JP2021516977A - 植物発現エンハンサ - Google Patents
植物発現エンハンサ Download PDFInfo
- Publication number
- JP2021516977A JP2021516977A JP2020547373A JP2020547373A JP2021516977A JP 2021516977 A JP2021516977 A JP 2021516977A JP 2020547373 A JP2020547373 A JP 2020547373A JP 2020547373 A JP2020547373 A JP 2020547373A JP 2021516977 A JP2021516977 A JP 2021516977A
- Authority
- JP
- Japan
- Prior art keywords
- seq
- protein
- plant
- nucleic acid
- expression
- 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
- 230000014509 gene expression Effects 0.000 title claims abstract description 306
- 239000003623 enhancer Substances 0.000 title claims abstract description 165
- 238000000034 method Methods 0.000 claims abstract description 61
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 298
- 102000004169 proteins and genes Human genes 0.000 claims description 259
- 150000007523 nucleic acids Chemical class 0.000 claims description 229
- 108020004707 nucleic acids Proteins 0.000 claims description 137
- 102000039446 nucleic acids Human genes 0.000 claims description 137
- 239000002773 nucleotide Substances 0.000 claims description 97
- 125000003729 nucleotide group Chemical group 0.000 claims description 97
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 95
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 87
- 210000004027 cell Anatomy 0.000 claims description 61
- 241001263478 Norovirus Species 0.000 claims description 55
- 206010022000 influenza Diseases 0.000 claims description 47
- 101710132601 Capsid protein Proteins 0.000 claims description 25
- 101710197658 Capsid protein VP1 Proteins 0.000 claims description 25
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 claims description 25
- 101710108545 Viral protein 1 Proteins 0.000 claims description 25
- 239000013598 vector Substances 0.000 claims description 24
- 101710154606 Hemagglutinin Proteins 0.000 claims description 10
- 101710093908 Outer capsid protein VP4 Proteins 0.000 claims description 10
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 claims description 10
- 101710176177 Protein A56 Proteins 0.000 claims description 10
- 230000001052 transient effect Effects 0.000 claims description 9
- 108010067390 Viral Proteins Proteins 0.000 claims description 8
- 239000000185 hemagglutinin Substances 0.000 claims description 8
- 241000723607 Comovirus Species 0.000 claims description 6
- 101710081079 Minor spike protein H Proteins 0.000 claims description 6
- 208000037798 influenza B Diseases 0.000 claims description 5
- 210000003743 erythrocyte Anatomy 0.000 claims description 4
- 101710186708 Agglutinin Proteins 0.000 claims 2
- 101710146024 Horcolin Proteins 0.000 claims 2
- 101710189395 Lectin Proteins 0.000 claims 2
- 101710179758 Mannose-specific lectin Proteins 0.000 claims 2
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 claims 2
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 description 262
- 235000018102 proteins Nutrition 0.000 description 240
- 241000723655 Cowpea mosaic virus Species 0.000 description 80
- 108010076504 Protein Sorting Signals Proteins 0.000 description 55
- 230000001105 regulatory effect Effects 0.000 description 47
- 108020003589 5' Untranslated Regions Proteins 0.000 description 38
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 33
- 230000001965 increasing effect Effects 0.000 description 27
- 150000001413 amino acids Chemical group 0.000 description 26
- 238000004519 manufacturing process Methods 0.000 description 26
- 229960004641 rituximab Drugs 0.000 description 22
- 239000000284 extract Substances 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 230000014616 translation Effects 0.000 description 14
- 238000001764 infiltration Methods 0.000 description 13
- 239000000419 plant extract Substances 0.000 description 13
