CN1513559A - A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine - Google Patents
A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine Download PDFInfo
- Publication number
- CN1513559A CN1513559A CNA031420990A CN03142099A CN1513559A CN 1513559 A CN1513559 A CN 1513559A CN A031420990 A CNA031420990 A CN A031420990A CN 03142099 A CN03142099 A CN 03142099A CN 1513559 A CN1513559 A CN 1513559A
- Authority
- CN
- China
- Prior art keywords
- cpg
- inoculation
- dna
- odn
- gene gun
- 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
- 238000011081 inoculation Methods 0.000 title claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 28
- 108010041986 DNA Vaccines Proteins 0.000 title claims abstract description 13
- 229940021995 DNA vaccine Drugs 0.000 title claims abstract description 13
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 10
- 230000024932 T cell mediated immunity Effects 0.000 title 1
- 239000013612 plasmid Substances 0.000 claims abstract description 46
- 229940046168 CpG oligodeoxynucleotide Drugs 0.000 claims abstract description 42
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000010931 gold Substances 0.000 claims abstract description 29
- 229910052737 gold Inorganic materials 0.000 claims abstract description 29
- 102100037850 Interferon gamma Human genes 0.000 claims abstract description 16
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 16
- 239000002671 adjuvant Substances 0.000 claims abstract description 15
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims abstract description 5
- 102000036639 antigens Human genes 0.000 claims abstract description 5
- 108091007433 antigens Proteins 0.000 claims abstract description 5
- 238000011238 DNA vaccination Methods 0.000 claims description 28
- 230000004044 response Effects 0.000 claims description 19
- 230000001900 immune effect Effects 0.000 claims description 7
- 230000003308 immunostimulating effect Effects 0.000 claims description 4
- 230000008676 import Effects 0.000 claims description 4
- 239000011261 inert gas Substances 0.000 claims description 4
- 210000002615 epidermis Anatomy 0.000 claims description 2
- 239000002245 particle Substances 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 14
- 230000003053 immunization Effects 0.000 abstract description 11
- 238000002649 immunization Methods 0.000 abstract description 11
- 241001465754 Metazoa Species 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 6
- 230000028993 immune response Effects 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 2
- 208000036142 Viral infection Diseases 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 230000002085 persistent effect Effects 0.000 abstract 1
- 230000009385 viral infection Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 21
- 230000036039 immunity Effects 0.000 description 16
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 14
- 210000003491 skin Anatomy 0.000 description 10
- 238000002255 vaccination Methods 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 238000010255 intramuscular injection Methods 0.000 description 7
- 239000007927 intramuscular injection Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 210000001015 abdomen Anatomy 0.000 description 5
- 230000002269 spontaneous effect Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 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
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 229910000906 Bronze Inorganic materials 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000010974 bronze Substances 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- KUNSUQLRTQLHQQ-UHFFFAOYSA-N copper tin Chemical compound [Cu].[Sn] KUNSUQLRTQLHQQ-UHFFFAOYSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 210000005081 epithelial layer Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 229960002275 pentobarbital sodium Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012113 quantitative test Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000007390 skin biopsy Methods 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108091029430 CpG site Proteins 0.000 description 1
- 241000233756 Fabriciana elisa Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000008884 pinocytosis Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940007046 shigella dysenteriae Drugs 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及生物技术领域，提供了一种可增强基因枪接种DNA疫苗诱生的Th1型免疫应答的方法，本发明也涉及制备质粒DNA－CpG寡聚核苷酸包裹金颗粒的方法。本发明在基因枪接种质粒DNA疫苗的同时共导入一定比例的合成的寡核苷酸CpG DNA(CpG ODN)作为佐剂。动物免疫实验证实：本发明能增强基因枪接种DNA疫苗的Th1型免疫应答水平，包括总IgG，IgG2a，特异性CTL活性，抗原特异的IFN－γ产生能力。本发明有利于克服基因枪接种DNA疫苗诱生低水平Th1型免疫应答的缺陷，可用于肿瘤及病毒持续性感染等的治疗。The invention relates to the field of biotechnology, and provides a method for enhancing the Th1 type immune response induced by gene gun inoculation with DNA vaccines, and also relates to a method for preparing plasmid DNA-CpG oligonucleotide-coated gold particles. In the present invention, a certain proportion of synthetic oligonucleotide CpG DNA (CpG ODN) is co-introduced as an adjuvant when the gene gun is inoculated with the plasmid DNA vaccine. Animal immunization experiment proves that the invention can enhance the Th1 type immune response level of gene gun inoculated DNA vaccine, including total IgG, IgG2a, specific CTL activity, and antigen-specific IFN-γ production ability. The invention is beneficial to overcome the defect of low-level Th1 type immune response induced by gene gun inoculation of DNA vaccine, and can be used for the treatment of tumors, persistent viral infections and the like.
