CN113430177B - Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody - Google Patents
Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody Download PDFInfo
- Publication number
- CN113430177B CN113430177B CN202110934059.8A CN202110934059A CN113430177B CN 113430177 B CN113430177 B CN 113430177B CN 202110934059 A CN202110934059 A CN 202110934059A CN 113430177 B CN113430177 B CN 113430177B
- Authority
- CN
- China
- Prior art keywords
- milk
- antibody
- epidemic diarrhea
- cow
- goat
- 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.)
- Active
Links
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 title claims abstract description 86
- 229940031551 inactivated vaccine Drugs 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 241000700605 Viruses Species 0.000 claims abstract description 74
- 230000003053 immunization Effects 0.000 claims abstract description 58
- 235000013336 milk Nutrition 0.000 claims abstract description 50
- 239000008267 milk Substances 0.000 claims abstract description 50
- 210000004080 milk Anatomy 0.000 claims abstract description 50
- 235000020247 cow milk Nutrition 0.000 claims abstract description 38
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 241001494479 Pecora Species 0.000 claims abstract description 19
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 235000020254 sheep milk Nutrition 0.000 claims abstract description 7
- 206010012735 Diarrhoea Diseases 0.000 claims description 133
- 235000020251 goat milk Nutrition 0.000 claims description 80
- 238000002649 immunization Methods 0.000 claims description 49
- 239000000243 solution Substances 0.000 claims description 43
- 238000003756 stirring Methods 0.000 claims description 40
- 238000001914 filtration Methods 0.000 claims description 32
- 230000003472 neutralizing effect Effects 0.000 claims description 28
- 210000003205 muscle Anatomy 0.000 claims description 25
- 230000036039 immunity Effects 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 241000283690 Bos taurus Species 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 238000011081 inoculation Methods 0.000 claims description 21
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 15
- 241000283707 Capra Species 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 13
- 238000002844 melting Methods 0.000 claims description 13
- 230000008018 melting Effects 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 239000005018 casein Substances 0.000 claims description 12
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 12
- 235000021240 caseins Nutrition 0.000 claims description 12
- 238000007710 freezing Methods 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 230000000415 inactivating effect Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 8
- 235000021243 milk fat Nutrition 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 239000008098 formaldehyde solution Substances 0.000 claims description 7
- 238000002255 vaccination Methods 0.000 claims description 5
- 238000010255 intramuscular injection Methods 0.000 claims description 4
- 239000007927 intramuscular injection Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 2
- 235000014121 butter Nutrition 0.000 claims description 2
- 235000019197 fats Nutrition 0.000 claims description 2
- 239000012982 microporous membrane Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- 230000001988 toxicity Effects 0.000 abstract description 8
- 206010067484 Adverse reaction Diseases 0.000 abstract description 3
- 230000006838 adverse reaction Effects 0.000 abstract description 3
- 230000009885 systemic effect Effects 0.000 abstract description 2
- 208000024891 symptom Diseases 0.000 description 39
- 238000011282 treatment Methods 0.000 description 38
- 238000012360 testing method Methods 0.000 description 32
- 230000002265 prevention Effects 0.000 description 30
- 210000004027 cell Anatomy 0.000 description 29
- 210000002969 egg yolk Anatomy 0.000 description 24
- 238000000855 fermentation Methods 0.000 description 23
- 230000004151 fermentation Effects 0.000 description 23
- 230000008673 vomiting Effects 0.000 description 23
- 102000002322 Egg Proteins Human genes 0.000 description 16
- 108010000912 Egg Proteins Proteins 0.000 description 16
- 235000013345 egg yolk Nutrition 0.000 description 15
- 206010047700 Vomiting Diseases 0.000 description 13
- 239000003053 toxin Substances 0.000 description 13
- 231100000765 toxin Toxicity 0.000 description 13
- 241000287828 Gallus gallus Species 0.000 description 12
- 235000013330 chicken meat Nutrition 0.000 description 12
- 201000010099 disease Diseases 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 12
- 230000002779 inactivation Effects 0.000 description 12
- 230000000120 cytopathologic effect Effects 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 10
- 210000004916 vomit Anatomy 0.000 description 10
- 210000003022 colostrum Anatomy 0.000 description 9
- 235000021277 colostrum Nutrition 0.000 description 9
- 230000018044 dehydration Effects 0.000 description 9
- 238000006297 dehydration reaction Methods 0.000 description 9
- 238000003556 assay Methods 0.000 description 8
- 235000013601 eggs Nutrition 0.000 description 8
- 230000003862 health status Effects 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 235000021050 feed intake Nutrition 0.000 description 7
- 210000003501 vero cell Anatomy 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 206010016256 fatigue Diseases 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 238000009175 antibody therapy Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- -1 ntibody Species 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241000282887 Suidae Species 0.000 description 4
- 239000005862 Whey Substances 0.000 description 4
- 102000007544 Whey Proteins Human genes 0.000 description 4
- 108010046377 Whey Proteins Proteins 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000521 hyperimmunizing effect Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000006386 neutralization reaction Methods 0.000 description 4
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 4
- 238000011076 safety test Methods 0.000 description 4
- 239000008227 sterile water for injection Substances 0.000 description 4
- 241000204031 Mycoplasma Species 0.000 description 3
- 241000204022 Mycoplasma gallisepticum Species 0.000 description 3
- 238000011887 Necropsy Methods 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 239000000645 desinfectant Substances 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010067482 No adverse event Diseases 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000003278 egg shell Anatomy 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 235000012631 food intake Nutrition 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229960000380 propiolactone Drugs 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011265 semifinished product Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- OUPZKGBUJRBPGC-UHFFFAOYSA-N 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione Chemical compound O=C1N(CC2OC2)C(=O)N(CC2OC2)C(=O)N1CC1CO1 OUPZKGBUJRBPGC-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
- 206010049244 Ankyloglossia congenital Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000272834 Cairina moschata Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 208000002979 Influenza in Birds Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000702665 Porcine rotavirus Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001165 anti-coccidial effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 229960002233 benzalkonium bromide Drugs 0.000 description 1
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 1
- 210000001172 blastoderm Anatomy 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- QSKWJTXWJJOJFP-UHFFFAOYSA-N chloroform;ethoxyethane Chemical compound ClC(Cl)Cl.CCOCC QSKWJTXWJJOJFP-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
-
- 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
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/04—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from milk
-
- 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
-
- 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
- C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- 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
- C12N2770/20011—Coronaviridae
- C12N2770/20021—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
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a porcine epidemic diarrhea virus strain, an inactivated vaccine, an antibody and a preparation method of the antibody, wherein the virus strain is named as a porcine epidemic diarrhea virus SDLC strain, the inactivated vaccine is prepared by using the porcine epidemic diarrhea virus SDLC strain as an inactivated antigen, the antibody is used for immunizing a cow or a sheep producing milk by using the porcine epidemic diarrhea virus inactivated vaccine, and the antibody is obtained by collecting the immunized cow or sheep milk and extracting the antibody. The porcine epidemic diarrhea virus strain provided by the invention is an epidemic strain of porcine epidemic diarrhea virus, the toxicity of the porcine epidemic diarrhea virus strain is higher than that of the common porcine epidemic diarrhea virus, but the porcine epidemic diarrhea virus antigen prepared by the porcine epidemic diarrhea virus strain has good safety, no local and systemic adverse reaction caused by vaccine occurs, and the porcine epidemic diarrhea virus antigen is suitable for preparing the antigen and the prepared antibody can obtain better effect.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a porcine epidemic diarrhea virus strain, an inactivated vaccine, an antibody and a preparation method of the antibody.
Background
Porcine Epidemic Diarrheic (PED) is an acute and highly contact digestive tract infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and has the main clinical symptoms of vomiting, severe diarrhea and dehydration. The disease mostly occurs in winter in China, and often occurs together with other diseases, pigs of all varieties and ages are susceptible to the virus, newborn suckling piglets are particularly serious, the morbidity and mortality can reach 100 percent at most, the mortality is gradually reduced along with the increase of the age of days, and fattening pigs and adult pigs are mostly diarrhea or recessive infection.
