CN1278301A - 新猪环病毒、疫苗及诊断试剂 - Google Patents
新猪环病毒、疫苗及诊断试剂 Download PDFInfo
- Publication number
- CN1278301A CN1278301A CN98810652A CN98810652A CN1278301A CN 1278301 A CN1278301 A CN 1278301A CN 98810652 A CN98810652 A CN 98810652A CN 98810652 A CN98810652 A CN 98810652A CN 1278301 A CN1278301 A CN 1278301A
- Authority
- CN
- China
- Prior art keywords
- virus
- vaccine
- porcine
- cell
- goods
- 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.)
- Granted
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 50
- 241001533384 Circovirus Species 0.000 title claims description 30
- 239000003153 chemical reaction reagent Substances 0.000 title description 11
- 239000000523 sample Substances 0.000 claims abstract description 32
- 241000202347 Porcine circovirus Species 0.000 claims description 38
- 241000700605 Viruses Species 0.000 claims description 37
- 210000004027 cell Anatomy 0.000 claims description 33
- 102000036639 antigens Human genes 0.000 claims description 30
- 108091007433 antigens Proteins 0.000 claims description 30
- 239000000427 antigen Substances 0.000 claims description 29
- 239000012634 fragment Substances 0.000 claims description 26
- 108020004414 DNA Proteins 0.000 claims description 21
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 20
- 208000011580 syndromic disease Diseases 0.000 claims description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 17
- 238000004113 cell culture Methods 0.000 claims description 16
- 229920001184 polypeptide Polymers 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 14
- 239000002671 adjuvant Substances 0.000 claims description 13
- 102100031725 Cortactin-binding protein 2 Human genes 0.000 claims description 11
- 101710197985 Probable protein Rev Proteins 0.000 claims description 11
- 230000000890 antigenic effect Effects 0.000 claims description 11
- 230000009849 deactivation Effects 0.000 claims description 10
- 101000748061 Acholeplasma phage L2 Uncharacterized 16.1 kDa protein Proteins 0.000 claims description 9
- 101000947615 Clostridium perfringens Uncharacterized 38.4 kDa protein Proteins 0.000 claims description 9
- 101000964391 Enterococcus faecalis UPF0145 protein Proteins 0.000 claims description 9
- 101000748063 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 11.1 kDa protein in rep-hol intergenic region Proteins 0.000 claims description 9
- 101000790840 Klebsiella pneumoniae Uncharacterized 49.5 kDa protein in cps region Proteins 0.000 claims description 9
- 101710110895 Uncharacterized 7.3 kDa protein in cox-rep intergenic region Proteins 0.000 claims description 8
- 101000818108 Acholeplasma phage L2 Uncharacterized 81.3 kDa protein Proteins 0.000 claims description 7
- 101000912350 Haemophilus phage HP1 (strain HP1c1) DNA N-6-adenine-methyltransferase Proteins 0.000 claims description 7
- 101000790844 Klebsiella pneumoniae Uncharacterized 24.8 kDa protein in cps region Proteins 0.000 claims description 7
- 238000013467 fragmentation Methods 0.000 claims description 7
- 238000006062 fragmentation reaction Methods 0.000 claims description 7
- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 claims description 5
- 241000188845 Porcine adenovirus Species 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 230000001717 pathogenic effect Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- 238000001727 in vivo Methods 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 4
- 229940031626 subunit vaccine Drugs 0.000 claims description 4
- 241000701447 unidentified baculovirus Species 0.000 claims description 4
- 241000204045 Mycoplasma hyopneumoniae Species 0.000 claims description 3
- 210000003292 kidney cell Anatomy 0.000 claims description 3
- 244000052769 pathogen Species 0.000 claims description 3
- 241000606750 Actinobacillus Species 0.000 claims description 2
- 201000008283 Atrophic Rhinitis Diseases 0.000 claims description 2
- 206010008631 Cholera Diseases 0.000 claims description 2
- 241000700662 Fowlpox virus Species 0.000 claims description 2
- 208000032923 Lobar pneumonia Diseases 0.000 claims description 2
- 241000009328 Perro Species 0.000 claims description 2
- 206010035664 Pneumonia Diseases 0.000 claims description 2
- 206010039088 Rhinitis atrophic Diseases 0.000 claims description 2
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims description 2
- 241000700618 Vaccinia virus Species 0.000 claims description 2
- 206010022000 influenza Diseases 0.000 claims description 2
- 241001529453 unidentified herpesvirus Species 0.000 claims description 2
- 230000002238 attenuated effect Effects 0.000 claims 1
- 239000012228 culture supernatant Substances 0.000 claims 1
- 238000000338 in vitro Methods 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 239000013598 vector Substances 0.000 claims 1
- 230000009385 viral infection Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 230000003612 virological effect Effects 0.000 abstract description 12
- 239000000203 mixture Substances 0.000 abstract description 7
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 238000002405 diagnostic procedure Methods 0.000 abstract description 3
- 238000010367 cloning Methods 0.000 abstract description 2
- 108091033319 polynucleotide Proteins 0.000 abstract description 2
- 102000040430 polynucleotide Human genes 0.000 abstract description 2
- 239000002157 polynucleotide Substances 0.000 abstract description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 description 39
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 23
- 108700026244 Open Reading Frames Proteins 0.000 description 21
- 238000005516 engineering process Methods 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 14
- 241001071864 Lethrinus laticaudis Species 0.000 description 13
- 239000002773 nucleotide Substances 0.000 description 13
- 125000003729 nucleotide group Chemical group 0.000 description 13
- 239000000839 emulsion Substances 0.000 description 12
- 230000000692 anti-sense effect Effects 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 230000010076 replication Effects 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 239000011521 glass Substances 0.000 description 9
- 238000013016 damping Methods 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 238000007901 in situ hybridization Methods 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 101000768957 Acholeplasma phage L2 Uncharacterized 37.2 kDa protein Proteins 0.000 description 7
- 101000823746 Acidianus ambivalens Uncharacterized 17.7 kDa protein in bps2 3'region Proteins 0.000 description 7
- 101000916369 Acidianus ambivalens Uncharacterized protein in sor 5'region Proteins 0.000 description 7
- 101000769342 Acinetobacter guillouiae Uncharacterized protein in rpoN-murA intergenic region Proteins 0.000 description 7
- 101000823696 Actinobacillus pleuropneumoniae Uncharacterized glycosyltransferase in aroQ 3'region Proteins 0.000 description 7
- 101000786513 Agrobacterium tumefaciens (strain 15955) Uncharacterized protein outside the virF region Proteins 0.000 description 7
- 101000618005 Alkalihalobacillus pseudofirmus (strain ATCC BAA-2126 / JCM 17055 / OF4) Uncharacterized protein BpOF4_00885 Proteins 0.000 description 7
- 102100020724 Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Human genes 0.000 description 7
- 101000967489 Azorhizobium caulinodans (strain ATCC 43989 / DSM 5975 / JCM 20966 / LMG 6465 / NBRC 14845 / NCIMB 13405 / ORS 571) Uncharacterized protein AZC_3924 Proteins 0.000 description 7
- 101000823761 Bacillus licheniformis Uncharacterized 9.4 kDa protein in flaL 3'region Proteins 0.000 description 7