Description
Technical field
The invention belongs to biological technical field, but relate to the new method of the cellullar immunologic response that a kind of enhancing gene rifle inoculation dna vaccination induces, and uses thereof.
Background technology
Dna vaccination (DNA vaccine) claims gene vaccine (Gene vaccine) again, it is in the nature extrinsic protein encoding gene fragment cloning on eucaryon plasmid DNA expression vector, and body produces specific cell and humoral immunization reaches the purpose of preventing and treating disease thereby can and bring out at the cell inner expression extrinsic protein behind the direct inoculation.Since it can overcome traditional vaccine the part defective, can bring out body simultaneously and produce specific cell and humoral immunization, and has preparation simply, advantages such as stable in properties, thereby be considered to third generation vaccine after attenuation, inactivated vaccine and recombinant vaccine subunit vaccine, a developing direction for following vaccine has obtained extensive concern and further investigation in recent years.The research report is arranged, and there is evident difference in dna vaccination induces immunne response in different animals intensity, and effect is comparatively sure in mouse model, and immune effect is then undesirable in large animal (as primate) and human body.The researching DNA vaccine is in the mechanism of allogenic animal difference not and how to make dna vaccination more effective focus that has become various countries scientist research in large animal, human body.
Known, the strategy that strengthens dna vaccination immunity efficient mainly comprises: screening high immunogenicity epi-position or use multivalence epitope, the targeting of optimization expression carrier, dna vaccination import, use adjuvant, seek best vaccination ways etc.Wherein the mode of dna vaccination inoculation is to its immunne response effect important influence that induces.The dna vaccination vaccination ways can be divided into by approach: intramuscular inoculation, cutaneous inoculation and mucosal vaccination; Can be divided into parcel gold grain-particle gun bombardment by the processing form of DNA, salt water dissolution-injector to inject and attenuation sramana/shigella dysenteriae or adenovirus etc. are vaccination ways such as the oral or suction of carrier.
The gene gun inoculation dna vaccination is that plasmid DNA is wrapped on the gold grain, utilize the impact of high-pressure inert gas that gold grain is directly banged in the cell, so it has higher transfection efficiency than syringe inocalation method.Gene gun inoculation only needs 1/100～1/1000 of syringe inoculation DNA consumption can induce the quite antibody response of level (Fynan EF et al.Proc Natl AcadSci USA, 1993; 90:11478-82).In view of this, it is generally acknowledged that the gene gun inoculation mode can overcome the poor efficiency of intramuscular injection inoculation dna vaccination in large animal and human body, the method for gene gun inoculation has been adopted in the experiment of dna vaccination in human body at present.
Level that there are some researches show type of immune response Th1 type immunne response that the gene gun inoculation dna vaccination induces based on the Th2 type extremely a little less than, then mainly induce Th1 type immunne response with syringe intramuscular injection or intradermal injection, (Pertmer TM et al.J Virol 1996; 70:6119-25; Feltquate DM et al.J Immunol, 1997; 158:2278-84).And Th1 type immunne response bacterium in removing viral persistent infection, anti-born of the same parents infects and antineoplastic immune in bringing into play important effect, the corresponding dna vaccination of gene gun inoculation induces the obstacle that low-level Th1 type immunne response becomes its application.