PEDV in China is mainly divided into two large groups, namely a classical virus strain group (G1 type) and a novel virus strain group (G2 type), wherein the classical virus strains mainly refer to vaccine strains represented by CV777 and traditional clinical virus strains, and most of the current epidemic virus strains are novel PEDV strains. Commercial PED vaccines in domestic markets are combined vaccines, mainly including a combined inactivated vaccine of porcine Transmissible Gastroenteritis (TGEV) and PEDV or a combined attenuated vaccine and a combined triple inactivated vaccine of PEDV, TGEV and porcine rotavirus. Although PEDV attenuated vaccines, inactivated vaccines and disease feedback play a role in the prevention and control of PED, the failure of vaccine immunization frequently occurs. Aiming at the problem that no effective treatment medicine exists for PED at present, the conventional treatment effect is poor, and particularly, the PED is popular in pig farms caused by the attack of lactating sows. Therefore, it is urgent to develop a method for effectively preventing and treating diarrhea in piglets, reducing the incidence rate of the piglet's epidemic diarrhea, and reducing the economic loss of farmers.
Disclosure of Invention
In order to solve the above problems, a porcine epidemic diarrhea virus strain, an inactivated vaccine, an antibody and a method for preparing the antibody have been proposed. In order to achieve the purpose, the invention provides the following technical scheme:
the porcine epidemic diarrhea virus strain is named as a porcine epidemic diarrhea virus SDLC strain, is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university at 14 months 5 and 2021, and has the preservation number of CCTCC No: and V202141.
The inactivated vaccine for the porcine epidemic diarrhea virus is prepared by using the SDLC strain of the porcine epidemic diarrhea virus as an inactivated antigen, and the virus content of virus liquid for preparing the vaccine is more than or equal to 10 7.5 TCID 50 /0.1ml。
The antibody is used for immunizing milk-producing cattle or sheep by using the porcine epidemic diarrhea virus inactivated vaccine, and the antibody is obtained by collecting the immunized milk or sheep milk and extracting the antibody.
The preparation method of the antibody for preventing and treating the piglet epidemic diarrhea is characterized by comprising the following steps of:
(1) immunizing milk-producing cattle or sheep by adopting the porcine epidemic diarrhea virus inactivated vaccine, and collecting sheep milk or cow milk after immunization;
(2) adjusting pH of goat milk or cow milk to 4.0-4.2, centrifuging to remove casein in goat milk or cow milk;
(3) adding alkali solution into goat milk or cow milk with casein removed to adjust pH to 6.2-6.5, heating to 50-70 deg.C, centrifuging to remove milk fat;
(4) heating goat milk or cow milk without butter fat to 60-70 deg.C, inactivating for 10-20 min, and cooling in water bath to below 30 deg.C;
(5) adding a formaldehyde solution into the goat milk or the cow milk cooled in the step (4) until the final concentration of formaldehyde is 0.5 per mill, fully stirring and uniformly mixing, and sealing for 50-80 minutes;
(6) and (5) carrying out deep filtration, ultrafiltration and sterilization filtration on the solution obtained in the step (5) to obtain the antibody for preventing and treating the piglet epidemic diarrhea.
Preferably, the step (1) of immunizing a goat producing milk with the inactivated vaccine against porcine epidemic diarrhea virus comprises the following specific steps of:
basic immunity, wherein 0.8ml of vaccine is injected into neck and back muscles of each sheep;
performing secondary immunization, performing vaccination 2 times 14 days after the primary immunization, and performing intramuscular injection of 1.0ml on the back of the neck of each sheep;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 1.5ml is injected into the neck and back muscles of each sheep;
maintaining immunity, and when the neutralizing titer of porcine epidemic diarrhea antibody in goat milk reaches 1:512, maintaining inoculation for 1 time, wherein the neck and back of each goat is injected with 1.5ml of vaccine through muscle;
collecting goat milk, performing three times of intensified immunization, starting to collect the goat milk 14 days later, extracting the antibody, wherein the neutralizing titer of the porcine epidemic diarrhea antibody in the goat milk is not less than 1:512, and storing the goat milk at-20 ℃ for freezing.
Preferably, in the step (1), the step of collecting the milk comprises the following steps:
basic immunity, wherein 1.5ml of vaccine is injected into the neck and back of each cow;
performing secondary immunization, wherein the 2 nd inoculation is performed 14 days after the primary immunization, and 2.0ml is injected into the neck and back muscles of each cow;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 2.5ml is injected into the neck and back muscles of each cow;
maintaining immunity, and when the neutralizing titer of porcine epidemic diarrhea antibody in milk reaches 1:1024, maintaining inoculation for 1 time, wherein each cow is injected with 2.5ml of vaccine in neck and back muscles;
collecting milk, starting to collect milk 14 days after three times of enhanced immunity, extracting antibodies, determining that the neutralizing titer of the porcine epidemic diarrhea antibodies in the milk is not less than 1:1024, and freezing and storing the milk at the temperature of-20 ℃.
Preferably, the collected goat milk or cow milk is placed at the temperature of minus 20 ℃ for freezing and storing after the collection of the goat milk or cow milk, the frozen goat milk or cow milk is taken out one day ahead of time before the pH is adjusted in the step (2) and placed in a melting tank for melting, the melted goat milk or cow milk is poured into a sterilized container on the next day, a hydrochloric acid solution is added while stirring to adjust the pH value to 4.0-4.2, after the pH value is detected to be stable, a centrifugal machine is used for centrifugally removing casein in the cow milk, and the centrifuged goat milk or cow milk is collected into another container.
Preferably, in step (3), the sodium hydroxide solution is added to the goat or cow milk without casein while stirring, the pH is adjusted to 6.2-6.5, then the goat or cow milk is heated to 60 ℃ while stirring, and the goat or cow milk at 60 ℃ is centrifuged to remove the milk fat.
Preferably, in the step (6), the solution obtained in the step (5) is filtered and clarified by a K-type multi-layer plate frame, an ultrafiltration membrane with the molecular weight cutoff of 1000KD is used for filtering and removing viruses, and the qualified antibody is filtered and sterilized by a 0.22-micron microporous filter membrane.
Has the advantages that:
(1) the porcine epidemic diarrhea virus strain provided by the invention is an epidemic strain of porcine epidemic diarrhea virus, the toxicity of the porcine epidemic diarrhea virus strain is higher than that of the common porcine epidemic diarrhea virus, but the porcine epidemic diarrhea virus antibody prepared by the porcine epidemic diarrhea virus strain has good safety, no local and systemic adverse reaction caused by the antibody occurs, and the porcine epidemic diarrhea virus antibody is suitable for preparing the antibody and can obtain better effect.
(2) The antibody for preventing and treating piglet epidemic diarrhea provided by the invention has stable and effective indexes through analysis of property, safety test and efficacy test data in a storage life test.
(3) The antibody for preventing and treating the piglet epidemic diarrhea can provide effective immune protection and treatment for piglets, and has good commercial development prospect.
Drawings
FIG. 1 is an agarose gel electrophoresis image.
FIG. 2 is a graph of results of animal regression experiments.
In the figure: 1. detecting a sample, 2, a positive control, 3, a negative control, 4, a marker, A, an attacking group piglet, B, a control group piglet, C, an attacking group piglet intestinal tract, and D, a control group piglet intestinal tract.
Detailed Description
In order to make the technical solutions of the present invention better understood, the following description of the technical solutions of the present invention with reference to the accompanying drawings of the present invention is made clearly and completely, and other similar embodiments obtained by a person of ordinary skill in the art without any creative effort based on the embodiments in the present application shall fall within the protection scope of the present application.
Example 1 isolation and identification of porcine epidemic diarrhea Virus
1. Sample collection
Water sample of a piglet 2 days old in a certain pig farm in a chatting area of Shandong province is diarrhea, vomit and then dehydrated to die, and disease materials such as small intestine, content and the like of the dead sick pig are collected, PEDV (preliminary detection of the disease materials) is positive, viruses such as TGEV (triglycidyl isocyanurate virus) are negative, and the piglet is stored at the temperature of-80 ℃ for later use.
2. Viral isolation and identification
2.1 treatment of disease material selecting small intestine and content of dead sick pigs with typical disease symptoms according to the mass-volume ratio of 1:3, adding sterile PBS containing double antibodies (1000U penicillin +1000ug streptomycin/ml), processing into tissue homogenate by a high-speed dispersion homogenizer, repeatedly freezing and thawing for 2 times, 12000r/min, centrifuging for 10min, and filtering and sterilizing the supernatant by a 0.22um filter for later use.