- 230000004186 co-expression Effects 0.000 description 12
- 230000001939 inductive effect Effects 0.000 description 12
- 230000008595 infiltration Effects 0.000 description 12
- 238000013519 translation Methods 0.000 description 11
- 238000013518 transcription Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 239000000306 component Substances 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 241000208125 Nicotiana Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000013599 cloning vector Substances 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 7
- 108091026890 Coding region Proteins 0.000 description 7
- 230000001976 improved effect Effects 0.000 description 7
- 210000002729 polyribosome Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000000411 inducer Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 241000207746 Nicotiana benthamiana Species 0.000 description 5
- -1 antibodies Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 239000000287 crude extract Substances 0.000 description 5
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 5
- 229960004359 iodixanol Drugs 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 230000010474 transient expression Effects 0.000 description 5
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 4
- 241000272525 Anas platyrhynchos Species 0.000 description 4
- 241000219195 Arabidopsis thaliana Species 0.000 description 4
- 244000105624 Arachis hypogaea Species 0.000 description 4
- 208000006339 Caliciviridae Infections Diseases 0.000 description 4
- 235000019750 Crude protein Nutrition 0.000 description 4
- 108090000051 Plastocyanin Proteins 0.000 description 4
- 108091081024 Start codon Proteins 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion 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
- 230000035772 mutation Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 235000020232 peanut Nutrition 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 235000004252 protein component Nutrition 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000219194 Arabidopsis Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 240000004658 Medicago sativa Species 0.000 description 3
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 3
- 241000532184 Norovirus GII Species 0.000 description 3
- 108091005461 Nucleic proteins Proteins 0.000 description 3
- 101150084101 RNA2 gene Proteins 0.000 description 3
- 101100353432 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP2 gene Proteins 0.000 description 3
- 108091058545 Secretory proteins Proteins 0.000 description 3
- 102000040739 Secretory proteins Human genes 0.000 description 3
- 240000006394 Sorghum bicolor Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 210000003763 chloroplast Anatomy 0.000 description 3
- 238000000326 densiometry Methods 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 239000003630 growth substance Substances 0.000 description 3
- 230000035931 haemagglutination Effects 0.000 description 3
- 208000037797 influenza A Diseases 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000005026 transcription initiation Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 235000006576 Althaea officinalis Nutrition 0.000 description 2
- 101100523932 Arabidopsis thaliana RD21A gene Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 241001529459 Enterovirus A71 Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000017278 Glutaredoxin Human genes 0.000 description 2
- 108050005205 Glutaredoxin Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 108090000862 Ion Channels Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 241000208134 Nicotiana rustica Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- 101900024951 Tomato bushy stunt virus RNA silencing suppressor p19 Proteins 0.000 description 2