- 101000819719 Bacillus methanolicus Uncharacterized N-acetyltransferase in lysA 3'region Proteins 0.000 description 7
- 101000789586 Bacillus subtilis (strain 168) UPF0702 transmembrane protein YkjA Proteins 0.000 description 7
- 101000792624 Bacillus subtilis (strain 168) Uncharacterized protein YbxH Proteins 0.000 description 7
- 101000790792 Bacillus subtilis (strain 168) Uncharacterized protein YckC Proteins 0.000 description 7
- 101000819705 Bacillus subtilis (strain 168) Uncharacterized protein YlxR Proteins 0.000 description 7
- 101000948218 Bacillus subtilis (strain 168) Uncharacterized protein YtxJ Proteins 0.000 description 7
- 101000718627 Bacillus thuringiensis subsp. kurstaki Putative RNA polymerase sigma-G factor Proteins 0.000 description 7
- 101000641200 Bombyx mori densovirus Putative non-structural protein Proteins 0.000 description 7
- 101000947633 Claviceps purpurea Uncharacterized 13.8 kDa protein Proteins 0.000 description 7
- 101000948901 Enterobacteria phage T4 Uncharacterized 16.0 kDa protein in segB-ipI intergenic region Proteins 0.000 description 7
- 101000805958 Equine herpesvirus 4 (strain 1942) Virion protein US10 homolog Proteins 0.000 description 7
- 101000790442 Escherichia coli Insertion element IS2 uncharacterized 11.1 kDa protein Proteins 0.000 description 7
- 101000788354 Escherichia phage P2 Uncharacterized 8.2 kDa protein in gpA 5'region Proteins 0.000 description 7
- 101000770304 Frankia alni UPF0460 protein in nifX-nifW intergenic region Proteins 0.000 description 7
- 101000797344 Geobacillus stearothermophilus Putative tRNA (cytidine(34)-2'-O)-methyltransferase Proteins 0.000 description 7
- 101000748410 Geobacillus stearothermophilus Uncharacterized protein in fumA 3'region Proteins 0.000 description 7
- 101000772675 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) UPF0438 protein HI_0847 Proteins 0.000 description 7
- 101000631019 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Uncharacterized protein HI_0350 Proteins 0.000 description 7
- 101000768938 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.9 kDa protein in int-C1 intergenic region Proteins 0.000 description 7
- 101000785414 Homo sapiens Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Proteins 0.000 description 7
- 101000782488 Junonia coenia densovirus (isolate pBRJ/1990) Putative non-structural protein NS2 Proteins 0.000 description 7
- 101000811523 Klebsiella pneumoniae Uncharacterized 55.8 kDa protein in cps region Proteins 0.000 description 7
- 101000818409 Lactococcus lactis subsp. lactis Uncharacterized HTH-type transcriptional regulator in lacX 3'region Proteins 0.000 description 7
- 101000878851 Leptolyngbya boryana Putative Fe(2+) transport protein A Proteins 0.000 description 7
- 101000758828 Methanosarcina barkeri (strain Fusaro / DSM 804) Uncharacterized protein Mbar_A1602 Proteins 0.000 description 7
- 101001122401 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF3 Proteins 0.000 description 7
- 101001055788 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) Pentapeptide repeat protein MfpA Proteins 0.000 description 7
- 101000740670 Orgyia pseudotsugata multicapsid polyhedrosis virus Protein C42 Proteins 0.000 description 7
- 101000769182 Photorhabdus luminescens Uncharacterized protein in pnp 3'region Proteins 0.000 description 7
- 101000961392 Pseudescherichia vulneris Uncharacterized 29.9 kDa protein in crtE 3'region Proteins 0.000 description 7
- 101000731030 Pseudomonas oleovorans Poly(3-hydroxyalkanoate) polymerase 2 Proteins 0.000 description 7
- 101001065485 Pseudomonas putida Probable fatty acid methyltransferase Proteins 0.000 description 7
- 101000711023 Rhizobium leguminosarum bv. trifolii Uncharacterized protein in tfuA 3'region Proteins 0.000 description 7
- 101000948156 Rhodococcus erythropolis Uncharacterized 47.3 kDa protein in thcA 5'region Proteins 0.000 description 7
- 101000917565 Rhodococcus fascians Uncharacterized 33.6 kDa protein in fasciation locus Proteins 0.000 description 7
- 101000790284 Saimiriine herpesvirus 2 (strain 488) Uncharacterized 9.5 kDa protein in DHFR 3'region Proteins 0.000 description 7
- 101000936719 Streptococcus gordonii Accessory Sec system protein Asp3 Proteins 0.000 description 7
- 101000788499 Streptomyces coelicolor Uncharacterized oxidoreductase in mprA 5'region Proteins 0.000 description 7
- 101001102841 Streptomyces griseus Purine nucleoside phosphorylase ORF3 Proteins 0.000 description 7
- 101000708557 Streptomyces lincolnensis Uncharacterized 17.2 kDa protein in melC2-rnhH intergenic region Proteins 0.000 description 7
- 101000649826 Thermotoga neapolitana Putative anti-sigma factor antagonist TM1081 homolog Proteins 0.000 description 7
- 101000827562 Vibrio alginolyticus Uncharacterized protein in proC 3'region Proteins 0.000 description 7
- 101000778915 Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) Uncharacterized membrane protein VP2115 Proteins 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 6
- 229940031551 inactivated vaccine Drugs 0.000 description 6
- 239000000376 reactant Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 208000007502 anemia Diseases 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 3
- 238000009007 Diagnostic Kit Methods 0.000 description 3
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 3
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 3
- 102100024407 Jouberin Human genes 0.000 description 3
- 241000710778 Pestivirus Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 150000002337 glycosamines Chemical class 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N squalane Chemical compound CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000276427 Poecilia reticulata Species 0.000 description 2
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 2
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- WXNRAKRZUCLRBP-UHFFFAOYSA-N avridine Chemical compound CCCCCCCCCCCCCCCCCCN(CCCN(CCO)CCO)CCCCCCCCCCCCCCCCCC WXNRAKRZUCLRBP-UHFFFAOYSA-N 0.000 description 2
- 229950010555 avridine Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000012631 diagnostic technique Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229960001484 edetic acid Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008521 reorganization Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012882 sequential analysis Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 1
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- PIGTXFOGKFOFTO-FVFWYJKVSA-N (2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8R,8aR,12aS,14aR,14bR)-8a-carboxy-4-formyl-8-hydroxy-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound O([C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)C[C@@H](O)[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O PIGTXFOGKFOFTO-FVFWYJKVSA-N 0.000 description 1
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101000621943 Acholeplasma phage L2 Probable integrase/recombinase Proteins 0.000 description 1
- 101000818123 Acholeplasma phage L2 Uncharacterized 17.2 kDa protein Proteins 0.000 description 1
- 101000818089 Acholeplasma phage L2 Uncharacterized 25.6 kDa protein Proteins 0.000 description 1
- 101000977065 Acidithiobacillus ferridurans Uncharacterized 11.6 kDa protein in mobS 3'region Proteins 0.000 description 1
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 101000618348 Allochromatium vinosum (strain ATCC 17899 / DSM 180 / NBRC 103801 / NCIMB 10441 / D) Uncharacterized protein Alvin_0065 Proteins 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 101000781117 Autographa californica nuclear polyhedrosis virus Uncharacterized 12.4 kDa protein in CTL-LEF2 intergenic region Proteins 0.000 description 1
- 101000770875 Autographa californica nuclear polyhedrosis virus Uncharacterized 14.2 kDa protein in PK1-LEF1 intergenic region Proteins 0.000 description 1
- 101000708323 Azospirillum brasilense Uncharacterized 28.8 kDa protein in nifR3-like 5'region Proteins 0.000 description 1
- 101000770311 Azotobacter chroococcum mcd 1 Uncharacterized 19.8 kDa protein in nifW 5'region Proteins 0.000 description 1
- 101000748761 Bacillus subtilis (strain 168) Uncharacterized MFS-type transporter YcxA Proteins 0.000 description 1
- 101000765620 Bacillus subtilis (strain 168) Uncharacterized protein YlxP Proteins 0.000 description 1
- 101000916134 Bacillus subtilis (strain 168) Uncharacterized protein YqxJ Proteins 0.000 description 1
- 241000725138 Banana bunchy top virus Species 0.000 description 1
- 241001302800 Beak and feather disease virus Species 0.000 description 1
- 101000754349 Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) UPF0065 protein BP0148 Proteins 0.000 description 1
- 101000827633 Caldicellulosiruptor sp. (strain Rt8B.4) Uncharacterized 23.9 kDa protein in xynA 3'region Proteins 0.000 description 1
- 101000736909 Campylobacter jejuni Probable nucleotidyltransferase Proteins 0.000 description 1
- 241001533399 Circoviridae Species 0.000 description 1
- 101000947628 Claviceps purpurea Uncharacterized 11.8 kDa protein Proteins 0.000 description 1
- 101000686796 Clostridium perfringens Replication protein Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 101000861180 Cupriavidus necator (strain ATCC 17699 / DSM 428 / KCTC 22496 / NCIMB 10442 / H16 / Stanier 337) Uncharacterized protein H16_B0147 Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000788129 Escherichia coli Uncharacterized protein in sul1 3'region Proteins 0.000 description 1
- 101000788370 Escherichia phage P2 Uncharacterized 12.9 kDa protein in GpA 3'region Proteins 0.000 description 1
- 208000035126 Facies Diseases 0.000 description 1
- 101000787096 Geobacillus stearothermophilus Uncharacterized protein in gldA 3'region Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 1
- 101000818121 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 18.2 kDa protein in rep-hol intergenic region Proteins 0.000 description 1
- 101000976889 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 19.2 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101000748060 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.3 kDa protein in rep-hol intergenic region Proteins 0.000 description 1
- 101000623276 Herpetosiphon aurantiacus Uncharacterized 10.2 kDa protein in HgiBIM 5'region Proteins 0.000 description 1
- 101000623175 Herpetosiphon aurantiacus Uncharacterized 10.2 kDa protein in HgiCIIM 5'region Proteins 0.000 description 1
- 101000626850 Herpetosiphon aurantiacus Uncharacterized 10.2 kDa protein in HgiEIM 5'region Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 101000827627 Klebsiella pneumoniae Putative low molecular weight protein-tyrosine-phosphatase Proteins 0.000 description 1
- 101000790842 Klebsiella pneumoniae Uncharacterized 65.4 kDa protein in cps region Proteins 0.000 description 1
- 101000768313 Klebsiella pneumoniae Uncharacterized membrane protein in cps region Proteins 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 101000804418 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) Uncharacterized protein MTH_1463 Proteins 0.000 description 1
- 101001130841 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF5 Proteins 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 101710087110 ORF6 protein Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 101000781204 Orgyia pseudotsugata multicapsid polyhedrosis virus Uncharacterized 36.6 kDa protein Proteins 0.000 description 1
- 101000770870 Orgyia pseudotsugata multicapsid polyhedrosis virus Uncharacterized 37.2 kDa protein Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 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
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102100040307 Protein FAM3B Human genes 0.000 description 1
- 101000974028 Rhizobium leguminosarum bv. viciae (strain 3841) Putative cystathionine beta-lyase Proteins 0.000 description 1
- 101000756519 Rhodobacter capsulatus (strain ATCC BAA-309 / NBRC 16581 / SB1003) Uncharacterized protein RCAP_rcc00048 Proteins 0.000 description 1
- 101000948219 Rhodococcus erythropolis Uncharacterized 11.5 kDa protein in thcD 3'region Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101000936711 Streptococcus gordonii Accessory secretory protein Asp4 Proteins 0.000 description 1
- 101000929863 Streptomyces cinnamonensis Monensin polyketide synthase putative ketoacyl reductase Proteins 0.000 description 1
- 101000788468 Streptomyces coelicolor Uncharacterized protein in mprR 3'region Proteins 0.000 description 1
- 101000845085 Streptomyces violaceoruber Granaticin polyketide synthase putative ketoacyl reductase 1 Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- 101000711771 Thiocystis violacea Uncharacterized 76.5 kDa protein in phbC 3'region Proteins 0.000 description 1
- 241000710914 Totivirus Species 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 101710198378 Uncharacterized 10.8 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101710134973 Uncharacterized 9.7 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101710095001 Uncharacterized protein in nifU 5'region Proteins 0.000 description 1
- 101000711318 Vibrio alginolyticus Uncharacterized 11.6 kDa protein in scrR 3'region Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910000318 alkali metal phosphate Inorganic materials 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000002565 arteriole Anatomy 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 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 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003725 endotheliocyte Anatomy 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 208000018555 lymphatic system disease Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- JXTPJDDICSTXJX-UHFFFAOYSA-N n-Triacontane Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC JXTPJDDICSTXJX-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 229940081969 saccharomyces cerevisiae Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 229940032094 squalane Drugs 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.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](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000012153 swine disease Diseases 0.000 description 1
- 229940032021 tetramune Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
- 238000001262 western blot Methods 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
- A23C19/0321—Propionic acid bacteria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
- A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
-
- 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
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- 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
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16741—Use of virus, viral particle or viral elements as a vector
- C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
- C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10021—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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10041—Use of virus, viral particle or viral elements as a vector
- C12N2750/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10061—Methods of inactivation or attenuation
- C12N2750/10064—Methods of inactivation or attenuation by serial passage
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种从肺或神经节样本中分离的新型猪环病毒株,所述样本获自患有断乳后多系统性消瘦综合症(PMWS)的牲畜。本发明涉及这些毒株的纯化制品、减毒疫苗或灭活疫苗、重组活体疫苗、质粒疫苗和亚单位疫苗,以及诊断试剂和诊断方法。本发明还涉及可用于在体外表达载体中制备亚单位疫苗的DNA片段,或者整合入病毒或质粒型体内表达载体中的序列。
Description
本发明涉及一种引起PMWS综合症(猪多系统性消瘦综合症,也称为断乳后多系统性消瘦综合症)的新型猪环病毒株(porcine circovirus简称为PCV),涉及检测其的试剂和方法,涉及免疫接种方法及疫苗,和涉及制备这些试剂和疫苗的方法。
最初,在猪肾细胞系PK/15中检测到PCV为非细胞致病性污染物。此病毒与幼禽贫血病毒(简称为CAV)和PBFDV病毒(Pscittacine Beakand feather disease Virus)被分类为环病毒科(Circocuridae)。此病毒为小型无被膜病毒(从15-24nm),共同特征为含有1.76-2.31kb的环状单链DNA形式的基因组。最初认为该基因组编码一种约30kDa的多肽(Todd等人,病毒学文献(Arch Virol),1991,117;129-135)。然而,最近的工作显示其更为复杂的转录(Meehan B.M.等人,1997,78:221-227)。此外,在三种类型已知环病毒之间的核苷酸序列或共同抗原决定簇中无明显同源性。
一直认为来源于PK/15细胞的PCV无致病性。其序列由MeehanB.M.等人,普通病毒学杂志(J.Gen.Virol.),1997,78:221-227可知。直到最近有人意识到PCV株可能为致病性的,与PMWS综合症相关(Gupi P.S.Nayar等人,加拿大兽医杂志(Can.Vet.J.),1997,38:385-387;Clark E.G.,美国猪应用协会进展(Proe.Am.Assoc.SwinePrac.),1997;499-501)。Nayar等人利用PCR技术从患有PMWS综合症的猪中检测到PCV DNA。此前,尚未有野生型PCV毒株被分离和纯化。
在加拿大、美国和法国检出的PMWS综合症临床上的特征为体重逐渐下降,出现诸如呼吸急促、呼吸困难和黄疸的临床表现。从病理学角度而言,这些症状是淋巴细胞或肉芽肿浸润、淋巴结病的临床表现,和更罕见的肝炎、淋巴细胞性或肉芽肿性肾炎(Clark E.G.,美国猪应用协会进展1997;499-501;La Semaine Veterinaire No.26,La SemaineVeterinaire增刊,1996(834);La Semaine Veterinaire1997(857):54;Gupi P.S.Nayar等人,加拿大兽医杂志,1997,38:385-387)。
本申请人从肺或神经节样本中成功分离了五种新PCV毒株,此后称为根据本发明的环病毒,其中这些样本获自位于加拿大、美国(加利福尼亚)和法国(Brittany)的农场。已经从患有PMWS综合症的猪的损伤处检出这些病毒,而在健康猪中未检出。
此外,本申请人测定了这些毒株中的4株之基因组序列,即从加拿大、美国获得的毒株以及从法国获得的两株毒株。在核苷酸水平上这些毒株相互之间显示出强烈的同源性,超过96%;而与PK/15毒株之间同源性较低,约76%。因此,认为新毒株为一种新型猪环病毒的代表,此处称为II型,I型由PK/15代表。
因此,本发明的主题为分离的或纯化制品形式的如上述的II型猪环病毒。
本发明涉及可从患有PMWS综合症病猪中的生理学样本或组织,尤其是损伤组织样本中分离的,尤其是依照实施例所述的方法分离的任何猪环病毒,特别是涉及II型环病毒。
本发明的主题更具体的是五种毒株的纯化制品,其已保藏在ECACC(欧洲动物细胞保藏中心,应用微生物及研究中心,Porton Down,Salisbury,Wiltshire SP40JG,英国):保藏号V97100219(此处称为Imp.1008PCV);保藏号V97100218(此处称为Imp.1010PCV);保藏号V97100217(此处称为Imp.999PCV);于1997年10月2日(星期四)保藏,以及保藏号V98011608(此处称为Imp.1011-48285);保藏号V98011609(此处称为Imp.1011-48121);于1998年1月16日(星期五)保藏。
本发明涉及从患病猪分离的猪环病毒,和/或与本发明毒株具有显著血清学相似性的环病毒,和/或在严格条件下可与本发明的毒株交叉杂交的环病毒,所述严格条件是指例如不能与PCV PK/15毒株杂交。
从患有PMWS综合症的猪中的生理学样本或组织,尤其是损伤组织样本中分离的病毒株可有利地在细胞系中增殖,尤其是猪肾细胞系,特别是无污染物(如PCV以及瘟病毒、猪腺病毒和猪细小病毒)的PK/15细胞中,用于其复制或具体地产生抗原,包括完整抗原(如病毒)和/或抗原亚单位(如多肽)。
非常明显而出人意料的是,已证明这些分离物在PK/15细胞培养物中产量很大,这无疑对病毒或抗原,尤其是对灭活疫苗的产生十分有利。
本发明的主题还涉及环病毒制品,其中在体外培养的细胞特别是细胞系(如:PK/15细胞)传代后分离环病毒,所述细胞或细胞系被至少一种根据本发明的环病毒感染,或者被可从患有PMWS综合症的猪的生理学样本或从组织样本(尤其是损伤组织)中分离的任一种猪环病毒感染。本发明的主题还包括培养物提取物或上清液,任选地,其通过标准技术纯化,以及通常任一种获自体外培养物的抗原性制品。
本发明的主题还涉及免疫活性组分和含有至少一种上述抗原的疫苗。
其可为基于减毒活体全病毒的免疫活性组分,或用这些活性组分制备的疫苗。可根据常规方法进行减毒,例如,通过在细胞上进行传代,优选在猪细胞特别是细胞系(如PK/15细胞)上传代,例如从50至150次,特别是约100次水平的传代。这些疫苗一般包括兽医学可接受的载体或稀释剂,任选地,兽医学可接受的佐剂,以及任选地一种冷冻干燥稳定剂。
这些疫苗优选地包含103-106 TCID50。
其可是免疫活性组分,或基于根据本发明环病毒抗原的灭活状态的疫苗。这些疫苗还包括兽医学可接受的载体或稀释剂,以及任选地兽医学可接受的佐剂。
根据本领域普通技术人员已知的技术,灭活根据本发明的环病毒以及可能存在的组分。灭活优选地通过化学方法进行,如通过使抗原接触一种化学药剂,如甲醛(福尔马林)、多聚甲醛、β-丙醇酸内酯、乙撑亚胺或其衍生物。灭活优选的方法为此处所述的接触化学药剂,特别是接触乙撑亚胺或β-丙醇酸内酯。
优选地,通过本领域技术人员周知的方法,根据本发明的灭活疫苗可添加佐剂有利地以乳液形式提供,例如油包水或水包油型。也可根据佐剂性质将常规佐剂化合物掺入活性组分中。
可用的佐剂中,以举例的方式,包括但不限于氢氧化铝、皂甙(如Quillaja皂甙或Quil A;参见“疫苗设计,亚单位疫苗剂佐剂方法”,1995,Michael F.Powel和Mark J.Newman编,Plennum出版社,New-York及London,210页)、Avridine(疫苗设计,148页)、DDA(溴化二甲基双十八烷基铵,疫苗设计,157页)、聚磷腈(疫苗设计,204页)、或备择的基于矿物油、角鲨烷(例如SPT乳液,疫苗设计,147页)、角鲨烯(例如MF59,疫苗设计,183页)的水包油型乳液,或基于可代谢油(优选根据WO-A-94 20071)的油包水型乳液,以及US-A-5,422,109中所述的乳液。也可选择佐剂的组合,例如Avridine或DDA与一种乳液组合。
这些疫苗优选地包含103-106 TCID50。
活疫苗佐剂可选自那些用于灭活疫苗的佐剂,优选乳液。可在用于灭活疫苗中所示的那些乳液还包括WO-A-9416681中所述的那些乳液。
至于冷冻干燥稳定剂,其以举例方式可为SPGA(Bovarnik等人,细菌学杂志(J.Bacterioligy),59,509,950)、碳水化合物(如山梨醇、甘露醇、淀粉、蔗糖、葡聚糖或葡萄糖)、蛋白质(白蛋白或酪蛋白),或这些化合物的衍生物,或如碱金属磷酸盐的缓冲液。
此外,申请人获得了分离株中4个的基因组,鉴定为SEQ ID NO:1-4和任选地SEQ ID No:6。
因此,本发明的主题是含有这些序列之一的部分或全部的DNA片段。不言而喻,本发明自然涵盖了等同序列,即不改变所述序列的功能或菌株特异性的序列,或由该序列编码的多肽之菌株特异性的序列。当然,由于密码子简并性造成的差异也包括在内。
本发明也涵盖了这样的等同序列,它们在高度严格条件下可与上述序列杂交,和/或与本发明的毒株有高度同源性,且属于上述II型环病毒组。
借助适当载体,这些序列及其片段可有利地用于多肽的体外或体内表达。
特别是,已经鉴定了在II型环病毒基因组序列上的可用于此方面的开放阅读框架,其形成了根据本发明的DNA片段。本发明涉及含有至少一个这些开放阅读框架(相应于氨基酸序列)的任何多肽。优选地,本发明涉及一种基本上由ORF4、ORF7、ORF10或ORF13组成的蛋白质。
为了在体外表达亚单位,作为一种表达方法可优选使用大肠杆菌或杆状病毒(US-A-4,745,051)。将编码序列或其片段整合入杆状病毒基因组(如,杆状病毒苜蓿银纹夜蛾多核型多角体病毒AcNPV),将后者在昆虫细胞中增殖,如草地夜蛾(Spodoptera frugiperda Sf9)(保藏号ATCC CRL 1711)。也可在真核细胞如酵母(如酿酒酵母(Saccharomycescerevisiae))或哺乳动物细胞(如CHO、BHK)中表达亚单位。
本发明的主题也涉及可通过这些表达方法制备,然后任选地根据传统技术纯化的多肽。本发明还涉及一种亚单位疫苗,其包括在兽医学可接受的载体或稀释剂之中的至少一种如此获得的多肽,或其片段,以及任选地兽医学可接受的佐剂。
为了用于制备重组活体疫苗的体内表达,在允许该多肽表达的条件下将编码序列或其片段插入一种合适的表达载体。至于合适的载体,根据本领域技术人员周知的技术可使用活病毒,优选地能在猪中增殖、对猪无致病性(天然无致病性或赋予此性质)。尤其是可使用猪疱疹病毒如假狂犬病病毒、猪腺病毒、疱疹病毒、痘病毒属,特别是牛痘病毒、禽痘病毒、犬痘病毒和猪痘病毒。也可使用质粒DNA作为载体(WO-A-9011092,WO-A-93 19813,WO-A-94 21797,WO-A-95 20660)。
因此,本发明主题也涉及如此制备的载体和重组活体疫苗或质粒疫苗(多核苷酸或DNA疫苗),此外,疫苗包括兽医学可接受的载体或稀释剂。
根据本发明的疫苗(活体减毒疫苗、灭活疫苗、亚单位疫苗、重组疫苗和质粒疫苗)可包括一种或多种(2或3种)根据本发明环病毒之中的一种或多种的一种或多种活性组分(抗原)。
对于每一上述疫苗类型,本发明也提供了针对猪环病毒的免疫接种与针对其它猪疾病(特别是可与PMWS综合征相关的疾病)的免疫接种的联合免疫接种疫苗。因此,根据本发明的疫苗,特别是灭活疫苗可包含相应于其它猪病原体的效价。这些其它猪病原体中,优选地包括PRRS(猪生殖和呼吸综合征)(本领域技术人员可参见WO-A-93/07898,WO-A-94/18311,FR-A-2 709 966;C.chareyre等人,第15届IPVS大会论文集,Birmingham,英国,1998年7月5-9日,139页;此处引入作为参考)和/或猪肺炎支原体(Mycoplasma hyopneumonia)(本领域技术人员可参见EP-A-597 852;EP-A-550 477;EP-A-571 648;O.Martinon等人,157,284和285页以及G.Reynaud等人,150页,均在上述第15届IPVS大会论文集,此处引入作为参考)。其它可能感兴趣的疫苗可包括大叶性肺炎放线杆菌(Actinobacillus pleuropneumoniae)、大肠杆菌、猪萎缩性鼻炎以及伪狂犬病(Aujeszky病)、猪霍乱、猪流感。
本发明的主题还涉及一种方法,从而可在猪中诱导针对本发明环病毒的免疫反应。该方法特别是一种在猪中有效的方法。
该方法可向猪一次或多次施用上述疫苗。也可在同一免疫接种方案中联合上述数种类型的疫苗。
该方法不仅可向成年猪施用,也可向幼猪或怀孕雌猪施用。对后者的免疫接种可对新生猪赋予被动免疫(母本抗体)。
本发明还提供了在猪中诊断根据本发明的环病毒的可能性。因此,本发明的主题还包括利用下述试剂的诊断试验和与其相关的方法。
对不同环病毒序列的了解使得确定共同序列成为可能,这使其可制备能识别所有已知猪环病毒的反应物片段。
为了进行特异性诊断,本领域普通技术人员能选择这样的序列片段,其相应于与对应的PK/15环病毒序列没有或几乎没有同源性的区域。
本领域普通技术人员可通过序列比较选择符合其意愿的反应物片段。
第一种反应物片段存在于此处公开的序列及其片段中,其可特别地在周知的杂交或PCR(聚合酶链反应)技术中用作探针或引物。
第二种反应物片段存在于由来自病毒的这些序列编码的多肽或在载体帮助下表达的多肽(见上),或根据传统用于肽合成的技术通过化学途径合成的多肽。
第三种和第四种反应物片段分别存在于多克隆和单克隆抗体,其可根据传统方法从病毒提取多肽或片段、或由DNA序列编码的多肽或其片段制备。
这第二、三和四种反应物片段可用于本发明主题的诊断方法,其中对获自待测猪的生理学液体样本(血液、血浆、血清等)或组织样本(神经中枢、肝脏、肺、肾等)进行试验,通过搜寻检测抗原自身或针对该抗原的抗体,检验是否存在特异于根据本发明环病毒的抗原。
根据本发明的抗原和抗体可用于任何已知的实验室诊断技术。
然而,优选地在可由兽医、饲养者或牲畜主现场直接使用的技术中使用这些抗原和抗体。本领域技术人员可使用多种实验室和现场技术,因此可有利地改造这些抗原和/或抗体以适于作为诊断试剂的应用。
可在本发明的框架中优选使用的诊断技术为蛋白质印迹、免疫荧光、ELISA和免疫层析。
就免疫层析的使用而言,专业人士可特别参见Robert f.Zurk等人,临床化学(Clin.Chem.),31/7,1144-1150(1985),以及下列专利或专利申请:WO-A-88/08 534、WO-A-91/12528、EP-A-291 176、EP-A-299 428、EP-A-291 194、EP-A-284 232、US-A-5 120 643、US-A-5 030 558、US-A-5 266 497、US-A-4 740 468、US-A-5 266 497、US-A-4 855 240、US-A-5451 504、US-A-5 141 850、US-A-5 232 035和US-A-5 238 652。
因此,优选通过间接试验(通过竞争或通过替换)在样本中检测特异性抗体。为此,使用抗原自身,或保留了抗体识别位点的该抗原片段作为检测试剂。可有利地使用过氧化物酶标记或特定标记进行标记,优选用胶体金。
也期望在特异于该抗原的标记抗体的帮助下检测样本中的抗原本身。标记有利地为如上所述。
对于特异于可特别用于竞争或替换的抗原之抗体,或特异于用来检测抗原自身的抗体,应理解为特异于抗原的多克隆抗体或单克隆抗体、这些抗体的片段,优选为Fab或F(ab)’2片段。
本发明的另一方面是特异于根据本发明的抗原的单克隆或多克隆抗体,这些抗体可特别用作诊断试剂,在生理学液体样本或组织样本中检测抗原,或者甚至在这样的样本或样品中检测抗体。本发明还包括这些抗体的免疫功能性片段,特别是Fab和F(ab)’2片段。
抗体可通过常规技术制备。可特别参见“抗体,实验室手册”,1988,冷泉港实验室,美国,或J.W.Goding。“单克隆抗体:原理与实践”,学院出版社,其内容此处引入作为参考。
也可特别的以自知方法进行小鼠脾细胞与适当的骨髓瘤细胞的融合,其中小鼠用抗原或至少一种该抗原的片段免疫过。
本发明的主题还包括一种特异于抗原的单克隆或多克隆抗体(特别是小鼠或兔抗体)制品,其优选为纯或基本上纯,或甚至为粗品。
本发明还可以确定目标表位,特别是基于此处描述的DNA序列,确定是否存在免疫用的表位或诊断用的表位。从根据本发明的环病毒基因组的DNA序列,本领域技术人员可根据已知方法,例如一种适当的计算机程序或PEPSCAN确定表位。表位是蛋白质的免疫决定区域,是暴露在蛋白质表面的区域。由此,其可通过抗体识别,因而可特别用于诊断领域,用于诊断目的之抗体制备或者用于可用作诊断试剂的相应肽的制备。
一个表位至少是具有8-9个氨基酸的肽。通常优选最少为13-25个氨基酸。
本领域技术人员因此可使用一种或多种这些技术以及其它可能的技术,发现可用于诊断目的的肽或抗体的表位。
本发明的主题还包括诊断试剂盒,其中含有该抗原和/或特异于该抗原的多克隆或单克隆抗体。特别是相应于上述诊断技术的诊断试剂盒。
以下,本发明将以非限制性实施方案结合附图更详细的进行描述,其中:
图1.Imp.1011-48121株的基因组DNA序列。
图2.Imp.1011-48285株的基因组DNA序列。
图3.Imp.999株的基因组DNA序列。
图4.Imp.1010株的基因组DNA序列。
图5.根据图1-4的4个序列与PCVPK/15株序列的比较。
图6.在1997年10月3日法国首次递交的文件中的Imp.999株的基因组DNA序列。
图7.图6序列与PK/15株序列的比较。
序列表SEQ ID
SEQ ID NO:1 Imp.1011-48121株的基因组DNA序列。
SEQ ID NO:2 Imp.1011-48285株的基因组DNA序列。
SEQ ID NO:3 Imp.999株的基因组DNA序列。
SEQ ID NO:4 Imp.1010株的基因组DNA序列。
SEQ ID NO:5 PCV PK/15株基因组的DNA序列。
SEQ ID NO:6在1997年10月3日法国首次递交的文件中的Imp.999株的基因组DNA序列。
实施例实施例1猪环病毒株的培养及分离
从法国、加拿大和美国小猪的肺和淋巴结收集组织样本。这些小猪均显示典型的断乳后多系统消瘦综合症的临床症状。为便于病毒分离,尸检后立即将组织样本于-70℃冷冻。
为了病毒分离,在含有Earle盐(EMEM,BioWhittaker英国有限公司,Wokingham,英国)、青霉素(100IU/ml)和链霉素(100μg/ml)的基本培养基(MEM-SA培养基)中,通过利用无菌马达和杵将组织样本与无菌沙研磨,制备含有约15%组织样本的悬浮液。然后将此研磨制品加入MEM-SA,4℃下于3000g离心30分钟,收获上清液。在接种细胞培养物之前,100μl体积的氯仿加入各个2ml上清液中,室温下连续混合10分钟。然后将此混合物转移入微量离心管中,3000g离心10分钟,收获上清液。将此上清液用作病毒分离实验的接种物。
所有的病毒分离研究均在PK/15细胞培养物上进行,该培养物已知无猪环病毒(PCV)、瘟病毒、猪腺病毒和猪细小病毒污染(Allan G.等人,初乳停断小猪的猪环病毒实验性感染的病原体及猪胎材料的检查,兽医微生物(Vet.Microbiol.),1995,44,49-64)。
根据下列技术进行猪环病毒的分离:
通过从生长至铺满的培养物用胰蛋白酶(胰蛋白酶-依地酸混合物)消化分离PK/15细胞单层,以终浓度约400,000个细胞/ml加入含有15%胎牛血清无瘟病毒污染的MEM-SA培养基(=MEM-G培养基)。然后将此细胞悬浮液的10ml等份与2ml上述接种物等份混合,最终将混合物以6ml等份培养于两个25cm2的Falcon烧瓶中。在含10%CO2的氛围中37℃培养18小时。
培养后,用300mM D-葡糖胺(Cat #G48175,Sigma-Aldich化学有限公司,Poole,英国)处理半铺满单层的培养液(Tischer I.等人,病毒学文献,1987,96:39-57),然后37℃继续培养48-72小时。后一培养结束后,每个接种物的两个Falcon烧瓶之一经过连续3次冻/融循环。所剩Falcon烧瓶之PK/15细胞用胰蛋白酶-依地酸溶液处理,重悬于20mlMEM-G培养基中,以约400,000个细胞/ml浓度接种入75cm2的Falcon中。通过加入经过冻/融循环获得的相应裂解物5ml,“超感染”新鲜接种的烧瓶培养物。实施例2用于通过免疫荧光或原位杂交检测猪环病毒的细胞培养物样本的制备
收集5ml超感染的悬浮液,接种入直径55mm的带有无菌无脂玻璃盖玻片的培养皿。在烧瓶中及玻璃盖玻片上的培养物于37℃培养,如实施例1所述用葡糖胺处理。用葡糖胺处理后24-48小时收集玻璃盖玻片上的培养物,用丙酮室温下固定10分钟,或用10%缓冲的甲醛固定4小时。固定后,所有玻璃盖玻片在硅胶上于-70℃储存,直至用于原位杂交研究和免疫细胞化学标记研究。实施例3通过原位杂交检测PCV的技术
对从患病猪收集的且用甲醛固定的组织进行原位杂交,也对固定于玻璃盖玻片上之为了病毒分离(见实施例2)接种的细胞培养物制品进行原位杂交。
使用相应于PK/15猪环病毒(PCV)以及相应于传染性禽贫血病毒(CAV)的完整基因组探针。使用质粒pPCV1作为PCV的特异性病毒DNA来源,其中含有以单一1.7千碱基对(kbp)插入片段形式克隆的PCV基因组复制形式(Meehan B.等人,猪环病毒DNA基因组序列:与植物环病毒的亲和性,普通病毒学杂志,1997,78:221-227)。使用含有2.3kbp复制形式的禽环病毒病毒CAV之类似质粒pCAA1作为阴性对照。使用两个质粒各自的甘油储藏原种用于根据碱裂解技术制备和纯化质粒(Sambrook J.等人,分子克隆:实验室手册,第二版,冷泉港实验室,纽约,1989),使其可用作模板制备探针。从上述纯化的质粒以及根据供应商说明使用市售非放射性标记试剂盒(DIG DNA标记试剂盒,Boehringer Mannheim,Lewes,英国)从随机六核苷酸引物制备代表PCV和CAV完整基因组的环病毒探针(每个探针1μg)。
在用于原位杂交前将地高辛标记的探针加入体积为50-100μl的无菌水中。
制备包埋在石蜡中用甲醛固定的患猪组织样本以及用甲醛固定的感染细胞培养物制品,用于按如下技术检测PCV核酸:
从包埋于石蜡中的组织切下厚度为5μm的切片,除去石蜡,在浓度递减的连续乙醇溶液中再水合。用甲醛固定的组织切片和细胞培养物在37℃下于0.5%蛋白酶K之0.05M Tris-HCl溶液中(含有5mM EDTA,pH7.6)分别温育15分钟和5分钟。然后将载玻片置于1%甘氨酸的无菌蒸馏水溶液中30秒,用0.01M PBS缓冲液(磷酸缓冲盐液,pH7.2)洗涤两次,最后用无菌蒸馏水洗涤5分钟。将其空气干燥,与探针接触。
每一组织/探针制品加盖洁净无脂玻璃盖玻片,置于90℃炉中10分钟,然后与冰块接触1分钟,最后于37℃培养18小时。将制品短暂浸入2X柠檬酸钠(SSC)缓冲液(pH7.0),以去除保护性玻璃盖玻片,然后于2 X SSC缓冲液中洗涤两次5分钟,最后于PBS缓冲液中洗涤两次5分钟。
经过洗涤后,将制品浸入0.1M马来酸、0.15M NaCl(pH1.5)(马来酸缓冲液)10分钟,然后于37℃在1%封闭试剂(Cat.#1096176,Boehringer Mannheim,英国,Lewis,East Sussex,英国)的马来酸缓冲液溶液中温育20分钟。
然后,制品与稀释于封闭缓冲液中的抗地高辛单克隆抗体1/250溶液(Boehringer Mannheim)37℃下温育1小时,于PBS中洗涤,最后与生物素酰化抗小鼠免疫球蛋白抗体37℃下温育30分钟。制品在PBS中洗涤,通过用0.5%过氧化氢的PBS溶液室温下处理20分钟封闭内源过氧化物酶活性。再次用PBS洗涤制品,用即配即用的3-氨基-9-二乙基咔唑(AEC)底物处理(Cambridge Bioscience,Cambridge,英国)。
最终用自来水洗涤后,将制品用苏木精复染,在自来水中“变蓝”,用封固液(GVA Mount,Cambridge Bioscience,Cambridge,英国)封固于显微镜玻璃盖玻片上。实验对照包括了在获自患病猪和非患病猪的样本上使用非相关阴性探针(CAV)和阳性探针(PCV)。实施例4:通过免疫荧光检测PCV的技术
使用1/100稀释的成体猪血清集合通过间接免疫荧光技术(IIF)进行丙酮固定的所有细胞培养物制品的初次筛选。该血清集合包含来自北爱尔兰的25只成体猪的血清,其中已知含有针对多种猪病毒(包括PCV、猪细小病毒、猪腺病毒和PRRS病毒)的抗体。通过将稀释于PBS中的血清37℃下接触细胞培养物1小时,进行IIF技术,然后在PBS中洗涤两次。再用1/80稀释的与荧光素异硫氰酸结合的兔抗猪免疫球蛋白抗体PBS溶液染色细胞培养物1小时,用PBS洗涤,在紫外光下显微镜观察之前用甘油缓冲液封固。实施例5:对患病猪组织原位杂交的结果
使用由获自法国、加拿大和加利福尼亚小猪的用甲醛固定的组织的PCV基因组探针进行原位杂交,其中小猪患有多系统消瘦综合症,结果显示在数个研究的损伤组织中存在与损伤相关的PCV核酸。当对获自非病患猪的组织使用PCV探针时或者对患病猪使用CAV探针时未观察到信号。在浸润入加利福尼亚小猪肺部损伤中的大量单核细胞之胞浆和细胞核中检出PCV核酸。在肺细胞、支气管和细支气管上皮细胞以及在微动脉、小囊突和淋巴管的内皮细胞中也证实PCV核酸的存在。
在患病法国猪中,在大量滤泡淋巴细胞和淋巴结窦状(小管)内单核细胞的胞浆中也存在PCV核酸。在临时合胞体中也存在PCV核酸。根据这些检测结果,选择加利福尼亚猪肺、法国猪肠系膜淋巴结和加拿大猪器官用于分离新型猪环病毒株。实施例6:新型猪环病毒株细胞培养物及免疫荧光检测结果
在用获自法国小猪(Imp.1008株)、加利福尼亚小猪(Imp.999株)和加拿大小猪(Imp.1010株)接种的细胞培养物中未观察到致细胞病变效应,这些猪显示多系统消瘦的临床症状。然而,获自接种细胞培养物的制品的免疫标记在用丙酮固定及用猪多克隆血清集合染色后,利用加利福尼亚小猪肺(Imp.999株)、法国小猪肠系膜淋巴结(Imp.1008株)、和加拿大小猪器官(Imp.1010株)接种的培养物的大量细胞中显示核荧光。实施例7:猪环病毒基因组DNA的提取
利用感染的PK/15细胞培养物(10只75cm2 Falcon)制备新型猪环病毒毒株的复制形式(见实施例1),该细胞培养物培养72-76小时后收获,用葡糖胺处理,如CAV复制形式克隆时所述(Todd.D等人,利用克隆DNA探针斑点杂交检测禽贫血病毒,临床微生物杂志,1991,29:933-939)。根据改进的Hirt技术提取这些复制形式的双链DNA(Hirt B.从感染细胞培养物中选择性提取多病毒DNA,分子生物学杂志,1967,36:365-369),如Molitor所述(Molitor T.W.等人,猪细小病毒DNA:两种病毒分离物的基因组及复制形式的表征,病毒学,1984,137:241-254)。实施例8:猪环病毒Imp.999株基因组复制形式的限制性图谱
依供应商说明,用S1核酸酶(Amersham)处理根据Hirt技术提取的DNA(1-5μg),然后用多种限制酶(Boehringer Mannheim,英国,Lewis,East Sussex,英国)酶切此DNA,如Todd等人所述,在存在溴乙锭时于1.5%琼脂糖凝胶上电泳分离酶切产物(禽贫血病毒的纯化和生化表征,普通病毒学杂志,1990,71:819-823)。从Imp.999株培养物提取的DNA具有单一EcoRI位点、两个SacI位点,不含有任何PstI位点。因此,此限制性图谱不同于PCV PK/15毒株的限制性图谱,该毒株中含有PstI位点,而不含有任何EcoRI位点。实施例9:猪环病毒Imp.999株基因组的克隆
用限制性酶EcoRI酶切PCV Imp.999毒株双链复制形式产生的约1.8kbp限制性片段,使用Qiagen市售试剂盒(QIAEXII凝胶提取试剂盒,Cat#20021,QIAGEN Ltd.,Crawley,West Sussex,英国)在1.5%琼脂糖凝胶上电泳分离酶切产物(见实施例3)。然后根据标准克隆技术(Sambrook J.等人,分子克隆:实验室手册,第二版,冷泉港实验室,纽约,1989),将此EcoRI-EcoRI限制性片段与已经用相同限制性酶消化并去磷酸化的载体pGEM-7(Promega,医学供应公司,都柏林,爱尔兰)连接。根据标准技术,用获得的质粒转化大肠杆菌JM109宿主菌株(Stratagene,La Jolla,美国)。还将PCV Imp.999毒株EcoRI-EcoRI限制性片段克隆入载体pBlueScript SK+的EcoRI位点(Stratagene,La Jolla,美国)。在每一宿主菌株获得的克隆中,筛选了含有期望片段大小的至少两个克隆。然后培养所得克隆,根据标准质粒制备和纯化技术,以小体积(2ml)或大体积(250ml)纯化含有完整Imp.999毒株基因组的质粒。实施例10:PCV Imp.999毒株基因组DNA(双链复制形式)的序列测定
根据Sanger双脱氧核苷酸技术使用测序试剂盒“AmpliTaq DNA聚合酶FS”(Cat#402070 PE Applied Biosystems,Warrington,英国)以及依制造商说明用Applied Biosystems ABI373A自动测序仪测定两个EcoRI Imp.999克隆(克隆pGEM-7/2和克隆pGEM-7/8)的核苷酸序列。用M13“正向”和“反向”通用引物进行初步测序反应。根据“DNA步行”技术进行如下测序反应。由Life Technologies(Inchinnan BusinessPark,Paisley,英国)合成下列序列测定所需的寡核苷酸。
用MacDNASIS 3.2版软件装配产生的序列并分析(Cat#22020101,Appligene,Durham,英国)。通过由“国家生物工程信息中心”服务器(NCBI,Bethesda,MD,美国)可得的BLAST序列对比工具分析开放阅读框架。
最初从克隆pGEM-7/8获得的完整序列(EcoRI-EcoRI片段)(SEQID NO:6)示于图6。其任意地从EcoRI位点中的G之后开始,从核苷酸角度而言存在几个不确定的地方。
然后优化序列测定,SEQ ID NO:3(图3)给出了此毒株的总序列,其任意地从EcoRI位点开始,即G为第一个核苷酸。
采用类似方法获得其它三种根据本发明的分离物之序列(见SEQ IDNO:1、2和4,以及图1、2和4)。
这四个毒株的基因组大小为:
Imp.1011-48121 1767核苷酸
Imp.1011-48285 1767核苷酸
Imp.999 1768核苷酸
Imp.1010 1768核苷酸实施例11:PCV Imp.999毒株序列分析
当从Imp.999毒株产生的序列用于同GenBank数据库中含有的序列进行同源性测试时,仅检测出与PK/15株(保藏号Y09921和U49186)序列在核酸水平上有约76%的同源性(见图5)。
在氨基酸水平,与数据库的6相序列翻译同源性测试(在NCBI服务器上的BLAST序列对比工具)可证实:与相应于理论上的BBTV病毒之复制酶的开放阅读框架有94%同源性,其类似于由GenBankU49186序列编码的植物环病毒(GenBank鉴定号1841515)。
其它数据库中含有的序列与从PCV Imp.999毒株产生的序列无明显的同源性。
收集自患有多系统消瘦综合症的加利福尼亚小猪损伤组织之培养的Imp.999毒株所产生的序列分析清楚表明,此病毒分离物是一种新型猪环病毒株。实施例12:序列的比较分析
与PCV PK/15株序列进行4株新PCV毒株核苷酸序列的序列比较(图5)。建立考虑了4株新毒株和已有PK/15毒株的同源性矩阵。结果如下:
1:Imp.1011-48121
2:Imp.1011-48285
3:Imp.999
4:Imp.1010
5:PK/15
1 2 3 4 5
1 | 1.0000 | 0.9977 | 0.9615 | 0.9621 | 0.7600 |
2 | 1.0000 | 0.9621 | 0.9632 | 0.7594 | |
3 | 1.0000 | 0.9949 | 0.7560 | ||
4 | 1.0000 | 0.7566 | |||
5 | 1.0000 |
两株法国毒株Imp.1011-48121与Imp.1011-48285的同源性超过99%(0.9977)。
两株北美毒株Imp.999与Imp.1010之间的同源性也大于99%(0.9949)。法国毒株与北美毒株之间的同源性略大于96%。
所有这些毒株与PK/15的同源性在75%-76%之间。
由此可推知,根据本发明的毒株代表了一种不同于由PK/15株代表的类型的新猪环病毒类型。从显示PMWS综合症的猪分离的这一新类型称为II型猪环病毒,PK/15代表I型。虽然它们事实上分离自十分不同的地理区域,属于此II型的毒株之间显示明显的同源性。实施例13:由新PCV毒株基因组编码的蛋白质的分析
认为Imp.1010分离物的核苷酸序列是与多系统消瘦综合症相关的其它环病毒毒株的代表。用BLAST序列对比工具以及MacVecter 6.0软件程序组合(牛津分子组,牛津OX4 4GA,英国),详细分析此序列(Altschul等人,分子生物学杂志,1990,215:403-410)。在此序列(环状基因组)上可检测到体积大于20个氨基酸的13个开放阅读框架(ORF)。这13个ORF为:
名称 | 起始 | 终止 | 链 | ORF大小(核苷酸(nt)) | 蛋白质大小(氨基酸(aa)) |
ORF1 | 103 | 210 | 有义 | 108nt | 35aa |
ORF2 | 1180 | 1317 | 有义 | 138nt | 45aa |
ORF3 | 1363 | 1524 | 有义 | 162nt | 53aa |
ORF4 | 398 | 1342 | 有义 | 945nt | 314aa |
ORF5 | 900 | 1079 | 有义 | 180nt | 59aa |
ORF6 | 1254 | 1334 | 有义 | 81nt | 26aa |
ORF7 | 1018 | 704 | 反义 | 315nt | 104aa |
ORF8 | 439 | 311 | 反义 | 129nt | 42aa |
ORF9 | 190 | 101 | 反义 | 90nt | 29aa |
ORF10 | 912 | 733 | 反义 | 180nt | 59aa |
ORF11 | 645 | 565 | 反义 | 81nt | 26aa |
ORF12 | 1100 | 1035 | 反义 | 66nt | 21aa |
ORF13 | 314 | 1381 | 反义 | 702nt | 213aa |
每个ORF的起始和终止位点参见图4(SEQ ID NO:4)的1010毒株基因组。ORF1-13的界限与999毒株相同。它们1011-48121毒株及1011-48285毒株中ORF的界限也与999毒株中相同,除了ORF3和13:ORF3 1432-1539,有义,108nt,35aaORF13 314-1377,反义 705nt,234aa。
13个ORF中,4个与位于克隆病毒PCV PK/15的基因组之类似ORF具有明显同源性。分析了所有与多系统消瘦综合症有关的环病毒分离物基因组上的每个开放阅读框架。这4个ORF如下:
名称 | 起始 | 终止 | 链 | ORF大小(nt) | 蛋白质大小(aa) | 分子量 |
ORF4 | 398 | 1342 | 有义 | 945nt | 314aa | 37.7 kDa |
ORF7 | 1018 | 704 | 反义 | 315nt | 104aa | 11.8kDa |
ORF10 | 912 | 733 | 反义 | 180nt | 59aa | 6.5 kDa |
ORF13 | 314 | 1381 | 反义 | 702nt | 213aa | 27.8 kDa |
每个ORF的起始和终止位置参见图4(SEQ ID NO:4)的序列。ORF的大小(以核苷酸计=nt)包括终止密码子。
PCV Imp.1010与PCV PK-15分离株的基因组排列之间的比较鉴定了两种病毒基因组中保守的4个ORF。下表为观察到的同源性程度:
ORF Imp.1010/ORF PCV PK-15 | 同源性百分比 |
ORF4/ORF1 | 86% |
ORF13/ORF3 | 66.4% |
ORF7/ORF3 | 61.5%(在重叠水平(104aa)) |
ORF10/ORF4 | 83%(在重叠水平(59aa)) |
在ORF4 Imp.1010与ORF1 PK-15之间观察到最大序列相同性(86%同源性)。由于此蛋白质可能参与了病毒DNA复制,且为病毒复制的关键,预计有这样的同源性(Meehan等人,普通病毒学杂志,1997,78:221-227;Mankertz等人,普通病毒学杂志,1998,79:381-384)。
在ORF13 Imp.1010与ORF2 PK-15之间序列相同性不甚强(66.4%同源性),但这两个ORF各自的确存在高度保守的N-末端基本区域,其与CAV禽环病毒的主要结构蛋白质之N-末端区域相同(Meehan等人,病毒学文献,1992,124:301-319)。此外,在ORF7 Imp.1010与ORF3PK-15之间以及ORF10 Imp.1010与ORF4 PK-15之间观察到许多差异。每个情况中,当与PCV PK-15的ORF3及ORF4比较时,Imp.1010分离株的ORF 7和ORF10的C-末端存在缺失。在ORF7/ORF3以及ORF10/ORF4的N-末端区域水平上观察到最大序列同源性,分别为61.5%同源性(在重叠水平)和83%同源性(在重叠水平)。
由于猪环病毒基因组特别紧密,其基因组排列似乎相当复杂。主要结构蛋白质可能由位于猪环病毒基因组相同链上的数个阅读框架剪接产生。因此,上表中的任一开放阅读框架(ORF1-ORF13)可代表所有或部分由II型猪环病毒编码的抗原性蛋白质,因此可能是一种可用于特异性诊断和/或用于免疫接种的抗原。因此,本发明涉及任一包含至少一种上述ORF的蛋白质。优选地,本发明涉及一种基本上由ORF4、ORF7、ORF10或ORF13组成的蛋白质。实施例14:由新毒株克隆的PCV基因组的传染特性
根据由Meehan等人所述的技术,将含有Imp.999分离株的完整基因组(复制形式)的质粒pGEM-7/8转染入PK/15细胞(用禽贫血病毒感染的细胞之病毒DNA的表征:克隆的复制形式的序列分析及克隆的基因组片段的转染能力,病毒学文献,1992,124:301-309)。对非污染的PK/15细胞上转染后的第一次传代细胞进行免疫荧光分析(见实施例4),显示克隆pGEM7/8的质粒能诱导传染性PCV病毒的产生。含有传染性PCV遗传物质的克隆的获得,允许为制备经修饰的PCV病毒(在猪中减毒的或缺陷型的)对病毒基因组进行任何有用的操作,其中经修饰的PCV病毒可用于制备减毒或重组疫苗,或用于制备用作诊断试剂盒的抗原。实施例15:体外培养制备PCV抗原
根据如实施例1的相同方法进行非污染PK/15细胞的培养和病毒操作。在37℃培养4天后经胰蛋白酶消化并计数后,收集感染细胞。以每ml中400,000个感染的细胞接种下一代细胞。实施例16:病毒抗原的灭活
病毒培养结束后,收集感染的细胞,用超声波(Branson Sonifier)或借助转子-定子型胶体磨(UltraTurrax,IKA)裂解细胞。悬浮液在3700g下离心30分钟。用0.1%乙撑亚胺于37℃下灭活病毒悬液18小时,或用0.5%β-丙醇酸内酯于28℃灭活24小时。如果在灭活前病毒滴度不足,利用300kDa截流分子量的膜通过超滤浓缩病毒悬浮液(MilliporePTMK300)。灭活的病毒悬浮液储存于5℃。实施例17:以基于矿物油的乳液形式制备疫苗
根据如下配方制备疫苗:
灭活猪环病毒悬浮液:250ml
MontanideISA 70(SEPPIC):750ml
分开单独对水相和油相过滤除菌。通过借助Silverson涡轮乳化机混合和匀浆化组分制备乳液。
一只疫苗剂量含有约107.5 TCID50。对皮内施用,一只疫苗剂量体积为0.5ml,对肌内途径给药为2ml。实施例18:可代谢性油基乳液形式的疫苗制备
根据下列配方制备疫苗:
灭活猪环病毒悬浮液:200ml
Dehymuls HRE 7(Henkel):60ml
Radia 7204(Oleofina):740ml
分开单独对水相和油相过滤除菌。借助Silverson涡轮乳化机混合和匀浆化组分制备乳液。
一只疫苗剂量含有约107.5 TCID50。对于肌内途径给药,一只疫苗剂量体积为2ml。实施例19:与美国和法国PCV病毒株有关及与污染有超免疫血清(PCV-T)的PK/15的间接免疫荧光结果,以及从PK/15制备的单克隆抗体F99表和从加拿大株(PCV-C)制备的超免疫血清
*在间接免疫荧光中产生阳性反应的血清或单克隆抗体的最终稀释度之倒数。
病毒 | |||
PK/15 | 美国 | 法国 | |
PCV-T抗血清PCV-C抗血清F99 1H4F994B10F99 2B7F99 2E12F99 1C9F99 2E1F99 1H4 | ≥6400200≥10 000≥10 000≥10 000≥10 000≥10 000≥10 000≥10 000 | 200≥6400<100<100100<100<100<100100 | 800≥6400100<100<100<100100<100<100 |
Claims (29)
1.II型猪环病毒的纯化制品。
2.猪环病毒的纯化制品,其选自保藏于ECACC保藏号为V97100219、V97100218、V97100217、V98011608、V98011609的制品。
3.由细胞产生且从细胞培养物中体外分离的猪环病毒制品,其中所述细胞已用一种猪环病毒感染,该病毒能从患有PMWS综合症的猪的生理学样本或组织样本,特别是损伤组织中分离出。
4.根据权利要求3的猪环病毒制品,其从猪肾细胞系中产生并分离。
5.根据权利要求4的猪环病毒制品,其从未经PCV污染的PK/15细胞中产生并分离。
6.一种培养物提取物或上清液,其收集自用根据权利要求1的环病毒感染的细胞之体外细胞培养物。
7.一种抗原性制品,其收集自用根据权利要求1的环病毒感染的细胞之体外细胞培养物。
8.一种疫苗,其包含根据权利要求7的抗原性制品,或根据权利要求6的培养物上清液或提取物,包含猪环病毒作为抗原。
9.根据权利要求8的疫苗,其特征在于疫苗包括在兽医学可接受的载体或稀释剂中的减毒活完整抗原,以及任选地兽医学可接受的佐剂和任选地冷冻干燥稳定剂。
10.根据权利要求9的疫苗,其特征在于抗原为灭活的,且疫苗还包含兽医学可接受的载体或稀释剂,以及任选地兽医学可接受的佐剂。
11.一种DNA片段,其含有选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:6的序列。
12.一种DNA片段,其含有选自ORF1-13的ORF。
13.根据权利要求12的DNA片段,其特征在于含有选自ORF4、ORF7、ORF10和ORF13的ORF。
14.一种多肽,其由根据权利要求11-13中任一项的DNA片段编码。
15.一种体外表达载体,包括整合入其基因组中的根据权利要求11-13中任一项的DNA序列或片段,以使其可在体外表达。
16.根据权利要求15的表达载体,其特征在于其选自大肠杆菌和杆状病毒。
17.一种由根据权利要求15或16的表达载体产生的,任选地经纯化的多肽。
18.一种亚单位疫苗,包括在兽医学可接受的载体或稀释剂中的至少一种根据权利要求14或17的多肽,以及任选地兽医学可接受的佐剂。
19.一种体内表达载体,包括整合入其基因组中的根据权利要求11-13中任一项的DNA片段,以使其可在体内表达。
20.根据权利要求19的表达载体,其特征在于其选自能在猪中复制而不会使该动物致病的活病毒和质粒。
21.根据权利要求20的表达载体,其特征在于病毒载体选自猪疱疹病毒,如假狂犬病病毒;猪腺病毒;痘病毒属,特别是牛痘病毒,禽痘病毒,犬痘病毒和猪痘病毒。
22.一种活体或质粒疫苗,其特征在于其包括在兽医学可接受的载体或稀释剂中的一种根据权利要求19-21中任一项的表达载体。
23.根据权利要求8-10、18和22中任一项的疫苗,其特征在于其包含如权利要求1-4中定义的数种猪环病毒抗原。
24.根据权利要求8-10、18、22和23中任一项的疫苗,其特征在于还包含至少一种相应于另一种猪病原体的其它效价。
25.根据权利要求24的疫苗,其特征在于包含至少一种下列的其它效价:PRRS、猪肺炎支原体、大叶性肺炎放线杆菌、大肠杆菌、萎缩性鼻炎、伪狂犬病、猪霍乱和猪流感。
26.根据权利要求24的疫苗,其特征在于包含至少一种选自PRRS和猪肺炎支原体的其它效价。
27.一种探针或引物,其包含全部或部分根据权利要求11-13中任一项的序列。
28.一种从根据权利要求1-5中任一项的环病毒、根据权利要求14或17的多肽或其片段制备的多克隆或单克隆抗体。
29.一种检测猪环病毒的方法,其中,在待测猪生理学液体或组织样本中进行检测,通过试图检测抗原本身或针对抗原的抗体进行抗原存在与否的测试。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN200910252636.4A CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR97/12382 | 1997-10-03 | ||
FR9712382A FR2769321B1 (fr) | 1997-10-03 | 1997-10-03 | Nouveaux circovirus porcins, vaccins et reactifs de diagnostics |
FR9800873A FR2769322B1 (fr) | 1997-10-03 | 1998-01-22 | Nouveaux circovirus porcins, vaccins et reactifs de diagnostic |
FR98/00873 | 1998-01-22 | ||
FR9803707A FR2776294B1 (fr) | 1998-03-20 | 1998-03-20 | Nouveaux circovirus porcins ; vaccins et reactifs de diagnostic |
FR98/03707 | 1998-03-20 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2008101690507A Division CN101445802B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
CN200910252636.4A Division CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1278301A true CN1278301A (zh) | 2000-12-27 |
CN100584948C CN100584948C (zh) | 2010-01-27 |
Family
ID=27253376
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN200910252636.4A Expired - Lifetime CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
CN98810652A Expired - Lifetime CN100584948C (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN200910252636.4A Expired - Lifetime CN101724606B (zh) | 1997-10-03 | 1998-10-01 | 新猪环病毒、疫苗及诊断试剂 |
Country Status (21)
Country | Link |
---|---|
US (1) | US6368601B1 (zh) |
EP (4) | EP1741785B1 (zh) |
JP (5) | JP5008218B2 (zh) |
KR (4) | KR100736129B1 (zh) |
CN (2) | CN101724606B (zh) |
AT (3) | ATE245191T1 (zh) |
AU (1) | AU756554C (zh) |
BR (1) | BR9812845B1 (zh) |
CA (1) | CA2305623C (zh) |
CY (3) | CY1110359T1 (zh) |
DE (7) | DE122005000009I1 (zh) |
DK (4) | DK1019510T3 (zh) |
ES (4) | ES2203984T3 (zh) |
FR (1) | FR05C0021I2 (zh) |
HK (1) | HK1095161A1 (zh) |
HU (2) | HU230340B1 (zh) |
NL (1) | NL300326I2 (zh) |
PL (1) | PL198506B1 (zh) |
PT (4) | PT1741785E (zh) |
UA (1) | UA78180C2 (zh) |
WO (1) | WO1999018214A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101688185B (zh) * | 2007-05-11 | 2013-05-22 | 淡马锡生命科学研究院有限公司 | 对2型猪环状构象病毒(pcv2)感染高度容许的均质细胞系的产生 |
Families Citing this family (56)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060029617A1 (en) * | 1997-10-03 | 2006-02-09 | Charreyre Catherine E | Porcine circovirus and Helicobacter combination vaccines and methods of use |
US7211379B2 (en) * | 1997-10-03 | 2007-05-01 | Merial Sas | Prevention of myocarditis, abortion and intrauterine infection associated with porcine circovirus-2 |
US6517843B1 (en) * | 1999-08-31 | 2003-02-11 | Merial | Reduction of porcine circovirus-2 viral load with inactivated PCV-2 |
US6391314B1 (en) * | 1997-10-03 | 2002-05-21 | Merial | Porcine circoviruses vaccines diagnostic reagents |
FR2781159B1 (fr) * | 1998-07-06 | 2000-10-06 | Merial Sas | Vaccin circovirus et parvovirus porcin |
US7192594B2 (en) * | 1997-10-03 | 2007-03-20 | Merial Limited | Postweaning multisystemic wasting syndrome and porcine circovirus from pigs |
US20040062775A1 (en) * | 1997-12-05 | 2004-04-01 | Agence Francaise De Securite Sanitaire Des Aliments | Circovirus sequences associated with piglet weight loss disease (PWD) |
FR2772047B1 (fr) * | 1997-12-05 | 2004-04-09 | Ct Nat D Etudes Veterinaires E | Sequence genomique et polypeptides de circovirus associe a la maladie de l'amaigrissement du porcelet (map), applications au diagnostic et a la prevention et/ou au traitement de l'infection |
JP3795751B2 (ja) * | 1997-12-11 | 2006-07-12 | ユニバーシティ オブ サスカッチェワン | ブタからの離乳後多全身系消耗症候群ウイルス |
EP1816200B1 (en) * | 1997-12-11 | 2016-03-09 | Merial | Postweaning multisystemic wasting syndrome virus for pigs |
US6943152B1 (en) | 1999-06-10 | 2005-09-13 | Merial | DNA vaccine-PCV |
US6497883B1 (en) * | 1999-06-10 | 2002-12-24 | Merial | Porcine circovirus recombinant poxvirus vaccine |
ES2170622B1 (es) * | 1999-12-03 | 2004-05-16 | Consejo Superior De Investigaciones Cientificas | Clones y vectores infectivos derivados de coronavirus y sus aplicaciones. |
JP2004503234A (ja) * | 2000-06-15 | 2004-02-05 | パーデュー・リサーチ・ファウンデーション | ブタの先天性振せん用ワクチン |
US7018638B2 (en) * | 2000-12-19 | 2006-03-28 | Wyeth | Mycoplasma hyopneumoniae bacterin vaccine |
WO2002077210A2 (en) | 2001-03-27 | 2002-10-03 | University Of Saskatchewan | Methods to culture circovirus |
MXPA04000589A (es) | 2001-07-20 | 2005-06-17 | Univ Georgia Res Found | Virus de la rabia atenuado con mutacion de nucleoproteina en el sitio de fosforilacion para vacunacion contra la rabia y terapia genetica en el snc. |
US7279166B2 (en) | 2001-12-12 | 2007-10-09 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
US7276353B2 (en) * | 2001-12-12 | 2007-10-02 | Virginia Tech Intellectual Properties, Inc. | Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof |
AU2003263390B2 (en) * | 2002-08-26 | 2008-12-18 | Zoetis Services Llc | Vaccine for respiratory and reproductive system infections in cattle |
KR20040021988A (ko) * | 2002-09-06 | 2004-03-11 | 서상희 | 바이러스 증식용 돼지 신장 상피 세포주, 및 이의 용도 |
UA95602C2 (ru) | 2004-12-30 | 2011-08-25 | Берингер Ингельхейм Ветмедика, Инк. | Иммуногенная композиция цвс2 и способы приготовления такой композиции |
US7700285B1 (en) | 2005-12-29 | 2010-04-20 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
US7833707B2 (en) * | 2004-12-30 | 2010-11-16 | Boehringer Ingelheim Vetmedica, Inc. | Methods of overexpression and recovery of porcine circovirus type 2 ORF2 |
US8834891B2 (en) | 2005-03-14 | 2014-09-16 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising Lawsonia intracellularis |
DK1869471T3 (en) * | 2005-04-13 | 2016-08-01 | Merial Inc | PROCEDURE FOR MANUFACTURING PIG CIRCOVIRUS |
EP1792996A1 (en) * | 2005-12-01 | 2007-06-06 | Consejo Superior de Investigaciones Cientificas | Nucleic acid sequences encoding vaccines against Porcine reproductive and respiratory syndrome virus (PRRSV) |
ES2572736T3 (es) | 2005-12-29 | 2016-06-02 | Boehringer Ingelheim Vetmedica, Inc. | Uso de una composición inmunógena para atenuar síntomas clínicos en cerdos |
KR101436794B1 (ko) * | 2005-12-29 | 2014-09-04 | 베링거잉겔하임베트메디카인코퍼레이티드 | 다가 pcv2 면역원성 조성물 및 이러한 조성물의 제조방법 |
KR100870077B1 (ko) * | 2006-10-27 | 2008-11-25 | 권영득 | 이유후 전신소모성증후군(pmws)의 치료용 복합제조성물 |
US20100129397A1 (en) | 2006-12-11 | 2010-05-27 | Boehringer Ingelheim Vetmedica, Inc. | Effective method of treatment of porcine circovirus and lawsonia intracellularis infections |
DK2094872T4 (da) * | 2006-12-15 | 2020-05-18 | Boehringer Ingelheim Animal Health Usa Inc | Behandling af anti-PCV2-antistofseropositive svin med PCV2-antigen |
EP1941903A1 (en) | 2007-01-03 | 2008-07-09 | Boehringer Ingelheim Vetmedica Gmbh | Prophylaxis and treatment of PRDC |
EP1958644A1 (en) | 2007-02-13 | 2008-08-20 | Boehringer Ingelheim Vetmedica Gmbh | Prevention and treatment of sub-clinical pcvd |
KR100948127B1 (ko) * | 2007-06-27 | 2010-03-18 | 주식회사 중앙백신연구소 | 복합호흡기 질환 돼지의 조직유제를 함유하는 백신 조성물 |
US20090017064A1 (en) * | 2007-07-10 | 2009-01-15 | Wyeth | Methods and Compositions for Immunizing Pigs Against Porcine Circovirus |
US7829274B2 (en) | 2007-09-04 | 2010-11-09 | Boehringer Ingelheim Vetmedica, Inc. | Reduction of concomitant infections in pigs by the use of PCV2 antigen |
NZ586238A (en) * | 2007-12-21 | 2012-10-26 | Wyeth Llc | Methods and compositions for immunizing pigs against porcine circovirus |
AU2008347235A1 (en) * | 2007-12-31 | 2009-07-16 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 ORF2 virus like particle with foreign amino acid insertion |
US20090317423A1 (en) * | 2008-01-23 | 2009-12-24 | Boehringer Ingelheim Vetmedica, Inc. | Pcv2 mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions |
AR078253A1 (es) * | 2009-09-02 | 2011-10-26 | Boehringer Ingelheim Vetmed | Metodos para reducir la actividad antivirica en composiciones pcv-2 y composiciones pcv-2 con mejor inmunogenicidad |
TWI508974B (zh) | 2010-12-22 | 2015-11-21 | Sbc Virbac Ltd | 豬第二型環狀病毒(Porcine Circovirus Type 2)、含彼之免疫組合物、檢測套組及其應用 |
EP2564869A1 (en) | 2011-09-02 | 2013-03-06 | Ceva Sante Animale | Synthetic capsid proteins and uses thereof |
EP2789346A1 (en) | 2013-04-11 | 2014-10-15 | CEVA Santé Animale SA | Fusion polypeptides and vaccines |
US9649370B2 (en) | 2013-05-08 | 2017-05-16 | Protatek International, Inc. | Vaccine for PCV2 and mycoplasma |
CA2925281C (en) | 2013-09-25 | 2022-05-03 | Zoetis Services Llc | Pcv2b divergent vaccine composition and methods of use |
JP6778104B2 (ja) | 2013-10-02 | 2020-10-28 | ベーリンガー・インゲルハイム・アニマル・ヘルス・ユーエスエー・インコーポレイテッドBoehringer Ingelheim Animal Health Usa Inc. | Pcv2 orf2タンパク質変種および前記を含むウイルス様粒子 |
CN104383527A (zh) * | 2014-11-18 | 2015-03-04 | 天津瑞普生物技术股份有限公司 | 一种猪圆环病毒2型灭活冻干疫苗的制备方法 |
EP3034609A1 (en) | 2014-12-19 | 2016-06-22 | Ceva Sante Animale | Recombinant swinepox virus and vaccines |
US9944904B2 (en) | 2015-05-14 | 2018-04-17 | Merial Inc. | Method for porcine circovirus production and PCV2 vaccines |
EP3254692A1 (en) | 2016-06-10 | 2017-12-13 | Ceva Sante Animale | Multivalent recombinant spv |
EP3720485A1 (en) | 2017-12-08 | 2020-10-14 | Ceva Sante Animale | Recombinant swinepox virus and vaccines |
JP7162072B2 (ja) | 2018-03-26 | 2022-10-27 | ベーリンガー インゲルハイム アニマル ヘルス ユーエスエイ インコーポレイテッド | 免疫原性組成物を製造する方法 |
EP3801609A1 (en) | 2018-06-11 | 2021-04-14 | Ceva Santé Animale | Vaccination against porcine circoviruses |
KR102124260B1 (ko) * | 2018-10-19 | 2020-06-26 | 대한민국(관리부서 질병관리본부장) | 신속 면역크로마토그래피법을 이용한 콜레라균 진단·탐지 키트 및 이를 위한 특이항체와 항체생산세포주 |
EP4185321A2 (en) | 2020-07-24 | 2023-05-31 | Boehringer Ingelheim Animal Health USA Inc. | Combination porcine vaccine |
Family Cites Families (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4745051A (en) | 1983-05-27 | 1988-05-17 | The Texas A&M University System | Method for producing a recombinant baculovirus expression vector |
US4740468A (en) | 1985-02-14 | 1988-04-26 | Syntex (U.S.A.) Inc. | Concentrating immunochemical test device and method |
US5232835A (en) | 1986-11-07 | 1993-08-03 | Syntex (U.S.A.) Inc. | Qualitative immunochromatographic method and device |
US5030558A (en) | 1986-11-07 | 1991-07-09 | Syntex (U.S.A.) Inc. | Qualitative immunochromatographic method and device |
CA1303983C (en) | 1987-03-27 | 1992-06-23 | Robert W. Rosenstein | Solid phase assay |
ATE225509T1 (de) | 1987-04-27 | 2002-10-15 | Inverness Medical Switzerland | Testgerät zur durchführung von spezifischen bindungsprüfungen |
US4855240A (en) | 1987-05-13 | 1989-08-08 | Becton Dickinson And Company | Solid phase assay employing capillary flow |
US5120643A (en) | 1987-07-13 | 1992-06-09 | Abbott Laboratories | Process for immunochromatography with colloidal particles |
EP0310317B1 (en) * | 1987-09-28 | 1993-12-15 | Beecham Inc. | Inactivation of viruses and bacteria |
DK0465529T3 (da) | 1989-03-21 | 1998-10-05 | Vical Inc | Ekspression af exogene polynukleotidsekvenser i et hvirveldyr |
FR2649013B1 (fr) | 1989-07-03 | 1991-10-25 | Seppic Sa | Vaccins et vecteurs de principes actifs fluides contenant une huile metabolisable |
US5141850A (en) | 1990-02-07 | 1992-08-25 | Hygeia Sciences, Inc. | Porous strip form assay device method |
AU657907B2 (en) | 1990-05-29 | 1995-03-30 | Wyeth Holdings Corporation | Mycoplasma hyopneumoniae vaccine |
US5238652A (en) | 1990-06-20 | 1993-08-24 | Drug Screening Systems, Inc. | Analytical test devices for competition assay for drugs of non-protein antigens using immunochromatographic techniques |
US5565205A (en) | 1990-08-16 | 1996-10-15 | Solvay Animal Health, Inc. | Inactivated Mycoplasma hypopneumoniae bacterin and method of use thereof |
US5266497A (en) | 1990-08-31 | 1993-11-30 | Japan Synthetic Rubber Co., Ltd. | Immunochromatographic assay with improved colored latex |
US5451504A (en) | 1991-07-29 | 1995-09-19 | Serex, Inc. | Method and device for detecting the presence of analyte in a sample |
EP0610250B2 (en) | 1991-10-14 | 2008-10-29 | Intervet International BV | Porcine reproductive respiratory syndrome (prrs) vaccine and diagnostic |
US5338543A (en) * | 1992-02-27 | 1994-08-16 | Ambico, Inc. | Thimerosal inactivated mycoplasma hyopneumoniae vaccine |
DE9204303U1 (zh) | 1992-03-30 | 1992-06-25 | Marx, Guenter Wolfgang, 7953 Bad Schussenried, De | |
EP0571648A1 (en) | 1992-05-26 | 1993-12-01 | Chung-Nan Weng | Vaccine protecting against mycoplasmal pneumonia |
US6592873B1 (en) * | 1992-10-30 | 2003-07-15 | Iowa State University Research Foundation, Inc. | Polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus (PRRSV) and proteins encoded by the polynucleic acids |
FR2700957B1 (fr) | 1993-01-29 | 1995-03-03 | Seppic Sa | Composition de vaccin sous-unitaire recombinant vivant et procédé de préparation. |
DE69426228T2 (de) | 1993-02-08 | 2001-03-01 | Bayer Ag | Verfahren zum Vermehren des das Reproduktions- und Atemwegs-Syndrom verursachenden Schweinevirus und dessen Verwendung als Impfstoff. |
FR2702373B1 (fr) | 1993-03-08 | 1996-06-07 | Rhone Merieux | Emulsions vaccinales fluides eau-dans-l'huile contenant une huile métabolisable. |
EP0620277A1 (en) | 1993-03-18 | 1994-10-19 | Merck & Co. Inc. | Nucleic acid pharmaceuticals |
ES2074950B1 (es) | 1993-09-17 | 1996-03-16 | Iberica Cyanamid | Vacuna para la prevencion de la enfermedad reproductiva y respiratoria de la cerda. |
ATE475668T1 (de) | 1994-01-27 | 2010-08-15 | Univ Massachusetts Medical | Immunisierung durch impfung von dns transkriptionseinheit |
PT676467E (pt) * | 1994-04-11 | 2002-02-28 | Akzo Nobel Nv | Estirpes europeias de vacina do virus do sindroma reprodutivo e respiratorio porcino (prrsv) |
WO1996004091A1 (en) * | 1994-08-05 | 1996-02-15 | Schneider Karl P | Self-chamfering drill bit |
FR2781159B1 (fr) * | 1998-07-06 | 2000-10-06 | Merial Sas | Vaccin circovirus et parvovirus porcin |
FR2772047B1 (fr) * | 1997-12-05 | 2004-04-09 | Ct Nat D Etudes Veterinaires E | Sequence genomique et polypeptides de circovirus associe a la maladie de l'amaigrissement du porcelet (map), applications au diagnostic et a la prevention et/ou au traitement de l'infection |
JP3795751B2 (ja) * | 1997-12-11 | 2006-07-12 | ユニバーシティ オブ サスカッチェワン | ブタからの離乳後多全身系消耗症候群ウイルス |
KR100298125B1 (ko) * | 1999-04-15 | 2001-09-13 | 정명식 | 구리의 화학 증착에 유용한 유기 구리 전구체 |
-
1998
- 1998-01-10 UA UA2000042372A patent/UA78180C2/uk unknown
- 1998-05-21 US US09/082,558 patent/US6368601B1/en not_active Expired - Lifetime
- 1998-10-01 PL PL339631A patent/PL198506B1/pl unknown
- 1998-10-01 JP JP2000515010A patent/JP5008218B2/ja not_active Expired - Lifetime
- 1998-10-01 CN CN200910252636.4A patent/CN101724606B/zh not_active Expired - Lifetime
- 1998-10-01 HU HU0800330A patent/HU230340B1/hu unknown
- 1998-10-01 PT PT50149988T patent/PT1741785E/pt unknown
- 1998-10-01 DK DK98946547T patent/DK1019510T3/da active
- 1998-10-01 KR KR1020007003628A patent/KR100736129B1/ko not_active IP Right Cessation
- 1998-10-01 ES ES98946547T patent/ES2203984T3/es not_active Expired - Lifetime
- 1998-10-01 EP EP05014998.8A patent/EP1741785B1/fr not_active Expired - Lifetime
- 1998-10-01 DE DE200512000009 patent/DE122005000009I1/de active Pending
- 1998-10-01 WO PCT/FR1998/002107 patent/WO1999018214A1/fr not_active Application Discontinuation
- 1998-10-01 CA CA002305623A patent/CA2305623C/en not_active Expired - Lifetime
- 1998-10-01 DE DE05014998T patent/DE05014998T1/de active Pending
- 1998-10-01 KR KR1020087004310A patent/KR100904852B1/ko not_active IP Right Cessation
- 1998-10-01 EP EP02017134A patent/EP1281760B9/fr not_active Expired - Lifetime
- 1998-10-01 KR KR1020097009112A patent/KR100944144B1/ko not_active IP Right Cessation
- 1998-10-01 DE DE69816460T patent/DE69816460T2/de not_active Expired - Lifetime
- 1998-10-01 ES ES02017134T patent/ES2246001T3/es not_active Expired - Lifetime
- 1998-10-01 AT AT98946547T patent/ATE245191T1/de active
- 1998-10-01 AU AU93555/98A patent/AU756554C/en not_active Expired
- 1998-10-01 CN CN98810652A patent/CN100584948C/zh not_active Expired - Lifetime
- 1998-10-01 EP EP98946547A patent/EP1019510B1/fr not_active Expired - Lifetime
- 1998-10-01 EP EP03016998A patent/EP1386617B1/fr not_active Expired - Lifetime
- 1998-10-01 AT AT03016998T patent/ATE387914T1/de active
- 1998-10-01 HU HU0003756A patent/HU226247B1/hu active Protection Beyond IP Right Term
- 1998-10-01 DK DK02017134T patent/DK1281760T5/da active
- 1998-10-01 PT PT03016998T patent/PT1386617E/pt unknown
- 1998-10-01 ES ES05014998.8T patent/ES2524359T3/es not_active Expired - Lifetime
- 1998-10-01 DE DE122008000069C patent/DE122008000069I1/de active Pending
- 1998-10-01 ES ES03016998T patent/ES2302885T3/es not_active Expired - Lifetime
- 1998-10-01 AT AT02017134T patent/ATE299531T1/de active
- 1998-10-01 DK DK03016998T patent/DK1386617T3/da active
- 1998-10-01 PT PT98946547T patent/PT1019510E/pt unknown
- 1998-10-01 DK DK05014998.8T patent/DK1741785T3/da active
- 1998-10-01 PT PT02017134T patent/PT1281760E/pt unknown
- 1998-10-01 KR KR1020067010174A patent/KR100854615B1/ko not_active IP Right Cessation
- 1998-10-01 DE DE69830858T patent/DE69830858T4/de not_active Expired - Lifetime
- 1998-10-01 BR BRPI9812845-0A patent/BR9812845B1/pt not_active IP Right Cessation
- 1998-10-01 DE DE69839227T patent/DE69839227T2/de not_active Expired - Lifetime
- 1998-10-01 DE DE69830858A patent/DE69830858D1/de not_active Expired - Lifetime
-
2005
- 2005-04-26 FR FR05C0021C patent/FR05C0021I2/fr active Active
- 2005-10-07 CY CY20051101221T patent/CY1110359T1/el unknown
-
2007
- 2007-01-31 HK HK07101074.0A patent/HK1095161A1/zh not_active IP Right Cessation
- 2007-09-28 JP JP2007254101A patent/JP4680245B2/ja not_active Expired - Lifetime
- 2007-12-18 NL NL300326C patent/NL300326I2/nl unknown
-
2008
- 2008-06-03 CY CY20081100583T patent/CY1108113T1/el unknown
-
2011
- 2011-01-13 JP JP2011004965A patent/JP5362750B2/ja not_active Expired - Lifetime
-
2012
- 2012-03-16 JP JP2012060619A patent/JP2012193179A/ja not_active Ceased
-
2014
- 2014-11-13 CY CY20141100947T patent/CY1115749T1/el unknown
- 2014-12-18 JP JP2014255849A patent/JP5869658B2/ja not_active Expired - Lifetime
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101688185B (zh) * | 2007-05-11 | 2013-05-22 | 淡马锡生命科学研究院有限公司 | 对2型猪环状构象病毒(pcv2)感染高度容许的均质细胞系的产生 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100584948C (zh) | 新猪环病毒、疫苗及诊断试剂 | |
CN1168502C (zh) | 猪环状病毒和细小病毒疫苗 | |
AU2005220241B2 (en) | Reduction of porcine circovirus-2 viral load | |
US6391314B1 (en) | Porcine circoviruses vaccines diagnostic reagents | |
US20060029617A1 (en) | Porcine circovirus and Helicobacter combination vaccines and methods of use | |
RU2283862C2 (ru) | Цирковирус свиней типа ii и его применение | |
CN101445802B (zh) | 新猪环病毒、疫苗及诊断试剂 | |
AU2003248395B2 (en) | Porcine CircoViruses, vaccines and diagnostic reagents | |
AU2002302120B2 (en) | Porcine circoviruses, vaccines and diagnostic reagents | |
AU2006202480B2 (en) | Porcine circo viruses, vaccines and diagnostic reagents I | |
ELLIS et al. | Patent 2604705 Summary | |
MXPA00003263A (en) | Porcine circoviruses, vaccines and diagnostic reagents |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CX01 | Expiry of patent term |
Granted publication date: 20100127 |
|
CX01 | Expiry of patent term |