CN103865884A - Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof - Google Patents
Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof Download PDFInfo
- Publication number
- CN103865884A CN103865884A CN201210530208.5A CN201210530208A CN103865884A CN 103865884 A CN103865884 A CN 103865884A CN 201210530208 A CN201210530208 A CN 201210530208A CN 103865884 A CN103865884 A CN 103865884A
- Authority
- CN
- China
- Prior art keywords
- dhav
- strain
- viral hepatitis
- duck
- duck viral
- 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
- 241000272525 Anas platyrhynchos Species 0.000 title claims abstract description 138
- 206010019799 Hepatitis viral Diseases 0.000 title claims abstract description 70
- 201000001862 viral hepatitis Diseases 0.000 title claims abstract description 65
- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 241000700605 Viruses Species 0.000 claims abstract description 101
- 241001557656 Duck hepatitis A virus 1 Species 0.000 claims abstract description 41
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 238000003756 stirring Methods 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229960005486 vaccine Drugs 0.000 claims abstract description 22
- 235000013601 eggs Nutrition 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- 241000287828 Gallus gallus Species 0.000 claims abstract description 19
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 14
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 14
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 239000000427 antigen Substances 0.000 claims abstract description 11
- 102000036639 antigens Human genes 0.000 claims abstract description 11
- 108091007433 antigens Proteins 0.000 claims abstract description 11
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 239000012153 distilled water Substances 0.000 claims abstract description 9
- 235000013345 egg yolk Nutrition 0.000 claims abstract description 8
- 230000002779 inactivation Effects 0.000 claims abstract description 8
- 238000005070 sampling Methods 0.000 claims abstract description 6
- 210000003278 egg shell Anatomy 0.000 claims abstract description 5
- 238000001471 micro-filtration Methods 0.000 claims abstract description 5
- 238000009928 pasteurization Methods 0.000 claims abstract description 5
- 239000012530 fluid Substances 0.000 claims abstract description 3
- 241001557661 Duck hepatitis A virus 3 Species 0.000 claims abstract 7
- 238000006386 neutralization reaction Methods 0.000 claims description 30
- 101710197658 Capsid protein VP1 Proteins 0.000 claims description 24
- 101710132601 Capsid protein Proteins 0.000 claims description 22
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 claims description 22
- 101710108545 Viral protein 1 Proteins 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 19
- 101150024766 VP1 gene Proteins 0.000 claims description 18
- 230000009849 deactivation Effects 0.000 claims description 18
- 230000036039 immunity Effects 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 13
- 210000002257 embryonic structure Anatomy 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 12
- 230000002265 prevention Effects 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000005352 clarification Methods 0.000 claims description 5
- 238000004821 distillation Methods 0.000 claims description 5
- 239000011229 interlayer Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 108091035707 Consensus sequence Proteins 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 18
- 230000003053 immunization Effects 0.000 abstract description 11
- 238000002649 immunization Methods 0.000 abstract description 10
- 230000000521 hyperimmunizing effect Effects 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000003306 harvesting Methods 0.000 abstract description 3
- 230000003472 neutralizing effect Effects 0.000 abstract description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 abstract 2
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 abstract 1
- 230000000249 desinfective effect Effects 0.000 abstract 1
- 230000020477 pH reduction Effects 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 37
- 238000012360 testing method Methods 0.000 description 35
- 208000006454 hepatitis Diseases 0.000 description 25
- 231100000283 hepatitis Toxicity 0.000 description 25
- 235000001014 amino acid Nutrition 0.000 description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 18
- 238000010255 intramuscular injection Methods 0.000 description 18
- 239000007927 intramuscular injection Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 17
- 238000002347 injection Methods 0.000 description 15
- 239000007924 injection Substances 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 14
- 235000013330 chicken meat Nutrition 0.000 description 13
- 239000003921 oil Substances 0.000 description 13
- 238000003757 reverse transcription PCR Methods 0.000 description 13
- 238000010790 dilution Methods 0.000 description 12
- 239000012895 dilution Substances 0.000 description 12
- 230000003612 virological effect Effects 0.000 description 12
- 230000005847 immunogenicity Effects 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 239000002574 poison Substances 0.000 description 10
- 231100000614 poison Toxicity 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 241001557655 Duck hepatitis A virus 2 Species 0.000 description 8
- 125000003275 alpha amino acid group Chemical group 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 8
- 230000009182 swimming Effects 0.000 description 8
- 230000000890 antigenic effect Effects 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000013207 serial dilution Methods 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- 208000009701 Embryo Loss Diseases 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 238000004945 emulsification Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000012797 qualification Methods 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000009736 wetting Methods 0.000 description 5
- 241000709664 Picornaviridae Species 0.000 description 4
- 108020000999 Viral RNA Proteins 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 239000008098 formaldehyde solution Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 208000032843 Hemorrhage Diseases 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229940031567 attenuated vaccine Drugs 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000000384 rearing effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 2
- 241000469171 Duck astrovirus Species 0.000 description 2
- 241001451766 Duck astrovirus 1 Species 0.000 description 2
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 2
- 206010019842 Hepatomegaly Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 101710106388 Structural protein VP1 Proteins 0.000 description 2
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical class [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 206010030899 opisthotonus Diseases 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 206010049244 Ankyloglossia congenital Diseases 0.000 description 1
- 241001651353 Avihepatovirus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010015856 Extrasystoles Diseases 0.000 description 1
- 241000709715 Hepatovirus Species 0.000 description 1
- 206010020565 Hyperaemia Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010048469 Kidney enlargement Diseases 0.000 description 1
- 101710175243 Major antigen Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000027645 antigenic variation Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 210000002298 blastodisc Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000013016 learning Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32421—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Communicable Diseases (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a duck viral hepatitis bivalent yolk antibody and a preparation method thereof, wherein the bivalent yolk antibody comprises a duck viral hepatitis DHAV-1 type antibody and a duck viral hepatitis DHAV-3 type antibody. The preparation method comprises: (1) adopting a duck viral hepatitis DHAV-1 type strain and a duck viral hepatitis DHAV-3 type strain to respectively vaccinate SPF chicken embryo and susceptible duck embryo, harvesting allantoic fluid, mixing the harvested virus liquids according to a certain ratio, carrying out formaldehyde inactivation, and preparing a vaccine; (2) adopting the vaccine to immunize laying hens, sampling after immunization to determine whether the neutralizing titer of the anti-DHAV-1 type antigen antibody and the anti-DHAV-3 type antigen antibody in the chicken hyperimmune egg yolk is more than or equal to 1:8192, and collecting the hyperimmune egg of the chicken; and (3) disinfecting the eggshell of the hyperimmune egg, collecting the egg yolk, adding the equal volume of distilled water, uniformly stirring and mixing, carrying out low temperature pasteurization inactivation, adopting an acidification distilled water method to purify, adopting an octanoic acid method to purify, and carrying out micro-filtration and ultra-filtration. The duck viral hepatitis bivalent yolk antibody has characteristics of low cost and high titer, and can be provided for effectively controlling duck viral hepatitis caused by DHAV-1 and DHAV-3 so as to obtain significant social benefits.
Description
Technical field
The present invention relates to a kind of high IgY Isolated from Immunized Egg Yolk, specifically, relate to a kind of yolk antibody of duck viral hepatitis.
Background technology
Duck viral hepatitis (Duck Virus Hepatitis, DVH) is that the duck hepatitis virus (Duck Hepatitis Virus, DHV) that belonged to by Picornaviridae enterovirus causes that the one of duckling is acute, height lethality, deadly infectious disease.This disease occurs throughout the year, only infects 3 week duckling in age under state of nature, often causes great financial loss, is threatening the sound development of foster duck industry.OIE (OIE) incorporates duck viral hepatitis into category-B Animal diseases into.