- 108700009124 Transcription Initiation Site Proteins 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000006837 decompression Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003262 industrial enzyme Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000008121 plant development Effects 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 101710171175 30S ribosomal protein S19 Proteins 0.000 description 1
- 101150073246 AGL1 gene Proteins 0.000 description 1
- 101710197633 Actin-1 Proteins 0.000 description 1
- 108090000104 Actin-related protein 3 Proteins 0.000 description 1
- 235000009434 Actinidia chinensis Nutrition 0.000 description 1
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- ZHQQRIUYLMXDPP-SSDOTTSWSA-N Actinidine Natural products C1=NC=C(C)C2=C1[C@H](C)CC2 ZHQQRIUYLMXDPP-SSDOTTSWSA-N 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 241001293113 Anas clypeata Species 0.000 description 1
- 101100274514 Arabidopsis thaliana CKL11 gene Proteins 0.000 description 1
- 101100071649 Arabidopsis thaliana HSPRO2 gene Proteins 0.000 description 1
- 108010027344 Basic Helix-Loop-Helix Transcription Factors Proteins 0.000 description 1
- 102000018720 Basic Helix-Loop-Helix Transcription Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 240000000385 Brassica napus var. napus Species 0.000 description 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101150064755 CKI1 gene Proteins 0.000 description 1
- OHDLZTDSTWANIC-UHFFFAOYSA-N C[I]1CCCC1 Chemical compound C[I]1CCCC1 OHDLZTDSTWANIC-UHFFFAOYSA-N 0.000 description 1
- 241000714198 Caliciviridae Species 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108010049994 Chloroplast Proteins Proteins 0.000 description 1
- 241001168968 Chroicocephalus ridibundus Species 0.000 description 1
- 241000631130 Chrysophyllum argenteum Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 101710173948 Cysteine proteinase 3 Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102100024452 DNA-directed RNA polymerase III subunit RPC1 Human genes 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 101710157092 Defensin J1-2 Proteins 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 208000010772 Dog disease Diseases 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108010019957 Escherichia coli periplasmic proteinase Proteins 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101710180399 Glycine-rich protein Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000689002 Homo sapiens DNA-directed RNA polymerase III subunit RPC1 Proteins 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 235000021506 Ipomoea Nutrition 0.000 description 1
- 241000207783 Ipomoea Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 241000272168 Laridae Species 0.000 description 1
- 241001494920 Larus argentatus Species 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 244000211187 Lepidium sativum Species 0.000 description 1
- 235000007849 Lepidium sativum Nutrition 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101710082270 Metallothionein-like protein 1 Proteins 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 241000234295 Musa Species 0.000 description 1
- 101150005851 NOS gene Proteins 0.000 description 1
- 101710202365 Napin Proteins 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 244000061322 Nicotiana alata Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710091688 Patatin Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010081996 Photosystem I Protein Complex Proteins 0.000 description 1
- 108700037249 Photosystem I reaction center subunit V Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 101710107464 Probable pyruvate, phosphate dikinase regulatory protein, chloroplastic Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 1
