CN1579553A - Ii型猪圆环病毒核酸疫苗的制备方法及其应用 - Google Patents
Ii型猪圆环病毒核酸疫苗的制备方法及其应用 Download PDFInfo
- Publication number
- CN1579553A CN1579553A CN 200410018528 CN200410018528A CN1579553A CN 1579553 A CN1579553 A CN 1579553A CN 200410018528 CN200410018528 CN 200410018528 CN 200410018528 A CN200410018528 A CN 200410018528A CN 1579553 A CN1579553 A CN 1579553A
- Authority
- CN
- China
- Prior art keywords
- pcv2
- orf2
- pci
- nucleic acid
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 229960005486 vaccine Drugs 0.000 title description 3
- 210000004027 cell Anatomy 0.000 claims abstract description 36
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 claims abstract description 29
- 229940023146 nucleic acid vaccine Drugs 0.000 claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 108090000978 Interleukin-4 Proteins 0.000 claims abstract description 14
- 101000833492 Homo sapiens Jouberin Proteins 0.000 claims abstract description 12
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 claims abstract description 12
- 102100024407 Jouberin Human genes 0.000 claims abstract description 12
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 9
- 101000768957 Acholeplasma phage L2 Uncharacterized 37.2 kDa protein Proteins 0.000 claims abstract description 8
- 101000823746 Acidianus ambivalens Uncharacterized 17.7 kDa protein in bps2 3'region Proteins 0.000 claims abstract description 8
- 101000916369 Acidianus ambivalens Uncharacterized protein in sor 5'region Proteins 0.000 claims abstract description 8
- 101000769342 Acinetobacter guillouiae Uncharacterized protein in rpoN-murA intergenic region Proteins 0.000 claims abstract description 8
- 101000823696 Actinobacillus pleuropneumoniae Uncharacterized glycosyltransferase in aroQ 3'region Proteins 0.000 claims abstract description 8
- 101000786513 Agrobacterium tumefaciens (strain 15955) Uncharacterized protein outside the virF region Proteins 0.000 claims abstract description 8
- 101000618005 Alkalihalobacillus pseudofirmus (strain ATCC BAA-2126 / JCM 17055 / OF4) Uncharacterized protein BpOF4_00885 Proteins 0.000 claims abstract description 8
- 102100020724 Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Human genes 0.000 claims abstract description 8
- 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 claims abstract description 8
- 101000823761 Bacillus licheniformis Uncharacterized 9.4 kDa protein in flaL 3'region Proteins 0.000 claims abstract description 8
- 101000819719 Bacillus methanolicus Uncharacterized N-acetyltransferase in lysA 3'region Proteins 0.000 claims abstract description 8
- 101000789586 Bacillus subtilis (strain 168) UPF0702 transmembrane protein YkjA Proteins 0.000 claims abstract description 8
- 101000792624 Bacillus subtilis (strain 168) Uncharacterized protein YbxH Proteins 0.000 claims abstract description 8
- 101000790792 Bacillus subtilis (strain 168) Uncharacterized protein YckC Proteins 0.000 claims abstract description 8
- 101000819705 Bacillus subtilis (strain 168) Uncharacterized protein YlxR Proteins 0.000 claims abstract description 8
- 101000948218 Bacillus subtilis (strain 168) Uncharacterized protein YtxJ Proteins 0.000 claims abstract description 8
- 101000718627 Bacillus thuringiensis subsp. kurstaki Putative RNA polymerase sigma-G factor Proteins 0.000 claims abstract description 8
- 101000641200 Bombyx mori densovirus Putative non-structural protein Proteins 0.000 claims abstract description 8
- 101000947633 Claviceps purpurea Uncharacterized 13.8 kDa protein Proteins 0.000 claims abstract description 8
- 101000948901 Enterobacteria phage T4 Uncharacterized 16.0 kDa protein in segB-ipI intergenic region Proteins 0.000 claims abstract description 8
- 101000805958 Equine herpesvirus 4 (strain 1942) Virion protein US10 homolog Proteins 0.000 claims abstract description 8
- 101000790442 Escherichia coli Insertion element IS2 uncharacterized 11.1 kDa protein Proteins 0.000 claims abstract description 8
- 101000788354 Escherichia phage P2 Uncharacterized 8.2 kDa protein in gpA 5'region Proteins 0.000 claims abstract description 8