" CpG structure " (CpG motif) is notion (the Krieg AM that Krieg AM etc. proposes, et al.Nature.1995,374:6522:546-549): promptly be respectively the specific oligonucleotides sequence of two purine (5 ' side) and two pyrimidines (3 ' side) in the middle of some for unmethylated CpG dinucleotide, both sides, as 5 '-AACGTT-3 ', 5 '-GACGTC-3 ', 5 '-AGCGCT-3 ' etc.A large amount of in vitro studies are found, this ad hoc structure has activation to mammal cotton dress system, can stimulate M φ, DC, NK, T and bone-marrow-derived lymphocyte, and make it to secrete IL-2, IL-6, IL-12 and IFN-γ etc., make immunoreation change (Stacey KJ, et al.J Immunol.1996,157:5:2116-2122 to the Th1 type; Sparwasser T, et al.Eur J Immunol.1998,28:6:2045-2054; Yamamoto S, et al.Jpn J Cancer Res.1998,79:7:866-873; Bendigs S, et al.Eur J Immunol.1999,29 (4): 1209-1218).(Sato Y such as Sato, et al.Science.1996,273:5273:352-354) find, do not contain the CpG structure that in ampicillin resistance gene, exists in the kalamycin resistance gene, the less immunogenic of respective carrier, whether the existence of inferring the CpG structure in the carrier framework has thus determined the immunogenicity of dna vaccination.They further insert the immune effect of this carrier of CpG topology discovery can the raising in the carrier that contains that resistant gene of card.Yuan Zhenghong etc. (Chinese patent publication number CN 1382492A) disclose to insert in dna vaccine vector has the active CpG motifs of differential stimulus can strengthen its specific immunity originality to human immune system.Use when Weeratna etc. have reported inoculation protein vaccine HBsAg CpG ODN as adjuvant than freund 's incomplete adjuvant (FIA) and Titermax Gold etc. stronger induce immunne response and toxicity is littler, and when CpG ODN mixes use with TH2 type adjuvant, can overcome the deflection of their TH2 type adjuvant.
Gene gun inoculation DNA consumption only for syringe inoculation consumption 1/100 so that still less, the amount of the CpG motif that the former contains also only for the latter 1/100 so that still less.This may be to cause gene gun inoculation dna vaccination immunne response to be one of reason of Th2 type deflection.Still the new method of not having at present enhancing gene rifle inoculation dna vaccination adjuvant.
Have the identification receptor Toll sample receptor 9 (TLR9) of report CpG to be positioned at cell, CpG-ODN need go into born of the same parents could activate downstream signal transduction (Ahmad-Nejad P et al.EurJ Immunol, 2002; 32:1958-68).If then mainly be positioned at the extracellular with syringe in the local inoculation of bombardment CpG-ODN, processes such as the endocytosis/pinocytosis of still needing enter in the cell.Mix with plasmid DNA in the situation of back with the syringe inoculation at CpG-ODN, Weeratna etc. report (Weeratna R et al.Antisense Nucleic Acid Drug Dev1998; 8:351-6) ODN and plasmid DNA may be competed into born of the same parents receptor mutually, thereby not only may cause the antigen amount minimizing of expressing but also the CpG effect is suppressed.
In addition, the effect of CpG-ODN in the gene gun inoculation dna vaccination also can be different and influenced with the ratio of plasmid DNA because of CpG-ODN.It is 50 μ g/1 μ g that CpG-ODN and plasmid DNA ratio are used in existing research, but but a large amount of CpG intense stimulus cytokine secretions and (the Tan Y et al.Hum Gene Ther1999 that causes that antigen gene expression is suppressed in the plasmid; 10:2153-61).
Summary of the invention
But the new method that the purpose of this invention is to provide the cellullar immunologic response that a kind of enhancing gene rifle inoculation dna vaccination induces, a kind of method that can enhancing gene rifle inoculation dna vaccination induces the Th1 immunne response, and practical use.
The present invention can inoculate the adjuvant strategies that dna vaccination induces the Th1 immunne response by the enhancing gene rifle.