2.2 Virus isolation Vero cells are cultured by DMEM nutrient solution containing 10% fetal calf serum, after the cells grow to a single layer, the nutrient solution is discarded, PBS is used for washing for 2 times, the treated tissue fluid in 2.1 is inoculated, pancreatin with the final concentration of 7.5ug/mL is added at the same time, the cells are placed in a 37 ℃ and 5% CO2 incubator for adsorption for 1 hour, the tissue fluid is discarded, serum-free DMEM maintenance fluid (the pancreatin with the final concentration of 7.5ug/mL) is added, the cells are cultured in a 37 ℃ and 5% CO2 incubator, Cytopathy (CPE) is observed once every 8 hours, if no CPE is generated, the cells are frozen and thawed 2 times after 72 hours of inoculation, the cells and the culture fluid are collected for blind transfer, the CPE appears when blind transfer to the 6 th generation is observed, and the virus fluid is collected for continuous subculture after the cells are frozen and thawed for 2 times. The separated virus strain is named as SDLC strain, is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university at 14/5/2021, and has the preservation number of CCTCC No: v202141.
2.3 PCR identification 200ul of isolate virus is taken, Solaibo RNA extraction kit is used for extracting virus RNA, after reverse transcription to cDNA, PCR amplification is carried out by PEDV specific primer, and the PCR reaction system is 25 ul.
The mixture was added to a 0.5m1 PCR tube in sequence
10×Ex Taq Mix---------------------12.5u1
Upstream primer (20pmol) - - - - - - - - - - - - - - - - -1u1
Downstream primer (20pmol) - - - - - - - - - - - - - - - - -1u1
cDNA-----------------------------------2ul
Supplementing the system with DEPC water to 25ul, setting a PCR reaction program to preheat at 94 ℃ for 2min, preheat at 94 ℃ for 30s, stretch at 55 ℃ for 30s, and stretch at 72 ℃ for 5min for 30 cycles, and after the PCR is finished, observing the result by 1% agarose gel electrophoresis, wherein the result is shown in figure 1, in the figure, 1 corresponds to a detection sample, 2 corresponds to a positive control, 3 corresponds to a negative control, 4 corresponds to a marker, and a PEDV specific band appears at 500bp as can be seen in figure 1.
2.4 measurement of Virus content isolates were serially diluted 10-fold and 10 were taken -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 6 dilutions, inoculating Vero cells with good growth vigor into a 96-well culture plate, inoculating 6 wells with 0.1ml of each dilution, simultaneously setting virus positive control wells and blank cell control wells, 37 ℃ and 5% CO 2 Culturing in an incubator, observing for 1-2 times every day, and continuously observing for 7 days. Carefully observing the pathological condition of cells, judging the cells to be infected by CPE, and calculating TCID by Reed-Muench method 50 PEDV SDLC strain with virus content of 10 7.5 ～10 8.5 TCID 50 Between/0.1 ml.
2.5 Virus specificity assay the 15 th passage virus solution was diluted to 200TCID in DMEM medium 50 0.1mL of the virus-free Vero cell culture medium is mixed with equivalent PEDV specific serum, the mixture is placed in a water bath at 37 ℃ for neutralization for 1 hour, 4 holes of a 24-hole cell plate full of Vero cell monolayers are inoculated, 4 holes of virus positive control cells and 4 holes of negative control cells are arranged at the same time, the temperature is 37 ℃, and 5 percent CO is added 2 Culturing in an incubator for 120h, observing cytopathic effect (CPE), wherein the neutralization group and the negative control group have no cytopathic effect, and the virus control group has cytopathic effect, which indicates that the virus is specific and can be neutralized by PEDV specific positive serum.
2.6 characterization of physicochemical Properties
2.6.1 Ether and chloroform sensitivity test cell culture virus solution was taken, treated with Ether and chloroform, respectively, and untreated virus controls were set, and TCID of treated and control groups was determined 50 The results show that the virus content of the virus liquid of the treatment group is 10 5.0 ～10 5.5 TCID 50 0.1ml, and a viral control virus content of 10 7.5 TCID 50 0.1ml, the virus content is reduced by more than 2 titers, which indicates that the virus is sensitive to the chloroform ether and is enveloped virus.
2.6.2 acid resistance test cell culture virus solution was taken and the pH of the virus solution was adjusted to 0.1mol/L hydrochloric acid3.0, setting physiological saline as control, placing in a constant temperature incubator at 37 deg.C for 2 hours, and adding 5.6% NaHCO 3 The treated virus solution was adjusted to pH 7.2, and then TCID was measured for the test group and the control group 50 The results show that after acid treatment, the virus content is reduced to 10 4.5 TCID 50 0.1ml, the virus titer is reduced by more than 2 titers, which indicates that the virus is not acid-resistant and can be inactivated under acidic conditions.
2.6.3 Heat resistance test the TCID of the test group and the control group was measured by dividing the cell culture virus solution into 3 tubes on average, placing 2 tubes in a water bath at 50 ℃ and 60 ℃ for 1 hour, and controlling the other tube 50 The results show that the virus content is 10 after the virus is bathed for 1 hour at 50 ℃ and 60 DEG C 5.5 TCID 50 /0.1ml，10 4.7 TCID 50 0.1ml, the virus titer dropped significantly, indicating that the virus was sensitive to heat.
2.7 animal regression test
Screening sow with negative PEDV pathogen and antibody, selecting 6 newborn piglets without colostrum after sow farrowing, dividing into toxin attacking group and control group, each group having 3 piglets, attacking 5 × 10 piglets in 3 days old 5 TCID 50 The PEDV SDLC strain virus liquid is orally taken in a toxin counteracting way, and meanwhile, a DMEM culture solution is orally taken in a control group. And (3) observing whether the piglets have symptoms such as water-sample diarrhea, vomiting, death and the like after challenge, wherein the result is shown in figure 2, A in the figure is the piglets of the challenge group, B is the piglets of the control group, C is the intestinal tract of the piglets of the challenge group, and D is the intestinal tract of the piglets of the control group, the results show that all aspects of the control group are normal, 2 piglets in the challenge group 12 hours after challenge have diarrhea symptoms, 3 piglets after 15 hours have vomiting and diarrhea symptoms, and the diarrhea is in a yellow water-sample shape. The piglets take food intermittently, and after 1 day, the food intake enthusiasm and the food intake recover, but the piglets always have diarrhea and vomiting phenomena, and the piglets gradually get thin. After 5 days of challenge, 3 piglets all died, and the results of the necropsy were shown in fig. 2C and D for piglets in the control group and the challenge group, where fig. 2D is the control group and the necropsy result is normal, and fig. 2C is the challenge group and the necropsy result in the challenge group showing small intestine swelling, filling with pale yellow liquid, thinning of intestinal wall, and individual small intestineThe intestinal mucosa has bleeding points, mesenteric lymph node edema and short small intestinal villi, and is an obvious symptom of porcine epidemic diarrhea virus infection. Anus swabs of 3 piglets are collected and PEDV detection is carried out, and as a result, the 3 piglets are positive to PEDV.
EXAMPLE 2 preparation of the vaccine
1. Preparation of porcine epidemic diarrhea virus SDLC strain antigen
1.1 cell preparation cell tube is taken out from liquid nitrogen tank and put into 37 ℃ water bath for melting, Vero cell is moved into a centrifuge tube filled with 10ml serum-free culture medium, centrifuged for 5 minutes at 1000r/min, supernatant is poured off, cell is suspended by DMEM culture solution containing 10% bovine serum, 37 ℃ and 5% CO 2 In the culture, cells were digested with trypsin when they grew into a monolayer, and subcultured.
1.2, virus inoculation when the cells grow into a good monolayer, discarding the original culture solution, adding the PEDV seed virus obtained by separation in the example 1 according to the amount of 1-2 percent of the total volume, simultaneously adding 7.5ug/ml pancreatin, culturing at 37 ℃, and observing the pathological condition of the cells every 8 hours.
1.3, harvesting cell culture solution when 80% of cells have pathological changes, repeatedly freezing and thawing for 2 times, centrifuging to remove cell debris, and reserving a sample for detecting the toxin value. Meanwhile, the sterile test is carried out according to the current Chinese veterinary pharmacopoeia, and the sterile growth is required. Storing at below-20 deg.C.
2. Inactivation of virus liquid of SDLC strain, semi-finished product inspection and preparation of vaccine
2.1 inactivating, namely placing the PEDV SDLC strain virus liquid into an inactivation bottle, adding beta-propiolactone (BPL) according to 0.025-0.05 percent of the total volume, inactivating for 18-24h at 4 ℃, then carrying out water bath for 2h at 37 ℃, finishing inactivation, and storing at 2-8 ℃ for no more than 1 month.
2.2 inspection of the semi-finished product
2.21 sterile examination inactivated virus solution was collected and examined according to 2015 edition of appendix of Chinese veterinary pharmacopoeia for sterile growth.
2.22 inactivation test the inactivated virus solution was inoculated into 6 wells of well-grown Vero cells (24-well plate) with 0.2ml per well, supplemented with maintenance solution to 2.0ml at 37 deg.C and 5% CO 2 Incubator, cell observation 2 times a day, observationThe inactivation was considered complete when no cytopathic effect appeared for 168 hours, and no cytopathic effect appeared in all cell wells in the blind generation.
2.23 measurement of Virus content cell sap before inactivation was serially diluted 10-fold with cell maintenance fluid, 10 cells were taken -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 6 dilutions were used to inoculate full monolayers of Vero cells (96 well cell plates), and each dilution was used to inoculate 6 wells, 0.1ml per well. Setting virus positive control hole and cell negative control hole at 37 deg.C and 5% CO 2 Culturing in an incubator, observing for 1-2 times every day, and continuously observing for 168 hours. Carefully observing the pathological condition of cells, judging the cells to be infected by CPE, and calculating the TCID of the virus seeds by a Reed-Muench method 50 . The virus content is more than or equal to 10 7.5 TCID 50 0.1ml of virus solution can be used for preparing the vaccine.
2.3 preparation of inactivated vaccine against PEDV
Emulsifying the inactivated virus solution and an oil adjuvant according to the proportion of 3: 7, and emulsifying to prepare the water-in-oil emulsion inactivated vaccine. Quantitatively subpackaging, covering and sealing, sticking a label, and storing at 2-8 ℃.
And 2.4, subpackaging quantitatively, sealing by covering, sticking a label, and storing at 2-8 ℃.
Example 3 preparation and examination of goat milk antibody against porcine epidemic diarrhea
1. Porcine epidemic diarrhea virus inactivated vaccine immunization
Basic immunity, wherein 0.8ml of vaccine is injected into neck and back muscles of each sheep;
performing secondary immunization, performing vaccination 2 times 14 days after the primary immunization, and performing intramuscular injection of 1.0ml on the back of the neck of each sheep;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 1.5ml is injected into the neck and back muscles of each sheep;
maintaining immunity, when neutralizing titer of porcine epidemic diarrhea antibody in sheep milk reaches 1:512, maintaining vaccination for 1 time, and injecting vaccine 1.5ml into neck and back muscle of each sheep.
Collecting goat milk, performing three times of intensified immunization, starting to collect the goat milk 14 days later, extracting the antibody, wherein the neutralizing titer of the porcine epidemic diarrhea antibody in the goat milk is not less than 1:512, and storing the goat milk at-20 ℃ for freezing.
2. Antibody extraction
2.1 taking the PEDV hyperimmune antibody goat milk out of a refrigeration house at the temperature of-20 ℃ one day in advance, and putting the goat milk into a melting tank for melting.
2.2 pouring the melted goat milk into a sterilized 500L fermentation tank the next day, adding 1mol/L hydrochloric acid solution while stirring to adjust the pH value to 4.0-4.2.
2.3 after the pH value is detected to be stable, centrifuging by using a horizontal decanter centrifuge to remove casein in the goat milk, and collecting the centrifuged goat milk to a second 500L sterile fermentation tank.
2.4 adding 1mol/L sodium hydroxide solution while stirring, and adjusting the pH value to 6.2-6.5.
2.5 heating the goat milk to 60 ℃ while stirring in a fermentation tank, and centrifuging the goat milk at 60 ℃ by a butterfly centrifuge to remove the milk fat.
2.6 adding the whey centrifuged by the butterfly centrifuge into a 500L sterile fermentation tank washed by the first sterile water for injection, stirring and mixing uniformly, heating the fermentation tank to 65 ℃ while stirring, preserving heat and inactivating for 15 minutes, and cooling in water bath to below 30 ℃ after inactivation is finished.
2.7 adding 10% formaldehyde solution into the 500L fermentation tank while stirring to the final concentration of 0.5 ‰, stirring, sealing for 60 min.
2.8 the deep filtration is clarified by K-type multi-layer plate-frame filtration.
2.9 Ultrafiltration the virus was removed by filtration through an ultrafiltration membrane with a molecular weight cut-off of 1000 kD.
2.10 sterilizing and filtering, filtering and sterilizing the qualified antibody by using a 0.22um microporous filter membrane, and subpackaging with 100 ml/bottle in an aseptic manner to obtain the porcine epidemic diarrhea goat milk antibody.
3. Antibody assay
3.1 the purity is detected according to the current pharmacopoeia of the people's republic of China, and the virus-free antibacterial disinfectant has no bacterial, mycoplasma and exogenous virus pollution.
3.2 the formaldehyde residue is detected according to the current pharmacopoeia of the people's republic of China, and the formaldehyde residue is in accordance with the regulations.
3.3 safety test 5 healthy piglets aged 3 days, 10ml of the oral administration is carried out on each piglet, 14 days of observation are carried out, 5/5 are healthy and alive, and no adverse reaction is caused.
3.4 efficacy test
3.4.1 detection of neutralizing potency of antibody was detected according to the current pharmacopoeia of the people's republic of China, and the antibody results were recorded.
The result of the measurement of the neutralizing antibody titer shows that the neutralizing titer of the diarrhea antibody is not less than 1: 32.
3.4.2 antibody prevention test
Screening sows to be born with PEDV pathogeny and antibody negative, randomly selecting 6 newborn piglets which do not eat colostrum after the sows lay piglets, randomly dividing the piglets into two groups, taking 3 piglets in a prevention group, 3 piglets in a control group, taking 5ml diarrhea antibody orally for each piglet in the morning and evening of 3 piglets in the prevention group when the piglets are 2 days old, taking the same dosage of physiological saline orally for each piglet in a toxicity attacking group, after 24 hours, respectively attacking the piglets in the prevention group and the toxicity attacking group, and taking 5 multiplied by 10 orally for each piglet 5 TCID 50 Viral solutions of PEDV SDLC strain. After the challenge, the health status of each piglet was observed, and the main observation indexes were: and (4) observing whether the piglets have watery diarrhea, vomit, death and the like for one week.
The antibody prevention test result shows that the piglets of the challenge control group all have the symptoms of vomiting and diarrhea 12 hours after the challenge, the diarrhea is yellow water-like, and then the piglets gradually become thin and finally die after dehydration. After the prevention group piglets are attacked by the toxin, 1 piglet has the symptoms of reduced feed intake, slight lassitude and the like, but can recover quickly, and has no diarrhea and vomiting in the whole observation period, so that the antibody is effective in preventing the piglet diarrhea.
3.4.3 antibody therapy assays
Screening sows to be born with PEDV pathogen and antibody as negative, after the sows lay piglets, randomly selecting 6 newborn piglets without colostrum, randomly dividing into two groups, 3 piglets in the challenge group, 3 piglets in the treatment group, and when the piglets are 2 days old, respectively carrying out challenge on each piglet of each group, and orally taking 5 × 10 piglets 5 TCID 50 Viral solutions of PEDV SDLC strain. After the piglet has symptoms and is about to counteract toxic pathogen 12In the treatment group, 3 piglets in the early, middle and late stages of each piglet are orally taken with 5ml diarrhea antibody respectively for 3 days continuously. The control group was not subjected to any treatment. The health status of each piglet was observed, with the main observation indicators: whether the piglets have watery diarrhea, vomit, death and other symptoms.
The antibody treatment test result shows that the piglets have diarrhea symptoms 12 hours after the toxin attack, the treatment group is given the antibody treatment, the diarrhea symptoms of the piglets in the treatment group are lighter in the early stage of the treatment compared with the control group, and the diarrhea is basically controlled after 3 days. The control group piglets all showed symptoms of vomiting and diarrhea after challenge, and 2/3 died due to dehydration after one week, which indicates that the antibody is effective in treating piglet diarrhea.
Example 4 preparation and examination of porcine epidemic diarrhea milk antibody
1. Porcine epidemic diarrhea inactivated vaccine immunity
Basic immunity, injecting vaccine 1.5ml into neck and back muscles of each cow;
performing secondary immunization, wherein the 2 nd inoculation is performed 14 days after the primary immunization, and 2.0ml is injected into the neck and back muscles of each cow;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 2.5ml is injected into the neck and back muscles of each cow;
maintaining immunity, when neutralizing titer of porcine epidemic diarrhea antibody in milk reaches 1:1024, maintaining inoculation for 1 time, and injecting vaccine 2.5ml into neck and back of each cow.
Collecting milk, starting to collect milk 14 days after three times of enhanced immunity, extracting antibodies, determining that the neutralizing titer of porcine epidemic diarrhea antibodies in the milk is not less than 1:1024, and freezing the milk at-20 ℃.
2 antibody extraction
2.1 taking the PEDV hyperimmune antibody milk out of a freezer at-20 ℃ one day in advance, and putting the milk into a melting tank for melting.
2.2 the next day, the melted milk was poured into a sterilized 500L fermenter, and 1mol/L hydrochloric acid solution was added with stirring to adjust the pH to 4.0-4.2.
2.3 after the pH value is detected to be stable, a horizontal screw centrifuge is used for centrifuging to remove casein in the milk, and the centrifuged milk is collected to a second 500L sterile fermentation tank.
2.4 adding 1mol/L sodium hydroxide solution while stirring, and adjusting the pH value to 6.2-6.5.
2.5 heating the milk to 60 ℃ while stirring in the fermentation tank, and centrifuging the 60 ℃ milk by a butterfly centrifuge to remove the milk fat.
2.6 adding the whey centrifuged by the butterfly centrifuge into a 500L sterile fermentation tank washed by the first sterile water for injection, stirring and mixing uniformly, heating the fermentation tank to 65 ℃ while stirring, preserving heat and inactivating for 15 minutes, and cooling in water bath to below 30 ℃ after inactivation is finished.
2.7 adding 10% formaldehyde solution into the 500L fermentation tank while stirring to the final concentration of 0.5 ‰, stirring, sealing for 60 min.
2.8 the deep filtration is clarified by K-type multi-layer plate-frame filtration.
2.9 Ultrafiltration the virus was removed by filtration through an ultrafiltration membrane with a molecular weight cut-off of 1000 kD.
2.10 sterilizing and filtering, filtering and sterilizing the qualified antibody by using a 0.22um microporous filter membrane, and subpackaging with 100 ml/bottle in an aseptic manner to obtain the porcine epidemic diarrhea milk antibody.
3. Antibody assay
3.1 the purity is detected according to the current pharmacopoeia of the people's republic of China, and the virus-free antibacterial disinfectant has no bacterial, mycoplasma and exogenous virus pollution.
3.2 the formaldehyde residue is detected according to the current pharmacopoeia of the people's republic of China, and the formaldehyde residue is in accordance with the regulations.
3.3 safety test 5 healthy piglets of 3 days old, orally take 10ml each, observe for 14 days, 5/5 are healthy and alive, have not any adverse reaction.
3.4 efficacy test
3.4.1 detection of neutralizing potency of antibody was detected according to the current pharmacopoeia of the people's republic of China, and the antibody results were recorded.
The result of the measurement of the neutralizing antibody titer shows that the neutralizing titer of the diarrhea antibody is not less than 1: 36.
3.4.2 antibody prevention test
Screening sows to be born with PEDV pathogeny and antibody negative, randomly selecting 6 newborn piglets which do not eat colostrum after the sows lay piglets, randomly dividing the piglets into two groups, taking 5ml diarrhea antibody orally for each piglet in the morning and evening in the 3 prevention groups and the 3 control groups respectively when the piglets are 2 days old, taking physiological saline with the same dose orally for each piglet in the 3 challenge groups, respectively carrying out challenge on the piglets in the prevention groups and the challenge groups after 24 hours, and taking 5 multiplied by 105TCID50 PEDV SDLC strain virus liquid orally for each piglet. After the challenge, the health status of each piglet was observed, and the main observation indexes were: and (4) observing whether the piglets have watery diarrhea, vomit, death and the like for one week.
The antibody prevention test result shows that the piglets of the challenge control group all have the symptoms of vomiting and diarrhea about 12 hours after the challenge, the diarrhea is yellow water-like, and then the piglets gradually thin and die after dehydration. After the prevention piglets are attacked by the toxin, the symptoms of reduced feed intake, slight lassitude and the like appear, but the piglets recover quickly, and the diarrhea and the vomiting do not appear in the whole observation period, which shows that the antibody is effective in preventing the diarrhea of the piglets.
3.4.3 antibody therapy
After the sows to be born with negative PEDV pathogeny and antibodies are screened, 6 newborn piglets which do not eat colostrums are randomly selected and randomly divided into two groups after the sows lay piglets, 3 piglets in a virus counteracting group and 3 piglets in a treatment group, when the piglets are 2 days old, each piglet in each group is respectively subjected to virus counteracting, and the virus liquid of the PEDV SDLC strain of 5 multiplied by 105TCID50 is orally taken by each piglet. After the piglets have symptoms and about 12 hours after the toxin is attacked, 5ml diarrhea antibodies are orally taken for 3 piglets in the treatment group respectively in the morning, the noon and the evening, and are continuously orally taken for 3 days. The control group was not subjected to any treatment. The health status of each piglet was observed, with the main observation indicators: and (4) whether the piglets have watery diarrhea, vomit, death and other symptoms.
The antibody treatment test result shows that the piglets have diarrhea symptoms 12 hours after the toxin attack, the treatment group is given the antibody treatment, the diarrhea symptoms of the piglets in the treatment group are lighter in the early stage of the treatment compared with the control group, and then the diarrhea is basically controlled. The piglets in the control group all have symptoms of vomiting and diarrhea after challenge, and 3/3 die due to dehydration after one week, which indicates that the antibody is effective in treating the diarrhea of the piglets.
Example 5 preparation and examination of porcine epidemic diarrhea refined yolk antibody
1. The laying hens should have the production performance of the commercial laying hens.
1.1 sampling blood for avian-free leukemia and avian influenza infection according to 0.5% of chicken flock, and all the samples should be negative.
1.2 the pullorum disease and mycoplasma gallisepticum are detected according to NY/T536-2002 'diagnosis technique for typhoid and pullorum disease' and NY/T553-2002 'diagnosis technique for mycoplasma gallisepticum disease', and the positive rate of pullorum disease and mycoplasma gallisepticum infection is less than or equal to 0.1%.
1.3 the construction of the feeding management chicken farm of the laying hens must meet the requirements of veterinary health epidemic prevention specifications. The chicken farm should leave the traffic road for more than 500 meters, and the entrance and exit roads should be separated. The material and the manure channel in the field are separated. The inlet and outlet of the chicken farm are provided with a disinfection pond. Isolation belts should be arranged in brooding houses and adult chicken houses. In addition, the chicken farm should be provided with manure treatment facilities, the whole-in and whole-out system is implemented, drinking water in the chicken farm should reach the sanitary standard, and feeding personnel should be sanitary and healthy.
1.4 according to the actual situation of local epidemic disease, related vaccines are timely inoculated according to a scientific immunization program, and according to the situation, antibacterial drugs are added into the feed according to the conventional method for preventing and treating bacterial infection, anticoccidial drugs are added for preventing coccidium infection, and the like.
1.5 chickens are 100-150 days old.
2. Porcine epidemic diarrhea inactivated vaccine immunization
Basic immunization: 0.6ml of vaccine is injected into the breast of each chicken by muscle injection;
and (3) secondary immunization: inoculating for the 2 nd time 14 days after the basic immunization, and injecting 0.8ml of the vaccine into the chest part of each chicken;
three times of immunization: inoculating for the 3 rd time 14 days after the second immunization, and injecting 1.0ml of the vaccine into the breast of each chicken intramuscularly;
and (3) maintaining immunity: when the neutralizing antibody titer of the porcine epidemic diarrhea virus in the yolk is 1:1024, the inoculation is maintained for 1 time, and 1.0ml of vaccine is injected into the breast of each chicken by muscle.
Egg collection: and (3) sampling eggs 10 days after the three times of enhanced immunization, separating egg yolks, extracting antibodies, collecting high-immunity eggs, storing at 4-8 ℃ for no more than 10 days, wherein the neutralizing titer of the egg yolks of the porcine epidemic diarrhea viruses is more than or equal to 1: 1024.
3. Antibody production
3.1 Eggshell Disinfection
Soaking high immunity egg in 0.1% benzalkonium bromide water solution at 42 deg.C for 15 min for disinfection, selecting egg shell with serious pollution, sterilizing separately, washing with clear water, and soaking for disinfection once. And then, soaking the eggs in a water bath with the temperature of more than 95 ℃ for disinfection for 5 seconds, taking out the eggs, and airing or blow-drying the eggs for later use.
3.2 separating the yolk and manually or mechanically beating the egg, fully removing the egg white, blastoderm and frenulum, and collecting the yolk.
3.3 inactivation of I
Adding the egg yolk liquid into a pasteurization tank, adding appropriate amount of water for injection, stirring, heating to 65 deg.C, inactivating for 15 min, and cooling to below 30 deg.C in water bath.
3.4 acidification extraction
Adding injection water with the volume 4 times that of the original egg yolk into an acidification reaction tank, adjusting the pH value to 4.2 by using 1mol/L HCL solution, cooling the water to 2-4 ℃, then adding the inactivated I egg yolk liquid while stirring, and keeping the temperature at 4-8 ℃ for standing for 4 hours.