- 101000770304 Frankia alni UPF0460 protein in nifX-nifW intergenic region Proteins 0.000 claims abstract description 8
- 101000797344 Geobacillus stearothermophilus Putative tRNA (cytidine(34)-2'-O)-methyltransferase Proteins 0.000 claims abstract description 8
- 101000748410 Geobacillus stearothermophilus Uncharacterized protein in fumA 3'region Proteins 0.000 claims abstract description 8
- 101000772675 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) UPF0438 protein HI_0847 Proteins 0.000 claims abstract description 8
- 101000631019 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Uncharacterized protein HI_0350 Proteins 0.000 claims abstract description 8
- 101000768938 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.9 kDa protein in int-C1 intergenic region Proteins 0.000 claims abstract description 8
- 101000785414 Homo sapiens Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Proteins 0.000 claims abstract description 8
- 101000782488 Junonia coenia densovirus (isolate pBRJ/1990) Putative non-structural protein NS2 Proteins 0.000 claims abstract description 8
- 101000811523 Klebsiella pneumoniae Uncharacterized 55.8 kDa protein in cps region Proteins 0.000 claims abstract description 8
- 101000818409 Lactococcus lactis subsp. lactis Uncharacterized HTH-type transcriptional regulator in lacX 3'region Proteins 0.000 claims abstract description 8
- 101000878851 Leptolyngbya boryana Putative Fe(2+) transport protein A Proteins 0.000 claims abstract description 8
- 101000758828 Methanosarcina barkeri (strain Fusaro / DSM 804) Uncharacterized protein Mbar_A1602 Proteins 0.000 claims abstract description 8
- 101001122401 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF3 Proteins 0.000 claims abstract description 8
- 101001055788 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) Pentapeptide repeat protein MfpA Proteins 0.000 claims abstract description 8
- 101000740670 Orgyia pseudotsugata multicapsid polyhedrosis virus Protein C42 Proteins 0.000 claims abstract description 8
- 101000769182 Photorhabdus luminescens Uncharacterized protein in pnp 3'region Proteins 0.000 claims abstract description 8
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 claims abstract description 8
- 101710130262 Probable Vpr-like protein Proteins 0.000 claims abstract description 8
- 101000961392 Pseudescherichia vulneris Uncharacterized 29.9 kDa protein in crtE 3'region Proteins 0.000 claims abstract description 8
- 101000731030 Pseudomonas oleovorans Poly(3-hydroxyalkanoate) polymerase 2 Proteins 0.000 claims abstract description 8
- 101001065485 Pseudomonas putida Probable fatty acid methyltransferase Proteins 0.000 claims abstract description 8
- 101000711023 Rhizobium leguminosarum bv. trifolii Uncharacterized protein in tfuA 3'region Proteins 0.000 claims abstract description 8
- 101000948156 Rhodococcus erythropolis Uncharacterized 47.3 kDa protein in thcA 5'region Proteins 0.000 claims abstract description 8
- 101000917565 Rhodococcus fascians Uncharacterized 33.6 kDa protein in fasciation locus Proteins 0.000 claims abstract description 8
- 101000790284 Saimiriine herpesvirus 2 (strain 488) Uncharacterized 9.5 kDa protein in DHFR 3'region Proteins 0.000 claims abstract description 8
- 101000936719 Streptococcus gordonii Accessory Sec system protein Asp3 Proteins 0.000 claims abstract description 8
- 101000788499 Streptomyces coelicolor Uncharacterized oxidoreductase in mprA 5'region Proteins 0.000 claims abstract description 8
- 101001102841 Streptomyces griseus Purine nucleoside phosphorylase ORF3 Proteins 0.000 claims abstract description 8
- 101000708557 Streptomyces lincolnensis Uncharacterized 17.2 kDa protein in melC2-rnhH intergenic region Proteins 0.000 claims abstract description 8
- 101000649826 Thermotoga neapolitana Putative anti-sigma factor antagonist TM1081 homolog Proteins 0.000 claims abstract description 8
- 101000827562 Vibrio alginolyticus Uncharacterized protein in proC 3'region Proteins 0.000 claims abstract description 8
- 101000778915 Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) Uncharacterized membrane protein VP2115 Proteins 0.000 claims abstract description 8
- 238000013461 design Methods 0.000 claims abstract description 8
- 238000003757 reverse transcription PCR Methods 0.000 claims abstract description 5
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 claims abstract description 3
- 241001661006 Pepper cryptic virus 2 Species 0.000 claims abstract description 3
- 241001673669 Porcine circovirus 2 Species 0.000 claims abstract 13
- 239000013612 plasmid Substances 0.000 claims description 17
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 claims description 15
- 238000002649 immunization Methods 0.000 claims description 15
- 108090000695 Cytokines Proteins 0.000 claims description 14
- 230000036039 immunity Effects 0.000 claims description 14
- 230000003053 immunization Effects 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 230000004927 fusion Effects 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 11
- 230000028993 immune response Effects 0.000 claims description 8
- 102000000588 Interleukin-2 Human genes 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- 101150000233 ORF4 gene Proteins 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 6
- 241000699670 Mus sp. Species 0.000 claims description 5
- 101001044453 Sus scrofa Interferon gamma Proteins 0.000 claims description 5
- 239000002671 adjuvant Substances 0.000 claims description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 4
- 238000005215 recombination Methods 0.000 claims description 4
- 230000006798 recombination Effects 0.000 claims description 4
- 241000202347 Porcine circovirus Species 0.000 claims 5
- 241000700605 Viruses Species 0.000 abstract description 12
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 101710197985 Probable protein Rev Proteins 0.000 abstract description 3
- 238000004113 cell culture Methods 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 210000004369 blood Anatomy 0.000 abstract description 2
- 239000008280 blood Substances 0.000 abstract description 2
- 230000007969 cellular immunity Effects 0.000 abstract description 2
- 210000001728 clone cell Anatomy 0.000 abstract 1
- 238000011109 contamination Methods 0.000 abstract 1
- 230000008506 pathogenesis Effects 0.000 abstract 1
- 230000028327 secretion Effects 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 description 25
- 239000000047 product Substances 0.000 description 14
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 12
- 230000029087 digestion Effects 0.000 description 10
- 102000004388 Interleukin-4 Human genes 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 101100508549 Sus scrofa IL2 gene Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 108010074328 Interferon-gamma Proteins 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 231100000167 toxic agent Toxicity 0.000 description 4
- 239000003440 toxic substance Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000008521 reorganization Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 102100031725 Cortactin-binding protein 2 Human genes 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 229940028617 conventional vaccine Drugs 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000012757 fluorescence staining Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000003340 mental effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- LEBVLXFERQHONN-UHFFFAOYSA-N 1-butyl-N-(2,6-dimethylphenyl)piperidine-2-carboxamide Chemical compound CCCCN1CCCCC1C(=O)NC1=C(C)C=CC=C1C LEBVLXFERQHONN-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101150081923 IL4 gene Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102100040019 Interferon alpha-1/13 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003810 Interleukin-18 Human genes 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150068419 ORF 1 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101000848947 Platymeris rhadamanthus Venom protein family 1 protein 1 Proteins 0.000 description 1