Technical scheme of the present invention is that to import a certain proportion of immunostimulation element in gene gun inoculation plasmid DNA vaccine be that synthetic oligonucleotide CpG DNA (CpG ODN) is as adjuvant.Plasmid DNA and oligonucleotide CpG DNA are wrapped in the gold grain surface jointly, utilize the high-pressure inert gas bombardment, it is directly imported in the epidermal layer cells.
Described CpG-ODN and plasmid DNA ratio are 1: 1 to 10: 1.
For estimating the present invention is the effect of adjuvant strategies, adopt the plasmid DNA vaccine pYQF-2CpG/HBs of gene gun inoculation HBsAg expression to add immunization experiment in CpG ODN and the mice body, verified to add with specific humoral immunization behind the CpG ODN adjuvant (detecting anti-HBsAg in the serum) and cellular immunization (the IFN-γ secretion capacity of antigenic specificity and CTL activity).The result shows 1) the present invention adds the amount that increases CpG motif with CpG ODN and can strengthen it and induce Th1 type immunne response level in the gene gun inoculation dna vaccination.2) mode that CpG-ODN and plasmid DNA co-precipitation are imported with the particle gun bombardment on gold grain then than gene gun inoculation plasmid DNA in the mode inoculated with syringe after mixing with plasmid DNA with the mode of syringe inoculation CpG-ODN or CpG-ODN of part effective, the inventive method can make it directly enter in the cell, thereby easier activation downstream signal transduction, and avoided ODN and plasmid DNA to going into the competition of born of the same parents' receptor.
The present invention realizes by following method and step:
The present invention adopts plasmid pYQF/s-2CpG (CN 1382492A), has the CMV promoter, contains BGH transcription stop signals and kalamycin resistance gene.Insert the HBVs protein gene in multiple clone site, and on framework, inserted two synthetic CpG structures (worker company is given birth in Shanghai).Plasmid with Qiagen Plasmid Maxi Kit prepare in a large number, purification, be dissolved in normal saline.Described CpG-ODN sequence is: 5 '-TCAAAACGTTGATG-3 ', the GpC-ODN control sequence is: 5 '-TCAAAAGCTTGATG-3 '.
Particle gun bullet of the present invention adopts spermidine-calcium chloride pack that DNA is wrapped in (available from U.S. Bio-rad company) on the bronze that diameter is 1.0 μ m.Plasmid DNA is mixed with ODN, and weight ratio can be controlled in 5-10: 1 or 1: 1, and perhaps plasmid DNA is dissolved in ddH separately
2O joins mixing in the spermidine that contains gold grain, adds CaCl again
2DNA is deposited on the gold grain.After the washing with alcohol, add the dehydrated alcohol mixing, gold grain-DNA alcoholic solution is drawn onto in the particle gun plastic tube, air drying is made bullet.
Get the 6 all female Mus of BALB/c in age (available from Chinese Academy of Sciences's Shanghai Experimental Animal Center) animal immunes, grouping, gene gun inoculation; The intramuscular injection inoculation; Intradermal vaccination, 4 all booster immunizations behind the initial immunity, method is the same.
The distribution of its gold grain of skin biopsy specimen immediately after the observation particle gun bombarded back 3 hours or bombarded.
Detect immunity back anti-HBsAg antibody and subclass, the ELISA method detects vein serum wherein IgG antibody and subclass IgG1, IgG2a, and indirect method ELISA detects IgG total serum titre.
Detect immunity back cellular immune function, adopt IFN-γ ELISA detection by quantitative test kit (available from the luxuriant first Science and Technology Ltd. in Shanghai) to detect IFN-γ level.Carry out Calcein fluorescent labeling CTL killing experiments, the fluorescence measurement instrument detects fluorescent value.Calculate kill rate and specific killing rate by following formula,
Kill rate=(FI of the spontaneous release of FI-of test group)/(FI-of positive control
The FI of spontaneous release) * 100%.
The specific killing rate is the value that the value of P815-s deducts P815.