- 101710193696 Putative pyruvate, phosphate dikinase regulatory protein Proteins 0.000 description 1
- 235000001537 Ribes X gardonianum Nutrition 0.000 description 1
- 235000001535 Ribes X utile Nutrition 0.000 description 1
- 235000016919 Ribes petraeum Nutrition 0.000 description 1
- 244000281247 Ribes rubrum Species 0.000 description 1
- 235000002355 Ribes spicatum Nutrition 0.000 description 1
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 101100397775 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) YCK2 gene Proteins 0.000 description 1
- 244000082988 Secale cereale Species 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 235000007230 Sorghum bicolor Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 101100137946 Synechocystis sp. (strain PCC 6803 / Kazusa) psaK1 gene Proteins 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 240000006909 Tilia x europaea Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 102100033598 Triosephosphate isomerase Human genes 0.000 description 1
- 101710194411 Triosephosphate isomerase 1 Proteins 0.000 description 1
- 102100028262 U6 snRNA-associated Sm-like protein LSm4 Human genes 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 108090000350 actinidain Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000008472 epithelial growth Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 244000309711 non-enveloped viruses Species 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000009894 physiological stress Effects 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108020003519 protein disulfide isomerase Proteins 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 235000005974 protein supplement Nutrition 0.000 description 1
- 229940116540 protein supplement Drugs 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 101150071134 psaK gene Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000701 toxic element Toxicity 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000011179 visual inspection Methods 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/16011—Caliciviridae
- C12N2770/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/16011—Caliciviridae
- C12N2770/16023—Virus like particles [VLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/18011—Comoviridae
- C12N2770/18041—Use of virus, viral particle or viral elements as a vector
- C12N2770/18043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
nbMT78(配列番号1);
nbATL75(配列番号2);
nbDJ46(配列番号3);
nbCHP79(配列番号4);
nbEN42(配列番号5);
atHSP69(配列番号6);
atGRP62(配列番号7);
atPK65(配列番号8);
atRP46(配列番号9);
nb30S72(配列番号10);
nbGT61(配列番号11);
nbPV55(配列番号12);
nbPPI43(配列番号13);
nbPM64(配列番号14);
nbH2A86(配列番号15)、および
配列番号1〜15のいずれか1つに示されるヌクレオチド配列に90〜100%の配列同一性を有する核酸。ここで、発現エンハンサは、目的の核酸、例えば目的の異種核酸に機能的に連結されている場合、目的の核酸の発現をもたらす。さらに、発現エンハンサは、目的の核酸、例えば目的の異種核酸に連結されている場合、発現エンハンサに機能的に連結されていない、または例えば先行技術の発現エンハンサCPMV 160(配列番号16)に機能的に連結されている場合の目的の同じ核酸または異種核酸の発現レベルと比較した場合、目的の核酸または目的の異種核酸の発現レベルを増加させ得る。
nbMT78(配列番号1);ACACAATTTGCTTTAGTGATTAAACTTTCTTTTACAACAAATTAAAGGTCTATTATCTCCCAACAACATAAGAAAACA;
nbATL75(配列番号2);ATCTCCACCACCAAAAACCCTAATCGCCTCTCCGTTTCTTCATCAGATTCTCGGTTCTCTTCTTCTACAGCAACA;
nbDJ46(配列番号3);ACTCACCAAGAAAATAAACAAATTAAAGAATTTTAAGAAAAACAAG;
nbCHP79(配列番号4);ATTCTGCCCTCAGTTAACTAAATTATCTCTCTGATTAACAGTACTTTCTGATTTTCTGTGATTTCTACAAATCTGAGAC;
nbEN42(配列番号5);ACTTTTGTATAGCTCCATTGAAATAGAGAAAAGAAAATAGCC;
atHSP69(配列番号6);AAATTCAAAATTTAACACACAAACACAAACACACACACCAAAAAAAACACAGACCTTAAAAAAATAAAA;
atGRP62(配列番号7);ATAACAAAACAAGATTTTGAAGTAAAACATAAAAGAAAATAAACCCTAAGAATATATCGAAA;
atPK65(配列番号8);GCAAAAACAAAAATAAAAAAAACATCGCACAAGAAAATAAAAGATTTGTAGAATCAACTAAGAAA;
atRP46(配列番号9);AGAAACAAAAAGAATTAAAAAAAAAAAAAAAAAAAAGAATAAAGAA;
nb30S72(配列番号10);ATCTTTCCCTCAAAACCCTAGCCGCAGTCACTTCCGTAGGTGCTTACTTCGCTGTTAGTGCAATTCCAAACC;
nbGT61(配列番号11);ATCCAGAAGTAGGAATTCTTCAGTATAATCTAGGGTTTTTTGAAAAGCAAATTGATCGAAA;
nbPV55(配列番号12);AATTAAAGATCAATTCACTGTATCCCTCTTCTCCAAAAAAAACTCTGCTGTAGTC;
nbPPI43(配列番号13);ACAAATCGTACACAGCGAAAACCTCACTGAAATATTTAGAGAG;
nbPM64(配列番号14);AGAAAGATTTGTTTCCTCTGAAATAGTTTTACAGAGCCAGAAGAAGAAAAAGAAGAAGAGAGCA;および
nbH2A86(配列番号15);ACTCAACACTCAAATCGCAATCCAAAAGCTTCAATTTTTCCTAATACTTCTCTGTATTCAAGCTTCGTAAACTTTCATTCACATCA。
エンハンサ配列は、配列番号1〜15のいずれか1つ、または配列番号1〜15のいずれか1つに示す配列と100%、99%、98%、97%、96%、95%、90%、もしくはそれらの間の任意の量の配列同一性を示すヌクレオチド配列から選択され得、ここで、発現エンハンサは、目的の核酸に機能的に連結されている場合、目的の核酸の発現をもたらすか、または発現エンハンサに機能的に連結されていない同じ目的の核酸の発現レベル、もしくは例えば先行技術の発現エンハンサCPMV 160(配列番号16)に機能的に連結されている場合の発現レベルと比較して、目的の核酸の発現レベルを増加させる。配列番号1〜15に示すエンハンサ配列のそれぞれは、類似もしくは増加したエンハンサ活性をもたらすか、または発現エンハンサの別の有益な特性をもたらす発現エンハンサを生成するために、エンハンサ配列の1つまたは複数のヌクレオチドの欠失、挿入、および/または置換を含む、当業者に公知の方法を使用して改変され得る(例えば、参照により本明細書に組み込まれる、2013年7月15日にオンラインで公開されたDiamos et.al.,Frontiers in Plant Science.2016,vol7 pp.1−15;Dvir S.et.al.,2013,PNAS;Leppek K et.al.,2018,Nature Reviews Mol.Cell Biol.19:158−174参照)。例えば、有益な特性は、転写開始の改善、mRNA安定性の改善、mRNA翻訳の改善、またはそれらの組み合わせを含み得る。
mRNAを、標準的なフェノール−クロロホルムプロトコルを使用してさまざまなストレス下で非浸潤および浸潤バイオマスまたは細胞培養物から抽出し、スクロース勾配による標準遠心分離を使用して低および高翻訳状態のmRNAを分離した。各非ポリソームおよびポリソーム画分中のmRNAを、標準的なフェノール−クロロホルム抽出プロトコルを使用して抽出した。各ポリソームおよび非ポリソーム画分中に存在する各mRNAの5’UTRの開始配列の決定を、遺伝子発現のキャップ分析(CAGE)法を使用して実施した。望ましくない配列タグ(リボソームRNA、葉緑体RNAおよびキャップなしタグ)を除去した後、配列決定されたタグを、遺伝子同定のために参照ゲノムデータベース、例えばシロイヌナズナ(アラビドプシス・タリアナ(Arabidopsis thaliana))のTAIR(URL:arabidopsis.org/参照)またはニコチアナ・ベンサミアナ(Nicotiana benthamiana)のSolゲノムネットワーク(URL:solgenomics.net/参照)と比較した。各所与の遺伝子の配列決定されたタグの数を分析し、正規化した。翻訳状態を、ポリソーム画分で見出された正規化されたタグの数を各所与の遺伝子のタグの総数で除することにより、ポリソーム比(PR比)を確立することによって評価した。浸潤条件下で高翻訳状態の遺伝子mRNAを、潜在的な5’UTR候補を特定するために使用した。
nbMT78 5’UTRに融合したDasher蛍光タンパク質(Atum、カタログ番号FPB−27−609)をコードする配列を、以下のPCRベースの方法を使用して2X35Sプロモータ+CPMV 3’UTR/NOS発現系にクローニングした。1回目のPCRでは、Dasher蛍光タンパク質を含む断片を、Dasher遺伝子配列(配列番号20;図16B)を鋳型として用いて、プライマーnbMT78_Dasher.c(配列番号17)およびIF−Dasher(27−609).r(配列番号18)を使用して増幅した。1回目の増幅からのPCR産物(表2のF1)を鋳型として用いて、IF−nbMT78.c(配列番号19)およびIF−Dasher(27−609).r(配列番号18)をプライマーとして使用してatMT78 5’UTR配列を付加した。最終的なPCR産物(表2のF2)を、In−Fusionクローニングシステム(Clontech,Mountain View,CA)を使用して2X35Sプロモータ+CPMV 3’UTR/NOS発現系にクローニングした。構築物番号1666(図6A)をAatIIおよびStuI制限酵素で消化し、線状化したプラスミドをIn−Fusionアセンブリ反応に使用した。構築物番号1666は、2X35Sプロモータ+CPMV 3’UTR/NOSベースの発現カセット中の目的の遺伝子の「In−fusion」クローニングのためのアクセプタプラスミドである。これは、アルファルファプラストシアニン遺伝子プロモータおよびターミネータの下でサイレンシングのTBSV P19サプレッサを共発現させるための遺伝子構築物も組み込んでいる。骨格はpCAMBIAバイナリプラスミドであり、左のt−DNA境界から右のt−DNA境界までの配列は、配列番号22に示されているとおりである。得られた構築物に番号4467を与えた(配列番号75;配列番号図16E)。Dasher蛍光タンパク質のアミノ酸配列を配列番号21に示す。構築物4467の表示を図6Bに示す。
アグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)のトランスフェクション
アグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)AGL1株を、D’Aoust et al.,2008(Plant Biotech.J.6:930−40)に記載されている方法を使用して、異なる発現ベクターを用いたエレクトロポレーションによってトランスフェクト(形質転換)した。トランスフェクトしたアグロバクテリウム(Agrobacterium)を、10mM 2−(N−モルホリノ)エタンスルホン酸(MES)および50μg/mlカナマイシンpH5.6を添加したLB培地で0.6〜1.6のOD600まで増殖させ、100μlアリコートで凍結した。
N.ベンサミアナ(N.benthamiana)植物を、市販のピートモス基質を満たした苗用木箱で種子から生育させた。植物を、16/8の光周期および25℃日中/20℃夜間の温度管理計画下に温室内で生育させた。播種の3週間後、個々の小植物を摘み取り、鉢に移植し、同じ環境条件下でさらに3週間、温室内で生育させた。
インキュベーション後、植物の地上部を収穫し、−80℃で凍結し、細かく砕いた。全可溶性タンパク質を、2容量の冷50mMトリス緩衝液pH8.0+500mM NaCl、0.4μg/mlメタビスルファイトおよび1mMフェニルメタンスルホニルフルオリド中で凍結破砕した植物材料の各試料を均質化することによって(Polytron)抽出した。均質化後、スラリーを4℃で10分間、10,000gで遠心分離し、これらの清澄化した粗抽出物(上清)を分析のために保存した。
赤血球凝集反応アッセイは、Nayak and Reichl(2004,J.Virol.Methods 122:9−15)によって記載されている方法に基づいた。100μLのPBSを含むV底96ウェルマイクロタイタプレート中で試験試料(100μL)の段階2倍希釈液を作製し、ウェルあたり100μLの希釈試料にした。100μLの0.25%シチメンチョウ赤血球懸濁液(Bio Link Inc.,Syracuse,NY;すべてのB株、H1、H5およびH7の場合)または0.5%モルモット赤血球懸濁液(H3の場合)を各ウェルに添加し、プレートを室温で2時間インキュベートした。完全な赤血球凝集を示す最も高い希釈の逆数をHA活性として記録した。
リツキシマブ発現分析のために、150mM NaClを含む150mMトリス、pH7.4緩衝液での機械的抽出によって葉から粗タンパク質抽出物(2gバイオマス/EU)を生成し、完全に構築されたH2L2型の抗体に対応するバンドのゲルデンシトメトリ定量化のために、非還元条件下にSDS−PAGEで抽出物を電気泳動した。タンパク質電気泳動をBio−RadのStain−Freeゲルで行い、画像分析とインゲル定量化のためのImage Labソフトウェアを含むGel Doc XR+システムを使用して、ゲルイメージングシステムを実施した。