- 101000848946 Platymeris rhadamanthus Venom protein family 1 protein 2 Proteins 0.000 description 1
- 101100406532 Porcine circovirus 2 ORF3 gene Proteins 0.000 description 1
- 101100028161 Porcine circovirus 2 ORF4 gene Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101100125863 Sus scrofa IL4 gene Proteins 0.000 description 1
- 101001043834 Sus scrofa Interleukin-2 Proteins 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960003150 bupivacaine Drugs 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 208000026498 progressive emaciation Diseases 0.000 description 1
- 210000003314 quadriceps muscle Anatomy 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000012153 swine disease Diseases 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种II型猪圆环病毒核酸疫苗的制备方法及其应用。制备方法为：1)设计特异性引物，以PCV2杭州株(HZ0201)的基因组为模板，通过PCR的方法克隆PCV－2的ORF1、ORF2、ORF3和ORF4基因，与pCI－neo构建为真核表达载体；2)分离猪外周血单个核细胞，通过RT－PCR方法克隆猪IFN、IL－2和IL－4基因，并与pCI－neo构建为真核表达载体；3)以上述重组载体为基础，分别构建PCV2 ORF2与PCV2的其它基因或与猪细胞因子基因的融合表达载体；本发明的优点：(1)不需培养病毒，生产周期短；(2)不需细胞培养，有效防止其它猪源病毒的污染；(3)不表达对机体有害的致病蛋白，安全性高；(4)可同时激活体液免疫与细胞免疫应答。
Description
技术领域
本发明涉及生物新技术领域，尤其涉及一种II型猪圆环病毒核酸疫苗的制备方法及其应用。
背景技术
断奶仔猪多系统衰竭综合征(PMWS)是1997年在加拿大首先发生一种新的猪病，表现进行性消瘦、呼吸困难、皮肤苍白、腹泻、黄疸。1998年，Ellis等首次分离到该病的病原，为II型猪圆环病毒(PCV2)。郎洪武等(2000)对北京、河北、山东、天津、江西、吉林、河南7省(市)22个猪群的559份血清检测表明，PCV2总阳性率为42.9％。周继勇等对浙江省杭州地区28个猪场采集了1677份猪血清，用IFA试验检测血清中PCV2抗体，结果PCV2抗体总阳性率为57.25％，表明PCV2在我国的流行情况也比较严重。PMWS对养猪业造成重大经济损失，PCV2是疫苗的研制成为急需解决的问题。
常规疫苗包括灭活苗或弱毒苗，但由于PCV2在细胞培养中不产生细胞病变，难以产生高滴度的病毒粒子，用常规技术制备PCV2疫苗十分不便，至今尚无II型猪圆环病毒常规疫苗可用。核酸免疫是一种新型的免疫技术，它是将编码抗原基因的表达性载体通过肌肉注射的方法给予动物，诱导机体产生免疫应答。
细胞因子佐剂对核酸疫苗的增强作用越来越受到人们的重视，根据产生的Th细胞亚群的不同，细胞因子可分为Th1型细胞因子(IL-2，IFN-γ，IL-12，IL-15，IL-18)和Th2型细胞因子(IL-4，IL-5，IL-6，IL-10)两类。细胞因子基因佐剂的应用对核酸免疫技术起了巨大的推动作用，能有效克服某些核酸疫苗免疫效力不足问题。IL-2和IFN-γ是常见的Th1型细胞因子，IL-4是常见的Th2型细胞因子，可以作为基因佐剂来增强PCV2核酸疫苗的免疫效力。我们用真核表达载体pCI-neo构建的PCV2基因、猪细胞因子、以及融合表达载体，转染PK-15细胞后，经间接免疫荧光(IFA)、酶联免疫吸附试验(ELISA)或Westernblot检测，证明能在PK-15细胞中表达。在此基础上，将其作为核酸疫苗分别免疫4-6周龄猪，发现能诱导产生针对PCV2的体液免疫和细胞免疫反应。表明我们构建的真核表达质粒可作为核酸疫苗来使用。
发明内容
本发明的目的是提供一种II型猪圆环病毒核酸疫苗的制备方法及其应用。
制备方法为：
1)设计特异性引物，以PCV2杭州株(HZ0201)的基因组为模板，通过PCR的方法克隆PCV-2的ORF1、ORF2、ORF3和ORF4基因，与pCI-neo构建为真核表达载体；
2)分离猪外周血单个核细胞，通过RT-PCR方法克隆猪IFN、IL-2和IL-4基因，并与pCI-neo构建为真核表达载体；
3)以上述重组载体为基础，分别构建PCV2 ORF2与PCV2的其它基因或与猪细胞因子基因的融合表达载体；
所构建的核酸疫苗免疫小鼠后，能诱导机体产生针对PCV2的体液免疫及细胞免疫反应。
所构建的核酸疫苗免疫仔猪后，能诱导机体产生针对PCV2的体液免疫及细胞免疫反应。
本发明的优点：
(1)不需培养病毒，生产周期短；
(2)不需细胞培养，有效防止其它猪源病毒的污染；
(3)不表达对机体有害的致病蛋白，安全性高；
(4)可同时激活体液免疫与细胞免疫应答；
附图说明
图1，真核表达载体pCI-neo的结构示意图；
图2，pCI-PCV2-ORF1重组质粒的鉴定，1.MluI和SalI双酶切，切出约945bp大小片段；2.以F1P1/F1P2作为引物的PCR产物：960bp；3.以PC1/PC2作为引物的PCR产物：1045bp；M.DNA marker；
图3，猪IFN-γ、IL2、IL4基因的RT-PCR结果，分别扩增出大小为501bp、465bp、402bp的特异性条带；M.DNA marker；
图4，pCI-pIL2重组质粒的鉴定，1.MluI和SalI双酶切：约465bp；2，3.以PIL21/PIL22为引物的PCR产物：约480bp；4.以PC1/PC2为引物的PCR产物：约587bp；M.DNA marker；
图5，pCI-pIL4重组质粒的鉴定，1.MluI和SalI双酶切：约412bp片段；2.以PIL41/PIL42为引物的PCR产物：约420bp；3.以PC1/PC2为引物进行的PCR产物：约524bp；M.DNA marker；
图6，pCI-pIFN重组质粒的鉴定，1.MluI和SalI双酶切：约507bp；2.以IF1/IF2为引物的PCR产物：519bp；3：以PC1/PC2为引物的PCR产物：约597bp；M.DNAmarker；
图7，pCI-PCV2-ORF1转染细胞的IFA检测(200×)，以猪PCV2多抗血清为一抗、FITC标记的羊抗鼠IgG为二抗，IFA法检测转染48h后的PK15，发现细胞的胞浆中呈现特异性的荧光染色；
图8.pCI-PCV2-ORF2转染细胞的IFA检测(200×)，以猪PCV2多抗血清为一抗、FITC标记的羊抗鼠IgG为二抗，IFA法检测转染48h后的PK15，发现细胞的胞浆中呈现特异性的荧光染色。
具体实施方式
本发明所说PCV2 ORF2与PCV2其它基因的融合表达载体的构建：是将PCV2ORF2与PCV2 ORF1、ORF3、ORF4基因相连接，并亚克隆于真核表达载体pCI-neo，构建重组质粒pCI-PCV2-ORF2-ORF1、pCI-PCV2-ORF2-ORF3、pCI-PCV2-ORF2-ORF4作为PCV2核酸疫苗。
PCV2 ORF2与猪细胞因子基因的融合表达载体构建：是将PCV2 ORF2与猪IFN-γ、IL-2、IL-4基因相连接，并亚克隆于真核表达载体pCI-neo，构建重组质粒pCI-PCV2-ORF2-pIFN、pCI-PCV2-ORF2-pIL2、pCI-PCV2-ORF2-pIL4作为PCV2核酸疫苗。
本发明性能的测定：
1)将纯化的重组质粒，用脂质体转染PK15细胞，转染后收集培养不同时间(24h、48h、72h)的细胞上清液进行ELISA检测或活性试验，对培养48h后的细胞进行IFA或Western blot检测，证明重组载体在真核细胞中的可以正确表达。
2)用制备重组载体免疫6-8周龄BALB/C雄鼠，间隔2周免疫三次，证明重组质粒可诱导机体产生针对PCV2的体液免疫及细胞免疫反应。
3)用制备的重组载体免疫4-6周龄仔猪，间隔2周免疫3次，证明重组质粒可诱导机体产生针对PCV2的体液免疫及细胞免疫反应，同时对猪无致病性。
实施例1：II型猪圆环病毒核酸ORF1~ORF4真核表达载体的构建
根据PCV2毒株HZ0201的基因序列，分别设计PCV2 ORF1、ORF2、ORF3、ORF4基因的特异性引物，引物序列如下：
PCV2 ORF1基因引物：
f1p1：ATAACGCGTCATGCCCAGCAAGAAG
f1p2：GCGGTCGACGACTCAGTAATTTATTTCATATGG
PCV2 ORF2基因引物：
f2ms1：GCGGTCGACTCATTAAGGGTTAAGTGGG