Duck hepatitis virus has and is divided in history serotype I type, II type and III type, there is no antigen dependency between three.Wherein I type duck viral hepatitis is worldwide distribution.II type duck viral hepatitis is nineteen sixty-five to be found in Britain, and after 20th century, the mid-80 broke out, this area also has no the generation of this disease again.III type is found in In Changdao Area, USA New York for 1969.II type, III type have been re-classified as duck Astrovirus member at present, are called duck Astrovirus 1 type (duck astrovirus 1, DAstV-1), duck Astrovirus 2 types (DAstV-2).2009, (the The International Committee on Taxonomy of Viruses of ICTV, ICTV) the new fowl hepatovirus (Avihepatovirus) of setting up in Picornaviridae, traditional serum I type duck hepatitis virus belongs to DHAV (DHAV) wherein, be called as DHAV-1, DHAV also comprises that two other serotype is DHAV-2, DHAV-3, DHAV-2 and DHAV-3 respectively with DHAV-1 serum-free cross-neutralization.Wang L etc. (2008) are referred to as A type (DHAV-A), Type B (DHAV-B), C type (DHAV-C) by gene type.Research shows, the genotype of distinguishing according to capsid protein encoding sequence exists corresponding relation with the serotype that adopts neutralization test to distinguish.
Chinese scholars has been carried out research with regard to the epidemic status of DHV serotype.2007, Tseng broke out bacterial isolate in the sick duck body of duck viral hepatitis from 1989-1990 Taiwan, prove a strain New Type Duck Hepatitis Virus by neutralization test, and N-DHV(can be called DHAV-2), be only found at present Taiwan.2007, Korea S Kim etc. were separated to a strain virus within 2003 and 2004, breaking out in the sick duck body of duck viral hepatitis, different from the serotype of DHV-I, are called DHV-AP(and are called again DHAV-3).
In the past China popular be mainly I type duck hepatitis virus, adopt the yellow antibody of traditional attenuated vaccine and high-immunity egg to carry out prevention and the treatment of I type DHV.But in recent years, constantly there is the duck group who uses I type duck hepatitis attenuated vaccine or the yellow antibody of high-immunity egg to prevent or to fail to respond to any medical treatment to break out doubtful duck hepatitis, suspected for Novel duck hepatitis.And in recent years, Korea S novel (DHAV-3) distributes comparatively extensive in the area of China.(separation of novel DHV and the preliminary evaluation such as Su Jingliang, China animal doctor science and technology, 2002,32(1): 15 ~ 16) through pathogen separation qualification and the serum neutralization test of duck embryo, be separated to the picornavirus of learning cross-immune reaction with DHV-I, DHV-III serum-free, be called New Type Duck Hepatitis Virus; (separation of New Type Duck Hepatitis Virus and the preliminary evaluation such as yellow Anguo, Guangxi animal and veterinary, 2003,19(5): 198 ~ 199) also from the morbidity duck group of the similar I type duck viral hepatitis that can not cure or prevent with the yellow antibody of I type duck viral hepatitis high-immunity egg or I type duck hepatitis attenuated vaccine, be separated to New Type Duck Hepatitis Virus.(novel DHV epidemiology survey and the immune protection test such as Liu Jian subsequently, China animal doctor magazine, 2006,42 (2): 3 ~ 6) from ground censorships such as Beijing, Hebei, Shandong and Guangxi and have the duckling liver of the hemorrhage characteristics of lesion of obvious liver and be separated to 9 strain virus strains, by it is carried out to serological identification, find that wherein 7 strains are New Type Duck Hepatitis Virus, 2 strains are I type duck hepatitis virus.These results of study just can disclose many regional ducklings in recent years and after generation duck viral hepatitis, inject hyper-immune serum or the invalid reason of the yellow Antybody therapy of high-immunity egg.
Result of study has shown China multiple areas popular DHAV-1 and DHAV-3 simultaneously, and lacks cross protection between the two, and the caused DVH of DHAV-3 is also lacked to effective measure of control.
Summary of the invention
To produce the upper active demand to the effective Prevention Technique of DVH in order meeting, to control in time the generation of domestic DVH, the present invention utilizes traditional I type (DHAV-1) duck hepatitis virus and novel (DHAV-3) duck hepatitis virus to prepare divalence yolk antibody.
For this reason, the invention provides a kind of duck viral hepatitis virus strain, the sequence of described virus strain VP1 gene coding amino acid comprise as shown in Figure 6 there are 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D.
Preferably, described duck viral hepatitis virus strain is duck viral hepatitis virus SD1 strain, and described SD1 strain preservation address is Chinese Typical Representative culture collection center, and deposit number is CCTCCNO.V201225.
The invention provides a kind of DNA sequence dna, it contains in fact the nucleotide sequence of seq No.1, term in the present invention " contains in fact " and refers to that nucleotides sequence of the present invention is listed in and keep in the scope of its function, and sequence number 1 base sequence can replace, insert or the variation such as disappearance.
The invention provides a kind of VP1 antigen protein, described VP1 antigen protein comprises the duck viral hepatitis virus VP 1 consensus sequence with 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D as shown in Figure 6.
Main purpose of the present invention is to provide a kind of duck viral hepatitis divalence yolk antibody, and described divalence yolk antibody comprises duck viral hepatitis DHAV-1 type and duck viral hepatitis DHAV-3 type yolk antibody.Described antibody titer is high and cost is lower, can effectively control the duck viral hepatitis of domestic popular different serotypes at present.
Another object of the present invention is to provide a kind of preparation method of duck viral hepatitis divalence yolk antibody, described method safety, be easy to use, described preparation method comprises:
(1) adopt duck viral hepatitis DHAV-1 type strain and DHAV-3 type strain to inoculate respectively 9 age in days SPF chicken embryos and 10 age in days susceptible duck embryos, then gather in the crops allantoic fluid, the virus liquid of described results is mixed in proportion, formalin-inactivated is also prepared vaccine;
(2) use the vaccine immunity laying hen of described preparation, after immunity sampling measure anti-DHAV-1 type in chicken high-immunity egg Huang, the neutralization of DHAV-3 type antigen-antibody and tire >=1: 8192, collect afterwards the high-immunity egg of described chicken;
(3) by described high-immunity egg eggshell sterilization, collect yolk, described yolk adds equal-volume distilled water, stirring and evenly mixing, low temperature pasteurization; Acidifying distillation water law is purified, sad method is purified; Micro-filtration, ultrafiltration.
Preferably, described duck viral hepatitis DHAV-1 type strain is that DRL-62 strain, described DHAV-3 type strain are SD1.
Preferably, described immune programme for children is: when described laying hen 120 age in days, intramuscular injection 1ml/ only, within 14th, wait afterwards dosage intramuscular injection, described two exempt to exempt from waiting dosage intramuscular injection to carry out three after 10 days again, and described three exempt from latter 10 days again with waiting dosage intramuscular injection to carry out reinforced immunological the 4th time.
Preferably, described low temperature pasteurization condition is 62.5 DEG C of heat inactivation 30min;
Preferably, described acidifying distillation water law purification step is: first in interlayer retort, add the sterile purified water that is equivalent to 6 times of volumes of described yolk, described distilled water pH value is 4.2, and be cooled to 4 DEG C, then, the described egg yolk liquid of deactivation is added, stir, 4 DEG C of standing 4h, centrifugation supernatant liquor;
Described sad method purification step is: it is sad to add by 0.2% volume of described supernatant liquor total amount, stirs, and room temperature is placed 4h, is filtered to clarification, then adds saturated formaldehyde solution by 0.1% of described filtrate total amount, stirs, and room temperature is placed 24h.
Preferably, described micro-filtration uses 0.22 μ m micropore filter element filtration sterilization, and the ultrafiltration membrance filter that described ultrafiltration is 1000KDa with molecular weight cut-off is except virus.
Still a further object of the present invention has been to provide the application of duck viral hepatitis divalence yolk antibody in the medicine of preparation prevention and treatment duck viral hepatitis.
An also object of the present invention has been to provide the vaccine composition of a kind of prevention and treatment duck viral hepatitis, and what described vaccine composition comprised immunity amount comprises the prepared antigen of duck virus hepatitis strains and the pharmaceutically acceptable carrier with 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D as shown in Figure 6 by VP1 gene coding amino acid gene order.
Preferably, described duck viral hepatitis virus is SD1 strain.
The vaccine composition of described prevention and treatment duck viral hepatitis uses this area ordinary method preparation.
The application of vaccine composition described in the present invention also provides in the medicine of preparation prevention and treatment duck viral hepatitis.
Technique effect
1. the present invention has adopted the multiple inactivation technology such as physics, chemistry, use acidified water dilution method in conjunction with modern biotechnologies such as sad method, high speed centrifugation, ultrafiltration, immunoglobulin (Ig) in yolk is carried out to effective separation and purification, in yolk antibody, do not contain any objectionable impurities and exogenous pollution, safe, when injecting immune duck group on carcass quality without impact, can suitability for industrialized production.
2. the duck virus hepatitis strains that the present invention obtains, there is good immunogenicity, the inactivated vaccine of preparation, antibody generation speed is fast, and antibody titer is high, and security is good, the yolk antibody of preparing with this can be prevented and treated infecting of DHAV-3 C-type virus C effectively, preservation, transport (2 ~ 8 DEG C) and easy to use, result of use is stable, the generation of prevention and control duck viral hepatitis effectively.
3. when contriver obtains in institute the VP1 gene order of duck virus hepatitis strains SD1 strain, surprisingly find that VP1 gene coding amino acid gene order wherein comprises as shown in Figure 6 there is 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D have vital role to its immunogenicity.Prove in subsequent embodiment, other duck virus hepatitis strains VP1 gene order corresponding identical site that suddenlyd change, also has same effect.
4. divalence yolk antibody provided by the invention, cost is low, the height of tiring, experimentation on animals shows that the duck viral hepatitis that can cause DHAV-1 and DHAV-3 accomplishes effective control, can obtain significant social benefit.
Brief description of the drawings
Fig. 1 is for utilizing DHV I type type strain primer RT-PCR to detect DRL-62 strain and SD1 strain VP1 result;
Fig. 2 is for utilizing DHV Korea S novel strain primer RT-PCR to detect DRL-62 strain and SD1 strain VP1 result;
Fig. 3 is SD1 VP1 gene and the analysis of DHV Reference strains nucleotide sequence homology;
Fig. 4 is SD1 VP1 gene and the analysis of DHV Reference strains amino acid sequence homology;
Fig. 5 is SD1 VP1 gene and DHV Reference strains nucleotide sequence evolutionary tree;
Fig. 6 is SD1 VP1 gene and DHV Reference strains aminoacid sequence compare of analysis;
Fig. 7 is SD1 VP1 albumen wetting ability, antigenic index, surperficial accessibility analysis.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation method of duck viral hepatitis divalence yolk antibody adopts the breeding of following step: duck hepatitis virus DHAV-1, DHAV-3 kind poison, the preparation of immunity vaccine, the immunity of laying hen, the extraction of yolk antibody.In the embodiment of the present invention, selected DHAV-1 strain is DRL-62 strain, and DHAV-3 strain is SD1 strain, and its concrete grammar is:
1. the source duck hepatitis virus DRL-62 strain (ATCC preservation, preserving number VR-1313) of seed culture of viruses; SD1 strain is separated by Pulaike Biological Engineering Co., Ltd., (CCTCC preservation, preserving number: CCTCC NO.V201225).
2. seed culture of viruses breeding is done dilution in 1: 100 by duck hepatitis virus DRL-62 strain seed culture of viruses with sterilizing PBS solution, through allantoic cavity inoculation 9 ~ 11 age in days SPF chicken embryos, every embryo 0.1ml.Put 37 DEG C and continue to hatch, discard the dead germ in 48 hours, gather in the crops 48～96 hours dead chicken embryos, put 2～8 DEG C cooling 12～24 hours.After blastochyle aseptic inspection is mixed, quantitative separating, freezing preservation.Dated harvest date, Virus passages etc.
Duck hepatitis virus SD1 strain seed culture of viruses is done to dilution in 1: 100 with sterilizing PBS solution, through allantoic cavity inoculation 10 ~ 12 age in days susceptible duck embryos, every embryo 0.2ml.Put 37 DEG C and continue to hatch, discard the dead germ in 48 hours, gather in the crops 48～96 hours dead duck embryos, put 2～8 DEG C cooling 12～24 hours.After duck blastochyle aseptic inspection is mixed, quantitative separating, freezing preservation.Dated harvest date, Virus passages etc.
3. the preparation of deactivation vaccine for immunity:
The results of 3.1 virus liquids are by centrifugal 30 minutes 3500 revs/min of the virus liquids of results, draw supernatant liquor in sterilising vessel, add 10% formaldehyde solution, with adding with shaking, it is fully mixed, the final concentration of formaldehyde solution is 0.15%, in airtight rearmounted 37 DEG C of incubators, acts on 24 hours (jolting during this time 3～4 times).
3.2 immunity are prepared with the oil phase of preparing of deactivation vaccine: get 94 parts of injection white oils, after part mixing of Si Ben-806, add 2 parts of aluminum stearates, heat while stirring for subsequent use to transparent rear autoclaving, be oil phase.Water preparation: DRL-62 strain antigen is mixed with the ratio of 1:1 with SD1 strain antigen, add sterilizing tween-80 by 4% of antigen, shake well, dissolves tween-80 completely, is water.Emulsification: get 2 parts of oil phases and put in oil phase tank, start motor and stir, slowly add 1 part of water, then with 3400 revs/min of emulsifications 10 minutes.Before emulsification stops, add 1% Thiomersalate, making its final concentration is 0.01%.Sample 3000 revs/min centrifugal 15 minutes, should be not stratified.Packing: quantitative separating, seals.
4. immunity
4.1 immune programme for children laying hen 120 1ml/ of deactivation oil seepage intramuscular injection for age in days left and right, after 14 days, use deactivation vaccine intramuscular injection 1ml/ only, two exempt from only to carry out three with deactivation vaccine intramuscular injection 1ml/ again after 10 days exempts from, and three exempt from only to carry out reinforced immunological the 4th time with deactivation vaccine intramuscular injection 1ml/ again in latter 10 days.
4.2 adopt egg measures DRL-62 antibody in chicken high-immunity egg Huang, the neutralization of SD1 antibody every sampling on the 10th and tires from four exempt from >=and 1: 8192 for qualified, can collect afterwards high-immunity egg, puts 4 DEG C of storages for subsequent use.
5. the preparation of yolk antibody
Eggshell is sterilized in results and the deactivation of 5.1 yolk, takes craft or machinery to beat eggs.Fully remove egg white (in vain), blastodisc and frenulum, collect yolk.Fully stir and make yolk be even paste, add equal-volume through 121 DEG C of 30min sterilizings cooling distilled water, stirring and evenly mixing, 62.5 DEG C of heat inactivation 30min.
The acidifying distillation water law of 5.2 antibody is purified first in interlayer retort, to add and is equivalent to the sterile purified water that the pH value of 6 times of volumes of former yolk is 4.2, and is cooled to 4 DEG C.Then, the egg yolk liquid of deactivation is added, limit edged stirs, 4 DEG C of standing 4h.Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also proceeds in another retort.
It is sad that the sad method purification of 5.3 antibody adds by 0.2% (volume ratio) of total amount, stirs, and room temperature is placed 4h.With after filter-cloth filtering, then be filtered to clarification with K type multilayer sheet frame, then add saturated formaldehyde solution in above-mentioned filtrate by 0.1% of total amount, fully stirring and evenly mixing, room temperature is placed 24h, and jolting is during this time for several times.
0.22 μ m micropore filter element filtration sterilization for 5.4 filtration sterilizations.The ultrafiltration membrance filter that is 1000KDa with molecular weight cut-off is except virus.
5.5 antibody titers are measured neutralization test
DHV DRL-62 strain is diluted to every 0.1ml containing 200ELD
50, mix with the antibody to be checked of 2 times of serial dilutions of equivalent, 37 DEG C act on 1 hour, shaken several times during this time.5 piece of 9 age in days SPF chicken embryo of each extent of dilution inoculation, every embryo 0.2ml, separately establishes each 5 pieces of virus control group that virus liquid mixes with equivalent sterilizing PBS and PBS blank group, puts 37 DEG C of cultivations 168 hours, records the chicken embryo death number of 48～168 hours, result of determination.Blank group should be all good for and be lived, and virus control group should be all dead.
DHV SD1 strain is diluted to every 0.1ml containing 200ELD
50, mix with the antibody to be checked of 2 times of serial dilutions of equivalent, 37 DEG C act on 1 hour, shaken several times during this time.5 piece of 10 age in days susceptible duck embryo of each extent of dilution inoculation, every embryo 0.2ml, separately establishes each 5 pieces of virus control group that virus liquid mixes with equivalent sterilizing PBS and PBS blank group, puts 37 DEG C of cultivations 168 hours, records the duck embryo death toll of 48～168 hours, result of determination.Blank group should be all good for and be lived, and virus control group should be all dead.
The neutralization that the highest antibody extension rate that can make 50% chicken (duck) embryo that death does not occur is this antibody is tired.
The 5.6 yolk antibody freeze-drying of purifying are to purifying and yolk antibody liquid through being up to the standards adds lyophilized vaccine to be sub-packed in cillin bottle to enter freeze drying box; in freeze-drying process, temperature is minimum is down to-40 DEG C; at this temperature, keep 4h; goods are with 1.0 DEG C/h intensification, 18 ~ 20h; to substantially dry; be warming up to 25 DEG C with 4 ~ 6 DEG C/h again and be dried to complete freeze-drying, tamponade outlet, is stored in 2 ~ 8 DEG C.
Embodiment 1DHV SD1 strain isolated molecular biology identification
1. the virus that duck hepatitis virus SD1 strain virus is new isolation identification, it has following characteristics:
The clinical symptom that 1.1DHAV-3 causes with DHAV-1 is very similar, and main manifestations is: duckling morbidity suddenly, occurs obvious nervous symptoms very soon, twitch, and very fast dead, after death duckling is opisthotonus posture.Pathological change shows as liver enlargement, and there are a large amount of blutpunktes and blood spots in liver surface, and along with the prolongation of death time, and hemorrhage performance is more for obviously.
The 1.2 multipair special primers of DHV genome sequence design of having delivered according to GenBank, RT-PCR method amplification VP1 gene, has carried out Sequence analysis to the VP1 gene of strain isolated SD1 routinely.Result shows, the VP1 sequence similarity of the VP1 gene of SD1 and China and Korea S DHAV-3 is the highest, all lower with the similarity of DHAV-1 and Taiwan DHAV-2.Nucleotide sequence and Evolution analysis result show that the genetic distance between separation poison and China and Korea S DHAV-3 is nearest, and and have larger difference between DHAV-1 and Taiwan DHAV-2.
The compare of analysis of 1.3VP1 aminoacid sequence and wetting ability, antigenic index and surperficial accessibility analysis show; between DHV SD1 strain isolated and other domestic DHAV-3, the variation of amino acid sites is all positioned at hydrophilic region and accessibility region; in addition jointly point out on DHV SD1 strain isolated VPl albumen and may have diverse epitope with the difference of DHAV-1, also will be embodied on immune protective simultaneously.
1.4 serum cross neutralizations show between DHV SD1 strain isolated and DHAV-1 that, without cross-protection, the two adheres to different serotype separately.Between SD1 strain isolated and the domestic DHAV-3 of separation strain, have cross-protection, and in mutual and provide protection SD1 strain be better than contrasting strain.
1.5 physicochemical property qualification results demonstrations, this strain can be resisted the processing of chloroform, ether, heat, pancreatin, acid.
This virus strain immunogenicity is good, and four exempt from sampling in latter 10 days measures in chicken high-immunity egg Huang DRL-62 antibody neutralization 1:17620, the neutralization of the SD1 antibody 1:13932 that tires that tires; Therapeutic test shows that divalence yolk antibody can effectively control the at present generation of the duck viral hepatitis of domestic popular different serotypes, and protection ratio reaches 80%～100%.
Duck viral hepatitis SD1 of the present invention strain was deposited in Chinese Typical Representative culture collection center on 06 08th, 2012.Depositary institution is called for short: CCTCC, deposit number: CCTCCNO.V201225, Classification And Nomenclature: duck viral hepatitis virus, English name is Duck hepatitis virus SD1 Strain, Latin name is called Tarpeiapulli, depositary institution address: typical case's culture collection center, Wuhan City, Hubei Province.
2.RT-PCR
From sick duck liver, isolated viral extracts RNA, DHAV-1 strain DRL-62 strain, the Korea S DHAV-3 strain N-DHV pnca gene group complete sequence announced according to GenBank, use following primer pair VP1 sequence to carry out the method for RT-PCR(with reference to Dalian precious biotechnology company limited reverse transcription test kit specification sheets):
1. DHAV-1 type upstream primer: 5 '-GGTGATTCTAACCAGTTGG-3 ',
Downstream primer: 5 '-TTCAATTTCCAGATTGAGT-3 ';
2. DHAV-3 type upstream primer: 5 '-CAGATGGCCGCCAATGATCAG-3 ',
Downstream primer: 5 '-GTCTCTGACATTTCGAAATTGGTATGA-3 '.
Positive amplified production send the English Weihe River prompt base (Shanghai) trade Co., Ltd to check order.
3. gene sequencing
Use DNAStar7.1 analysis software that other DHV strain (in the table 1) nucleotide sequence logging in isolated strain VP1 gene nucleotide series and I type DHV type strain DRL-62 and GenBank is carried out to homology comparative analysis, and use the method drawing system evolutionary tree in Megalign, carry out phylogenetic analysis.
Table 1 Reference strains and GenBank accession number thereof
4. molecular biology identification result
4.1RT-PCR result
Result is referring to Fig. 1, Fig. 2, utilize DHV VP1 Auele Specific Primer to carry out RT-PCR to DRL-62 strain and strain isolated, in Fig. 1, M swimming lane is DL2000Marker, swimming lane 1 utilizes DHAV-1 type type strain primer RT-PCR result for DRL-62 strain, swimming lane 2 utilizes DHAV-1 type type strain primer RT-PCR result, the negative contrast of swimming lane 3 for SD1 strain isolated;
In Fig. 2, M swimming lane is DL2000Marker, and swimming lane 1 is DHAV-3 type primer RT-PCR result for SD1 strain isolated, and swimming lane 2 is DHAV-3 type primer RT-PCR result for DRL-62 strain, the negative contrast of swimming lane 3.
Interpretation of result: utilize DHAV-1 type type strain primer, DRL-62 strain can amplify about 700bp size specific band; Utilizing DHAV-3 type primer amplification, there is specific band at about 750bp place in visible strain isolated.
4.2VP1 the sequencing results
4.2.1DHV the sequencing result of SD1 VP1 gene is referring to sequence SEQ 1:
4.2.2DHV the amino acid sequence homology analysis of SD1 strain VP1 gene nucleotide and coding thereof
Referring to Fig. 3, Fig. 4, sequential analysis discovery, DHV SD1 strain isolated is only about 69% with DHV I type standard strain DRL-62 strain, the Chinese DHV of separation I type MY strain (DHAV-1) Nucleotide similarity, and amino acid similarity is respectively 77.3%, 76.5%; And and the Chinese Nucleotide similarity that separates DHAV-3 type strain such as DHV FS strain, C-YDF strain respectively between 93.1% ~ 97.9%, amino acid similarity reaches 97.5 ~ 99.2%; And the novel DHV(DHAV-3 of Korea S) between nucleotide sequence similarity be 93.1%, amino acid similarity is respectively 92.5%, 92.9%; And the novel DHV(DHAV-2 in Taiwan) between nucleotide sequence similarity be only 72.7%, amino acid similarity is respectively 80.5%, 80.1%.
Referring to Fig. 5, Evolution analysis result further shows, DHV SD1 strain isolated and DHAV-3 China, Korea S's strain are in a branch, and genetic distance is nearest, and and has larger difference between DHAV-1 and DHAV-2 strain.Show that DHV SD1 strain isolated and domestic DHAV-3 and Korea S DHAV-3 are respectively in two subbranches, the domestic DHAV-3 that the DHV SD1 strain isolated of this research and other GenBank log in is again respectively in the middle of Liang Ge branch simultaneously.
4.2.3VP1 the compare of analysis of aminoacid sequence and wetting ability, antigenic index and surperficial accessibility analysis
VP1 is the main antigenic gene of picornavirus, and albumen major part is exposed to viral surface, is major structural protein, contains the multiple epitopes that can induce T, bone-marrow-derived lymphocyte reaction, can induce body to produce the neutralizing antibody of protectiveness.Because VP1 is positioned at surface, selective pressure is large, and aberration rate is high, and some is amino acid whose also will cause the change of antigen site, thereby antigenic variation exerts an influence to strain.
In Picornaviridae, structural protein VP1 has conservative aminoacid sequence RGD, and it is fulfiling the critical function that adherent cell is combined with cell receptor, can also induce virus to produce neutralizing antibody.From the aminoacid sequence of deriving, can find out in the VP1 sequence of DHAV-1 type strain and not there is RGD sequence, but SGD, corresponding DHAV-3 is all QSD.This variation shows that novel DHV and the combination of cell receptor may be different with FMDV etc.
Referring to Fig. 6, DHV SD1 strain isolated and DHAV-1 relatively show, strain isolated has 2 amino acid GG to insert at 143,144, and variation region is mainly at 140 ~ 146,180 ~ 200,212 ~ 219.The novel DHV of DHV SD1 strain isolated and Korea S (NC-009750) relatively shows, has 18 amino acids and morphs, and hypervariable region is mainly at 178 ~ 196.DHAV-2 relatively shows with Taiwan, at 2 amino acid DG of 51,52 strain isolated disappearances, has 3 aminoacid insertion of GGG at 143 ~ 145, and 185 have 1 aminoacid insertion L.DHV SD1 strain isolated with China other DHAV-3 strain (10 strain) relatively show, mainly exist for point mutation, wherein SD1 strain the 49th amino acids is T, other each strain is G or S.196 amino acids are N, and in contrast strain, 7 strains are D.207 amino acids are E, and in contrast strain, having 4 strains is K.
Referring to Fig. 7, VP1 gene is analyzed by Protean program.Carry out hydrophilic region analysis with Kyte-Doolittle method respectively, carry out amino acid with Emini scheme and be positioned at molecular surface probability analysis, carry out antigenic index analysis with Jameson-Wolf method.Result shows, the areal distribution that VP1 albumen wetting ability is high is extensive, the region high to antigenic index is similar, wherein 131 ~ 141,195 ~ 205,210 ~ 224 3 section antigenic indexs, wetting ability peak value, surperficial accessibility value are high, and consistent with main flexible region distribution, infer that this section of region may be the major antigen peptide section of VP1.Between DHV SD1 strain isolated and other domestic DHAV-3, the variation of amino acid sites is all positioned at hydrophilic region and accessibility region; in addition jointly point out on DHV SD1 strain isolated VPl albumen and may have diverse epitope with the difference of DHAV-1, also will be embodied on immune protective simultaneously.This research discovery, the strain with 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D has good immunogenicity to duck viral hepatitis.
Fixed virus dilute serum, does 2 times of serial dilutions with physiological saline respectively by DHV DRL-62 strain, SD1 strain isolated positive serum.DRL-62 strain, SD1 strain isolated virus liquid containing 200ELD50/0.2ml are respectively got to 1.0ml dilution positive serum balanced mix different from each strain respectively, 37 DEG C of effect 1h.Meanwhile, establish virus and physiological saline contrast.Each neutralization group and control group are inoculated to 5 pieces of duck embryos through allantoic cavity, 0.2m1/ embryo, 37 DEG C of cultivations, observe 7, record each group of dead germ number.Tire taking the highest serum extent of dilution of 50% duck embryo protection as the neutralization of serum.
The strain of neutralization test result display separation can neutralize by separated strain positive serum completely, can not be by DHV standard I type duck hepatitis positive serum and protection, and test group is all dead; After DRL-62 positive serum and strain isolated effect, neutralization is tired as 1:16; After SD1 positive serum and the effect of DRL-62 strain, neutralize the <1:8 that tires, show strain isolated and the cross-protection of DRL-62 strain serum-free, the results are shown in Table 2.Calculate the relationship value between different strains
wherein R1 is serum 1 to the neutralization of virus 2 ratio of the neutralization of virus 1 being tired with serum l of tiring; R2 is serum 2 to the neutralization of the viral l ratio of the neutralization of virus 2 being tired with serum 2 of tiring.) with reference to foot and mouth disease virus according to relationship value (R), (when R value 70% is above, two strains subtypes are identical to distinguish the differentiation standard of serotype and hypotype; R value is 32 ~ 70% time, and two strains are different hypotypes; R value is below 10%, and strain is different type), the R value between DRL-62 strain and strain isolated (<0.74%) below 10%, does not belong to same serotype, is a kind of new serotype.
Table 2 serum cross neutralization result
230 5 age in days susceptible ducklings are divided into 5 groups at random, 1,2,3,4 group every group 50,5,6,7 groups every group 10.SD1, GD strain (national veterinary microorganism culture presevation administrative center preserving number: CVCC AV321) positive serum (self-control) is diluted to respectively to neutralization tires as 1:32,1:64,1:128,1:256,1:512.1, every intramuscular injection SD1 positive serum 1.0m1 of 2 groups of ducklings, each 10 of injections of tiring; 3, every intramuscular injection GD strain positive serum 1.0m1 of 4 groups of ducklings, each 10 of injections of tiring.After 24 hours, every subcutaneous injection 100LD of 1,3,5 group of duckling
50sD1 strain 0.2ml, every intramuscular injection 100LD of 2,4,6 groups of ducklings
50gD strain 0.2ml, it is blank that the 7th group of duckling do not do any injection.Isolated rearing respectively, observes 10, records each group of death toll.
Result shows, SD1 strain, GD strain all can be protected by serum separately; In the time that SD1 positive serum antibody titer is 1:128, after passive immunization duckling, 7/10 protection can be produced to the attack of GD strain, in the time that SD1 tires as 1:256,10/10 protection can be produced; In the time that GD positive serum antibody titer is 1:256, after passive immunization duckling, 5/10 protection can be produced to the attack of SD1 strain, in the time that SD1 tires as 1:512,9/10 protection can be produced.Visible SD1 serum is better than the protection of GD serum to SD1 strain to the protection effect of GD strain, the results are shown in Table 3.
Table 3 passive immunization protection test result 1
Note: in table, ratio is duckling survival ratio, and 10 groups of experimental animal less than cause for there is non-specific death.
Design and synthesize 5 pairs of special primers according to DHAV-3GD strain whole genome sequence, application RT-PCR method is divided 5 sections of full genome cDNAs of amplification DHV.The cDNA overlapping fragments of amplification is distinguished to directed cloning to carrier pBluescript II KS(+) in, DHAV-3GD Full_length cDNA plasmid obtained.Plasmid checks order and serves marine life engineering corporation, and design mutant primer utilizes the overlapping extension mutagenesis of PCR mediation to VP1 the 49th bit codon GGT → ACT, 196 bit codon GAT → AAT, and 207 bit codon AAG → GAG carry out rite-directed mutagenesis.Through order-checking qualification, rite-directed mutagenesis is accurate, does not change other sequences.Each fragment is connected and obtains DHAV-3GD Full_length cDNA clone.Utilize Nru I by full-length cDNA plasmid linearization, utilize SP6 RNA polymerase system to transcribe in vitro, obtain viral RNA.Utilize the extracting of phenol/chloroform, the method for isopropanol precipitating, removes the impurity such as the enzyme in reverse transcription system, thereby improves the efficiency of transfection.In the time that BHK-21 cell density is 80% in Tissue Culture Plate, carry out transfection according to DMRI-C transfection reagent specification sheets.After 72h, inoculate BHK-21 cell by after transfection viral RNA cell freeze-thaw 1 time, save out viral GD-BHK.After rescue virus inoculation 10 age in days duck embryos, can make it lethal.Serum neutralization test shows to save virus and can be neutralized by GD positive serum.Sequencing result shows that rescue virus has 8 amino acid whose differences with parent's poison, has no this 8 reports that amino acid is relevant with virus virulence.
Utilize rescue virus immunity SPF chicken to prepare positive serum, neutralization is tired and is diluted to respectively: 1:32,1:64,1:128,1:256,1:512.1, every intramuscular injection GD of 2 groups of ducklings saves malicious positive serum 1.0m1, each 10 of injections of tiring.After 24 hours, 1,2 group of duckling is used respectively 100LD
50sD1 strain, GD strain are attacked, and 0.2ml/ only.Separately establish the blank group that SD1 strain, DG strain are attacked poison contrast and do not done any injection.The isolated rearing of each group, observes 10, records duckling death condition.
Result shows, in the time of tire >=1:64 of GD rescue strain serum neutralization, can produce more than 9/10 protection to SD1, in the time of tire >=1:256 of neutralization, can produce more than 9/10 protection to GD, in this result and passive immunization protection test 1, the protection effect of SD1 positive serum is close, the sudden change that GD rescue strain is described has changed its immunogenicity to a certain extent, make it obtain the attack poison close with SD1 strain and protect effect, there is the immunogenicity suitable with SD1 strain, because the sudden change of GD rescue strain is the same with SD1 strain, the 49-T at structural protein VP1, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, there is identical variation in tri-conserved sites of 199-D.There is important impact in this mutational site on duck viral hepatitis virus immunity originality as can be seen here, and not only SD1 strain is because of the corresponding site that suddenlys change, and immunogenicity strengthens, and is suddenling change behind the corresponding site of GD strain, and its immunogenicity is also corresponding, and change occurred.Thereby illustrated there is 49-T, 196-N, the strain of tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D has good immunogenicity.
Table 4 passive immunization protection test result 2
Note: in table, ratio is duckling survival ratio, and 10 groups of experimental animal less than cause for there is non-specific death.
The physicochemical property qualification of embodiment 5DHV SD1 strain
1. ether sensitivity test gets the duck blastochyle obtaining, and 3000 revs/min of centrifugal 15min remove larger particles.Draw supernatant liquor 1.6ml, move into respectively in 2 sterilizing bottles, every bottle of 0.8ml, and in wherein adding anesthesia ether 0.2ml in one bottle, in another bottle, do not add diethyl ether in contrast.Two bottles all use soft rubber ball jam-pack rearmounted 4 DEG C 24 hours, vibration frequently during this time.Add the bottle of ether, be now divided into two-layer clearly: upper strata is ether, lower floor is virus liquid.Then draw virus liquid with capillary pipet, move in another bottle, suitably piping and druming, volatilizees totally remaining ether.Finally get above-mentioned two parts of virus liquids and make respectively 10 times of serial dilutions, 10 piece of 10 non-duck embryo of exempting from of age in days of each extent of dilution inoculation, observes 168 hours, records duck embryo death condition.The relatively viral level of the virus liquid of ether processing and contrast virus liquid.
2. chloroform sensitivity test gets the duck blastochyle obtaining, and 3000 revs/min of centrifugal 15min remove larger particles.In virus liquid, add chloroform (analytical pure), making its final concentration is 4.8%, put 4 DEG C vibration mix 10 minutes, subsequently 500 revs/min centrifugal 5 minutes, draw supernatant liquid, measure viral level.Contrast virus liquid adds the sterile saline that is equal to chloroform amount, after same processing, measures viral level.The relatively viral level of the virus liquid of chloroform processing and contrast virus liquid.
3. acid resisting test gets the duck blastochyle obtaining, 3000 revs/min centrifugal 15 minutes, remove larger particles.Getting supernatant equivalent is sub-packed in 2 bottles.With the HCl of 0.1mol/L be 3.0 by the virus liquid adjust pH in 1 bottle, and in virus liquid in another 1 bottle, add be equal to acid amount sterile saline in contrast, put 37 DEG C of senses and do after 2 hours, then pH value is adjusted to 7.2 left and right with 5.6%NaHCO3 solution.Contrast adds the sterile saline that is equal to alkali number.Two bottles of virus liquids are made to 10 times of serial dilutions, and 10 piece of 10 non-duck embryo of exempting from of age in days of each extent of dilution inoculation, observes 168 hours, records duck embryo death condition.The viral level of more acid-treated virus liquid and contrast virus liquid.
4. thermal test gets the duck blastochyle obtaining, 3000 revs/min centrifugal 15 minutes, remove larger particles.Get supernatant equivalent and be sub-packed in 2 bottles, 1 bottle is placed in 50 DEG C of water-bath effects 30 minutes, and another 1 bottle does not deal with in contrast.Two bottles of virus liquids are made to 10 times of serial dilutions, and 10 piece of 10 non-duck embryo of exempting from of age in days of each extent of dilution inoculation, observes 168 hours, records duck embryo death condition.The viral level of more heat treated virus liquid and contrast virus liquid.
5. trypsinase sensitivity test gets the duck blastochyle obtaining, 3000 revs/min centrifugal 15 minutes, remove larger particles.Getting supernatant equivalent is sub-packed in 2 bottles, every bottle of 1.0ml, 1 bottle adds the trypsinase of 1.0ml 1% therein, and making final concentration is 0.5%, another bottle adds 1.0ml1640 substratum, with soft rubber ball jam-pack bottleneck, bottle is reversed several times, it is fully mixed, put 37 DEG C of effects 1 hour, add immediately inactivated fetal bovine serum 8.0ml, fully mix, stop the effect of pancreatin.Two bottles of virus liquids are made to 10 times of serial dilutions, and 10 piece of 10 non-duck embryo of exempting from of age in days of each extent of dilution inoculation, observes 168 hours, records duck embryo death condition.The relatively viral level of the virus liquid of trypsin treatment and contrast virus liquid.
Physicochemical property qualification result:
Carry out with reference to above test method, visible DHV SD1 strain isolated is after ether, chloroform, acid, heat, pancreatin are processed, and malicious valency is without obvious reduction, and result is referring to table 5, table 6, table 7, table 8.
Table 5 fatsolvent sensitization test result
Table 6 acid resisting test result
Table 7 thermal test result
Table 8 trypsinase sensitivity test result
Embodiment 6: the preparation of duck viral hepatitis divalence yolk antibody
Only, after two weeks, two exempt from the bigeminy deactivation oil seepage intramuscular injection 120 age in days laying hen 1ml/ that prepare with DRL-62, SD1 strain, and after 10 days, three exempt from, and three exempt within latter 10 days, to carry out the 4th time reinforced immunological, and each immunizing dose is exempted from head.Four exempt from sampling in latter 10 days measures in chicken high-immunity egg Huang DRL-62 antibody neutralization 1:17620, the neutralization of the SD1 antibody 1:13932 that tires that tires, and egg collection is put 4 DEG C of storages for subsequent use.The aseptic collection yolk of beating eggs.Add equal-volume through 121 DEG C of 30min sterilizings cooling distilled water, stirring and evenly mixing, 62.5 DEG C of heat inactivation 30min.In interlayer retort, add 6 volume sterile purified waters (pH value is 4.2), be cooled to after 4 DEG C, slowly add the egg yolk liquid of deactivation, limit edged stirs, 4 DEG C of standing 4h.Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also proceeds in another retort.It is sad to add by 0.2% (volume ratio) of total amount, stirs, and room temperature is placed 4h.After filter-cloth filtering, then be filtered to clarification with K type multilayer sheet frame, add saturated formaldehyde solution by 0.1% of total amount, fully stirring and evenly mixing, room temperature is placed 24h, and jolting is during this time for several times.With 0.22 μ m micropore filter element filtration sterilization.The ultrafiltration membrance filter that is 1000KDa with molecular weight cut-off is except virus.
Duck liver divalence yolk antibody (A group), DHAV-1 antibody (B group) and DHAV-3 antibody (C group) is intramuscular injection 1 age in days, 6 ages in days, 12 ages in days, 18 age in days ducklings respectively, 10 of every group of every ages in days.Only, 6 ages in days, 12 ages in days, 18 age in days duckling injection of antibodies 1.0ml/ are only for 1 age in days duckling injection of antibodies 0.5ml/.24 hours every duckling 100LD after injection of antibodies
50dRL-62, SD1 strong virus attack.Attack poison and establish each 10 of virus control and blank simultaneously.Each test group is separately raised, and observes 10, records duckling death toll.
Result demonstration, duck liver divalence yolk antibody can be protected all strong living of each age in days duckling, uses separately DHAV-1 antibody and DHAV-3 antibody obviously can not resist the attack of DHAV-1, DHAV-3.The results are shown in Table 9.
Table 9 preventive trial result
Note: in table, ratio is duckling survival ratio, and 10 groups of experimental animal less than cause for there is non-specific death.
8.1: test is divided into 3 groups of A, B, C, artificial challenge DHAV-1 and DHAV-3 under 60 4 age in days duckling laboratory conditions of every group of random selection.A group duckling is divided into 3 groups, and 20 every group, respectively at infecting latter 24 hours, 36 hours, 48 hours intramuscular injection duck viral hepatitis divalence yolk antibodies, 1.0ml/ only; B group, the operation of C group, with A group, are used respectively DHAV-1 and DHAV-3 duck viral hepatitis antibody to treat.Test is established 10 ducklings and is not done injection for blank.Do not treat contrast after establishing 10 infection for every group.Each group is separately raised, and observes 10, observes and record duckling death condition.
Result shows, infects and treats with antibody for latter 24 hours, and all ducklings do not occur dead within the observation period again, within latter 48 hours, use Antybody therapy group and attack poison, and duckling curative ratio obviously reduces.Visible duckling is more early used antibody after infecting, and can greatly improve protection effect.Result shows simultaneously, only cannot produce effective protection with DHAV-1 or DHAV-3 duck liver antibody.Blank is lived entirely, and not treatment group all death afterwards on the 10th, the results are shown in Table 10.
Table 10 artificial challenge therapeutic test result
Note: in table, ratio is duckling survival ratio, and 10 groups of experimental animal less than cause for there is non-specific death.
8.2: the 6 age in days duckling morbidities of District, Xinyang Area, Henan Province duck field, performance lassitude, appetite stimulator, necking down, sleeping ground, both legs spasm before 7 ages in days start to occur death, be dead, opisthotonus, send the inspection of animal epidemic prevention and control center, Xinyang, cut open inspection and find liver enlargement, hemorrhage, kidney enlargement, hyperemia, spleen enlargement, gather liver organization and carry out RT-PCR detection, result is DHAV-1, the DHAV-3 duck viral hepatitis positive.8 ages in days use the subcutaneous injection of divalence yolk antibody, use respectively DHAV-1 and DHAV-3 duck liver antibody treatment group 1,2 in contrast simultaneously, observe 10.Result demonstration, there is not death in the beginning on the 5th from treating of 1622 ducklings for the treatment of group, coexists and live 1339, protection ratio is 81.6%, and randomized controlled treatment group 1,2 protection ratios are only respectively 6.3%, 57.8%, the results are shown in Table 11.Visible bivalent antibody can play comparatively desirable protection effect to current clinical multiple DHAV-1, DHAV-3 duck viral hepatitis, and uses separately DHAV-1 or DHAV-3 antibody cannot effectively control the generation of different serotypes duck viral hepatitis.
Table 11 natural occurrence therapeutic test result
The preparation of embodiment 9DHAV-3SD1 yolk antibody and protection effect test
The preparation of 1.DHAV-3SD1 yolk antibody
The preparation of 1.1 antigens is done dilution in 1: 100 by duck hepatitis virus SD1 strain seed culture of viruses with sterilizing PBS solution, inoculates 10 age in days susceptible duck embryos, every embryo 0.2ml through allantoic cavity.Putting 37 DEG C continues to hatch, discard the dead germ in 48 hours, gather in the crops 48～96 hours dead duck embryos, by inspection aseptic duck blastochyle mix latter 3500 revs/min centrifugal 30 minutes, draw supernatant liquor in sterilising vessel, add 10% formaldehyde solution, with adding with shaking, it is fully mixed, and the final concentration of formaldehyde solution is 0.15%, in airtight rearmounted 37 DEG C of incubators, acts on 24 hours (jolting during this time 3～4 times).
1.2 immunity are prepared with the oil phase of preparing of deactivation vaccine: get 94 parts of injection white oils, after part mixing of Si Ben-806, add 2 parts of aluminum stearates, heat while stirring for subsequent use to transparent rear autoclaving, be oil phase.Water preparation: add sterilizing tween-80 by 4% of antigen, shake well, dissolves tween-80 completely, is water.Emulsification: get 2 parts of oil phases and put in oil phase tank, start motor and stir, slowly add 1 part of water, then with 3400 revs/min of emulsifications 10 minutes.Before emulsification stops, add 1% Thiomersalate, making its final concentration is 0.01%.Sample 3000 revs/min centrifugal 15 minutes, should be not stratified.Packing: quantitative separating, seals.
1.3 Immune Laying Hens 120 about ages in days with deactivation oil seepage intramuscular injection 1ml/ only, after 14 days, use deactivation vaccine intramuscular injection 1ml/ only, two exempt from after 10 days and three exempt to use respectively deactivation vaccine intramuscular injection 1ml/ only after 10 days.When 1 tire >=1: 8192 egg collection of antibody neutralization of SD in yolk, put 4 DEG C of storages for subsequent use.
1.4 eggshell is sterilized in the preparation of yolk antibody, collects yolk.Fully stir and make yolk be even paste, add equal-volume through 121 DEG C of 30min sterilizings cooling distilled water, stirring and evenly mixing, 62.5 DEG C of heat inactivation 30min.First in interlayer retort, add and be equivalent to the sterile purified water that the pH value of 6 times of volumes of former yolk is 4.2, and be cooled to 4 DEG C.Then, the egg yolk liquid of deactivation is added, limit edged stirs, 4 DEG C of standing 4h.Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also proceeds in another retort.It is sad to add by 0.2% (volume ratio) of total amount, stirs, and room temperature is placed 4h.With after filter-cloth filtering, then be filtered to clarification with K type multilayer sheet frame, then add saturated formaldehyde solution in above-mentioned filtrate by 0.1% of total amount, fully stirring and evenly mixing, room temperature is placed 24h, and jolting is during this time for several times.With 0.22 μ m micropore filter element filtration sterilization.The ultrafiltration membrance filter that is 1000KDa with molecular weight cut-off is except virus.Obtain SD1 yolk antibody.
2.DHV SD1 yolk antibody is attacked poison protection effect
4 age in days ducklings are divided into 1,2,3 group, 1,2 group every group 10,3 groups 5.1 group injection of antibodies 0.5ml/, 24 hours 1,2 group every duckling 100LD after injection of antibodies
50sD1 strong virus attack.3 groups is blank group.Each test group is separately raised, and observes 10, records duckling death toll.
Result demonstration, after SD1 antibody can protect immune group to attack poison, duckling all survives, and virus control group is all dead, and blank group is all survived.The results are shown in Table 12.
Table 12SD1 antibody protest test result
| Group | Immune group | Virus control group | Blank group |
| |
10/10 | 0/10 | 5/5 |
Embodiment 10DHAV-3SD1 VP1 protein immunization effect test
Extract test kit specification sheets by Trizol viral RNA and extract viral RNA from the virus liquid of duck embryo results.RT-PCR amplification DHAV-3SD1VP1 gene order.VP1 gene clone is arrived in expression vector pET-32a (+), screening prokaryotic expression carrier pET-32a-VP1, transform e. coli bl21 cell and carry out great expression, inclusion body protein is through cracking, washing, purifying, renaturation, the concentrated DHAV-3SD1 VP1 albumen that obtains.Uv-absorbing standard measure protein concentration is 850 micrograms/ml by 1.2 method seedling in embodiment 9,10 of immune SPF chickens, subcutaneous injection 1.0ml/ by the dilution of obtained albumen.Three exempt from latter 10 days blood sampling separation of serum, 56 DEG C of deactivations.4 age in days ducklings are divided into 4 groups, 10 every group.1 group of injection recombinant protein (VP1) antiserum(antisera), 0.5ml/ is only; 2 groups of injection SD1 antiserum(antisera)s, 0.5ml/ is only; Do not inject any serum for immune group not for 3 groups; Next day 1,2,3 groups of duckling SD1 strong virus attacks, 4 groups as blank group.The isolated rearing of each group is observed 10.
Attack malicious result and show, SD1 antiserum(antisera) group duckling survival 10/10; Recombinant protein antiserum(antisera) group duckling survival 5/10; Immune group duckling is all not dead.DHV SD1 restructuring VP1 protein antiserum immunity duckling of the present invention, can partly resist the lethal attack of the strong poison of SD1, and visible DHVSD1 restructuring VP1 albumen has suitable immunogenicity, makes duckling adaptive immune protection.
Table 13DHAV-3SD1 VP1 protein immunization effect
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (12)
1. a duck viral hepatitis virus strain, the sequence of described virus strain VP1 gene coding amino acid comprises have 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D as shown in figure six.
2. duck viral hepatitis virus according to claim 1, described duck viral hepatitis virus is duck viral hepatitis virus SD1 strain, and described SD1 strain preservation address is Chinese Typical Representative culture collection center, and deposit number is CCTCC NO.V201225.
3. a DNA sequence dna, it contains in fact the nucleotide sequence of seq No.1.
4. a VP1 antigen protein, described VP1 antigen protein comprises the duck viral hepatitis virus VP 1 consensus sequence with 49-T, 196-N, tri-aminoacid replacement sites of 207-E and 197-Q, 198-S, tri-conserved sites of 199-D as shown in figure six.
5. a duck viral hepatitis divalence yolk antibody, is characterized in that, described divalence yolk antibody comprises duck viral hepatitis DHAV-1 type and duck viral hepatitis DHAV-3 type yolk antibody.
6. a preparation method for duck viral hepatitis divalence yolk antibody, described preparation method comprises:
(1) adopt duck viral hepatitis DHAV-1 type strain and DHAV-3 type strain to inoculate respectively 9 age in days SPF chicken embryos and 10 age in days susceptible duck embryos, then gather in the crops allantoic fluid, the virus liquid of described results is mixed in proportion, formalin-inactivated is also prepared vaccine;
(2) use the vaccine immunity laying hen of described preparation, after immunity sampling measure anti-DHAV-1 type in chicken high-immunity egg Huang, the neutralization of DHAV-3 type antigen-antibody and tire >=1: 8192, collect afterwards the high-immunity egg of described chicken;
(3) by described high-immunity egg eggshell sterilization, collect yolk, described yolk adds equal-volume distilled water, stirring and evenly mixing, low temperature pasteurization; Acidifying distillation water law is purified, sad method is purified; Micro-filtration, ultrafiltration.
7. preparation method according to claim 6, wherein, described duck viral hepatitis DHAV-1 type strain is that DRL-62 strain, described DHAV-3 type strain are SD1 strain.
8. preparation method according to claim 6, wherein, described low temperature pasteurization condition is 62.5 DEG C of heat inactivation 30min.
9. preparation method according to claim 6, wherein, described acidifying distillation water law purification step is: first in interlayer retort, add be equivalent to 6 times of volumes of described yolk sterile purified water, described distilled water pH value is 4.2, and is cooled to 4 DEG C, then, the described egg yolk liquid of deactivation is added, stir 4 DEG C of standing 4h, centrifugation supernatant liquor;
Described sad method purification step is: it is sad to add by 0.2% volume of described supernatant liquor total amount, stirs, and room temperature is placed 4h, is filtered to clarification, then adds saturated formaldehyde solution by 0.1% of described filtrate total amount, stirs, and room temperature is placed 24h.
10. preparation method according to claim 6, wherein, described micro-filtration uses 0.22 μ m micropore filter element filtration sterilization, and the ultrafiltration membrance filter that described ultrafiltration is 1000KDa with molecular weight cut-off is except virus.
11. application in the medicine of preparation prevention and treatment duck viral hepatitis according to the duck viral hepatitis divalence yolk antibody described in claim 5 ~ 10 any one.
The vaccine compositions of 12. 1 kinds of preventions and treatment duck viral hepatitis, described vaccine composition comprises the antigen that prepared by strain described in claim 1 or claim 2 and acceptable carrier pharmaceutically.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710026081.6A CN106854647B (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof |
| CN201210530208.5A CN103865884A (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210530208.5A CN103865884A (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710026081.6A Division CN106854647B (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103865884A true CN103865884A (en) | 2014-06-18 |
Family
ID=50904895
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710026081.6A Active CN106854647B (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof |
| CN201210530208.5A Pending CN103865884A (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710026081.6A Active CN106854647B (en) | 2012-12-11 | 2012-12-11 | Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN106854647B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215762A (en) * | 2014-09-28 | 2014-12-17 | 武汉中博生物股份有限公司 | Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody |
| CN104530232A (en) * | 2014-12-23 | 2015-04-22 | 山东信得科技股份有限公司 | Preparation method of refined egg yolk antibody for duck viral hepatitis |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109232735B (en) * | 2017-07-10 | 2022-02-25 | 普莱柯生物工程股份有限公司 | Avian adenovirus egg yolk antibody, and preparation method and application thereof |
| CN111110839B (en) * | 2020-01-06 | 2022-10-04 | 青岛易邦生物工程有限公司 | Goose astrovirus bivalent inactivated vaccine for preventing gosling gout |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102078609A (en) * | 2010-12-23 | 2011-06-01 | 天津康莱森生物科技集团有限公司 | Method for preparing duck virus hepatitis refined heterogenous yolk antibody |
| CN102127531A (en) * | 2010-12-22 | 2011-07-20 | 山东省农业科学院家禽研究所 | Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN102174476A (en) * | 2010-12-29 | 2011-09-07 | 中国科学院微生物研究所 | Inactivated vaccine for preventing duck virus hepatitis and preparation method thereof |
| CN102716485A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Dual hyper-immune egg yolk antibody injection for duck virus hepatitis and duck plague and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845095A (en) * | 2010-05-10 | 2010-09-29 | 洛阳普莱柯生物工程有限公司 | Method for preparing double yolk antibody of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus |
| CN102716476B (en) * | 2012-05-31 | 2015-04-22 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
-
2012
- 2012-12-11 CN CN201710026081.6A patent/CN106854647B/en active Active
- 2012-12-11 CN CN201210530208.5A patent/CN103865884A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127531A (en) * | 2010-12-22 | 2011-07-20 | 山东省农业科学院家禽研究所 | Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN102078609A (en) * | 2010-12-23 | 2011-06-01 | 天津康莱森生物科技集团有限公司 | Method for preparing duck virus hepatitis refined heterogenous yolk antibody |
| CN102174476A (en) * | 2010-12-29 | 2011-09-07 | 中国科学院微生物研究所 | Inactivated vaccine for preventing duck virus hepatitis and preparation method thereof |
| CN102716485A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Dual hyper-immune egg yolk antibody injection for duck virus hepatitis and duck plague and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215762A (en) * | 2014-09-28 | 2014-12-17 | 武汉中博生物股份有限公司 | Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody |
| CN104215762B (en) * | 2014-09-28 | 2016-07-06 | 武汉中博生物股份有限公司 | A kind of indirect ELISA method detecting Serotype-3 DHAV antibody and test kit |
| CN104530232A (en) * | 2014-12-23 | 2015-04-22 | 山东信得科技股份有限公司 | Preparation method of refined egg yolk antibody for duck viral hepatitis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106854647B (en) | 2021-01-15 |
| CN106854647A (en) | 2017-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108558995B (en) | Yolk antibody for preventing and treating novel goose astrovirus and preparation method thereof | |
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| CN101514334A (en) | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof | |
| CN110872578B (en) | Variant infectious bursal disease virus, subunit vaccine, preparation method and application thereof | |
| CN102851257A (en) | Attenuated vaccine strain for avian infectious bronchitis virus and application thereof | |
| CN107177001A (en) | It is a kind of to prevent and treat Yolk antibody of Porcine Epidemic Diarrhea and preparation method thereof | |
| CN108912227B (en) | Yolk antibody for resisting duck reovirus as well as preparation method and application thereof | |
| CN114561366B (en) | Goat kuraria virus isolate and application thereof | |
| CN114807060B (en) | Coxsackie virus A6 type strain and immunogenic composition and application thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN103865884A (en) | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof | |
| CN108676092B (en) | Egg yolk antibody for preventing and treating novel duck reovirus and preparation method thereof | |
| CN105274064B (en) | A kind of duck tembusu virus attenuated vaccine strain and its application | |
| CN108465107B (en) | Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine | |
| CN112280750B (en) | Novel goose astrovirus with cross-species transmission capability and application thereof | |
| CN112341539B (en) | Yolk antibody for preventing and treating novel goose astrovirus with cross-species transmission capability and preparation method thereof | |
| CN109207436B (en) | Group I type 4 avian adenovirus strain and application thereof | |
| CN108913666A (en) | A kind of duck reovirus leading to duck spleen necrosis and its inactivated vaccine and application | |
| CN103705918B (en) | Porcine epidemic diarrhea resisting virus hyper-immune serum and preparation method thereof | |
| CN104130981A (en) | Application of avian infectious bronchitis virus vaccine strain in preparation of inactivated vaccine | |
| CN108939063B (en) | Muscovy duck triple inactivated vaccine | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN113278595B (en) | Duck adenovirus type 3 strain, duck adenovirus egg yolk antibody, and preparation methods and application thereof | |
| CN107365382B (en) | Egg yolk antibody of duck adenovirus type 2 and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140618 |