2容量の抽出緩衝液(100mMリン酸緩衝液pH7.2+150 mM NaCl)を含むブレンダでの機械的抽出によって、凍結バイオマスからタンパク質を抽出した。スラリーを大きな孔のナイロンフィルタで濾過して大きな破片を除去し、4℃で5分間、5000gで遠心分離した。上清を収集し、5000gで30分間(4°C)、再度遠心分離して、さらなる破片を除去した。次に上清を不連続なイオジキサノール密度勾配に充填する。分析密度勾配遠心分離を次のように行った:酢酸緩衝液中の不連続なイオジキサノール密度勾配(45%のイオジキサノール1ml、35%2ml、33%2ml、31%2ml、29%2mlおよび25%5ml)を含む38mlチューブを調製し、ウイルス様粒子を含む25mlの抽出物で覆った。勾配を175000gで4時間遠心分離した(4℃)。遠心分離後、1mlの画分を下から上に収集し、タンパク質染色またはウエスタンブロットと組み合わせたSDS−PAGEによって画分を分析した。
N.ベンサミアナ(N.benthamiana)の葉を、例2に記載されているように、目的のタンパク質の発現を可能にするために、定義された発現エンハンサに機能的に連結された目的のタンパク質をコードする発現ベクターを含むアグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)を用いて減圧浸潤し、葉を目的のタンパク質の産生について調べた。浸潤の9日後(DPI)、葉のホモジネートから全粗タンパク質抽出物を調製し、血球凝集素力価を上記のように測定した。
本明細書に記載される発現エンハンサはまた、目的の多量体タンパク質、例えば抗体の作製のために使用され得る。N.ベンサミアナ(N.benthamiana)の葉を、例2に記載されているように、目的のタンパク質の発現を可能にするために、定義された発現エンハンサに機能的に連結された目的のタンパク質をコードする発現ベクターを含むアグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)を用いて減圧浸潤し、葉を目的のタンパク質の産生について調べた。浸潤の9日後(DPI)、葉のホモジネートから全粗タンパク質抽出物を調製し、SDS−PAGEで分離し、上記のように、完全なIgGのゲルデンシトメトリによって発現レベルを決定した。
配列表2 <223>発現エンハンサnbATL75
配列表3〜4 <223>発現エンハンサ
配列表5 <223>発現エンハンサnbEN42
配列表6 <223>発現エンハンサatHSP69
配列表7 <223>発現エンハンサatGRP62
配列表8 <223>発現エンハンサatPK65
配列表9 <223>発現エンハンサatRP46
配列表10 <223>発現エンハンサnb30S72
配列表11 <223>発現エンハンサnbGT61
配列表12 <223>発現エンハンサnbPV55
配列表13 <223>発現エンハンサnbPPI43
配列表14 <223>発現エンハンサnbPM64
配列表15 <223>発現エンハンサnbH2A86
配列表16 <223>CPMV 160
配列表17 <223>プライマーnbMT78_Dasher.c
配列表18 <223>プライマーIF−Dasher(27−609).r
配列表19 <223>プライマーIF−nbMT78.c
配列表20 <223>プライマーCPMV 160 5’UTR−Dasherの核酸配列
配列表21 <223>Dasherプライマーのアミノ酸配列
配列表22 <223>T−DNAの左境界から右境界までのクローニングベクター(Dasher用)1666
配列表23 <223>プライマーIF−(2X35S+C)_CPMV160.c
配列表24 <223>プライマーnbGT61_Dasher.c
配列表25 <223>プライマーIF−nbGT61.c
配列表26 <223>プライマーnbATL75_Dasher.c
配列表27 <223>プライマーIF−nbATL75.c
配列表28 <223>プライマーnbDJ46_Dasher.c
配列表29 <223>プライマーIF−nbDJ46.c
配列表30 <223>プライマーnbCHP79_Dasher.c
配列表31 <223>プライマーIF−nbCHP79.c
配列表32 <223>プライマーnbEN42_Dasher.c
配列表33 <223>プライマーIF−nbEN42.c
配列表34 <223>プライマーnb30S72_Dasher.c
配列表35 <223>プライマーIF−nb30S72.c
配列表36 <223>プライマーnbPV55_Dasher.c
配列表37 <223>プライマーIF−nbPV55.c
配列表38 <223>プライマーnbPPI43_Dasher.c
配列表39 <223>プライマーIF−nbPPI43.c
配列表40 <223>プライマーnbPM64_Dasher.c
配列表41 <223>プライマーIF−nbPM64.c
配列表42 <223>プライマーnbH2A86_Dasher.c
配列表43 <223>プライマーIF−nbH2A86.c
配列表44 <223>プライマーatHSP69_Dasher.c
配列表45 <223>プライマーIF−atHSP69.c
配列表46 <223>プライマーatGRP62_Dasher.c
配列表47 <223>プライマーIF−atGRP62.c
配列表48 <223>プライマーatPK65_Dasher.c
配列表49 <223>プライマーIF−atPK65.c
配列表50 <223>プライマーIF−atRP46_Dasher.c
配列表51 <223>プライマーIF−H1cTMCT.s1−4r
配列表52 <223>プライマーnbGT61_SpPDI.c
配列表53 <223>プライマーnbATL75_SpPDI.c
配列表54 <223>プライマーnbDJ46_SpPDI.c
配列表55 <223>プライマーnbCHP79_SpPDI.c
配列表56 <223>プライマーnbEN42_SpPDI.c
配列表57 <223>プライマーnb30S72_SpPDI.c
配列表58 <223>プライマーnbMT78_SpPDI.c
配列表59 <223>プライマーnbPV55_SpPDI.c
配列表60 <223>プライマーnbPPI43_SpPDI.c
配列表61 <223>プライマーnbPM64_SpPDI.c
配列表62 <223>プライマーnbH2A86_SpPDI.c
配列表63 <223>プライマーatHSP69_SpPDI.c
配列表64 <223>プライマーatGRP62_SpPDI.c
配列表65 <223>atPK65_SpPDI.c
配列表66 <223>プライマーIF−atRP46_SpPDI.c
配列表67 <223>プライマーIF−H3_Swi_13.r
配列表68 <223>プライマーIF−GII4Syd12VP1.r
配列表69 <223>プライマーnbATL75+GII4Syd12.c
配列表70 <223>プライマーnbCHP79+GII4Syd12.c
配列表71 <223>プライマーnbMT78+GII4Syd12.c
配列表72 <223>プライマーatHSP69+GII4Syd12.c
配列表73 <223>プライマーIF−HC(Ritux).s1−6r
配列表74 <223>IF−LC(Ritux).s1−6r
配列表75 <223>2X35SプロモータからNOSターミネータまでの構築物4467(Dasher)
配列表76 <223>T−DNAの左境界から右境界までのクローニングベクター(H3およびHA B用)4160
配列表77 <223>T−DNAの左境界から右境界までのクローニングベクター(ノロウイルスVP1用)4170
配列表78 <223>Dasherプライマーの核酸配列
配列表79 <223>プライマーCPMV 160 5’UTR−PDI+H1 Calの核酸配列
配列表80 <223>PDI+H1 Calの核酸配列
配列表81 <223>PDI+H1 Calのアミノ酸配列
配列表82 <223>CPMV 160 5’UTR−PDI+H1 Michの核酸配列
配列表83 <223>PDI+H1 Michの核酸配列
配列表84 <223>PDI+H1 Michのアミノ酸配列
配列表85 <223>CPMV 160 5’UTR−PDI+H3 HKの核酸配列
配列表86 <223>PDI+H3 HKの核酸配列
配列表87 <223>PDI+H3 HKのアミノ酸配列
配列表88 <223>CPMV 160 5’UTR−PDI+HA B Briの核酸配列
配列表89 <223>PDI+HA B Briの核酸配列
配列表90 <223>PDI+HA B Briのアミノ酸配列
配列表91 <223>CPMV 160 5’UTR−PDI+HA B Phuの核酸配列
配列表92 <223>PDI+HA B Phuの核酸配列
配列表93 <223>PDI+HA B Phuのアミノ酸配列
配列表94 <223>プライマーCPMV 160 5’UTR−VP1(GII.4)の核酸配列
配列表95 <223>VP1(GII.4)の核酸配列
配列表96 <223>VP1(GII.4)のアミノ酸配列
配列表97 <223>CPMV 160 5’UTR−PDI+リツキシマブHCの核酸配列
配列表98 <223>PDI+リツキシマブHCの核酸配列
配列表99 <223>プライマーPDI+リツキシマブHCのアミノ酸配列
配列表100 <223>CPMV 160 5’UTR−PDI+リツキシマブLCの核酸配列
配列表101 <223>PDI+リツキシマブLCの核酸配列
配列表102 <223>PDI+リツキシマブLCのアミノ酸配列
Claims (19)
- 植物、植物の一部または植物細胞において活性な単離された発現エンハンサであって、
nbMT78(配列番号1);
nbATL75(配列番号2);
nbDJ46(配列番号3);
nbCHP79(配列番号4);
nbEN42(配列番号5);
atHSP69(配列番号6);
atGRP62(配列番号7);
atPK65(配列番号8);
atRP46(配列番号9);
nb30S72(配列番号10);
nbGT61(配列番号11);
nbPV55(配列番号12);
nbPPI43(配列番号13);
nbPM64(配列番号14);
nbH2A86(配列番号15)、および
配列番号1〜15のいずれか1つに示されるヌクレオチド配列に90〜100%の配列同一性を有する核酸
からなる群より選択され、
目的の核酸に機能的に連結されている場合、目的の核酸の発現をもたらす、上記単離された発現エンハンサ。 - 目的のタンパク質をコードする異種ヌクレオチド配列と機能的に連結された、請求項1に記載の単離された発現エンハンサを含む核酸配列。
- 異種ヌクレオチド配列がウイルスタンパク質または抗体をコードする、請求項2に記載の核酸配列。
- ウイルスタンパク質がインフルエンザタンパク質またはノロウイルスタンパク質である、請求項3に記載の核酸。
- インフルエンザタンパク質が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、およびインフルエンザB型赤血球凝集素の群から選択される赤血球凝集素タンパク質である、請求項4に記載の核酸。
- ノロウイルスタンパク質が、GI.1、GI.2、GI.3、GI.5、GI.7、GII.1、GII.2、GII.3、GII.4、GII.5、GII.6、GII.7、GII.12、GII.13、GII.14、GII.17およびGII.21の群から選択されるVP1、VP2、またはそれらの組み合わせである、請求項4に記載の核酸。
- 請求項2に記載の核酸配列の1つまたは複数を含む植物発現系。
- 請求項3に記載の核酸配列の1つまたは複数を含む植物発現系。
- 請求項4に記載の核酸配列の1つまたは複数を含む植物発現系。
- 請求項5に記載の核酸配列の1つまたは複数を含む植物発現系。
- 請求項6に記載の核酸配列の1つまたは複数を含む植物発現系。
- コモウイルス3’UTRをさらに含む、請求項7に記載の植物発現系。
- 植物、植物の一部、または植物細胞において目的のタンパク質を生産する方法であって、
植物、植物の一部、または植物細胞に、安定なまたは一過性の方法で、核酸配列の1つまたは複数を含む請求項8に記載のベクターを導入すること、および、
目的のタンパク質をコードする異種ヌクレオチド配列のそれぞれの発現を可能にする条件下で植物または植物の一部をインキュベートすること
を含む上記方法。 - 目的のタンパク質がウイルスタンパク質である、請求項13に記載の方法。
- ウイルスタンパク質がインフルエンザタンパク質またはノロウイルスタンパク質である、請求項14に記載の方法。
- インフルエンザタンパク質が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、およびインフルエンザB型赤血球凝集素の群から選択される赤血球凝集素タンパク質である、請求項15に記載の方法。
- ノロウイルスタンパク質が、GI.1、GI.2、GI.3、GI.5、GI.7、GII.1、GII.2、GII.3、GII.4、GII.5、GII.6、GII.7、GII.12、GII.13、GII.14、GII.17およびGII.21の群から選択されるVP1、VP2、またはそれらの組み合わせである、請求項15に記載の方法。
- 目的のタンパク質が多量体タンパク質であり、および導入工程が、それぞれが多量体タンパク質の成分をコードする2つまたは2つを超える核酸配列を共発現させることを含む、請求項13に記載の方法。
- 請求項8に記載のベクターで一過性に形質転換または安定に形質転換された植物、植物の一部、または植物細胞。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862643053P | 2018-03-14 | 2018-03-14 | |
US62/643,053 | 2018-03-14 | ||
PCT/CA2019/050317 WO2019173924A1 (en) | 2018-03-14 | 2019-03-14 | Plant expression enhancer |
Publications (3)
Publication Number | Publication Date |
---|---|
JP2021516977A true JP2021516977A (ja) | 2021-07-15 |
JP7423875B2 JP7423875B2 (ja) | 2024-01-30 |
JP7423875B6 JP7423875B6 (ja) | 2024-03-15 |
Family
ID=67908652
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2020547373A Active JP7423875B6 (ja) | 2018-03-14 | 2019-03-14 | 植物発現エンハンサ |
Country Status (10)
Country | Link |
---|---|
US (1) | US20210246462A1 (ja) |
EP (1) | EP3765618A4 (ja) |
JP (1) | JP7423875B6 (ja) |
CN (1) | CN112400021A (ja) |
AU (1) | AU2019233860A1 (ja) |
BR (1) | BR112020018639A2 (ja) |
CA (1) | CA3093592A1 (ja) |
MX (1) | MX2020009495A (ja) |
RU (1) | RU2020132255A (ja) |
WO (1) | WO2019173924A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021222832A1 (en) * | 2020-05-01 | 2021-11-04 | Meso Scale Technologies, Llc. | Viral serology ace2 competition assays |
WO2024014976A1 (ru) * | 2022-07-13 | 2024-01-18 | Институт Полиомиелита, Федеральный Научный Центр Исследований И Разработки Иммунобиологических Препаратов Им. М.П. Чумакова Российской Академии Наук, Фгану | Инфильтрация целевых компонентов в ткани вегетирующего растения |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016175132A1 (ja) * | 2015-04-30 | 2016-11-03 | 国立大学法人 奈良先端科学技術大学院大学 | 植物において組み換えタンパク質の高発現を可能にする5'utrをコードするdna分子 |
JP2017501738A (ja) * | 2014-01-10 | 2017-01-19 | メディカゴ インコーポレイテッド | Cpmvエンハンサーエレメント |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2614527T3 (es) * | 2010-11-04 | 2017-05-31 | Medicago Inc. | Sistema de expresión en plantas |
TWI526539B (zh) * | 2010-12-22 | 2016-03-21 | 苜蓿股份有限公司 | 植物中生產類病毒顆粒(vlp)的方法及以該方法生產之vlp |
GB201107468D0 (en) * | 2011-05-04 | 2011-06-15 | Plant Bioscience Ltd | Protein expression systems |
NZ622731A (en) * | 2011-09-30 | 2016-04-29 | Medicago Inc | Increasing virus-like particle yield in plants |
SG11201507928QA (en) * | 2013-03-28 | 2015-10-29 | Medicago Inc | Influenza virus-like particle production in plants |
EP3122883B1 (en) * | 2014-03-27 | 2022-03-02 | Medicago Inc. | Modified cpmv enhancer elements |
US20210393765A1 (en) * | 2019-02-19 | 2021-12-23 | Medicago Inc. | Rotavirus vp7 fusion proteins and rotavirus-like particles comprising them |
-
2019
- 2019-03-14 AU AU2019233860A patent/AU2019233860A1/en active Pending
- 2019-03-14 CA CA3093592A patent/CA3093592A1/en active Pending
- 2019-03-14 MX MX2020009495A patent/MX2020009495A/es unknown
- 2019-03-14 WO PCT/CA2019/050317 patent/WO2019173924A1/en active Search and Examination
- 2019-03-14 BR BR112020018639-2A patent/BR112020018639A2/pt unknown
- 2019-03-14 EP EP19767538.2A patent/EP3765618A4/en active Pending
- 2019-03-14 US US16/980,266 patent/US20210246462A1/en active Pending
- 2019-03-14 CN CN201980032546.6A patent/CN112400021A/zh active Pending
- 2019-03-14 JP JP2020547373A patent/JP7423875B6/ja active Active
- 2019-03-14 RU RU2020132255A patent/RU2020132255A/ru unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017501738A (ja) * | 2014-01-10 | 2017-01-19 | メディカゴ インコーポレイテッド | Cpmvエンハンサーエレメント |
WO2016175132A1 (ja) * | 2015-04-30 | 2016-11-03 | 国立大学法人 奈良先端科学技術大学院大学 | 植物において組み換えタンパク質の高発現を可能にする5'utrをコードするdna分子 |
Also Published As
Publication number | Publication date |
---|---|
RU2020132255A (ru) | 2022-04-15 |
MX2020009495A (es) | 2020-12-07 |
CA3093592A1 (en) | 2019-09-19 |
BR112020018639A2 (pt) | 2020-12-29 |
AU2019233860A1 (en) | 2020-10-15 |
EP3765618A4 (en) | 2022-01-05 |
CN112400021A (zh) | 2021-02-23 |
US20210246462A1 (en) | 2021-08-12 |
JP7423875B6 (ja) | 2024-03-15 |
JP7423875B2 (ja) | 2024-01-30 |
WO2019173924A1 (en) | 2019-09-19 |
EP3765618A1 (en) | 2021-01-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2011325827B2 (en) | Plant expression system | |
US11884929B2 (en) | CPMV enhancer elements | |
JP6599354B2 (ja) | 改変されたcpmvエンハンサーエレメント | |
JP7423875B6 (ja) | 植物発現エンハンサ | |
JP7512546B2 (ja) | 内因性植物発現エンハンサー | |
RU2809237C2 (ru) | Мутанты гемагглютинина вируса гриппа |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A524 | Written submission of copy of amendment under article 19 pct |
Free format text: JAPANESE INTERMEDIATE CODE: A525 Effective date: 20201027 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220303 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20230124 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230131 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230420 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230623 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230911 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231020 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20231116 |
|
A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20231205 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20231214 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20231205 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7423875 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
S631 | Written request for registration of reclamation of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313631 |