f2ms2：TATACGCGTTTATGACGTATCCAAGGAGG
PCV2 ORF3基因引物：
f3P1：TAAGTCGACCTTACTGATGGAGTGTGG
f3P2：ATAACGCGTATGGTAACCATCCCAC
PCV2 ORF4基因引物：
f4P1：TATGTCGACTCTCAGGGACAACGG
f4P2：ATAACGCGTCAATGACGTGTACATTAGTCT
以上的上、下游引物分别引入MluI和SalI位点。
同时根据pCI-neo载体序列，在其多克隆位点附近分别设计上下游引物，用于重组载体的鉴定：
PC1：GAGTACTTAATACGAC
PC2：CGAAGCATTAACC
以抽提的PCV2基因组DNA为模板，扩增PCV2的以上基因。PCR程序为：95℃变性10min，后按95℃ 1min，48℃ 1min，72℃ 90sec进行30个循环，最后72℃延伸10min。
用1％的琼脂糖凝胶电泳检测PCR产物并回收纯化分别为945bp、702bp、315bp、180bp的特异性片段，用MluI和SalI双酶切，与同样经MluI和SalI双酶切并回收纯化的pCI-neo载体用T4 DNA连接酶进行连接反应，转化E.coliTOP10感受态细胞，在含Amp的LB培养基平板上挑取阳性克隆，经酶切、PCR和测序鉴定，正确构建pCI-PCV2-ORF1、pCI-PCV2-ORF2、pCI-PCV2-ORF3、pCI-PCV2-ORF4真核表达载体。
实施例2：猪IL2、IL4和IFN-γ真核表达载体的构建
采取健康大约克猪血10ml，用淋巴细胞分离液分离淋巴细胞，后悬浮于RPMl1640综合培养基中，将细胞悬液稀释至2×106/ml，加conA终浓度至10mg/ml，置于细胞培养板中，CO2培养箱中37℃培养48h。然后取培养细胞用Trizol Reagent提取总RNA。根据已发表的猪IL2、IL4和IFN-γcDNA序列，设计上下游引物：
猪IL2引物：
PIL21：ATAACGCGTCAATGTATAAGATGCAG
PIL22：TCAGTCGACTTATCAAGTCAGTGTTG
猪IL4引物：
PIL41：ATAACGCGTGCTCTATTCATGGG
PIL42：TATGTCGACTTCAACACTTTGAGTAT
猪IFN-γ引物：
IF1：ATAACGCGTACAATGAGTTATACAAC
IF2：TAGGTCGACACAATTATTTTGATGCT
以上的上、下游引物分别引入MluI和SalI位点。
按RevertAidTM First Strand cDNA Synthesis Kit说明书，以Olig(dT)18为引物，M-MuLV逆转录酶作用下反转录合成猪IL-2 cDNA第一链；反转录产物在94℃ 40s，45℃ 40s，72℃ 50s的条件下进行PCR扩增，共30个循环。最后72℃延伸10min，PCR产物经1.5％的琼脂糖凝胶电泳分析并回收，应分别扩增出大小为465bp、402bp、501bp的特异性条带。用MluI和SalI双酶切RT-PCR扩增产物，与同样经MluI和SalI双酶切回收纯化的pCI-neo载体连接，转化E.coli TOP10感受态细胞，在含Amp的LB培养基平板上挑取阳性克隆。经酶切、PCR和测序鉴定，正确构建pCI-pIL2、pCI-pIL4和pCI-pIFN真核表达载体。
实施例3：PCV2 ORF2与PCV2其它基因的融合表达载体构建
设计引物，以PCV2基因组为模板，通过PCR方法扩增PCV2 ORF2基因，扩增产物用XhoI和MluI双酶切，与同样酶切的pCI-PCV2-ORF1、pCI-PCV2-ORF3、pCI-PCV2-ORF4载体相连，构建为pCI-PCV2-ORF2-ORF1、pCI-PCV2-ORF2-ORF3、pCI-PCV2-ORF2-ORF4融合表达载体。
PCV2 ORF2的扩增引物分别为：
pCI-PCV2-ORF2-ORF1：
f2xm1：TCTCTCGAGTATGACGTATCCAAGGAGGC
f2xm21：TAGACGCGTAAAGGGTTAAGTGGGGGGT
pCI-PCV2-ORF2-ORF3：
f2xm1：TCTCTCGAGTATGACGTATCCAAGGAGGC
f2xm20：TAGACGCGTAGGGTTAAGTGGGGGGT
pCI-PCV2-ORF2-ORF4：
f2xm1：TCTCTCGAGTATGACGTATCCAAGGAGGC
f2xm22：TAGACGCGTAAGGGTTAAGTGGGGGGT
PCR程序为：95℃变性10min，后按94℃ 40s，52℃ 40s，72℃ 50s进行35个循环，最后72℃延伸10min。
实施例4：PCV2 ORF2与猪细胞因子基因的融合表达载体构建
设计引物，以PCV2基因组为模板，通过PCR方法扩增PCV2 ORF2基因。将扩增产物用XhoI和MluI双酶切，与同样酶切的pCI-pIL2、pCI-pIL4、pCI-pIFN载体连接，构建为pCI-PCV2-ORF2-pIL2、pCI-PCV2-ORF2-pIL4、pCI-PCV2-ORF2-pIFN融合表达载体。
构建pCI-PCV2-ORF2-pIL2的扩增引物同pCI-PCV2-ORF2-ORF4，为f2xm1/f2xm22；构建pCI-PCV2-ORF2-pIL4、pCI-PCV2-ORF2-pIFN的扩增引物同pCI-PCV2-ORF2-ORF3，为f2xm1/f2xm20，PCR程序同实施例3。
实施例5：重组表达载体在真核细胞中的表达检测
以1ug纯化质粒加2ul Invitrogen公司的Lipofectamine Reagent转染试剂，按说明书转染2×105 PK15细胞。为检测PCV2 ORF1、ORF2、ORF3和ORF4及其融合表达产物，分别以PCV2 ORF1~4特异性单抗和PCV2病毒多抗为一抗，按常规进行间接免疫荧光(IFA)、酶联免疫吸附试验(ELISA)或Western blot，检测PCV2 ORF1~4编码产物在真核细胞中的表达。为检测猪IL2、IL4和IFN-γ基因及其融合表达产物，分别以猪IL2、IL4和IFN-γ特异性单抗为一抗，按常规进行IFA试验，检测以上猪细胞因子基因在真核细胞中的表达。检测结果表明，所构建的重组载体均能在真核细胞中正确表达。
实施例6：重组猪IL2、IL4的生物学活性检测
用MTT法检测PK15细胞表达的重组pCI-pIL2、pCI-pIL4的生物学活性：分离猪外周血单个核细胞(PBMC)，制备1×106~5×106悬液，加conA至终浓度20μg/ml，分装于96孔板中，诱导培养3-5天后，每孔加入100μl倍比稀释的PK15细胞培养上清，每个样品设3-5个重复，继续培养72h，加入5mg/ml的MTT溶液20μl，继续培养4h。每孔加入100μl含0.04mol/LHCl的异丙醇，置培养箱恒温反应2小时，取出细胞培养板，室温下放置20min，用酶联仪测定A490光密度值。表明重组猪IL2、IL4具有生物学活性。
实施例7：PCV2核酸疫苗对小鼠的免疫效果检测
大规模制备质粒，紫外分光光度法测定DNA含量和纯度，调整DNA浓度为1mg/ml。免疫6-8周龄BALB/C雄鼠，方法为：后肢股四头肌每侧各肌注7.5g/L的布比卡因10μl，72h后每侧注射重组质粒50μl，即100μg质粒/只，每组6只-10只。每隔2周按相同方法注射相同剂量，共免疫三次。同时设空载体组作对照。免疫结束后第二周分别通过淋巴细胞增殖试验(LPA)、T淋巴细胞介导的细胞毒试验(CTL)和酶联免疫吸附试验(ELISA)检测免疫鼠的细胞免疫及体液免疫应答。检测结果表明免疫鼠可产生针对PCV2的特异性细胞免疫及体液免疫反应。
淋巴细胞增殖试验(LPA)的具体过程：基因免疫结束后第2周，取免疫小鼠的脾脏，制备脾淋巴细胞悬液，将细胞浓度调整为1×I06/ml，加入96孔板，每孔200ul，加入纯化的PCV2和ORF2蛋白进行刺激(另设空白对照)，每样平行三孔。37℃，5％CO2条件下培养68h后，每孔中加入5mg/mlMTT溶液20ul，37℃下5％CO2培养箱中继续培养4h，小心吸弃孔内培养上清，加入DMSO150ul/孔，振荡10min，测定各孔OD490值，计算增殖指数。增殖指数：抗原刺激组OD490值/空白对照组值OD490值。
T淋巴细胞介导的细胞毒试验(CTL)的具体过程：基因免疫结束后第2周进行检测，在24孔培养板中加入2×I07/ml免疫鼠脾细胞悬液0.25ml，同时加入1ml RPM 11640培养液，置37℃5％条件下培养5d，收集细胞，洗2次，计活细胞数，调成5×I06/ml作为效应细胞备用。将接种PCV2的，处于对数生长期的PK15细胞，清洗2次，消化，悬浮为2×I05个/mL作为靶细胞。96孔培养板中加入效应细胞100μl和靶细胞100μl，每个样本设3个复孔。并设效应细胞自然释放孔、阴性对照(不加效应细胞)、最大释放孔(用100μl 1％NP40代替效应细胞)。37℃5％条件下培养6h后取出。200×g离心培养板10min，每孔吸100μl上清液，对应加入另一块96孔酶联检测板中，每孔加入新配制的LDH底物混合液100μl，，室温放置20min。在酶标仪上测各孔的OD值，检测波长为492nm，参考波长为650nm。特异性杀伤活性(细胞毒)的计算：先将3个复孔的OD值计算平均值，然后按计算细胞毒百分数：细胞毒％＝(试验孔OD值—靶细胞自然释放孔OD值/最大释放孔OD值—靶细胞自然释放孔OD值)×100％.
实施例8：PCV2核酸疫苗对猪的免疫效果检测
大规模制备质粒，免疫4-6周龄、PCV2抗体阴性猪，每只猪注射相应质粒200μg，免疫程序为间隔2周免疫3次，同时设空载体组作对照。首免后每周采血，通过ELISA检测血清中PCV2抗体效价；同时首次免疫后每周采血，通过流式细胞仪计数血液中的CD4+、CD8+T细胞的变化；免疫结束后第2周，取免疫猪的脾脏，检测淋巴细胞增殖活性(LPA)和T淋巴细胞介导的细胞毒(CTL)作用。结果表明PCV2核酸疫苗能有效介导机体对PCV2的特异性免疫反应。
实施例9：PCV2核酸疫苗对猪的安全性试验
为检测PCV2 ORF1~4核酸疫苗对猪是否安全，基因免疫后观察猪的精神状态、采食、体温、体重等变化；免疫结束后取实验猪的腹股沟淋巴结进行组织组织学检查，并用免疫组化(IHC)检测淋巴组织中的PCV2编码蛋白的分布。
结果表明PCV2核酸疫苗对猪的精神状态、采食、体温、体重等无明显影响，不产生明显组织学病变，有较高安全性。
Claims (5)
1.一种猪II型圆环病毒核酸疫苗的制备方法，其特征在于方法的步骤为：
1)设计特异性引物，以PCV2杭州株(HZ0201)的基因组为模板，通过PCR的方法克隆PCV-2的ORF1、ORF2、ORF3和ORF4基因，与pCI-neo构建为真核表达载体，以PCV2 ORF1、ORF2、ORF3、ORF4基因作为PCV2核酸疫苗的基本组成部分；
2)分离猪外周血单个核细胞，通过RT-PCR方法克隆猪IFN、IL-2和IL-4基因，并与pCI-neo构建为真核表达载体，以猪IFN-γ、IL-2、IL-4基因作为基因佐剂；
3)以上述重组载体为基础，分别构建PCV2 ORF2与PCV2的其它基因或与猪细胞因子基因的融合表达载体。
2.根据权利要求1所述的一种猪II型圆环病毒核酸疫苗的制备方法，其特征在于所说PCV2 ORF2与PCV2其它基因的融合表达载体的构建：是将PCV2 ORF2与PCV2 ORF1、ORF3、ORF4基因相连接，并亚克隆于真核表达载体pCI-neo，构建重组质粒pCI-PCV2-ORF2-ORF1、pCI-PCV2-ORF2-ORF3、pCI-PCV2-ORF2-ORF4作为PCV2核酸疫苗。
3.根据权利要求1所述的一种猪II型圆环病毒核酸疫苗的制备方法，其特征在于所说PCV2 ORF2与猪细胞因子基因的融合表达载体构建：是将PCV2 ORF2与猪IFN-γ、IL-2、IL-4基因相连接，并亚克隆于真核表达载体pCI-neo，构建重组质粒pCI-PCV2-ORF2-pIFN、pCI-PCV2-ORF2-pIL2、pCI-PCV2-ORF2-pIL4作为PCV2核酸疫苗。
4.一种猪II型圆环病毒核酸疫苗的应用，其特征在于：所构建的核酸疫苗免疫小鼠后，能诱导机体产生针对PCV2的体液免疫及细胞免疫反应。
5.一种猪II型圆环病毒核酸疫苗的应用，其特征在于：所构建的核酸疫苗免疫仔猪后，能诱导机体产生针对PCV2的体液免疫及细胞免疫反应。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410018528 CN1579553A (zh) | 2004-05-18 | 2004-05-18 | Ii型猪圆环病毒核酸疫苗的制备方法及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410018528 CN1579553A (zh) | 2004-05-18 | 2004-05-18 | Ii型猪圆环病毒核酸疫苗的制备方法及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1579553A true CN1579553A (zh) | 2005-02-16 |
Family
ID=34581833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410018528 Pending CN1579553A (zh) | 2004-05-18 | 2004-05-18 | Ii型猪圆环病毒核酸疫苗的制备方法及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1579553A (zh) |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328373C (zh) * | 2005-04-07 | 2007-07-25 | 南京农业大学 | 猪ⅱ型圆环病毒重组腺病毒 |
| CN100489092C (zh) * | 2006-06-16 | 2009-05-20 | 中国农业科学院哈尔滨兽医研究所 | 一种猪圆环病毒2型遗传标记毒株及其应用 |
| CN100594931C (zh) * | 2005-11-03 | 2010-03-24 | 中国农业科学院兰州兽医研究所 | 一种猪疫苗使用的pIFN-γ基因佐剂及制备方法 |
| JP2011508595A (ja) * | 2007-12-31 | 2011-03-17 | ベーリンガー インゲルハイム フェトメディカ インコーポレイテッド | 外来性アミノ酸の挿入を含むｐｃｖ２ｏｒｆ２ウイルス様粒子 |
| CN102127533A (zh) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | 重组猪圆环病毒2型Cap抗原的制备方法 |
| CN102274523A (zh) * | 2011-08-04 | 2011-12-14 | 河南农业大学 | 一种猪圆环病毒ⅱ型核酸疫苗及其制备方法 |
| CN102813918A (zh) * | 2012-08-13 | 2012-12-12 | 郑州后羿制药有限公司 | 一种猪圆环病毒2型重组亚单位疫苗及其制备方法 |
| CN103194454A (zh) * | 2013-04-08 | 2013-07-10 | 山东省农业科学院畜牧兽医研究所 | 高效表达的猪白细胞介素-2基因及其表达蛋白的用途 |
| US9011872B2 (en) | 2004-12-30 | 2015-04-21 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
| US9011868B2 (en) | 2005-12-29 | 2015-04-21 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| US9101561B2 (en) | 2005-12-29 | 2015-08-11 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent PCV2 immunogenic compositions and methods of producing such compositions |
| US9132186B2 (en) | 2007-09-04 | 2015-09-15 | Boehringer Ingelheim Vetmedica, Inc. | Reduction of concomitant infections in pigs by the use of PCV2 antigen |
| US9132187B2 (en) | 2007-02-13 | 2015-09-15 | Boehringer Ingelheim Vetmedica, Inc. | Prevention and treatment of sub-clinical PCVD |
| US9505808B2 (en) | 2013-10-02 | 2016-11-29 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 ORF2 protein variant and virus like particles composed thereof |
| CN106177945A (zh) * | 2016-08-25 | 2016-12-07 | 芜湖英特菲尔生物制品产业研究院有限公司 | 一种猪疫苗用免疫增强剂、其制备方法及应用 |
| US9517260B2 (en) | 2006-12-15 | 2016-12-13 | Boehringer Ingelheim Vetmedica, Inc. | Treatment of pigs with PCV2 antigen |
| US9522182B2 (en) | 2007-01-03 | 2016-12-20 | Boehringer Ingelheim Vetmedica, Inc. | Prophylaxis and treatment of PRDC |
| US9561270B2 (en) | 2009-09-02 | 2017-02-07 | Boehringer Ingelheim Vetmedica, Inc. | Methods of reducing virucidal activity in PCV-2 compositions and PCV-2 compositions with an improved immunogenicity |
| US9636389B2 (en) | 2006-12-11 | 2017-05-02 | Boehringer Ingelheim Vetmedica, Inc. | Effective method of treatment of porcine circovirus and Lawsonia intracellularis infections |
| US9669086B2 (en) | 2008-01-23 | 2017-06-06 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 Mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions |
| CN107469076A (zh) * | 2017-08-03 | 2017-12-15 | 中国水产科学研究院黑龙江水产研究所 | Il‑2蛋白在制备动物疫苗佐剂中的应用 |
| US9919041B2 (en) | 2004-12-30 | 2018-03-20 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
| CN108026538A (zh) * | 2015-12-28 | 2018-05-11 | 财团法人农业科技研究院 | 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 |
| RU2744193C2 (ru) * | 2015-10-16 | 2021-03-03 | Канзас Стейт Юниверсити Рисерч Фаундейшн | Иммуногенные композиции для иммунизации свиней против цирковируса типа 3 и способы их получения и применения |
-
2004
- 2004-05-18 CN CN 200410018528 patent/CN1579553A/zh active Pending
Cited By (41)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10576142B2 (en) | 2004-12-30 | 2020-03-03 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
| US9919041B2 (en) | 2004-12-30 | 2018-03-20 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
| US9011872B2 (en) | 2004-12-30 | 2015-04-21 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 immunogenic compositions and methods of producing such compositions |
| CN1328373C (zh) * | 2005-04-07 | 2007-07-25 | 南京农业大学 | 猪ⅱ型圆环病毒重组腺病毒 |
| CN100594931C (zh) * | 2005-11-03 | 2010-03-24 | 中国农业科学院兰州兽医研究所 | 一种猪疫苗使用的pIFN-γ基因佐剂及制备方法 |
| US9101561B2 (en) | 2005-12-29 | 2015-08-11 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent PCV2 immunogenic compositions and methods of producing such compositions |
| US9925255B2 (en) | 2005-12-29 | 2018-03-27 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent PCV2 immunogenic compositions and methods of producing such compositions |
| US10568955B2 (en) | 2005-12-29 | 2020-02-25 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| CN109125721A (zh) * | 2005-12-29 | 2019-01-04 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Pcv2免疫原性组合物用于减轻猪临床症状的用途 |
| US9987349B2 (en) | 2005-12-29 | 2018-06-05 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| US9669087B2 (en) | 2005-12-29 | 2017-06-06 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| US9011868B2 (en) | 2005-12-29 | 2015-04-21 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| US9610345B2 (en) | 2005-12-29 | 2017-04-04 | Boehringer Ingelheim Vetmedica, Inc. | Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs |
| US10624963B2 (en) | 2005-12-29 | 2020-04-21 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent PCV2 immunogenic compositions and methods of producing such compositions |
| US9925256B2 (en) | 2005-12-29 | 2018-03-27 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent PCV2 immunogenic compositions and methods of producing such compositions |
| CN100489092C (zh) * | 2006-06-16 | 2009-05-20 | 中国农业科学院哈尔滨兽医研究所 | 一种猪圆环病毒2型遗传标记毒株及其应用 |
| US9636389B2 (en) | 2006-12-11 | 2017-05-02 | Boehringer Ingelheim Vetmedica, Inc. | Effective method of treatment of porcine circovirus and Lawsonia intracellularis infections |
| US9517260B2 (en) | 2006-12-15 | 2016-12-13 | Boehringer Ingelheim Vetmedica, Inc. | Treatment of pigs with PCV2 antigen |
| US10780156B2 (en) | 2006-12-15 | 2020-09-22 | Boehringer Ingelheim Animal Health USA Inc. | Treatment of pigs with PCV2 antigen |
| CN107412761A (zh) * | 2006-12-15 | 2017-12-01 | 贝林格尔.英格海姆维特梅迪卡有限公司 | 以pcv2抗原治疗猪 |
| US9522182B2 (en) | 2007-01-03 | 2016-12-20 | Boehringer Ingelheim Vetmedica, Inc. | Prophylaxis and treatment of PRDC |
| US9132187B2 (en) | 2007-02-13 | 2015-09-15 | Boehringer Ingelheim Vetmedica, Inc. | Prevention and treatment of sub-clinical PCVD |
| US9555092B2 (en) | 2007-02-13 | 2017-01-31 | Boehringer Ingelheim Vetmedica, Inc. | Prevention and treatment of sub-clinical PCVD |