Handle through variance analysis and t inspection statistics, the result confirms that particle gun bombardment of the present invention can make the gold grain of parcel DNA directly enter in the epithelial layer cell, the adjuvant strategies energy enhancing gene rifle of the CpG ODN that the common CpG of importing ODN/ plasmid DNA ratio is 5-10/1 in the confirmation gene gun inoculation plasmid DNA vaccine in the mice body is inoculated the Th1 type immunne response level of dna vaccination, comprise total IgG, IgG2a, the specific CTL activity, the IFN-γ of antigen-specific produces ability.The inventive method helps overcoming the gene gun inoculation dna vaccination and produces the low defective of Th1 type immunne response level, potential adjuvant strategies when being a kind of human body applying gene rifle inoculation dna vaccination.
Description of drawings:
Fig. 1 is the used dna vaccination pYQF-CpG/HBs of the invention process example plasmid vector sketch map and full thio-modification CpG-ODN and GpC-ODN control sequence.
Fig. 2 is the distribution of particle gun bombardment back gold grain at skin histology:
Wherein, a, tissue slice H﹠amp; E colored light sem observation (amplifying 240 times); B, transmission electron microscope observing (amplifying 2850 times).
Fig. 3 is the position of particle gun bombardment back gold grain at cell:
Wherein, a, amplification are 6500 times; 11000 times of b, amplifications.
Fig. 4 is anti-HBs antibody response reaction after the immunity inoculation:
Wherein, a.ELISA detects IgG, IgG1 and IgG2a in the 8 all serum (1: 100) of immunity back; B. immune back mouse IgG antibody titre level.
Fig. 5 is a HBsAg specific cell immunoreaction after the immunity inoculation:
Wherein, HBsAg specific CTL reaction around behind a, the booster immunization.Mouse boosting cell is through HBsAg stimulated in vitro IFN-γ emission levels (pg/ml) after b, the immunity inoculation.
The specific embodiment
Adopt spermidine-calcium chloride pack that DNA is wrapped on the bronze that diameter is 1.0 μ m, preparation particle gun bullet.With 10 μ g plasmid DNA and 100 μ g or 10 μ g ODN, weight ratio can be controlled in 5-10: 1 or 1: 1, mix or 10 μ g plasmid DNA are dissolved in 100 μ l ddH separately
2O joins mixing in the 100 μ l 0.1M spermidines that contain the 5mg gold grain, adds 200 μ l 2.5M CaCl again
2DNA is deposited on the gold grain.Behind the ethanol thorough washing, add 100 μ l dehydrated alcohol mixings, per 10 μ l are a bullet.Gold grain-DNA alcoholic solution is drawn onto in the particle gun special plastic pipe, promptly makes bullet after the air drying.Wherein, every bullet carries the 0.5mg gold grain, 1 μ g DNA (8.34 * 10
-7μ mol), 10 μ g ODN (2.2 * 10
-3μ mol); Or the 0.5mg gold grain, 1 μ g DNA (8.34 * 10
-7μ mol), 1 μ g ODN (2.2 * 10
-4μ mol); Or the 0.5mg gold grain, 1 μ g DNA (8.34 * 10
-7μ mol).
Get the female Mus animal immune of 6 week BALB/c in age, be divided into nine groups at random, every group of 6 mices.1) gene gun inoculation: the Pericarpium Arecae hair is shaved only, uses particle gun (Xin Zhi Science and Technology Ltd.) with the pressure bombardment of 2.5MPa gold grain-DNA or gold grain-DNA-CpG ODN to be imported mouse part skin, local visible 2cm
2About the bombardment scope.2) intramuscular injection inoculation: after pentobarbital sodium (75mg/kg) anesthesia, in bilateral tibialis anterior inserting needle, slowly inject the plasmid DNA normal saline solution, every side 25 μ g/50ul with the 100u insulin syringe.) intradermal vaccination: mice back of the body tail end fur is shaved only, and after pentobarbital sodium (75mg/kg) anesthesia, with the parallel inserting needle skin with spinal column of 100u insulin syringe, as seen Intradermal heaves the skin mound.4 weeks behind the initial immunity, use the said method booster immunization, table 1 is experiment grouping, immunization ways and inoculation material.
Table 1
The group side of inoculation target position inoculation material
A particle gun Intradermal, skin of abdomen 1 μ g plasmid+1 μ g CpG-
B particle gun Intradermal, skin of abdomen 1 μ g plasmid+1 μ g
C particle gun Intradermal, skin of abdomen 1 μ g plasmid+10 μ g
D particle gun Intradermal, skin of abdomen 1 μ g plasmid+10 μ g
E particle gun Intradermal, skin of abdomen 1 μ g plasmid
The F syringe intramuscular injection shank shin outside 1 μ g plasmid
The G syringe intramuscular injection shank shin outside 50 μ g plasmids
H syringe Intradermal, skin of back 50 μ g plasmids
I syringe intramuscular injection shank shin outside normal saline
Wherein, every group of 6 BALB/c mouse.Plasmid DNA is PYQF-2CpG.1 μ g plasmid DNA contains 8.34 * 10
-7The mol molecule; 50 μ g plasmid DNA contain 4.17 * 10
-5μ mol molecule; 1 μ g ODN DNA contains 2.2 * 10
-4μ mol molecule; 10 μ g ODN DNA contain 2.2 * 10
-3μ mol molecule.
Observe the distribution of particle gun bombardment back gold grain: bombard local skin biopsy in back 3 hours and draw materials, 10% formalin fixed, paraffin embedding, tissue slice (6 μ m), H﹠amp; E dyeing, light microscopic is taken pictures under amplifying 240 times.Or biopsy is immediately drawn materials and is prepared into electron microscope specimen after the bombardment, ultrathin section (70-90nm), uranyl acetate and lead citrate dyeing, transmission electron microscope observing.
Fig. 2, Fig. 3 have shown the position of gold grain in histiocyte of particle gun bombardment back parcel DNA.The result shows, tissue slice H﹠amp; E colored light sem observation and transmission electron microscope show that gold grain mainly is positioned at epithelial layer, and minority can be deep into the upper strata of corium.Gold grain is positioned at endochylema or has directly entered karyon, does not have obvious film spline structure around the gold grain, shows that gold grain is that bombardment by high-pressure inert gas directly enters but not enters in the cell by processes such as endocytosis.
Immunity back anti-HBsAg antibody and subclass detect: the immunity day beginning is every the corner of the eyes venous plexus blood sampling behind the mouse orbit of 2 weeks, separation of serum, and the ELISA method detects wherein IgG antibody and subclass IgG1, IgG2a.With each mice serum equivalent mixing in every group, make gradient dilution, indirect method ELISA detects IgG total serum titre.Fig. 4 has shown HBsAg specific humoral immunity level after the immunity inoculation.Wherein the E group is gene gun inoculation 1 μ g plasmid DNA (8.34 * 10
-7μ mol) IgG2a level is 0.396 ± 0.287 far below syringe muscle or intradermal vaccination 50 μ g plasmid DNA (4.17 * 10
-5μ mol) (G group: 2.915 ± 0.117 and the H group: 2.269 ± 0.307).Gene gun inoculation adds 1 or 10 μ g contrast GpC-ODN can not increase IgG2a level (B group: 0.29 ± 0.153 and the D group: 0.354 ± 0.184).Gene gun inoculation adds CpG-ODN then can be increased anti--HBs IgG2a level (A group: 0.541 ± 0.234 and the C group: 1.545 ± 0.111), wherein the C group is for adding with 10 μ g CpG-ODN (2.2 * 10
-3μ mol) statistically significant difference there is (p＜0.01) than increasing of E group.Add with anti--HBsAg total IgG behind the CpG-ODN and also strengthen to some extent.Monitoring HBsAg-specific antibody titre, behind the booster immunization, C group IgG responsing reaction is faster morning stronger than A group or E group.In inoculation the 14th week of back, A group, C group, E group, G group and the specific IgG antibody titer of H group HBsAg were respectively 1: 2400, and 1: 4800,1: 2400,1: 4800,1: 4800.