3.5 centrifugal separation, freezing and centrifuging the acidified extract, removing precipitate, and taking supernatant for later use.
3.6 inactivation II adding octanoic acid to the final concentration of 0.02%, stirring and mixing, standing for 4 hours at room temperature.
3.7 the deep filtration is clarified by K-type multi-layer plate-frame filtration.
3.8 adding the inactivated III into a formaldehyde solution according to the final concentration of 0.05 percent, fully stirring and uniformly mixing, and sealing for 60 minutes.
3.9 Ultrafiltration to remove virus with a molecular weight cutoff of 1000KD ultrafiltration membrane.
3.10 blending the total mixture, adding Tween-80 to a final concentration of 0.02%, and adjusting the pH value to 6.8 by using 1mol/L sodium hydroxide solution.
3.11 sterilizing and filtering, filtering and sterilizing the qualified prepared antibody by using a 0.22um microporous filter membrane, and performing sterile subpackaging to obtain 100 ml/bottle of the novel Muscovy duck adenovirus refined egg yolk antibody.
4. Antibody assay
4.1 the purity is detected according to the current pharmacopoeia of the people's republic of China, and the virus-free antibacterial disinfectant has no bacterial, mycoplasma and exogenous virus pollution.
4.2 the octanoic acid and formaldehyde residue are respectively detected according to the current pharmacopoeia of the people's republic of China, and the regulations are met.
4.3 safety test 5 healthy piglets aged 3 days, 10ml of the oral administration is carried out on each piglet, 14 days of observation are carried out, 5/5 are healthy and alive, and no adverse reaction is caused.
4.4 efficacy test
4.4.1 detection of neutralizing potency of antibody was detected according to the current pharmacopoeia of the people's republic of China, and the antibody results were recorded.
The result of the measurement of the neutralizing antibody titer shows that the neutralizing titer of the diarrhea antibody is not less than 1: 64.
4.4.2 antibody prevention assay
Screening sows to be born with PEDV pathogeny and antibody negative, randomly selecting 6 newborn piglets which do not eat colostrum after the sows lay piglets, randomly dividing the piglets into two groups, taking 3 piglets in a prevention group, 3 piglets in a control group, taking 5ml diarrhea antibody orally for each piglet in the morning and evening of 3 piglets in the prevention group when the piglets are 2 days old, taking the same dosage of physiological saline orally for each piglet in a toxicity attacking group, after 24 hours, respectively attacking the piglets in the prevention group and the toxicity attacking group, and taking 5 multiplied by 10 orally for each piglet 5 TCID 50 Viral solutions of PEDV SDLC strain. After the challenge, the health status of each piglet was observed, and the main observation indexes were: and (4) observing whether the piglets have watery diarrhea, vomit, death and the like for one week.
The antibody prevention test result shows that the piglets of the challenge control group begin to have the symptoms of vomiting and diarrhea 12 hours after the challenge, the diarrhea is yellow water-like, and then the piglets gradually thin and die after dehydration. After the prevention piglets are attacked by the toxin, the symptoms of reduced feed intake, slight lassitude and the like appear, but the piglets recover quickly, and the diarrhea and the vomiting do not appear in the whole observation period, which shows that the antibody is effective in preventing the diarrhea of the piglets.
4.4.3 antibody therapy assay
Screening sows to be born with PEDV pathogen and antibody as negative, after the sows lay piglets, randomly selecting 6 newborn piglets without colostrum, randomly dividing into two groups, 3 piglets in the challenge group, 3 piglets in the treatment group, and when the piglets are 2 days old, respectively carrying out challenge on each piglet of each group, and orally taking 5 × 10 piglets 5 TCID 50 Virus liquid of PEDV SDLC strain. After the piglets have symptoms and are about 12 hours after the virus attack, 5ml diarrhea antibodies are orally taken by each piglet in the morning, in the middle and at the evening of each piglet of the 3 piglets in the treatment group for 3 days continuously. The control group was not subjected to any treatment. The health status of each piglet was observed, with the main observation indicators: and (4) whether the piglets have watery diarrhea, vomit, death and other symptoms.
The antibody treatment test results show that the piglets show diarrhea symptoms 12 hours after the challenge, and the diarrhea symptoms of the piglets in the treatment initial stage are lighter compared with those in the control group, and then the diarrhea is basically controlled. The piglets in the control group all have symptoms of vomiting and diarrhea after challenge, and 3/3 die due to dehydration after one week, which indicates that the antibody is effective in treating the diarrhea of the piglets.
Example 6 efficacy test of 3 different antibodies against porcine epidemic diarrhea
6.1 antibody prevention assay
Screening a to-be-born sow with negative PEDV pathogeny and antibody, after the sow produces piglets, randomly selecting 12 newborn piglets without colostrums, randomly dividing the newborn piglets into four groups, 3 prevention groups and a control group which are all 3 per group, wherein the 3 prevention groups respectively comprise a goat milk antibody group, a cow milk antibody group and a yolk antibody group, when the piglets are 2 days old, the 3 prevention groups respectively immunize the piglets to respectively obtain the goat milk antibody, the cow milk antibody and the yolk antibody with the PEDV neutralization titers of 32, each piglet is orally administered with 5ml of diarrhea antibody respectively in the morning and evening, each piglet in the challenge group is orally administered with the same dosage of physiological saline, after 24 hours, the piglets in the prevention group and the challenge group are respectively challenged, and each piglet is orally administered with 5 multiplied by 10 5 TCID 50 Virus liquid of PEDV SDLC strain. After the challenge, the health status of each piglet was observed, and the main observation indexes were: and (4) observing whether the piglets have watery diarrhea, vomit, death and the like for one week. Before attacking poison for each group of pigletsAnd weighing after the challenge test is finished, and calculating the weight increasing condition of the survival piglets.
TABLE 1 results of antibody prevention test
The antibody prevention test result shows that the piglets of the control group begin to have the symptoms of vomit and diarrhea after about 12 hours of toxin counteracting, the diarrhea is yellow water-like, and then gradually get thin, and finally die after dehydration. After the different antibodies prevent the piglets from attacking the toxin, the symptoms of feed intake reduction, lassitude and the like in different degrees appear. The specific results of the antibody prevention test are shown in table 1, and it can be seen from table 1 that after the piglets in 3 prevention groups are attacked by the toxin, the mental states and the feed intake of the piglets are different, the average weight change amount is different, the piglets in the goat milk antibody group have no clinical symptoms, the piglets in the cow milk antibody group have the conditions of reduced feed intake and lassitude, the piglets in the egg yolk antibody group all have the symptoms of reduced feed intake, lassitude and slight diarrhea, but the piglets recover to normal after 1 day. The average weight of piglets of the goat milk antibody group is increased by 1.2kg, the average weight of piglets of the cow milk antibody group is increased by 1.0kg, and the average weight of piglets of the egg yolk antibody group is increased by 0.6kg, so that the diarrhea antibody neutralization titers are both 32, and the diarrhea goat milk antibody group has better effect than the diarrhea goat milk antibody group and the diarrhea egg yolk antibody group.
6.2 antibody therapy test
Screening a to-be-born sow with negative PEDV pathogen and antibody, after the sow produces piglets, randomly selecting 12 newborn piglets which do not eat colostrums, randomly dividing the piglets into four groups, wherein a toxicity counteracting group comprises 3 piglets, a treatment group comprises 3 groups of goat milk antibody group, a cow milk antibody group and a yolk antibody group, 3 piglets are selected, when the piglets are 2 days old, each piglet is subjected to toxicity counteracting, and each piglet is orally taken by 5 multiplied by 10 5 TCID 50 Viral solutions of PEDV SDLC strain. When the piglets have symptoms, about 12 hours after the toxin attack, each piglet of 3 treatment groups is respectively orally taken with 5ml of diarrhea goat milk antibody, diarrhea goat milk antibody group and diarrhea egg yolk antibody in the morning, the noon and the evening, and the oral administration of the antibodies is continued until the complete control. Control group did not perform any taskAnd (4) processing. The health status of each piglet was observed, with the main observation indicators: whether the piglets have watery diarrhea, vomit, death and the like. Weighing each group of piglets before and 10 days after the challenge, and calculating the weight gain condition of the survival piglets.
The antibody treatment test results show that 12 hours after the challenge, piglets in each experimental group have diarrhea symptoms, the symptoms of a control group are continuously worsened, and 2/3 die due to dehydration after one week; the treatment group is given with antibody treatment, and compared with the control group, the diarrhea symptoms of the treatment group are relieved and obviously relieved in the early treatment period, and then the diarrhea of the piglets is gradually controlled and recovered to be normal, which shows that the diarrhea goat milk antibody, the diarrhea milk antibody and the diarrhea egg yolk antibody are all effective in treating the diarrhea of the piglets. The specific results of the antibody treatment tests are shown in table 2, and it can be seen from table 2 that after the piglets of 3 treatment groups respectively take the diarrhea goat milk antibody, the diarrhea goat milk antibody group and the diarrhea egg yolk antibody orally, the time required for alleviating and obviously reducing the diarrhea symptoms of the piglets, the time required for controlling the diarrhea and the average weight variation are different, the time required for alleviating and obviously reducing the diarrhea symptoms of the piglets is only 2 days at the shortest, the time required for controlling the diarrhea is only 3 days, the time required for alleviating and obviously reducing the diarrhea symptoms of the goat milk antibody group is only 3 days at the shortest, the time required for controlling the diarrhea is 5 days, the time required for alleviating and obviously reducing the diarrhea symptoms of the egg yolk antibody group is 5 days at the shortest, and the time required for controlling the diarrhea is 9 days; after 10 days of the challenge, the average weight of piglets in the goat milk antibody group increased 1.6kg, the average weight of piglets in the cow milk antibody group increased 1.1kg, and the average weight of piglets in the egg yolk antibody group decreased 0.2kg, so that the effect of the goat milk antibody group with diarrhea was better than that of the cow milk antibody group with diarrhea than that of the egg yolk antibody group with diarrhea.
TABLE 2 results of antibody therapy tests
Through the comparative tests of the diarrhea goat milk antibody, the diarrhea milk antibody and the diarrhea yolk for preventing and treating the diarrhea of the piglets, the neutralizing titer of the PEDV antibody is 1:32, the diarrhea goat milk antibody has the best effect in both the prevention and treatment tests of the diarrhea of the piglets, the diarrhea goat milk antibody has the second best effect, the diarrhea yolk antibody has the worst effect in the three antibodies, and the good prevention and treatment means that the loss of farmers can be reduced to the greatest extent.
Example 7 preparation of goat milk antibody against porcine epidemic diarrhea
1. Porcine epidemic diarrhea virus inactivated vaccine immunization
Basic immunity, wherein 0.8ml of vaccine is injected into neck and back muscles of each sheep;
performing secondary immunization, wherein the 2 nd inoculation is performed 14 days after the basic immunization, and the back of the neck of each sheep is injected with 1.0ml of intramuscular injection;
three times of immunization, wherein 3 rd time of inoculation is carried out 14 days after the second time of immunization, and 1.5ml of vaccine is injected into the neck and back of each sheep through muscle;
maintaining immunity, when neutralizing titer of porcine epidemic diarrhea antibody in sheep milk reaches 1:512, maintaining vaccination for 1 time, and injecting vaccine 1.5ml into neck and back muscle of each sheep.
Collecting goat milk, performing three times of intensified immunization, starting to collect the goat milk 14 days later, extracting the antibody, wherein the neutralizing titer of the porcine epidemic diarrhea antibody in the goat milk is not less than 1:512, and storing the goat milk at-20 ℃ for freezing.
2. Antibody extraction
2.1 taking the PEDV hyperimmune antibody goat milk out of a refrigeration house at the temperature of-20 ℃ one day in advance, and putting the goat milk into a melting tank for melting.
2.2 pouring the melted goat milk into a sterilized 500L fermentation tank the next day, adding 1mol/L hydrochloric acid solution while stirring to adjust the pH value to 4.0-4.2.
2.3 detecting that the pH value is stable, centrifuging by using a horizontal decanter centrifuge to remove casein in the goat milk, and collecting the centrifuged goat milk to a second 500L sterile fermentation tank.
2.4 adding 1mol/L sodium hydroxide solution while stirring, and adjusting the pH value to 6.2-6.5.
2.5 heating the goat milk to 50 ℃ while stirring in a fermentation tank, and centrifuging the goat milk at 50 ℃ by a butterfly centrifuge to remove the milk fat.
2.6 adding the whey centrifuged by the butterfly centrifuge into a 500L sterile fermentation tank washed by the first sterile water for injection, stirring and mixing uniformly, heating the fermentation tank to 60 ℃ while stirring, preserving heat and inactivating for 20 minutes, and cooling in water bath to below 30 ℃ after inactivation is finished.
2.7 adding 10% formaldehyde solution into the 500L fermentation tank while stirring to the final concentration of 0.5 ‰, stirring, sealing for 50 min.
2.8 the deep filtration is clarified by filtration with a K-type multi-layer plate frame.
2.9 Ultrafiltration the virus was removed by filtration through an ultrafiltration membrane with a molecular weight cut-off of 1000 kD.
2.10 sterilizing and filtering, filtering and sterilizing the qualified antibody by using a 0.22um microporous filter membrane, and subpackaging with 100 ml/bottle in an aseptic manner to obtain the porcine epidemic diarrhea goat milk antibody.
Example 8 preparation of porcine epidemic diarrhea milk antibody
1. Porcine epidemic diarrhea inactivated vaccine immunization
Basic immunity, wherein 1.5ml of vaccine is injected into the neck and back of each cow;
performing secondary immunization, wherein the 2 nd inoculation is performed 14 days after the basic immunization, and 2.0ml is injected into the neck and back muscles of each cow;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 2.5ml is injected into the neck and back muscles of each cow;
maintaining immunity, when neutralizing titer of porcine epidemic diarrhea antibody in milk reaches 1:1024, maintaining inoculation for 1 time, and injecting vaccine 2.5ml into neck and back of each cow.
Collecting milk, starting to collect milk 14 days after three times of enhanced immunity, extracting antibodies, determining that the neutralizing titer of porcine epidemic diarrhea antibodies in the milk is not less than 1:1024, and freezing the milk at-20 ℃.
2 antibody extraction
2.1 taking the PEDV hyperimmune antibody milk out of a freezer at-20 ℃ one day in advance, and putting the milk into a melting tank for melting.
2.2 pouring the melted milk into a sterilized 500L fermentation tank the next day, adding 1mol/L hydrochloric acid solution while stirring to adjust the pH value to 4.0-4.2.
2.3 after the pH value is detected to be stable, a horizontal screw centrifuge is used for centrifuging to remove casein in the milk, and the centrifuged milk is collected to a second 500L sterile fermentation tank.
2.4 adding 1mol/L sodium hydroxide solution while stirring, and adjusting the pH value to 6.2-6.5.
2.5 heating the milk to 70 ℃ while stirring in the fermentation tank, and centrifuging the milk at 70 ℃ by a butterfly centrifuge to remove the milk fat.
2.6 adding the whey centrifuged by the butterfly centrifuge into a 500L sterile fermentation tank washed by the first sterile water for injection, stirring and mixing uniformly, heating the fermentation tank to 70 ℃ while stirring, preserving heat and inactivating for 10 minutes, and cooling in water bath to below 30 ℃ after inactivation is finished.
2.7 adding 10% formaldehyde solution into the 500L fermentation tank while stirring to the final concentration of 0.5 ‰, stirring, sealing for 80 min.
2.8 the deep filtration is clarified by K-type multi-layer plate-frame filtration.
2.9 Ultrafiltration the virus was removed by filtration through an ultrafiltration membrane with a molecular weight cut-off of 1000 kD.
2.10 sterilizing and filtering, filtering and sterilizing the qualified antibody by using a 0.22um microporous filter membrane, and subpackaging with 100 ml/bottle in an aseptic manner to obtain the porcine epidemic diarrhea milk antibody.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. The porcine epidemic diarrhea virus strain is named as porcine epidemic diarrhea virus SDLC strain, is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university at 13 months 4 in 2021, and has the preservation number of CCTCC No: v202141.
2. The inactivated vaccine for porcine epidemic diarrhea virus is characterized by being prepared by using the SDLC strain for porcine epidemic diarrhea virus as the inactivated antigen according to claim 1, wherein the virus content of virus liquid for preparing the vaccine is more than or equal to 10 7.5 TCID 50 /0.1ml。
3. An antibody for preventing and treating piglet epidemic diarrhea, which is characterized in that the antibody is obtained by immunizing a milk-producing cow or sheep with the inactivated vaccine for porcine epidemic diarrhea virus of claim 2, and collecting the immunized cow or sheep milk and extracting the antibody.
4. The method for preparing an antibody for preventing and treating piglet epidemic diarrhea according to claim 3, wherein the preparation method comprises the following steps:
(1) immunizing milk-producing cattle or sheep by using the porcine epidemic diarrhea virus inactivated vaccine of claim 2, and collecting sheep milk or cow milk after immunization;
(2) adjusting the pH of the goat milk or cow milk to 4.0-4.2, and centrifuging to remove casein in the goat milk or cow milk;
(3) adding alkali into goat milk or cow milk without casein to adjust pH to 6.2-6.5, heating to 50-70 deg.C, centrifuging to remove milk fat;
(4) heating goat milk or cow milk without butter fat to 60-70 deg.C, inactivating for 10-20 min, and cooling in water bath to below 30 deg.C;
(5) adding a formaldehyde solution into the goat milk or the cow milk cooled in the step (4) until the final concentration of formaldehyde is 0.5 per mill, fully stirring and uniformly mixing, and sealing for 50-80 minutes;
(6) and (5) carrying out deep filtration, ultrafiltration and sterilization filtration on the solution obtained in the step (5) to obtain the antibody for preventing and treating the piglet epidemic diarrhea.
5. The method for preparing the antibody for preventing and treating the piglet epidemic diarrhea according to claim 4, wherein the porcine epidemic diarrhea virus inactivated vaccine in the step (1) is used for immunizing a goat producing milk, and the step of collecting the goat milk comprises the following steps:
basic immunity, wherein 0.8ml of vaccine is injected into neck and back muscles of each sheep;
performing secondary immunization, performing vaccination 2 times 14 days after the primary immunization, and performing intramuscular injection of 1.0ml on the back of the neck of each sheep;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 1.5ml is injected into the neck and back muscles of each sheep;
maintaining immunity, when neutralizing titer of porcine epidemic diarrhea antibody in goat milk reaches 1:512, maintaining inoculation for 1 time, and injecting vaccine 1.5ml into neck and back muscle of each goat;
collecting goat milk, starting to collect the goat milk 14 days after three times of boosting immunization, extracting the antibody, determining that the neutralizing titer of the porcine epidemic diarrhea antibody in the goat milk is not less than 1:512, and storing the goat milk at-20 ℃ for freezing.
6. The method for preparing the antibody for preventing and treating the piglet epidemic diarrhea according to claim 4, wherein the step (1) of immunizing the cows producing milk with the inactivated vaccine for porcine epidemic diarrhea virus comprises the following specific steps of:
basic immunity, wherein 1.5ml of vaccine is injected into the neck and back of each cow;
performing secondary immunization, wherein the 2 nd inoculation is performed 14 days after the primary immunization, and 2.0ml is injected into the neck and back muscles of each cow;
three times of immunization, wherein the 3 rd inoculation is carried out 14 days after the second immunization, and 2.5ml is injected into the neck and back muscles of each cow;
maintaining immunity, when the neutralizing titer of porcine epidemic diarrhea antibody in milk reaches 1:1024, maintaining inoculation for 1 time, and injecting vaccine 2.5ml into neck and back muscles of each cow;
collecting milk, starting to collect milk 14 days after three times of enhanced immunity, extracting antibodies, determining that the neutralizing titer of the porcine epidemic diarrhea antibodies in the milk is not less than 1:1024, and freezing and storing the milk at the temperature of-20 ℃.
7. The method according to claim 4, wherein the collected goat or cow's milk is frozen at-20 ℃ after collection, the frozen goat or cow's milk is taken out one day before the pH is adjusted in step (2) and placed in a melting tank for melting, the melted goat or cow's milk is poured into a sterilized container the next day, a hydrochloric acid solution is added while stirring to adjust the pH to 4.0-4.2, the casein in the cow's milk is removed by a centrifuge after the pH is detected to be stable, and the centrifuged goat or cow's milk is collected in another container.
8. The method for preparing antibody for preventing and treating piglet epidemic diarrhea according to claim 4, wherein in the step (3), the sodium hydroxide solution is added to the goat or cow's milk without casein while stirring, the pH is adjusted to 6.2-6.5, then the goat or cow's milk is heated to 60 ℃ while stirring, and the goat or cow's milk with 60 ℃ is centrifuged to remove the milk fat.
9. The method for preparing the antibody for preventing and treating piglet epidemic diarrhea according to claim 4, wherein in the step (6), the solution obtained in the step (5) is clarified by filtration through a K-type multi-layer plate frame, and is subjected to virus removal by ultrafiltration membrane with the molecular weight cutoff of 1000KD, and the antibody which is qualified in preparation is subjected to filtration sterilization through a 0.22um microporous membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110934059.8A CN113430177B (en) | 2021-08-16 | 2021-08-16 | Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110934059.8A CN113430177B (en) | 2021-08-16 | 2021-08-16 | Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113430177A CN113430177A (en) | 2021-09-24 |
| CN113430177B true CN113430177B (en) | 2022-09-02 |
Family
ID=77797654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110934059.8A Active CN113430177B (en) | 2021-08-16 | 2021-08-16 | Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113430177B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304416A (en) * | 2016-04-19 | 2017-10-31 | 北京大伟嘉生物技术股份有限公司 | One plant of Porcine epidemic diarrhea virus and its cultural method and application |
| JP6253210B1 (en) * | 2016-08-17 | 2017-12-27 | 一般財団法人日本生物科学研究所 | Method for preventing or treating swine epidemic diarrhea, vaccine, and vaccine kit |
| CN107050447B (en) * | 2016-11-14 | 2018-08-28 | 陕西诺威利华生物科技有限公司 | Porcine epidemic diarrhea virus inactivated vaccine and preparation method thereof |
| CN106591244B (en) * | 2016-12-29 | 2019-09-03 | 江苏省农业科学院 | A kind of Porcine epidemic diarrhea virus, inactivated vaccine and preparation method thereof |
-
2021
- 2021-08-16 CN CN202110934059.8A patent/CN113430177B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113430177A (en) | 2021-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111905100B (en) | Goose astrovirus bivalent inactivated vaccine and yolk antibody and preparation method thereof | |
| CN110251671B (en) | Preparation method of goose astrovirus egg yolk antigen-antibody complex | |
| CN109265540B (en) | Preparation and application of goose astrovirus egg yolk antibody | |
| CN108794627A (en) | A kind of preparation method of duck reovirus refined vitelline antibody | |
| CN114539396B (en) | Bovine enterovirus egg yolk antibody and preparation | |
| CN111000993A (en) | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof | |
| CN112996808A (en) | Compositions and methods for treating acute diarrhea and intestinal infections in animals | |
| CN110590941B (en) | Novel Muscovy duck adenovirus refined egg yolk antibody and preparation method thereof | |
| CN111533803B (en) | Muscovy duck adenovirus type 2 and type3 refined egg yolk antibody and preparation method thereof | |
| CN113430177B (en) | Porcine epidemic diarrhea virus strain, inactivated vaccine, antibody and preparation method of antibody | |
| CN106563125B (en) | Duck hepatitis A virus III type compound live vaccine and preparation method thereof | |
| CN105693854A (en) | Preparation method of refined yolk antibody against duck tembusu virus | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| CN108939063B (en) | Muscovy duck triple inactivated vaccine | |
| CN112552398B (en) | Duck viral hepatitis egg yolk antibody and preparation method thereof | |
| CN107365382B (en) | Egg yolk antibody of duck adenovirus type 2 and preparation method thereof | |
| CN112646750B (en) | Serum 18 type riemerella anatipestifer and application thereof | |
| CN116286670A (en) | Novel duck reovirus and application thereof in preparation of inactivated vaccine and egg yolk antibody | |
| CN112999343B (en) | Inactivated vaccine of goose astrovirus and preparation method thereof | |
| CN105831391B (en) | Traditional Chinese medicine micro-ecological granules for preventing and treating newcastle disease, feed and application thereof | |
| CN107982532B (en) | Duck hepatitis A virus antigen-antibody complex vaccine and preparation method thereof | |
| CN111110839A (en) | Goose astrovirus bivalent inactivated vaccine for preventing gosling gout | |
| CN111621450B (en) | Duck-source gallibacterium and application thereof | |
| CN111087467A (en) | Preparation method of duck virus hepatitis and duck tongue disease bigeminal polyvalent egg yolk antibody | |
| CN112679606B (en) | Hypericum suis high-immunity serum and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230523 Address after: 262200 Jiayue Town, Zhucheng City, Weifang City, Shandong Province Patentee after: SHANDONG SINDER ANIMAL VACCINE Co.,Ltd. Address before: 262200 no.195 Shungeng Road, Zhucheng City, Weifang City, Shandong Province Patentee before: SHANDONG SINDER TECHNOLOGY Co.,Ltd. |