| US9132186B2 (en) | 2007-09-04 | 2015-09-15 | Boehringer Ingelheim Vetmedica, Inc. | Reduction of concomitant infections in pigs by the use of PCV2 antigen |
| JP2011508595A (ja) * | 2007-12-31 | 2011-03-17 | ベーリンガー インゲルハイム フェトメディカ インコーポレイテッド | 外来性アミノ酸の挿入を含むｐｃｖ２ｏｒｆ２ウイルス様粒子 |
| US9669086B2 (en) | 2008-01-23 | 2017-06-06 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 Mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions |
| US9561270B2 (en) | 2009-09-02 | 2017-02-07 | Boehringer Ingelheim Vetmedica, Inc. | Methods of reducing virucidal activity in PCV-2 compositions and PCV-2 compositions with an improved immunogenicity |
| CN102127533A (zh) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | 重组猪圆环病毒2型Cap抗原的制备方法 |
| CN102127533B (zh) * | 2010-12-31 | 2012-07-18 | 华南农业大学 | 重组猪圆环病毒2型Cap抗原的制备方法 |
| CN102274523A (zh) * | 2011-08-04 | 2011-12-14 | 河南农业大学 | 一种猪圆环病毒ⅱ型核酸疫苗及其制备方法 |
| CN102813918B (zh) * | 2012-08-13 | 2014-05-28 | 郑州后羿制药有限公司 | 一种猪圆环病毒2型重组亚单位疫苗及其制备方法 |
| CN102813918A (zh) * | 2012-08-13 | 2012-12-12 | 郑州后羿制药有限公司 | 一种猪圆环病毒2型重组亚单位疫苗及其制备方法 |
| CN103194454A (zh) * | 2013-04-08 | 2013-07-10 | 山东省农业科学院畜牧兽医研究所 | 高效表达的猪白细胞介素-2基因及其表达蛋白的用途 |
| US11858963B2 (en) | 2013-10-02 | 2024-01-02 | Boehringer Ingelheim Animal Health USA Inc. | PCV2 ORF2 protein variant and virus like particles composed thereof |
| US9505808B2 (en) | 2013-10-02 | 2016-11-29 | Boehringer Ingelheim Vetmedica, Inc. | PCV2 ORF2 protein variant and virus like particles composed thereof |
| RU2744193C2 (ru) * | 2015-10-16 | 2021-03-03 | Канзас Стейт Юниверсити Рисерч Фаундейшн | Иммуногенные композиции для иммунизации свиней против цирковируса типа 3 и способы их получения и применения |
| CN108026538A (zh) * | 2015-12-28 | 2018-05-11 | 财团法人农业科技研究院 | 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 |
| CN108026538B (zh) * | 2015-12-28 | 2021-09-03 | 财团法人农业科技研究院 | 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 |
| CN106177945A (zh) * | 2016-08-25 | 2016-12-07 | 芜湖英特菲尔生物制品产业研究院有限公司 | 一种猪疫苗用免疫增强剂、其制备方法及应用 |
| CN107469076B (zh) * | 2017-08-03 | 2020-07-07 | 中国水产科学研究院黑龙江水产研究所 | Il-2蛋白在制备动物疫苗佐剂中的应用 |
| CN107469076A (zh) * | 2017-08-03 | 2017-12-15 | 中国水产科学研究院黑龙江水产研究所 | Il‑2蛋白在制备动物疫苗佐剂中的应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1579553A (zh) | Ii型猪圆环病毒核酸疫苗的制备方法及其应用 | |
| Fan et al. | Epimedium polysaccharide and propolis flavone can synergistically stimulate lymphocyte proliferation in vitro and enhance the immune responses to ND vaccine in chickens | |
| Feng et al. | Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity | |
| CN107739731B (zh) | 包含柯萨奇病毒a10驯化株的病毒组合物及其应用 | |
| CN103952379A (zh) | 重组猪伪狂犬病毒毒株及其制备方法 | |
| CN106267182A (zh) | 一种猪伪狂犬病病毒亚单位疫苗的制备方法及疫苗组合物和应用 | |
| CN116064548B (zh) | 一种新型CpG疫苗佐剂及其应用 | |
| Cheng et al. | Incorporation of GM-CSF or CD40L enhances the immunogenicity of hantaan virus-like particles | |
| CN108117583B (zh) | 一种免疫增强剂及其应用 | |
| CN112439056A (zh) | 基于自组装铁蛋白纳米抗原颗粒及由其制备的o型口蹄疫疫苗和应用 | |
| Su et al. | Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens | |
| CN102813921B (zh) | 一种口蹄疫疫苗新型复合氢氧化铝佐剂以及制备疫苗方法 | |
| Zhaolong et al. | Evaluation of a novel inactivated vaccine against duck circovirus in muscovy ducks | |
| CN115976108B (zh) | 一种表达PCV2、PCV3 Cap蛋白的重组伪狂犬病毒载体、构建方法与应用 | |
| CN102924576B (zh) | 幽门螺杆菌免疫显性表位肽及其制备方法和应用 | |
| CN108329394A (zh) | 一种鸭短喙矮小综合征疫苗及其制备方法 | |
| Li et al. | Immunogenicity assessment of rift valley fever virus virus-like particles in BALB/c mice | |
| CN101940787A (zh) | 一种提高猪繁殖与呼吸道障碍综合征灭活疫苗免疫效能的佐剂及其制备方法和用途 | |
| CN102218139A (zh) | 一种治疗和/或预防病毒感染的药物 | |
| CN1814285A (zh) | 一种新型治疗性乙肝疫苗组合物及其制备方法 | |
| CN1701120A (zh) | 用于基因表达的核酸构建体 | |
| CN106480003A (zh) | 基于cd4+t细胞免疫的幽门螺杆菌优势抗原组合及筛选方法 | |
| CN101049508A (zh) | 携带结核分支杆菌Ag85B的减毒伤寒杆菌载体疫苗 | |
| CN107326012A (zh) | 一种细胞系及其应用 | |
| Alsakini et al. | Adjuvant effects of novel water/oil emulsion formulations on immune responses against infectious bronchitis (IB) vaccine in mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |