitable for such tests are known per se .
- porcine blood protected against coagulation is contacted with a monoclonal antibody against pig erythrocytes, to which one of the above-described oligopeptides/expression products according to the invention is coupled.
- a monoclonal antibody against pig erythrocytes to which one of the above-described oligopeptides/expression products according to the invention is coupled.
- the blood contains antibodies against the oligopeptide/expression product, an agglutination of the pig erythocytes will take place.
- no agglutination will occur.
- Porcine serum can also be contacted with a nitrocellulos strip on which various oligopeptides/expression products according to the invention (in a purified or non-purified form) have been arranged.
- a labelled (e.g. peroxidase-coupled) anti-pig antibody washing again, and adding a substrate for the peroxidase, colouring can be obtained in the position of the oligopeptide, with which the antibodies present in the sample react.
- the invention is not limited to the oligopeptide 52-85, but includes the oligopeptide/expression product 78-238, and more generally all oligopeptides/expression products based on the amino acids 52-238 of gl which are immunologically functional
- MAbs monoclonal antibodies
- HRPO horse radish peroxidase
- antigenic domain A represented by MAbs 1, 3 and 5
- antigenic domain B represented by MAbs 4, 8 and 11
- antigenic domain C represented by MAbs 6 and 9
- antigenic domain D represented by MAb 7
- antigenic domain E represented by MAb 2
- antigenic domain F represented by MAb 10.
- MAb 9 competed for the binding of MAb 6-HRPO in a 1 : 1000 dilution of the competing MAb
- MAb 6 competed with conjugated MAb 9 only in a 1 : 50 dilution.
- Reciprocal competition between MAb 7 and the MAbs 1, 3 and 5 was only effected in a 1 : 50 dilution of the competing MAb. These results indicate that antigenic domains A and D are closely linked. Reciprocal competition was also found between MAbs 4 and 1 in a 1 : 50 dilution. MAbs 3 and 4 competed non-reciprocally only in a 1 : 50 dilution.
- the pEX plasmids express inserted genes in the form of a cro-B-galactosidase fusion protein.
- the expression of this gene is under the control of the lambda Pr promotor and is induced by inactivation of the temperature-sensitive cl repressor at 42°C.
- Escherichia coli bacteria strain pop 2136, Intitut Pasteur, Paris
- Plasmids linearized by a single digestion were dephosphorylated with calf intestine phosphatase.
- the expression of the pEX fusion proteins was induced by incubation of a 1.5 ml exponential culture (ODgoo about 0.25) of cells at 42°C for 90 min.
- the expression products were purified as follows: cells were spun down (5 min; 6000xg) , re-suspended in 100 ⁇ l 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, ' 15%
- the nitrocellulose sheets were washed twice in phosphate-buffered saline containing 0.5% (w/v) gelatin, 0.1% (w/v) Triton X-100 (PBS-GT) , and incubated with antibodies (MAb 1 : 1000 diluted in PBS-GT) at room temperature for 1 h.
- the filters were washed twice in PBS-GT for 5 min. and incubated with rabbit anti-mouse IgG-HRPO at room temperature for 1 h.
- the filters were washed twice in PBS-GT for-5 min. and once in phosphate-buffered saline for 5 min. , and incubated in substrate solution (3, 3 '-diamino-benzidine 0.5 mg/ml and 0.001% H 2 O 2 ) for approximately 5 min.
- the Ahalll-Nrul fragment was treated- with Nael, whereafter the resulting fragments were cloned, depending on the desired reading frame, in the Smal site of pEXl, pEX2, or pEX3.
- the plasmids p2 (with the sequence coding for the amino acids 52 to 305) , p3 (with the sequence coding for the amino acids 501 to 654) , p4 (with the sequence coding for the amino acids 305 to 411) , p5 (with the sequence coding for the amino acids 411 to 501) , and p6 (with the sequence coding for the amino acids -15 to 52) were obtained.
- MAb 2 (antigenic domain E) and MAbs 6 and 9 (antigenic domain C) only reacted with the fusion protein of plasmid ps2.
- the fusion protein of plasmid ps3 was not recognized by any of the tested gl MAbs (see Table 1) . These findings indicate that the antigenic domains A, B and D are located between the amino acids 52 and 78, and that the antigenic domains C and E are located between the amino acids 78 and 238.
- Overlapping nonapeptides covering amino acids 52-238 of the gl protein were synthesized and tested as described by Geysen et al. All MAbs were tested in the ELISA in a dilution of 1 : 150.
- the results indicate that antigenic domain D is a continuous domain, whereas the antigenic domain A and B are semi-continuous.
- the fact that, according to the immunoscreening, the antigenic domains C and E (belonging to the MAbs 2, 6 and 9) should be located on plasmid ps2, but the MAbs in question do not react with the fusion proteins of the plasmids ps3 and ps4, or with any nonapeptide in the PEPSCAN analysis appears to indicate that a local conformation is required for the recognition between these MAbs and their respective epitopes, which local conformation is present in the fusion protein of plasmid ps2, but not in the fusion proteins of plasmids ps3 and ps4, or in the synthetic nonapeptides .
- Fig. 1 shows the results of the competitive ELISA.
- Fig. 2 shows the cloning strategy of the entire sequence coding for gl and fragments thereof in pEX expression plasmids.
- the fat line indicates the open reading frame of gl.
- the numbers indicate amino acid positions.
- Fig. 3 shows the results of-the PEPSCAN analysis of the 33 nonapeptides covering the amino acids 52 to 84.
- the amino acid sequence is indicated in the one-letter code, with reference to the amino acid position.
- the horizontal lines below the amino acids sequence mark the peptides binding to the MAbs indicated.
- the fat lines indicate the peptides binding strongest to the MA s.
- the vertical lines indicate th OD value resulting from the reaction of the MAbs with the nonapeptides.
- Table 1 reactivity of MAbs with pEX expression proteins containing gl fragments and in the PEPSCAN analysis
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Abstract
The invention relates to novel oligopeptides/expression products derived from glycoprotein I of the Aujeszky's disease virus, to their use for diagnostic purposes or for producing polyclonal or monoclonal antibodies, and to recombinant genetic information coding for the novel oligopeptides/expression products.
Description
Title: Oligopeptides/expression products of the Aujesz y's disease virus, and recombinant polynucleotides coding therefor.
This invention relates to novel oligopeptides/expression products which can be used for diagnostic purposes or for the production of polyclonal or monoclonal antibodies.
More specifically, the present invention relates to nove oligopeptides/expression products derived from glycoprotein I (referred to hereinafter by the conventional abbreviation gl) of the Aujeszky's disease virus (ADV, sometimes referred to a pseudorabies virus or PRV) , and suitable for use in serological diagnostics directed_to the differentiation of animals (specifically pigs) infected by the wild-type virus from animals not infected by the wild-type virus expressing the gl, more specifically animals vaccinated against ADV with a vaccin not containing or expressing gl (a gl-negative vaccin) . The present invention relates to these oligopeptides/expression products themselves, to immunogenic compositions containing one or more of these novel oligopeptides/expression products in a form suitable for immunisation purposes, to the use of the novel oligopeptides/expression products for diagnostic purposes or for producing polyclonal or monoclonal antibodies, and to recombinant genetic information (recombinant polynucleotides, i.e. recombinant DNA or recombinant RNA) coding for the novel oligopeptides/expression products. It is known, for example, from European patent application EP-A-0 223 382 that.the disease of Aujeszky is caused by the herpes virus PRV (or ADV) . The virus causes huge losses in pig farms, especially due to fertility problems in sows, high mortality among piglets, and growth retardation among fatteners . All infected pigs form antibodies against the gl of ADV. These antibodies continue to be demonstrable in the blood for many years. Pigs vaccinated with gl-negative vaccins, on the other hand, do not form antibodies against gl of ADV.
To reduce economic losses, pigs are being vaccinated againt the disease on a large scale in many countries . However, vaccination alone is not sufficient to eradicate the ADV; the virus can continue to circulate in vaccinated pig populations. Therefore, to eradicate ADV in countries where vaccination is practised, it is of crucial importance to have a test to differentiate between infected and vaccinated pigs (in other words, pigs with antibodies against gl and pigs without antibodies against gl) . Tests for making such a distinction have been developed. These tests are based on a detection of antibodies against gl (Van Oirschot et al., J. Gen. Virol. 31, 1986, 1179-1182; Anonymous, Interviews 12., 1987, 25-32; Van Oirschot et al. , J. Virol. Meth. .22., 1988, 191-206; Eloit et al. , Vet.Rec. 124, 1989, 91-94) or a detection of antibodies against glycoprotein X (McMillen and McDonald, Proc. 1st Eur. Congr. Vet. Virol. 1989, p. 38) .
An article by Petrovskis et al. , J. Virol. 3D., 1986, 185- 193, shows that the full amino acid sequence of the glycoprotein gl is known. In the research on which the present invention is based, the sequence shown in this article has been used.
The above-mentioned European patent application EP-A- 0 223 382 discloses recombinant DNA containing the genetic information coding for gl of PRV, which consists of 577 amino acids. Although it also refers to fragments of gl exhibiting "pseudorabies virus activity", it does not make clear which fragments of the gl have such "pseudorabies virus activity", or which fragments or parts of the gl are immunologically functional. Both for diagnostic purposes and for the production of polyclonal and monoclonal antibodies with a high specific affinity to gl, it may be useful to use a suitable fragment instead of the complete protein.
Up until now, no oligopeptides/expression products of the gl have been described which are-capable of generating antibodies in the pig, the natural host of ADV. Such
oligopeptides/expression products could mean an important advance in the field of diagnostics. Such oligopeptides/expression products could be used for a., diagnostic test to differentiate infected pigs from pigs vaccinated with gl-negative vaccins . Such oligopeptides/expression products could also be used for the development of a so-called "pig-side" test in accordance with the principle described in an article by Kempt et al. , Scienc 241, 1988, 1352-1354. Such a test could be based upon a binding of the antigenic sequence of the oligopeptide/expression product by antibodies against the gl, present in infected animals. For—such a test, the oligopeptide/expression product would have to be coupled to a monoclonal antibody directed against pig erythrocytes. Another possibility would be for the oligopeptides/expression products to be used for generating an immune response within the framework of a process for producing polyclonal or monoclonal antibodies specific for the antigenic region(s) concerned, and, in turn, suitable for use in a diagnostic test.
According to the present invention, by means of the so-called PEPSCA method of Geysen et al. (Pxoc.Natl .Acad. Sci. USA £1, 1984, 3998-4002; Proc.Natl.Acad.Sci. USA 32., 1985, 178-182; " Synthetic peptides as antigens", 1986, 130- 149, Wiley; WO 84/03506 and WO 84/03564) and of monoclonal antibodies directed against the glycoprotein I of ADV, reactive sequences located on the glycoprotein I have been found which can be used for the above purposes in the form of oligopeptides/expression products. The present invention is in the first place embodied in an oligopeptide having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu Asp Ala Arg Gly Asp Gly Ala Val Val Ala Gly lie Trp Thr Phe
Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys Phe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu Ala Pro Glu Arg Gly lie Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gin Arg Glu Pro Pro lie Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp Arg Pro Pro Ala Pro Leu Ala Pro Val Gly Pro Ala Arg His Glu Pro Arg Phe His Ala Leu Gly Phe His Ser Gin Leu Phe Ser Pro which sequence corresponds to that of the amino acids 52-238 of the glycoprotein I (gl) of the Aujeszky's disease virus (ADV) , or an immunologically functional fragment or derivative thereof.
The term "oligopeptide" is used herein in a broad sense, and, specifically, should not be construed as being in any way limitative with regard to the way in which it has been obtained. The term includes both peptides prepared by chemical synthesis, and products obtained by the expression of relevant genetic information in suitable (micro)biological systems. Therefore, to leave no doubt, the phrase
"oligopeptides/expression products" is used in many places in the present text .
More specifically, the invention is embodied in an oligopeptide having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 52-85 of gl of ADV, or an immunologically functional fragment or derivative thereof.
As the oligopeptide in question is a relatively small one, chemical synthesis (by techniques known per se) will be a suitable way of obtaining the oligopeptide. Production by expression of relevant genetic information in a (micro) biological host, however, is also possible.
One possible embodiment of the present invention relates to an oligopeptide having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg which sequence corresponds to that of the amino acids 52-67 o gl of ADV, or an immunologically functional fragment or derivative thereof.
Another possible embodiment of the present invention concerns an oligopeptide having the following amino acid sequence (according to the three-letter code) Asn Gly Asp Asp Arg Arg Ala Gly -Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 62-85 o gl of ADV, or an immunologically functional fragment or derivative thereof.
A possibility within this embodiment is an oligopeptide having the following amino acid sequence (according tQ.the three-letter code) Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 68-85 o gl of ADV, or an immunologically functional fragment or derivative thereof. An important preferred embodiment of the present invention concerns an oligopeptide having the following amino acid sequence (according to the three-letter code) Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu Asp Ala Arg Gly 'Asp Gly Ala Val Val Ala Gly lie Trp Thr- Phe Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys Phe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu Ala Pro Glu Arg Gly lie Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gin Arg Glu Pro Pro lie Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp Arg Pro Pro Ala
Pro Leu Ala Pro Val Gly Pro Ala Arg His Glu Pro Arg Phe His Ala Leu Gly Phe His Ser Gin Leu Phe Ser Pro which sequence corresponds to that of the amino acids. 78-238 of gl of ADV, or an immunologically functional fragment oir derivative thereof.
Here again, the oligopeptide can be produced by both chemical synthesis and expression. In the case of this oligopeptide, however, production by expression of relevant genetic information is much preferred. The phrase "immunologically functional fragment or derivative" is used herein to designate parts or modified forms of the oligopeptide/expres-sion product which are also reactive with antibodies against gl . Fragments of the oligopeptide will be at least eight to nine amino acids long for them to have a specific reactivity with antibodies against gl. In the case of derivatives of the oligopeptide, such modifications of the oligopeptide are permitted that the reactivity is not reduced below one-fifth of the reactivity of the oligopeptide. Modifications include, for example, replacing a limited number of amino acids by other amino acids, deleting or adding (e.g. inserting) a limited number of amino acids, and chemical modifications, such as the introduction of substituents at a limited number of amino acids and blocking the terminal amino and carboxyl groups . Expression products which in addition to a sequence according to the present invention comprise a foreign protein fragment (i.e., an amino acid sequence not occurring in gl, such as an amino acid sequence of a protein different from gl) , in particular fusion proteins, are "also covered by this terminology.
The oligopeptide according to the present invention can be synthesized by known per se methods. They can also be obtained, however, by expression (e.g. in bacteria or in eukaryotic cells) of recombinant DNA comprising nucleotide sequences coding for one or more-oligopeptides according to the present invention.
Accordingly, the present invention also provides a recombinant polynucleotide comprising a nucleotide sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to the present invention, as defined hereinbefore.
More specifically, the invention provides recombinant DN comprising a DNA sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to the present invention. A recombinant DNA consisting of a vector portion and an insertion portion containing a DNA sequence coding for an oligopeptide or immunologically-functional fragment or derivative thereof in accordance with this invention is preferred. The present invention is further embodied in a host cell genetically engineered with the help of such a recombinant DN to be given the capacity of expressing an oligopeptide or immunologically functional fragment or derivative thereof in accordance with this invention. The nature of the host cell i relatively uncritical, and is mainly determined by considerations of production engineering and the available technical possibilities. Generally speaking, microorganisms, in particular bacteria (e.g. E . coli) will be selected, but eukaryotic cells, such as yeast cells, fungi, mammalian cells and plant cells can also be used.
As stated before, the present invention also relates to expression products that can be obtained by using such transformed host cells.
Naturally, an application of the oligopeptides/expression products in a process for producing polyclonal or monoclonal antibodies requires the oligopeptides/expression products in question to be used in a form in which they are capable of bringing about an immune reaction which includes a production of antibodies against the gl . The present invention is therefore further embodied in novel compositions comprising one or more
oligopeptides/expression products according to this invention brought into an immunogenic form. As well known to those skilled in the art, there are various methods of bringing a substance which is non-immunogenic per se into an immunogenic form. A first possibility is for the oligopeptides/expression products according to this invention to be coupled to a suitable carrier protein. For a chemical coupling, the C-terminus or N-terminus can be used for this purpose. It is well known to those skilled in the art what coupling methods and what carrier proteins are eligible. By way of example, we mention here the possibility of coupling the oligopeptide by means of a suitable coupling agent to KLH (keyhole limpet hemocyanin) or to BSA (bovine serum albumin) . Toxoids and liposomes are also suitable carriers . If desired, the oligopeptide is provided at the N-terminus or at the
C-terminus with extra amino acid suitable for such a coupling. According to the invention, compositions comprising an immunogenic conjugate of a protein and an oligopeptide according to the invention are primarily preferred. Another possibility is for the oligopeptide to be converted by cross-linking into some larger complex, or for the oligopeptide to be expressed by means of recombinant DNA engineering as a part of a (larger) protein. The invention is accordingly also embodied in compositions comprising an immunogenic complex or an immunogenic recombinant protein comprising an oligopeptide according to the invention.
The oligopeptides/expression products according to the invention may be used in combination with one or more carriers and/or adjuvants suitable for immunization purposes to ensure a strong immune response. Such carriers and adjuvants are known per se. Carriers such as poly-L-lysine, poly-L-glutamic acid, muramyldipeptide and murabutidine can be included in the composition.
Accordingly, the invention is naturally also embodied in novel compositions comprising one or more oligopeptides/expression products according to this invention
in a form suitable for generating an immune response, whereby antibodies are formed, in combination with at least one immun adjuvant. Suitable immuno adjuvants are known to those skille in the art. Among the suitable adjuvants are also, for example, aluminium hydroxide and other known adjuvants.
Diluents, such as distilled water, phosphate-buffered saline solutions, and buffer solutions (such as a citrate buffer) , suitable for the administration of the compositions, are also known per se. The invention also relates to the use of one or more oligopeptides/expression products according to the invention, or of one or more polyclonal or-monoclonal antibodies obtaine with the use of the oligopeptides/expression prodμcts for diagnostic purposes. More specifically, the invention include a test of pig sera by means of a set of oligopeptides/expression products according to this invention to determine whether the sera are from a pig infected with th ADV. Such tests can be instrumental to the control or eradication of the ADV in infected pig populations. Technique suitable for such tests are known per se . We mention here by way of example ELISAs, RIPAs, Western blots and dotblots. According to a more concrete example, porcine blood protected against coagulation is contacted with a monoclonal antibody against pig erythrocytes, to which one of the above-described oligopeptides/expression products according to the invention is coupled. When the blood contains antibodies against the oligopeptide/expression product, an agglutination of the pig erythocytes will take place. In the absence of such antibodie against the oligopetide/expression product, no agglutination will occur.-
Porcine serum can also be contacted with a nitrocellulos strip on which various oligopeptides/expression products according to the invention (in a purified or non-purified form) have been arranged. By successively washing, adding a labelled (e.g. peroxidase-coupled) anti-pig antibody, washing again, and adding a substrate for the peroxidase, colouring
can be obtained in the position of the oligopeptide, with which the antibodies present in the sample react.
The invention is illustrated in and by the following description of the tests carried out and the accompanying drawings. As regards the methodology and measuring methods used, reference is made, for the sake of brevity, to the above-cited publications by Geysen et al. The study included competitive ELISAs, immunoscreening of prokaryotic expression products and PEPSCAN analysis to trace reactive peptides of gl of the ADV. The result was that free binding sites for monoclonal antibodies against gl were found to be located in the N-terminal part of gl, between the amino acids 52 and 85. Accordingly, this part of the gl is an antigenic site on which several epitopes are located. As a consequence, there is a great chance that such a site is recognized by polyclonal antisera. This hypothesis was tested by investigating antisera of pigs which in the known gl-ELISA (Van Oirschot et al., 1988) were positive or negative in an indirect ELISA with oligopeptide 52-85 as an antigen. The sera were tested in the peptide ELISA in a 1 : 10 dilution. Eight positive sera were tested, of which five were field sera and three sera of a pig first vaccinated with a gl-negative vaccin and after three weeks challenged with virulent wild-type NIA-3 virus . The sera were collected at days 15, 18 and 21 after the challenge. Of the six negative sera, five came from the field, and one from an spf pig. The gl-seropositive sera all reacted with the oligopeptide, unlike the gl-seronegative sera. The results showed that pigs infected experimentally or in the field form antibodies specifically reacting with the oligopeptide used. Another result of the study was that two binding sites for monoclonal antibodies against gl were found in the N-terminal part of gl, namely between the amino acids 78 and 238. On the ground of the results of the "conventional" gl-ELISA (articles by Van Oirschot et al.) it was found that the epitopes located in this part are highly immunogenic to the pig. Antibodies against one or both epitopes remain
demonstrable very long after the infection, presumably even during the entire lifetime of the animal. For this reason the invention is not limited to the oligopeptide 52-85, but includes the oligopeptide/expression product 78-238, and more generally all oligopeptides/expression products based on the amino acids 52-238 of gl which are immunologically functional
Experiments conducted
Monoclonal antibodies In the study, use was made of a panel of eleven differen monoclonal antibodies (MAbs) , raised against the pseudorabies virus strains NIA-3 and Phylaxia- The MAbs were purified from ascites fluid by ammonium sulphate precipitation and diluted in phosphate-buffered saline to an ultimate protein concentration of about 7 mg/ml. The MAbs were conjugated with horse radish peroxidase (HRPO) essentially as described by Wilson and Nakane (in the book by Knapp, Holibar and Wick, eds, "Immunofluorescence and related staining techniques", Elsevier, Biochemical Press, Amsterdam, 1978, 215-224) .
Competitive ELISA
Dilution series of MAbs-HRPO conjugates in phosphate- buffered saline containing 0.05% Tween 80 were made to determine the highest dilution of conjugate which gives an OD450 of 1.5-2.0 in the absence of a second non-labelled antibody. Twice that concentration of conjugate was used in the competitive ELISA. Each of the competing unconjugated MAbs were diluted 1 : 50, 1 : 100, and 1 : 1000 in each of the MAb-HRPO dilutions. The mixtures were transferred to the wells (100 μl/well) of an ELISA plate coated with PRV-antigen. After incubation for 1 h at 37°C, the plates were washed with 0.05% Tween in deionized water, and 100 μl substrate
(3, 3 ' , 5, 5 '-tetramethylbenzidine or TMB, 1 mg/ml) was added. The reactions were stopped by adding 100 μl 0.5 M H2SO4, and the plates were read at 450 nm in a TITERTEK multiscan plate reader. The OD450 value in wells without competing antibody wa
set at 100%. A MAb dilution which decreased the OD450 value of the conjugated MAb by >50% was regarded as competitive.
Topographic analysis of the antigenic domains of σl The gl specificity of the MAbs was determined by a radio immuno precipitation assay and by SDS-PAGE (results not shown) . In the competitive ELISA all MAbs were found to inhibit the binding of the homologous MAb-HRPO by more than 50% (Fig. 1) . Using a 1 : 1000 dilution of the competing MAbs, six antigenic domains could be distinguished: antigenic domain A, represented by MAbs 1, 3 and 5; antigenic domain B, represented by MAbs 4, 8 and 11—antigenic domain C, represented by MAbs 6 and 9; antigenic domain D, represented by MAb 7; antigenic domain E, represented by MAb 2, and antigenic domain F, represented by MAb 10. MAb 9 competed for the binding of MAb 6-HRPO in a 1 : 1000 dilution of the competing MAb, whereas MAb 6 competed with conjugated MAb 9 only in a 1 : 50 dilution. Reciprocal competition between MAb 7 and the MAbs 1, 3 and 5 was only effected in a 1 : 50 dilution of the competing MAb. These results indicate that antigenic domains A and D are closely linked. Reciprocal competition was also found between MAbs 4 and 1 in a 1 : 50 dilution. MAbs 3 and 4 competed non-reciprocally only in a 1 : 50 dilution.
Recombinant DNA techniques
Unless mentioned otherwise, the recombinant DNA techniques were carried out essentially as described by Maniatis (in "Molecular Cloning': a Laboratory Manual", Cold Spring Harbor Laboratory, USA, 1982) or by Davis et al. (in "Basic Methods in Molecular Biology", Elsevier, New York, 1986) . Restriction enzymes and DNA modifying enzymes were used as prescribed by the manufacturer. Plasmid DNA was prepared using the alkaline lysis method (Maniatis 1982) . DNA restriction fragments were isolated from agarose gels by electroelution.
Construction of recombinant plasmids
The pEX plasmids express inserted genes in the form of a cro-B-galactosidase fusion protein. The expression of this gene is under the control of the lambda Pr promotor and is induced by inactivation of the temperature-sensitive cl repressor at 42°C. Escherichia coli bacteria (strain pop 2136, Intitut Pasteur, Paris) were transformed after a thermal shock of 5 min at 34°C and 2 min on ice. Plasmids linearized by a single digestion were dephosphorylated with calf intestine phosphatase. The 2055 bpp Ahalll-Nrul fragment (isolated from the BamHI-7 fragment of PRV strain NIA-3, see Quint et al., J.Gen.Virol. 33., 1987, 523-534) with the full gl coding sequence thereon, and fragments derived from the gl coding region were cloned into the correct reading frame into the
S al site of pEXl, pEX2, or pEX3. Fragments were made blunt if neceesary by a treatment with the Klenow fragment of DNA polymerase I .
Isolation of expression products
The expression of the pEX fusion proteins was induced by incubation of a 1.5 ml exponential culture (ODgoo about 0.25) of cells at 42°C for 90 min. The expression products were purified as follows: cells were spun down (5 min; 6000xg) , re-suspended in 100 μl 50 mM Tris-HCl (pH 8.0), 50 mM EDTA,' 15%
(w/v) sucrose and treated with lysozyme (1 mg/ml) for 10 min. After the addition of 140 μl 0.2% (w/v) Triton X-100 in 10 mM Tris-HCl (pH 8) , the DNA was degraded by adding 24 μl 1 M MgCl2 and 1 μl DNAse (10 mg/ml) : Incubation was at 37°C until the suspension was no longer viscous. After the insoluble expression proteins had been spun down, the pellet was resuspended in 250 μl phosphate-buffered saline.
Immunoscreening of expression products A 5 μl sample of resuspended proteins was dissolved in lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 10% glycerol,
2% SDS, 5% 2-mercaptoethanol, Laemmli, Nature 227, 1970, 680- 681) . The proteins were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%), followed by Western-blotting using the LKB-multiphor II Nova Blot system in 40 mM glycine, 50 mM Tris, 0.04% SDS (w/v) and 20% methanol (v/v) . The nitrocellulose sheets were washed twice in phosphate-buffered saline containing 0.5% (w/v) gelatin, 0.1% (w/v) Triton X-100 (PBS-GT) , and incubated with antibodies (MAb 1 : 1000 diluted in PBS-GT) at room temperature for 1 h. The filters were washed twice in PBS-GT for 5 min. and incubated with rabbit anti-mouse IgG-HRPO at room temperature for 1 h. The filters were washed twice in PBS-GT for-5 min. and once in phosphate-buffered saline for 5 min. , and incubated in substrate solution (3, 3 '-diamino-benzidine 0.5 mg/ml and 0.001% H2O2) for approximately 5 min.
Localization of antiσenic domains bv binding of σl-MAbs to gl-specific fusion proteins
An Ahalll-Nrul fragment containing the entire gl coding region was cloned in the proper reading frame into pEX2 plasmid to produce the plasmid pi (see Fig. 2) which contained an insert coding for 685 amino acids . As the insertion was effected at the 3' end of the cro-lac Z gene, expression resulted in a hybrid protein of 115 kDa (cro-B-galactosidase) plus the product of the inserted foreign gene. The predicted size of the expression protein of plasmid pi is 115+80 kDa (see Mettenleiter et al., J. Virol. JL2., 1985, 52-57) . Analysis of the hybrid protein with SDS-PAGE showed a protein band of about 190 kDa (the gel electrophoresis and Western blot results of the hybrid B-galactosidase proteins of the various pEX clones are not shown) . In a Western blot, ten of the eleven MAbs tested turned out to recognize the fusion protein of plasmid pi (Table 1) .
To map the antigenic domains on the protein, the Ahalll-Nrul fragment was treated- with Nael, whereafter the resulting fragments were cloned, depending on the desired
reading frame, in the Smal site of pEXl, pEX2, or pEX3. Thus the plasmids p2 (with the sequence coding for the amino acids 52 to 305) , p3 (with the sequence coding for the amino acids 501 to 654) , p4 (with the sequence coding for the amino acids 305 to 411) , p5 (with the sequence coding for the amino acids 411 to 501) , and p6 (with the sequence coding for the amino acids -15 to 52) were obtained. Only the fusion protein of plasmid p2 turned out to be recognized by the same MAbs that bound to the fusion protein of plasmid pi (containing the entire gl coding sequence) . These results indicated that the antigenic domains A, B, C, D and E were situated between the amino acids 52 and 305. Subsequently, fragments derived from the gl sequence of p2 were cloned in pEX (Fig. 2) . In a Western blot, the same 10 MAbs that recognized the fusion proteins of the plasmids pi and p2 reacted with the fusion proteins of the plasmid psl (containing the sequence coding for the amino acids 52 to 238) . The MAbs 1, 3 and 5 (antigenic domain A) , MAbs 4, 8 and 11 (antigenic domain B) and MAb 7 (antigenic domain D) reacted with the fusion proteins of the plasmid ps4 (containing the sequence coding for the amino acids 52 to 123) , but not with the fusion proteins of the plasmids ps2 (containing the sequence coding for the amino acids 78 to 238) and ps3 (with the sequence coding for the amino acids 123 to 238) . MAb 2 (antigenic domain E) and MAbs 6 and 9 (antigenic domain C) only reacted with the fusion protein of plasmid ps2. The fusion protein of plasmid ps3 was not recognized by any of the tested gl MAbs (see Table 1) . These findings indicate that the antigenic domains A, B and D are located between the amino acids 52 and 78, and that the antigenic domains C and E are located between the amino acids 78 and 238.
PEPSCAN
Overlapping nonapeptides covering amino acids 52-238 of the gl protein were synthesized and tested as described by
Geysen et al. All MAbs were tested in the ELISA in a dilution of 1 : 150.
Localization of the epitopes with the PEPSCAN method The 168 overlapping nonapeptides spanning the amino acids 52 to 238 of the psl plasmid were tested by the PEPSCAN method, using the gl MAbs. MAbs 1, 3 and 5 (antigenic domain A) reacted in the same way with peptides within the amino acid sequences 63-73 and 75-84. MAbs 4, 8 and 11 (antigenic domain B) reacted in the same way with peptides within the amino acid sequence 52-67. MAb 7 (antigenic domain D) recognized peptides within the amino acid sequence 6S-84 (see Fig.3) . MAbs 2, 6, 9 and 10, representing the antigenic domains C, E and F, did not react with any of the peptides investigated. The results indicate that antigenic domain D is a continuous domain, whereas the antigenic domain A and B are semi-continuous. The fact that, according to the immunoscreening, the antigenic domains C and E (belonging to the MAbs 2, 6 and 9) should be located on plasmid ps2, but the MAbs in question do not react with the fusion proteins of the plasmids ps3 and ps4, or with any nonapeptide in the PEPSCAN analysis appears to indicate that a local conformation is required for the recognition between these MAbs and their respective epitopes, which local conformation is present in the fusion protein of plasmid ps2, but not in the fusion proteins of plasmids ps3 and ps4, or in the synthetic nonapeptides .
Description of the drawings Fig. 1 shows the results of the competitive ELISA.
Fig. 2 shows the cloning strategy of the entire sequence coding for gl and fragments thereof in pEX expression plasmids. The fat line indicates the open reading frame of gl. The numbers indicate amino acid positions. Fig. 3 shows the results of-the PEPSCAN analysis of the 33 nonapeptides covering the amino acids 52 to 84. The amino
acid sequence is indicated in the one-letter code, with reference to the amino acid position. The horizontal lines below the amino acids sequence mark the peptides binding to the MAbs indicated. The fat lines indicate the peptides binding strongest to the MA s. The vertical lines indicate th OD value resulting from the reaction of the MAbs with the nonapeptides.
Table 1: reactivity of MAbs with pEX expression proteins containing gl fragments and in the PEPSCAN analysis
PEPSC N l ps2 ps3 ps4
+
+ + + + + +
Claims
1. Oligopeptide having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu Asp Ala Arg Gly Asp Gly Ala Val Val Ala Gly lie Trp Thr Phe Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys Phe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu Ala Pro Glu Arg Gly lie Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gin Arg Glu-Pro Pro lie Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp Arg Pro Pro Ala Pro Leu Ala Pro Val Gly Pro Ala Arg His Glu Pro Arg Phe His Ala Leu Gly Phe His Ser Gin Leu Phe Ser Pro which sequence corresponds to that of the amino acids 52-238 of the glycoprotein I (gl) of the Aujeszky's disease virus (ADV) , or an immunologically functional fragment or derivative thereof.
2. An oligopeptide according to claim 1, having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg
Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 52-85 of gl of ADV, or an immunologically functional fragment or derivative thereof.
3. An oligopeptide according to claim 2, and having the following amino acid sequence (according to the three-letter code)
Ala Gly Asp Asp Asp Leu Asp Gly Asp Leu Asn Gly Asp Asp Arg Arg 19
which sequence corresponds to that of the amino acids 52-67 o gl of ADV, or an immunologically functional fragment or derivative thereof.
4. An oligopeptide according to claim 2, and having the following amino acid sequence (according to the three-letter code)
Asn Gly Asp Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser
Leu Arg Glu Ala Pro Pro Ala His Leu which sequence corresponds to that of the amino acids 62-85 o gl of ADV, or an immunologically functional fragment or derivative thereof.
5 An oligopeptide according fee claim 4, having the following amino acid sequence (according to the three-letter code) Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala Pro Pro
Ala His Leu which sequence corresponds to that of the amino acids 68-85 of gl of ADV, or an immunologically functional fragment or derivative thereof.
6. An oligopeptide having the following amino acid sequence
(according to the three-letter code)
Arg Glu Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu Asp Ala Arg Gly Asp Gly Ala Val Val Ala Gly lie Trp Thr Phe Leu Pro Val Arg Gly Cys Asp Ala Val Ala Val Thr Met Val Cys Phe Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu Ala Pro Glu Arg Gly lie Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg Leu Gin Arg Glu Pro Pro lie Val Thr Pro Glu Arg Trp Ser Pro His Leu Thr Val Arg Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr' Thr Leu His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp.Arg Pro Pro Ala Pro Leu Ala Pro Val Gly Pro Ala Arg His Glu Pro Arg Phe His Ala Leu Gly Phe His Ser Gin Leu Phe Ser Pro which sequence corresponds to that of the amino acids 78-238 of gl of ADV, or an immunologically functional fragment o±r derivative thereof.
7. An oligopeptide according to any one of claims 1-6 in the form of an expression product obtained by expression of genetic information coding for the amino acid sequence of the oligopeptide.
8. A composition comprising at least one oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7, brought into an immunogenic form.
9. A composition as claimed in claim 8, comprising an immunogenic conjugate of a protein and an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7-=-
10. A composition as claimed in claim 8, comprising an immunogenic complex including an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7.
11. A composition as claimed in claim 8, comprising an immunogenic recombinant protein including an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7.
12. A recombinant polynucleotide comprising a nucleotide sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
13. Recombinant DNA comprising a DNA sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
14. Recombinant DNA consisting of a vector portion and an insertion portion containing a DNA sequence coding for an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
15 A host cell genetically engineered with the help of a recombinant DNA according to claim 14 to be given the capacity of expressing an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
16. Microorganisms genetically engineered with the help of a recombinant DNA according to claim 14 to be given the capacit of expressing an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
17. Bacteria genetically engineered with the help of a recombinant DNA according to claim 14 to be given the capacit of expressing an oligopeptide or immunologically functional fragment or derivative thereof according to any one of claims 1-7.
18. Eukaryotic cells genetically engineered with the help of a recombinant DNA according to eiaim 14 to be given the capacity of expressing an oligopeptide or immunologically functional fragment or derivative thereof according to any on of claims 1-7.
19. The use of an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claim 1-7 for diagnostic purposes .
20. The use of an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claim
1-7 for serologically distinguishing animals infected by ADV from animals not infected by ADV.
21. The use of an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claim 1-7 for serologically distinguishing pigs infected by ADV fro pigs not infected by ADV.
22.. The use of an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7 for producing polyclonal or'monoclonal antibodies having specific affinity to gl of ADV.
23. A kit for diagnostic examination for ADV infection, comprising an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7.
24. The use of polyclonal or monoclonal antibodies having a specific affinity to gl of ADV, and obtained using an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7, for diagnostic purposes.
25. A kit for a diagnostic test for ADV infection comprising polyclonal or monoclonal antibodies having a specific affinity to gl of ADV, and obtained using an oligopeptide or immunologically functional fragment or derivative thereof as claimed in any one of claims 1-7.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL8902414A NL8902414A (en) | 1989-09-28 | 1989-09-28 | OLIGOPEPTIDES / EXPRESSION PRODUCTS OF THE AUJESZKY'S DISEASE VIRUS AND CODING RECOMBINANT POLYNUCLEOTIDES. |
| NL8902414 | 1989-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1991004986A1 true WO1991004986A1 (en) | 1991-04-18 |
Family
ID=19855368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/NL1990/000143 WO1991004986A1 (en) | 1989-09-28 | 1990-09-28 | Oligopeptides/expression products of the aujeszky's disease virus, and recombinant polynucleotides coding therefor |
Country Status (2)
| Country | Link |
|---|---|
| NL (1) | NL8902414A (en) |
| WO (1) | WO1991004986A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5631316A (en) * | 1993-09-30 | 1997-05-20 | Exxon Chemical Patents Inc. | Tire innerliner comprising ester-functionalized elastomeric interpolymers of C4-C7 isomonoolefin and para-alkylstyrene |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0223382A1 (en) * | 1985-10-04 | 1987-05-27 | The Upjohn Company | Pseudorabies virus protein |
-
1989
- 1989-09-28 NL NL8902414A patent/NL8902414A/en not_active Application Discontinuation
-
1990
- 1990-09-28 WO PCT/NL1990/000143 patent/WO1991004986A1/en unknown
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0223382A1 (en) * | 1985-10-04 | 1987-05-27 | The Upjohn Company | Pseudorabies virus protein |
Non-Patent Citations (1)
| Title |
|---|
| Virology, volume 171,no. 2, August 1989, Academic Press, Inc., T.A. Kost et al.: "Biological evaluation of glycoproteins mapping to two distinct mRNAs within the BamHI fragment 7 of pseudorabies virus: expression of the coding regions by vaccinia virus", pages 365-376 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5631316A (en) * | 1993-09-30 | 1997-05-20 | Exxon Chemical Patents Inc. | Tire innerliner comprising ester-functionalized elastomeric interpolymers of C4-C7 isomonoolefin and para-alkylstyrene |
Also Published As
| Publication number | Publication date |
|---|---|
| NL8902414A (en) | 1991-04-16 |
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