Detect immunity back cellular immune function: every group of 2 mices of picking at random, to put to death, the aseptic spleen of getting is made single cell suspension.Adjusting concentration is 4 * 10
6/ ml detects the active usefulness of cytokine and CTL fully.IFN-γ detects to adding HBsAg 10 μ g/ml, cultivates and collects supernatant in 48 hours, adopts IFN-γ ELISA detection by quantitative test kit (available from the luxuriant first Science and Technology Ltd. in Shanghai) to detect IFN-γ level.Calcein fluorescent labeling CTL killing experiments: the gamma-rays deactivation exponential phase P815-s cell with 70 Gy is made irritation cell.Add irritation cell in splenocyte, ratio is 20: 1, cultivates 6 days, adds mIL-225u/ml and keeps.Stimulate the after effect cell with Ficoll separating medium purification, resuspended there not to be phenol red culture fluid.With 37 ℃ of water-bath labelling exponential phase P815-s of 5 ~ 10uMcalcein AM (available from U.S. Molecular Probe company), P815 cell, D-Hank ' s liquid are washed the back there not to be the resuspended labels targets cell of making of phenol red culture fluid.In culture plate at the bottom of 96 hole circles in effect/target ratio: 50/1,25/1,12.5/1,6.25/1,3.13/1,1.56/1 adds gradient dilution effector lymphocyte and the good target cell of labelling.If release group fully wherein adds lysis liquid, wherein add no phenol red culture fluid with spontaneous release group, hatch after 4.5 hours centrifugally, supernatant is moved in the new plate hole, the fluorescence measurement instrument detects fluorescent value.Calculate kill rate and specific killing rate by following formula,
Kill rate=(FI of the spontaneous release of FI-of test group)/(FI-of positive control
The FI of spontaneous release) * 100%.
The specific killing rate is the value that the value of P815-s deducts P815.
Fig. 5 has shown HBsAg specific cellular immunity level after the immunity inoculation.Wherein the G of syringe muscle or intradermal vaccination 50 μ g plasmid DNA group and H group have the CTL activity of higher level, and B group and the D group CTL activity of the E group of gene gun inoculation 1 μ g plasmid DNA or adding contrast GpC-ODN are all very low.And the C that gene gun inoculation adds with 10 μ g CpG-ODN organizes the active obviously raising of its CTL.Effect/target ratio is 50/1 o'clock, and A group, B group, C group, D group, E group, G group, H group and I group are respectively 7.89%, 3.44%, 21.84%, 4.17%, 4.02%, 31.47%, 33.15% and 4.14%.
Immunity back 8 all mouse boosting cells stimulate the releasability testing result of back IFN-γ to show at HBsAg, and E group antigenic specificity IFN-γ emission levels only is 236.4pg/ml.And G group and H group are respectively 2609.1pg/ml and 1878.5pg/ml.The B group and the D group IFN-γ level that add with GC-ODN do not increase even decline to some extent on the contrary, are respectively 95.0pg/ml and 159pg/ml.Organize its IFN-γ level and increase greatly and gene gun inoculation adds C with 10 μ g CpG-ODN, be 1046.2pg/ml.To organize its IFN-γ level be 258.1pg/ml but only add A with 1ug CpG-ODN, obviously do not raise than the E group.
Claims (6)
1, but a kind of enhancing gene rifle inoculation dna vaccination induces the method for cellullar immunologic response, it is characterized in that in gene gun inoculation plasmid DNA vaccine, import the immunostimulation element as adjuvant, its ratio is 1: 1-10, plasmid DNA and immunostimulation element are wrapped in the gold grain surface, with the high-pressure inert gas bombardment, directly import in the epidermal layer cells.
2, by the described method of claim 1, it is characterized in that described immunostimulation element is synthetic oligonucleotide CpG-ODN.
3, by claim 1 and 2 described methods, it is characterized in that described CpG-ODN and plasmid DNA vaccine ratio are 1: 1.
4, by claim 1 and 2 described methods, it is characterized in that described CpG-ODN and plasmid DNA vaccine ratio are 1: 10.
5, by claim 1 and 2 described methods, it is characterized in that described oligonucleotide CpG ODN when being used for the gene gun inoculation dna vaccination as adjuvant.
6, by claim 1 and 2 described methods, it is characterized in that described cellullar immunologic response is a Th1 type immunne response, comprise total IgG, IgG2a, specific CTL IFN-γ active and antigen-specific produces ability.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031420990A CN1513559A (en) | 2003-08-06 | 2003-08-06 | A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031420990A CN1513559A (en) | 2003-08-06 | 2003-08-06 | A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1513559A true CN1513559A (en) | 2004-07-21 |
Family
ID=34240359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031420990A Pending CN1513559A (en) | 2003-08-06 | 2003-08-06 | A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1513559A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007012285A1 (en) * | 2005-07-28 | 2007-02-01 | Changchun Huapu Biotechnology Co., Ltd. | Viral infection resistent single strand deoxynucleosides |
| CN103173485A (en) * | 2013-03-05 | 2013-06-26 | 东北师范大学 | Method for cultivating starch-content-increased transgenic plant through multi-gene transformation |
| CN114748614A (en) * | 2021-12-27 | 2022-07-15 | 上海药明生物医药有限公司 | Method for immunizing animals by using needleless injector |
-
2003
- 2003-08-06 CN CNA031420990A patent/CN1513559A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007012285A1 (en) * | 2005-07-28 | 2007-02-01 | Changchun Huapu Biotechnology Co., Ltd. | Viral infection resistent single strand deoxynucleosides |
| CN103173485A (en) * | 2013-03-05 | 2013-06-26 | 东北师范大学 | Method for cultivating starch-content-increased transgenic plant through multi-gene transformation |
| CN114748614A (en) * | 2021-12-27 | 2022-07-15 | 上海药明生物医药有限公司 | Method for immunizing animals by using needleless injector |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111848831B (en) | Application of cationic polymer modified by fluorine-containing compound in preparation of vaccine medicine | |
| CN104027324B (en) | Soluble microneedle vaccine patch and preparation method thereof | |
| Ebensen et al. | The bacterial second messenger cyclic diGMP exhibits potent adjuvant properties | |
| CN1575815A (en) | Aqueous immunologic adjuvant compositions of monophosphoryl lipid a | |
| CN112972670B (en) | Immunostimulatory compositions and uses thereof | |
| Wang et al. | Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice | |
| CN107456575A (en) | A kind of manganese dioxide nano adjuvant and preparation method thereof, application | |
| CN108714213A (en) | The preparation method and application of a kind of nanometer adjuvant of self assembly and the nano vaccine formed by the adjuvant | |
| Jin et al. | A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses | |
| JP2012524793A (en) | Hydrogel for combined delivery of immunomodulatory biomolecules | |
| CN1549728A (en) | Immunogenic compositions comprising antigens, genetic vectors and adjuvants in biodegradable microspheres | |
| CN101489588A (en) | Bioactive purified HspE7 compositions | |
| CN112089834B (en) | Preparation and Application of Graphene Oxide-Based Poria Cocos Nano-Adjuvants and Adjuvant/Antigen Co-delivery Vaccines | |
| CN117897487A (en) | Application of artificially synthesized CpG-containing single-chain deoxyoligonucleotide in vaccine | |
| CN110042103A (en) | A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation | |
| CN1513559A (en) | A method for enhancing the cellular immune response induced by gene gun inoculation of DNA vaccine | |
| CN1324661A (en) | Human hepatitis B nucleic acid vaccine | |
| CN116200394A (en) | CpG oligodeoxynucleotide and application thereof | |
| CN116763933A (en) | mRNA vaccine delivery vector, mRNA vaccine, preparation method and application thereof | |
| CN112089833A (en) | A universal CpG ODN nanoparticle adjuvant and its preparation method and application | |
| CN1692943A (en) | Preparation and Application of CpG DNA Molecular Anti-infection Immune Preparation | |
| US10232036B2 (en) | Vaccine formulation, preparation method therefor and use thereof | |
| CN105381458A (en) | Application of cationic polymer serving as vaccine adjuvant | |
| He et al. | Cistanche deserticola polysaccharide-functionalized dendritic fibrous nano-silica as oral delivery system for H9N2 vaccine to promote systemic and mucosal immune response | |
| CN114796480B (en) | A kind of preparation method of composite nanoparticle aluminum adjuvant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |