CN1063109A - Improvement to hormone and associated materials - Google Patents
Improvement to hormone and associated materials Download PDFInfo
- Publication number
- CN1063109A CN1063109A CN92100141A CN92100141A CN1063109A CN 1063109 A CN1063109 A CN 1063109A CN 92100141 A CN92100141 A CN 92100141A CN 92100141 A CN92100141 A CN 92100141A CN 1063109 A CN1063109 A CN 1063109A
- Authority
- CN
- China
- Prior art keywords
- analogue
- fish
- antibody
- amino acid
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940088597 hormone Drugs 0.000 title description 8
- 239000005556 hormone Substances 0.000 title description 8
- 230000006872 improvement Effects 0.000 title description 2
- 239000000463 material Substances 0.000 title description 2
- 241000251468 Actinopterygii Species 0.000 claims abstract description 92
- 238000000034 method Methods 0.000 claims abstract description 61
- NMJREATYWWNIKX-UHFFFAOYSA-N GnRH Chemical class C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CC(C)C)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 NMJREATYWWNIKX-UHFFFAOYSA-N 0.000 claims abstract description 24
- 150000001413 amino acids Chemical group 0.000 claims description 37
- 235000001014 amino acid Nutrition 0.000 claims description 36
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 23
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 claims description 21
- 229960005486 vaccine Drugs 0.000 claims description 20
- 239000000126 substance Substances 0.000 claims description 19
- 230000036039 immunity Effects 0.000 claims description 16
- 230000002441 reversible effect Effects 0.000 claims description 11
- 239000007790 solid phase Substances 0.000 claims description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 125000000539 amino acid group Chemical group 0.000 claims description 7
- 238000005516 engineering process Methods 0.000 claims description 7
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 claims description 5
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 5
- 230000035558 fertility Effects 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 230000036301 sexual development Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 150000008574 D-amino acids Chemical class 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 2
- 238000003379 elimination reaction Methods 0.000 claims description 2
- 230000000670 limiting effect Effects 0.000 claims description 2
- 239000000273 veterinary drug Substances 0.000 claims description 2
- 238000012797 qualification Methods 0.000 claims 7
- 235000009508 confectionery Nutrition 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 230000001568 sexual effect Effects 0.000 abstract description 8
- 235000019688 fish Nutrition 0.000 description 85
- 208000026487 Triploidy Diseases 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- -1 His Chemical compound 0.000 description 7
- 241000277275 Oncorhynchus mykiss Species 0.000 description 7
- 241001327647 Oncorhynchus mykiss gairdneri Species 0.000 description 7
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 150000003431 steroids Chemical class 0.000 description 6
- 241000277331 Salmonidae Species 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 4
- 241000972773 Aulopiformes Species 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 206010047400 Vibrio infections Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 229960005309 estradiol Drugs 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 240000003323 Centaurea nigra Species 0.000 description 3
- 235000002505 Centaurea nigra Nutrition 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 210000004907 gland Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 235000019515 salmon Nutrition 0.000 description 3
- 229960003604 testosterone Drugs 0.000 description 3
- 150000003573 thiols Chemical class 0.000 description 3
- 229960001005 tuberculin Drugs 0.000 description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 2
- 241000607525 Aeromonas salmonicida Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 206010017553 Furuncle Diseases 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 206010036590 Premature baby Diseases 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 241000544286 Vibrio anguillarum Species 0.000 description 2
- 241001135139 Vibrio ordalii Species 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000006480 benzoylation reaction Methods 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 229960003983 diphtheria toxoid Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 210000000750 endocrine system Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 108010074605 gamma-Globulins Proteins 0.000 description 2
- XLXSAKCOAKORKW-UHFFFAOYSA-N gonadorelin Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 XLXSAKCOAKORKW-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 2
- 229940126578 oral vaccine Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000005070 ripening Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- WTPMRQZHJLJSBO-XQALERBDSA-N 11-oxotestosterone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 WTPMRQZHJLJSBO-XQALERBDSA-N 0.000 description 1
- SLAMLWHELXOEJZ-UHFFFAOYSA-N 2-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1[N+]([O-])=O SLAMLWHELXOEJZ-UHFFFAOYSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 241001519451 Abramis brama Species 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- 241000252082 Anguilla anguilla Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 108700012941 GNRH1 Proteins 0.000 description 1
- 240000004859 Gamochaeta purpurea Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 241001417986 Hippoglossoides Species 0.000 description 1
- 101000904173 Homo sapiens Progonadoliberin-1 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000594011 Leuciscus leuciscus Species 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- 241000269908 Platichthys flesus Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 102100024028 Progonadoliberin-1 Human genes 0.000 description 1
- 241000157468 Reinhardtius hippoglossoides Species 0.000 description 1
- 241001481819 Sebastes marinus Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 101000996723 Sus scrofa Gonadotropin-releasing hormone receptor Proteins 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 241001148129 Yersinia ruckeri Species 0.000 description 1
- 240000008866 Ziziphus nummularia Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 235000013527 bean curd Nutrition 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 241001233037 catfish Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 210000003060 endolymph Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960005030 other vaccine in atc Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000003488 releasing hormone Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
-
- 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
Abstract
The invention discloses the method for making fish GnRH analogue, said analogue has following sequence:
pGlu—His—Trp—Ser—Tyr—W—Trp—Leu—Arg—Pro—Gly-X-Y-Z
W wherein, X, Y, Z is defined in the specification.Analogue can be connected on the carrier and and start sexual maturity and improve growth rate in order to for example delay.
Description
The extension analogue that the present invention relates to be called fish gonadotropin-releasing hormone (pGnRH) and be sometimes referred to as the fish hormone of fish luteinizing hormone-releasing hormone (pLHRH).Specifically, the present invention relates to add an analogue that comprises the formed pGnRH of short amino acid sequence of halfcystine or tyrosine residues at the C-terminal of natural pGnRH aminoacid sequence, under the unaltered basically situation of its conformation in solution, make its polypeptide conjugates be suitable for producing anti-pGnRH antibody, the invention still further relates to the method for immune fish.
The marketable fish aquaculture is the industry that develops rapidly all over the world.West Europe, North America and South America, Canada and East Coast all are the main grown places of fish, particularly Oncorhynchi.For example, the U.S. produced 185,000 tons of Oncorhynchi in 1989, be right after thereafter be catfish market.On the other hand, as the Norway of the Atlantic Ocean, West Europe Oncorhynchi major country of production, the output of 1989 to nineteen ninety is 120,000 tons.It is estimated that to the end of this century, Oncorhynchi market, the world increases by 7.5% every year on average, annual volume of business is 2,000,000,000 pounds.Though at present Europe is the main production area of ocean bream and perch, and culturing carp, also imagining ocean perch and big dace flounder in Eastern Europe and Africa, and in the long term to the business development of Hippoglossoides fish and cod.
In aquaculture, need the sexual maturity of control fish especially, so that stop: the somatocyte growth turns to the sexual gland growth, reduces the quality of fish and shows the commercial secondal sexual character of not expecting.Particularly for salmon fishes, precocious samlet and prematurity samlet can be ended to produce when not reaching the product size samlet and be brought serious economic impact, and reduce its marketable value because be eager to gather in the crops samlet in short duration.In addition, by plant's migration and contribute under the situation in natural gene storehouse, expectation suppresses the reproductivity of having cultured fish fish.
From in logic, problem is associated with the sexually matured fish of results.These can not be sold, and always have that a certain proportion of to have early the male fish of ripe advantage be everlasting ripe before other fishes.Want spended time and funds and isolate sexually matured fish from the prematurity fish, in case fish begins maturation, they more or less all will pass through same time and speed, thereby cause difficulty to results.Therefore, find a kind of sophisticated method of fish of controlling simultaneously to have suitable beyond doubt.
For example, since precocious, the ideal weight that usually can not reach the results fish caused, promptly being forced in not too suitable results in period, the control maturation then can be avoided the amount of missing suitable results period and can make fish long to the ideal size.
Up to now, guarantee to be unlikely that sexually matured effective way takes place is to culture sterile fish.Although carried out a large amount of experimental studies, but still just show that not the colony of two state properties realizes this purpose.
Existing several method is used to control the sexual maturity of fish, as comprises operation castration, triploidization and property control (back two kinds of methods are to belong to genetics technology).Past attempts has attempted with surgical method fish being castrated, but causes the damage of fish to abandon because of their incur great expenses and after carrying out this program eventually.
The triploid method is a kind of method that causes three group chromosomes to exist in the fish individuality, and male and female fish is sterile in order to induced triploid.Yet there are several shortcomings relevant with triploid.At first, this is the time-consuming method of trouble that a kind of needs just can be finished for a long time.For making triploid ovum survival, must carry out high-quality treating processes, all be alive though resulting fish be it seems, this survival rate has comprised the average loss amount of 15-20%, the growth velocity of triploid fish is slower than liploid fish beyond spawning season simultaneously.Near the spawning time time, between the male and female triploid very big-difference is arranged, wherein patrogenesis can be near normal inspection standard and all relevant second features, and female triploid does not then grow tangible ovary, and shows immature outward appearance when normal spawning.
Successfully use the inductive triploid and require to share only female technology (female-only technique), and this commercial be non-remunerative.The shortcoming of other relevant with triploid is, they and diploid compete in same pond and cause growing bad, and the growth of the female fish of triploid does not demonstrate and surpasses the female fish of ripe diploid.
Fish is the height two condition, and promptly they can be in a male and female change, and can controlled other change.For culturing purpose, as the viewpoint from the speed of growth, a kind of sex is normally preferable.Turbot and common eel are female than male example of growing greatlyyer.In samlet, childhood, male and female fish had the similar same speed of growth, but male afterwards fish has grown the feature that does not expect to have.In the ripening stage, male stopping growing, the brown and the love that becomes are picked a quarrel gradually, and its value as food and toy is just very poor.In addition, the rainbow trout (Salmo gairdneri) of raising and Atlantic Ocean Oncorhynchi ripe male in case put into the seawater cultivation pool, just can not be adapted to salt solution and cause death.Can use a kind of generation sex reversing, to obtain the method for all female colonies, but having comprised, it in fish meal, mixes hormone, make fish be not suitable as the food on the dining table, method such as illumination, the chemical sterilization of other control fish fertilizabilitys and to give hormone also all unreliable, uneconomical or be not suitable for the fish of production as food.
Controlled sophisticated immunological method is obtained some success on one's body Mammals.Yet, can not become the solid foundation of studying sashimi (raw fish) Neo-Confucianism to the research of mammalian hormones and immunological response.Fish is a cold blooded animal, and their immunity system can variation to some extent under different condition.Temperature promptly has remarkably influenced to the immune response of fish, shows higher immune response level when consequently in physiological range comparatively high temps being arranged.As if physiological low temperature can postpone the speed of antibody generation and can suppress the foundation of immunological memory, responsive especially to this T lymphocyte.
In addition, the immunity system of fish is subjected to the influence of the hormone that may rationally change.Hormonal readiness can and be arrested equipressure or quality such as influence that oxygen level is low etc. changes because of carrying.Go into sea and sexual maturing period (being two basic hormone change periods) at the troutlet effluent, fish is particularly responsive to disease because body endolymph cell number reduces.
Moreover fish only produces one type antibody molecule, and promptly IgM has five big antibody-likes in definite mammalian body.
The endocrine system that is exactly fish in addition is different in essence in mammiferous endocrine system, and for example the fish gonadotropin release by salmon flying fish such as Oncorhynchi and the generation of fermented red beancurd fish has following sequence:
PGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH
2(Sherwood et al, 1983; " Characterisation of a teleost GnRH ", PNAS USA80:2794-2798), the amino acid on its 7th and 8 is different from Mammals LHRH.
We have now found that, the analogue of a class fish GnRH can be connected on the appropriate carriers molecule and be used for immune fish, so can induce to produce the immunogenic antibody of anti-cross reactivity, the latter to GnRH have high-affinity and can in and endogenous GnRH.
According to an aspect of the present invention, we provide the analogue that the fish of following amino acid sequences GnRH is arranged:
pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z
Wherein W represents Gly or a D-amino acid
X represents a key, or one or more identical or different amino acid,
Y represents Cys or Tyr or does not have Y, and
Z does not exist or represents one or more identical or different amino acid, or 1 continuous or discrete peptide chain, and each discontinuities exists non-alpha amino acid multifunctional access head part or reverse conversion amino acid, condition be when W be Gly, when X and Y do not exist, have Z.
The residue at W place, position is Gly preferably, and it is naturally occurring residue; The residue at Y place, position is Cys preferably, because it is usually more special, and than being easier to receive on the carrier proteins with the C-terminal Tyr person of obtaining.
X preferably represents relatively small number purpose residue, as 1 to 15, better is 1 to 8, is more preferably 1 to 3, preferably has only one.Though can have any one or a plurality of amino acid on the X position, this amino-acid residue is Gly preferably.Certainly, other amino acid better be to have the amino acid of little side chain such as Ala, Ser, Asn and Val also to may reside on the X position, and this locational any amino-acid residue include within the scope of the invention.Be also to be noted that to comprise more than one identical or different residue on the X position, for example in following sequence:
pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-Gly-Ala-Y-Z。
Should be clear and definite be not have the pGnRH of amino-acid residue to be also included within the scope of the present invention on the X position.
In the preferred embodiment of the pGnRH according to the present invention, Z does not have amino-acid residue on the position, and has following sequence according to the particularly preferred example of analogue of the present invention:
pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Arg-Pro-Gly-Gly-Cys。
Yet we studies show that, comprise on the Z position when adding amino acid generally the conformation of molecule rest part being selected not have a significant effect.If the Z position has comprised such extension, so in preferred version of the present invention, this extension will comprise can be as one or more small segments of the protein sequence of T cell epitope.For example, the small segment of the aminoacid sequence of general formula 1-2-3-4, wherein 1 is that Gly or charged amino acid are (as Lys, His, Arg, Asp or Glu), the 2nd, hydrophobic amino acid is (as Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), the 3rd, hydrophobic amino acid (being defined as above) or uncharged polare Aminosaeren are (as Asn, Ser, Thr, Pro, Gln, Gly), and the 4th, polare Aminosaeren is (as Lys, Arg, His, Glu, Asp, Asn, Gln, Ser, Thr, Pro), as if can be used as T cell epitope (Rothbard, J.B.﹠amp at least some instances; Taylor, W.R, (1988), A sequence pattern in common to T-cell epitopes, The EMBO Journal7(1): 93-100).Similar small segment can be sequence 1 '-2 '-3 '-4 '-5 ', wherein 1 ' 1,2 ' suitable 2,3 ' and 4 that are equivalent to limit above ' be equivalent to 3,5 ' ibid to be equivalent to 4().Two kinds of forms are included in the general formula that limits below: pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-S-T-S ', wherein the T representative contains the peptide sequence small segment (most preferably less than 5 amino acid) of one or more T cell epitopes, it can be type or other structure that limits in the preceding paragraph, and can be separated by any length or the spacer small segment formed, S and S ' (wherein each can suitably and individually be left out) then represent the spacer small segment by any length and the aminoacid sequence formed.The length of S is most preferably less than 5 amino-acid residues, and comprises the residue that for example is selected from Gly, Ala, Pro, Asn, Thr, Ser or multifunction conjunction such as non-alpha amino acid.S ' is most preferably less than 10 amino-acid residues and can comprise multifunction conjunction as non-alpha amino acid; Draw in the amino acid of the S representative that S ' part preferably provides from above.Might represent a whole protein by S ', therefore just not necessarily will be connected on the carrier proteins.
It should be noted, when Z does not exist, can on the X position, provide T cell epitope above-mentioned.
The derivative of basic analogue also is included in the scope of the invention, and wherein Z is or comprises " oppositely reversing " amino acid.The bifunctional amine who promptly has the functional group that is equivalent to certain amino acid.For example one according to the present invention and comprise the amino acid whose analogue of reverse reversing and can have following general formula:
Wherein R is any functional group, is more preferably Gly, and A1 and A2 are more preferably the copy (but not necessarily identical) of one of analogue that this paper of being connected by its C-terminal limits separately.In particularly preferred embodiments, locational Tyr of Y or Cys should be replaced by the amino acid of any kind beyond the residue on the corresponding position of another copy among one of A1 or A2, and perhaps it can be left out.Therefore this dimeric connection is to realize by the side chain of one of residue on the Y position (also can be two residues).In addition, A1 and/or A2 can be natural pGnRH, and oppositely to reverse amino acid whose R can be Cys or Tyr.As previously discussed, the T cell epitope also can be included among the small segment Z.
Analogue can have following general formula:
Wherein B oppositely reverses amino acid, and A1 and A2 are identical or different, and show sequence under having:
pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z′
Wherein the definition of W, X and Y is the same, and Z ' is identical with aforesaid Z.Just it does not comprise reverse reversing amino acid.
Reverse reversing modification to peptide comprises that the one or more peptide bonds of reversing are to produce the analogue that enzyme liberating is had bigger resistance than initial molecule, and provide a kind of approach easily for producing branch's immunogen, for in wait until big immunogen, this branch's immunogen will contain the epitope of high density.In the reverse solution in enormous quantities that reverses analogue of short chain biologic activity peptide synthesizes, use these compounds to have very big potential value.
Another aspect of the present invention provides the method for making the fish GnRH analogue with following amino acid sequences:
pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z，
Wherein W represents Gly or D-amino acid,
X represents a key, or one or more identical or different amino acid,
Y represents Cys or Tyr, or does not exist, and
Z does not exist, or represents one or more identical or different amino acid, or continuous or discrete peptide chain, and each discontinuities exists the multifunctional shank of non-alpha amino acid or the amino acid that oppositely reverses, condition be when W be Gly, and there is Z in X and Y when not existing,
This method comprises to be used known chemistry, biology and/or each residue of recombinant technology coupling and separates this analogue.
Can use the peptide synthetic technology of standard, synthesize analogue of the present invention as the solid phase method of peptide synthesis (SPPS) that Merrifield introduces in 1963 (J.Am.Chem, Soc.85,2149).In this method, the peptide chain of growth is covalently bound to insoluble solid phase carrier by its C-terminal.Synthesize by in expected sequence, adding amino acid continuously.In this case, the intermediate steps of all purifying (these steps are for synthetic being absolutely necessary in the solution) all is reduced to simple flushing, because the by product of the Degradation of great majority reaction all has been dissolved in the reaction mixture.The advantage of SPPS is rapidly, relatively easily to finish and is a kind of partly or entirely automated method, uses SPPS might produce the peptide of tens grams.
Briefly, the SPPS by the Merrifield development in 1963 comprises following four-stage:
(A) by in entire synthesis process, all keeping stable covalent linkage, first protected amino acid is connected on the solid phase carrier;
(B) under all stable condition of the blocking group of any side chain functionalities, produce the free amine group acid groups of the residue that is connected again by going to protect;
(C) form amido linkage by condensation, make next protected amino acid and first amino acid coupling.Repeating step B and C finish up to building-up process;
(D) when synthetic finishing, by discharging peptide on the solid phase carrier and removing any amino acid side chain blocking group.
Describe the combination of various amino acid blocking groups in the document, but at present commonly used had only wherein two kinds of combinations.First kind is tert butoxy carbonyl (Boc)/benzyl composition, and it was just used by Merriifield from 1963, and still was widely adopted so far.Be surprisingly found out that recently fluorenyl methoxy phosphinylidyne (the Fmoc)/tertiary butyl system that is developed by people such as Sheppard has also obtained using widely (Sheppard, R.C, 1986, Science Tools, The LKB Instrument Journal33,9).Further example comprises by Arsady; R. wait the Fmoc-polymeric amide method (J.Chem of the use solid-phase resin of people's foundation; Soc.Perkin Trans; 1981,1,529-537); indivedual amino acid (the Atherton that are impregnated in fluorenyl methoxy phosphinylidyne (Fmoc) protection; E.et al, J.Chem, Soc.Perkin Trans; 1983; 1,65-73) and 9-fluorenyl methoxy phosphinylidyne (Fmoc) chemical method (as referring to Atherton, E.et al; 1985; J.Chem.Soc.Chem, Comm, 165).
In addition, the DNA and the RNA molecule of the sequence by genetic code coding each peptide analogs described herein are also included within the scope of the invention.These dna moleculars can be used, in order to produce analogue as herein described in suitable microorganism or eukaryotic cell culture expression system.In such system, synthesized similar molecule and in this document, addressed (as referring to No. 85307311.2, european patent application).
It should be noted, can not use such system directly to make the analogue that mixes reverse reversing amino acid derivative.But can be further purified basic analogue and use the peptide/organic chemistry method of standard to be connected on the reverse reversing amino acid.Describe on polyamides amine type resin solid phase synthesis recently and oppositely reversed the practical of peptide and novel method [Gazerro, H, Pinori, M.﹠amp easily; Verdini, A.S.(1990), A new general Procedure for the solid-phase synthesis of retro-inverso peptides, In " Innovation and Perspective in Solid Phase Synthesis " Ed.Roger Epton.SPC(UK) Ltd.Birmingham, UK].
Preferably analogue is connected on the carrier molecule, to obtain the analogue binding substances.Any small molecules or macromolecular carrier or microorganism can be used, but preferably following carrier commonly used: the key hole
The tuberculin albumen microorganism (PPD) of hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma Globulin (CGG), Toxoid,tetanus (TT), diphtheria toxoid (DPT), purifying, Muramyl dipeptide (MDP), soybean trypsin inhibitor (STT) and Toxins,exo-, cholera or its B subunit.It should be noted that and using under the situation of PPD, with the BCG vaccine cause more favourable (Lachmann, P.et al, 1986, In " Synthetic Peptides as Antigens ", Ciba Foundation Symposium119, pp.25-40).
The carrier of used LHRH analogue during as oneself immunization fish particularly preferably is following microorganism or macromole antigen as vaccine composition: aeromonas salmonicida (Aeromonas salm onici da) (furuncle ulcer disease), Yersinia ruckeri(ERM) and Vibrio anguillarum (Vibrio anguillarium) and Vibrio ordalii(vibriosis) mixture.
Recently, tested the sick effect (latter is caused by the non-bacterium aeromonas salmonicida that moves about of Gram-negative) of anti-furuncle ulcer of following five base type vaccines: whole being killed or destructive cell (vaccine), extracellular products (ECP) and ECP toxoid, whole killed cell ECP share, the antigen of live attenuated vaccine and purifying.Anti-some antigenic hyper-immuneserum that also will produce in fish and mammalian body in addition, is used for passive protection.In addition, successfully produced the vaccine (or vaccine) of anti-redmouth enteritis (enteric redmouth ERM) (this disease is caused by the Gram-negative bacterium Yersinia rukeri that moves about), it comprises the full bacterial cultures of formalin deactivation.The national on the Northern Hemisphere recently commodity vibriosis vaccine that uses comprises the mixture of the most common bacterial classification Vibrio anguillarum and V.ordalii.These vaccines are the cultures of simple deactivation that contain the mixture of full cell and ECP.
Another aspect of the present invention provides the pGnRH analogue that contains with good grounds the present invention or analogue binding substances and the veterinary drug vaccine with vehicle.
These vaccines can be used for oneself immune fish, and one side more of the present invention provides a kind of method that piscinity is grown that suppresses, this method comprises pGnRH analogue or the analogue binding substances that uses significant quantity to said fish, so that the anti-pGnRH antibody titers that is produced is enough to significantly reduce the biological efficiency of endogenous pGnRH.
Can use reagent and the method fully described in the document, make the binding substances that contains the analogue of Cys or Tyr on the Y position at an easy rate.For example, can use N-γ-maleoyl-imino-butyryl acyloxy succinimide (Calbiochem), by thioester bond the fish GnRH analogue that contains the Cys residue on the Y position is connected on the lysine side-chain of carrier protein, simultaneously can with the N-(4-benzene diazonium)-maleimide forms and is connected the diazonium key that the Y position contains the analogue of Tyr residue.
Can use methods known in the art to be easy to peptide and binding substances are purified to necessary degree, these methods are as comprising high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and FPLC.
Peptide-carrier binding substances of the present invention can be used for following purpose:
(1) immune any animal is used for the anti-fish GnRH antibody of following described specific needs with generation:
(2) fish is carried out oneself immunity of anti-fish GnRH, so that:
-the sexual development of slowing down, stop or disappearing
-control, restriction or elimination fecundity
-treatment GnRH dependency fish tumour
-carry out sashimi (raw fish) Neo-Confucianism/veterinary science research;
(3) as detecting as whether there being the diagnostic reagent of anti-fish GnRH antibody in the serum sample.
Anti-fish GnRH antibody at analogue also constitutes a part of the present invention.For example they can
1) as research sashimi (raw fish) Neo-Confucianism/immunologic research tool
2) as the immunogen that produces anti-alloantibody;
3) carry out passive immunization, so that:
-be implemented in a short time the sexual development of slowing down, stop or disappearing
-reach and control fecundity in a short time
-treatment GnRH dependency fish tumour
-disturb the activity of fish GnRH in addition.
(can carry out passive immunization) by polyclone, mono-clonal or the single area antibody that gives in fish or mammalian body, to produce
4) as the diagnostic reagent that whether has fish GnRH in test example such as the serum sample.
Obviously, above 2) in the anti-alloantibody mentioned also constitute a part of the present invention.
Can according to ordinary method preparation can with the chimeric form of the polyclone of synthetic polypeptide specific combination of the present invention or monoclonal antibody, these antibody or the distinctive form of fish (as referring to Morrison et al, (1984) PNAS(USA) 81,6851-6855; Riech-mann et al, (1988) Nature 332,323-327), and single area antibody (as referring to Ward, E.S, Gussow, D.Griffiths, A.D., Jones, P.and Winter, G.(1989) Nature 341,544-546).As mentioned above, these antibody also are considered to a part of the present invention.
Aspect anti-fish GnRH antibody of detection or fish GnRH, those skilled in the art can use various known immunoassay technologies, as using competition or competitive inhibition detection method or direct and indirect labelling method.The medicine box of the anti-fish GnRH of a kind of preferred detection antibody has comprised analogue of the present invention or analogue binding substances, marking tool and solid phase carrier.Similarly, a kind of medicine box that detects fish GnRH comprised can with antibody or Fab, marking tool and the solid phase carrier of the present invention's analogue specific combination.
Can use fish vaccine through several method, as injection, oral route and by submergence etc.
Injection is the most effectual way of vaccine inoculation in the abdomen control, simultaneously the adjuvant that improves the immune response degree easy to use.Shortcoming is the respective handling that fish need be anaesthetized, and will make the fish withstanding pressure like this and strengthen labour intensity.If but used syringe and the line production system that reuses, 1000 fishes per hour could be injected.But this is unsuitable for the fish less than 15 grams.
The oral vaccine inoculation is suitable for the fish administration to all size in enormous quantities, and the pressure that does not cause because of operation.Shortcoming is to need to use a large amount of vaccines, thereby has improved cost, and the using dosage of every fish of bad control.It is poor that oral vaccine is renderd a service, and causes the level of provide protection low and non-constant, and most of oral administrations are all at the vibriosis vaccine.
Preferred directly immersion method (D.I) is a kind of simple and rapid method, only need contact several seconds with vaccine.This method has now realized automatization and set up the schedule of operation of " bath " and " washing " conversion at vibriosis and ERM vaccine, and only comprises simply vaccine is poured in the accumulator tanks.Also need long duration of contact (about one hour, comprise the oxygenation of water and the pressure that close monitoring fish is subjected to) though this method will consume more vaccine, its labour intensity is littler than injection.
With containing (I) and the known analogue of more than one type of carrier molecule bonded, and/or the mixed vaccine submergence of (II) more than one analogues that combine with carrier molecule equally may be more favourable.In addition, can be with any peptide analogs, their binding substances and composition thereof is added in any suitable adjuvant or the release system and uses, and more than one adjuvant and release system can be lumped together, and forms so-called " super mixture ".Preferred adjuvants and release system comprise aluminium hydroxide (alum), microsphere, liposome, micella, rrna, ISCOMS, Brauns lipoprotein and full cell or microorganism fish vaccine composition.
Further describe the present invention by non-limiting example below.
Embodiment 1
Use the carboxyl terminal of the synthetic pGnRH of the present invention of standard solid-phase Fmoc method to extend form.In the presence of the trifluoroacetic acid by resin on cleavage of peptide, carry out purifying with gel-filtration, ion exchange chromatography and reverse high performance liquid chromatography (HPLC) then.By MBS(m-maleimide benzoyl-N-hydroxy-succinamide phenyl ester) peptide is connected on the various carriers.
Proved that chicken gamma Globulin (CGG) is a kind of good carriers for other vaccines of hardhead trout, and be used in the present embodiment.Also used PPD, because this is a kind of confirmed carrier (although needing to use BCG before immunity) in mammiferous experimental study.
With peptide: protein is 30-40: 1 molar ratio is coupled to the pGnRH analogue on the carrier, and does intraperitoneal injection with three different concentration to rainbow trout (Salmo gairdneri).
Yet the monoclonal anti trout immunoglobulin (Ig) that uses sour jujube root peroxidase (HRP) to connect is monitored serum antibody response with euzymelinked immunosorbent assay (ELISA) (ELISA), surveys once totally 10 weeks weekly.In some test of present embodiment,, or carry out booster shots with pre-immunity of carrier or use adjuvant.To determine to produce the optimum immuning program of anti-pGnRH antibody.
Embodiment 2
Treat sophisticated hardhead trout for one group by the optimum immuning program immunity of determining among the embodiment 1.With the monitoring of method described in the embodiment 1 antibody response, monitor the generation of the speed of growth and sex steroid (as testosterone) simultaneously.When experiment finishes fish is killed, remove gonadectomy and weigh, with determinacy body of gland index (GSI).Fixing sexual gland then is so that carry out Histological research with opticmicroscope.The fish that immunity is crossed has obviously good GSI than untreated control group fish.
Embodiment 3
Extend the rainbow trout (Salmo gairdneri) of analogue (sGnRHa-PPD) immune mature with the salmon gonadin releasing hormone on the protein derivatives that is coupled to the tuberculin purifying.
Method
A) sGnRH-Gly-Cys is connected on the PPD
In the benzoylation cellulose tube, make tuberculin PPD(1.008mg/ml+ phenol sanitas, Evans Limited) to the 0.9%NaCl liquid of under 12 ℃, dialysing, to remove phenol.Use polyoxyethylene glycol (pG20000) to handle then 1.5 hours, PPD is concentrated to 4.032mg/ml.At room temperature make PPD solution (2.5ml, containing 10.08mg PPD mixed 1 hour with 1.08mgN-γ-maleoyl-imino-butyryl acyloxy succinimide (GMBS) in 5.04ul dimethyl formamide (DMF), go up Sephadex G-25M PD-10 post then, and with 3.5ml buffer A (0.05M NaH
2PO
4, 0.14M NaCl, pH7.0) wash-out.Be dissolved in sGnRH-Gly-Cys that 4.687ml concentration in the distilled water is 3mg/ml and buffer A (that is, and 14,0616mg SGnRH-Gly-Cys dropwise is added among the PPD.Under nitrogen environment with solution mixed at room temperature 2 hours, and the free thiol content of estimation from start to finish.In the benzoylation cellulose tube, make binding substances to 12 ℃ of liquid of dialysing of distilled water, be stored in-20 ℃ then and descend standby.
B) free thiol analysis
Use reagent 5,5 ' one or two sulphur two (2-nitrobenzoic acid) (DTNS) and go back the free thiol content of ortho states gsh standard substance estimation binding substances.Use the microtiter plate variant of scaled Ell-manShi assay method, wherein in 50 μ l samples, add 2 μ l10mMDTNB and 250 μ l PBS(pH8), after 10 minutes in 405nm place mensuration light absorption ratio.
C) hardhead trout and immune programme for children
Use freezing printing tools difference 130 ripe rainbow trout (Salmo gairdneri)s of mark (the about 30cm of length) of various combinations.Write down length and the weight of every fish and get 600 μ l blood samples mensuration pre-immune serum antibody horizontal.In addition, detect the serum steroid levels, to determine the sex of fish.Fish is divided into two groups, and the sGnRH-Gly-Cys-PPD or the salt solution that are added in respectively in the Freund's complete adjuvant (FCA) are done intraperitoneal injection.Tell treatment group and control group male and female respectively again.Male fish is placed under light and the temperature condition.Femalely further be divided into two groups again: one group is placed under ambient light and the temperature condition (temperature can change) between 14 ℃ or lesser temps, and another group then places under ambient light and the 14 ℃ of conditions.Estimate the influence of temperature antagonism sGnRH-Gly-Cys antibody titers and any difference that in the fish maturation, causes with the one group of fish in back.In the whole experiment, with circumferentially-spaced the fish of each bar stamp is weighed and to detect and bloodletting.With the anti-sGnRH-Gly-Cys antibody (using sGnRH-Gly-Cys-BSA) in the ELISA method detection serum, and use RIA method detection type steroid hormone 17-and/or 11-ketone testosterone (11-ketotestosterone).
D) steroid analysis
Detect the concentration of 17-in the ripe fish serum (E2) and 11-ketone testosterone (11-KT) with radioimmunology.In this analysis, the steroid of radioactivity mark is attached on the limited amount steroid specific antibody, this interaction can partly be suppressed because of adding unlabelled steroid.
E) optimizing of anti-sGnRH-Gly-Cys elisa assay
The salmon class GnRH-Gly-Cys that will be attached on the bovine serum albumin (BSA) is used for the elisa assay method.The 10mgBSA that is added in the 0.5ml buffer A is merged with the 1mgGMBS that is added among the 5 μ lDMF, be coupled on the 6.246mgsGnRH-Gly-Cys at last.ELISA checkerboard detection method can be selected to be suitable for carrying out the sGnRH-Gly-Cys-BSA bag of anti-sGnRH-Gly-CysELISA analysis by concentration in test of maturity.
The result
ⅰ) anti-sGnRH-Gly-Cys antibody titers
(male and female) as shown in table 1 increased anti-sGnRH-Gly-Cys antibody titers with sGnRH-Gly-Cys-PPD immunity (2 μ g sGnRH-Gly-Cys/ fishes are added in intraperitoneal injection among the FCA) 8 weeks of back with respect to control group.
ⅱ) the level of rainbow trout (Salmo gairdneri) ripening period serum 17-β estradiol: with the influence of sGnRH-Gly-Cys-PPD immunity
As shown in table 2, in 4 weeks after immunity, the serum 17-β estradiol level of treatment group and control group adult female trout increases at the same rate.To immunity the 8th week of back, the 17-β estradiol level of control group still continues to increase at the same rate.But in 8 weeks after immunity, the sGnRH-Gly-Cys-PPD group in the ripe raun then comparison has much lower 17-β estradiol level (P＜0.05:t test) according to forming ripe raun.
ⅲ) the mutual relationship between antibody titers and steroid levels
Obviously, after the sGnRH-Gly-Cys-PPD immunity, the increase of antibody titers is that a small amount of increase with ripe rainbow trout (Salmo gairdneri) serum 17-level is mutually related, and data show that pGnRH-Gly-Cys-PPD is active and effective in postponing female rainbow trout (Salmo gairdneri) sexual maturity.
Claims (20)
1, make the method for the fish GnRH analogue that following amino acid sequences is arranged:
pGIu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z
Wherein W represents Gly or D-amino acid,
X represents a key, or one or more identical or different amino acid,
Y represents Cys or Tyr or does not exist, and
Z does not exist or represents one or more amino acid that can be identical or different, or continuous or discrete peptide chain, and each discontinuities exists the multifunctional shank of non-alpha amino acid or the amino acid that oppositely reverses, condition be when W be Gly, when X and Y did not have, Z existed,
This method comprises to be used known chemistry, biology and/or each residue of recombinant technology coupling and separates this analogue.
2, according to the process of claim 1 wherein that W is Gly.
3, according to the method for claim 1 or 2, wherein Y is Cys.
4, according to the method for aforementioned each claim, wherein X is one or more sweet amino-acid residues.
5, according to each method in the claim 1 to 3, wherein X or Z comprise at least one T cell epitope.
6, according to each the method in the aforementioned claim, wherein Z comprises reverse reversing amino acid.
7, according to the method for claim 6, wherein analogue has following general formula:
A
1-B-A
2
Wherein B oppositely reverses amino acid, A
1And A
2Be identical or different, and have following sequence:
pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z′
Wherein W, X and Y are as limiting in the claim 1, and Z ' is with the Z that limits in the claim 1, but it does not comprise reverse reversing amino acid.
8, according to the process of claim 1 wherein that analogue has sequence: pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Arg-Pro-Gly-Gly-Cys.
9, according to each the method in the aforementioned claim, it comprises analogue is connected on the carrier molecule.
10, make the method for vaccine, wherein with the analogue binding substances and the veterinary drug mixed with excipients that limit in each limited in the claim 1 to 8 analogue or the claim 9.
11, the medicine box of anti-fish GnRH antibody in the test sample, said medicine box comprise the analogue that limits in each of aforesaid right requirement 1 to 8, or the analogue binding substances, marking tool and the solid phase carrier that limit in the claim 9.
12, the analogue that limits in each of use claim 1 to 9 prepares the medicine of slow down, stop or disappearing piscinity growth or control fish fecundity.
13, according to the method for each or claim 8 in the claim 1 to 5, wherein analogue produces as peptide chain continuously with recombinant technology.
14, make specifically in conjunction with the antibody of the analogue of each qualification in the claim 1 to 7 or the method for Fab, this method comprises the analogue binding substances immunity that limits with the analogue of each qualification in the claim 1 to 8 or claim 9 by immune object, and separates the cell of formed antibody or generation antibody.
15, the medicine box of fish GnRH in the test sample, said medicine box comprise antibody or Fab, marking tool and the solid phase carrier that limits in the claim 14.
16, be used to make as the antibody that limits in the claim 15 or Fab and slow down, stop or the sexual development of the fish of disappearing or the medicine of control fish fecundity.
17, make method to the special anti-alloantibody of the antibody that limits in the claim 14 or Fab, this method comprises with the antibody that produces in accordance with the method for claim 14 or Fab immunity by immune object and separate formed antibody.
18, fish is carried out anti-fish GnRH immunity, to slow down, to stop or disappearing sexual development, or the method for control, restriction or elimination fecundity, this method comprises to fish throws analogue or the analogue binding substances of claim 9 qualification or antibody or the Fab that claim 14 limits that gives each qualification in the significant quantity claim 1 to 8.
19, according to the method for claim 18, wherein fish is immersed in the water of antibody that analogue binding substances that the analogue that contains each qualification in the claim 1 to 8 or claim 9 limit or claim 14 limit or Fab.
20, according to the method for claim 18, wherein the antibody or the Fab of the analogue binding substances of analogue that each limited in the claim 1 to 8 or claim 9 qualification or claim 15 qualification are that oral administration gives.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9100468.9 | 1991-01-09 | ||
| GB919100468A GB9100468D0 (en) | 1991-01-09 | 1991-01-09 | Improvements in and relating to hormones |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1063109A true CN1063109A (en) | 1992-07-29 |
Family
ID=10688207
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92100141A Pending CN1063109A (en) | 1991-01-09 | 1992-01-09 | Improvement to hormone and associated materials |
Country Status (17)
| Country | Link |
|---|---|
| EP (1) | EP0566611A1 (en) |
| JP (1) | JPH06504537A (en) |
| KR (1) | KR930703444A (en) |
| CN (1) | CN1063109A (en) |
| AU (1) | AU652611B2 (en) |
| CA (1) | CA2100057A1 (en) |
| DK (1) | DK80993A (en) |
| FI (1) | FI933138A0 (en) |
| GB (2) | GB9100468D0 (en) |
| HU (1) | HUT66829A (en) |
| IE (1) | IE920055A1 (en) |
| IS (1) | IS3802A (en) |
| NO (1) | NO932491D0 (en) |
| NZ (1) | NZ241240A (en) |
| PT (1) | PT99991A (en) |
| WO (1) | WO1992012247A1 (en) |
| ZW (1) | ZW392A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU667578B2 (en) * | 1992-08-27 | 1996-03-28 | Deakin Research Limited | Retro-, inverso-, and retro-inverso synthetic peptide analogues |
| US5688506A (en) | 1994-01-27 | 1997-11-18 | Aphton Corp. | Immunogens against gonadotropin releasing hormone |
| US5760000A (en) * | 1994-05-13 | 1998-06-02 | University Technologies International,Inc. | Inhibition of liver cancer by the use of GnRH and GnRH analogs |
| AU1765300A (en) * | 1998-12-22 | 2000-07-12 | Dalhousie University | Compositions and methods for reducing or preventing fertilization in fish and birds |
| GB0226179D0 (en) | 2002-11-09 | 2002-12-18 | Millar Robert P | Vaccine |
| NO20075894L (en) * | 2007-11-15 | 2009-05-18 | Thia Medica As | Reduced maturation in fish |
| JP6909160B2 (en) * | 2015-11-27 | 2021-07-28 | 日本水産株式会社 | Fish feed using cell-penetrating peptide and cell-penetrating peptide |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4443368A (en) * | 1982-11-01 | 1984-04-17 | The Salk Institute For Biological Studies | Peptides affecting gonadal function |
| US4410514A (en) * | 1982-12-06 | 1983-10-18 | The Salk Institute For Biological Studies | GnRH Agonists |
| US4608251A (en) * | 1984-11-09 | 1986-08-26 | Pitman-Moore, Inc. | LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues |
| GB8713240D0 (en) * | 1987-06-05 | 1987-07-08 | Proteus Biotech Ltd | Hormones |
| CA2112566A1 (en) * | 1991-07-01 | 1993-02-18 | Graham J. Moore | Non-desensitizing analogs of gnrh and other biologically active ligands |
-
1991
- 1991-01-09 GB GB919100468A patent/GB9100468D0/en active Pending
-
1992
- 1992-01-07 NZ NZ241240A patent/NZ241240A/en unknown
- 1992-01-08 JP JP4502565A patent/JPH06504537A/en active Pending
- 1992-01-08 IS IS3802A patent/IS3802A/en unknown
- 1992-01-08 IE IE005592A patent/IE920055A1/en not_active Application Discontinuation
- 1992-01-08 KR KR1019930702060A patent/KR930703444A/en not_active Application Discontinuation
- 1992-01-08 WO PCT/GB1992/000040 patent/WO1992012247A1/en not_active Application Discontinuation
- 1992-01-08 AU AU11617/92A patent/AU652611B2/en not_active Ceased
- 1992-01-08 CA CA002100057A patent/CA2100057A1/en not_active Abandoned
- 1992-01-08 EP EP92902189A patent/EP0566611A1/en not_active Withdrawn
- 1992-01-08 HU HU9301979A patent/HUT66829A/en unknown
- 1992-01-08 PT PT99991A patent/PT99991A/en not_active Application Discontinuation
- 1992-01-09 ZW ZW3/92A patent/ZW392A1/en unknown
- 1992-01-09 CN CN92100141A patent/CN1063109A/en active Pending
-
1993
- 1993-07-06 DK DK080993A patent/DK80993A/en not_active Application Discontinuation
- 1993-07-08 NO NO932491A patent/NO932491D0/en unknown
- 1993-07-08 GB GB9314141A patent/GB2267496B/en not_active Expired - Fee Related
- 1993-07-08 FI FI933138A patent/FI933138A0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| GB9100468D0 (en) | 1991-02-20 |
| NO932491L (en) | 1993-07-08 |
| AU652611B2 (en) | 1994-09-01 |
| NZ241240A (en) | 1992-11-25 |
| HU9301979D0 (en) | 1993-11-29 |
| FI933138A (en) | 1993-07-08 |
| PT99991A (en) | 1993-01-29 |
| CA2100057A1 (en) | 1992-07-10 |
| FI933138A0 (en) | 1993-07-08 |
| GB9314141D0 (en) | 1993-09-22 |
| KR930703444A (en) | 1993-11-30 |
| HUT66829A (en) | 1995-01-30 |
| GB2267496A (en) | 1993-12-08 |
| JPH06504537A (en) | 1994-05-26 |
| DK80993D0 (en) | 1993-07-06 |
| NO932491D0 (en) | 1993-07-08 |
| EP0566611A1 (en) | 1993-10-27 |
| ZW392A1 (en) | 1992-07-22 |
| DK80993A (en) | 1993-07-06 |
| GB2267496B (en) | 1994-09-07 |
| AU1161792A (en) | 1992-08-17 |
| IE920055A1 (en) | 1992-07-15 |
| IS3802A (en) | 1992-07-10 |
| WO1992012247A1 (en) | 1992-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5114713A (en) | P. falciparum cs-peptides as universal t-cell epitope | |
| RU2422460C2 (en) | Recovered peptide exhibiting specific anti-gdf-8 antibody binding activity, recovered nucleic acid molecule, expression vector, host cell, method for producing peptide, vaccine composition and method of eliciting anti-gdf-8 immune response, method of anti-gdf-8 antibody selection screening and method of gdf-8 activity down-regulation in animal | |
| US20070161545A1 (en) | Triple polypeptide complexes | |
| CN1063109A (en) | Improvement to hormone and associated materials | |
| CN107073086B (en) | Immune LHRH composition and application thereof in pigs | |
| EP0450715B1 (en) | Immunogenic compounds, the process for their synthesis and their use in the preparation of antimalaria vaccines | |
| HUT52787A (en) | Process for production of biologically active peptides | |
| AP71A (en) | Peptide structures, immunogens containing them and their uses in the control of fertility. | |
| JP2715472B2 (en) | Cyclic peptide | |
| WO1989000166A2 (en) | Growth hormone related peptide | |
| RU2034457C1 (en) | Method of agriculture animal productivity increase, preparation for its realization | |
| NZ255529A (en) | Antigenic molecules equivalent to part of a natural growth hormone, antibodies therefrom and pharmaceutical compositions | |
| AU2002339227A1 (en) | Triple polypeptide complexes | |
| US20060188513A1 (en) | Novel inhibin-related multiple antigenic peptide compositions that enhance production performance in avians | |
| CN1219548C (en) | Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody | |
| CA2329755A1 (en) | Synthetic somatostatin immunogen for growth promotion in farm animals | |
| CN1455781A (en) | Discrimination between GnRH-I and GnRH-II | |
| CN1090584A (en) | Synthetic polypeptide | |
| NO177858B (en) | Peptide | |
| HU210966B (en) | Method for the preparation of peptides and veterinary compositions containing them | |
| CS272790A2 (en) | Peptides with biological effect | |
| WO1996030405A1 (en) | Peptides with growth promotion properties | |
| MXPA00011752A (en) | Synthetic somatostatin immunogen for growth promotion in farm animals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |