EP0910650A1 - Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtained - Google Patents
Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtainedInfo
- Publication number
- EP0910650A1 EP0910650A1 EP97924066A EP97924066A EP0910650A1 EP 0910650 A1 EP0910650 A1 EP 0910650A1 EP 97924066 A EP97924066 A EP 97924066A EP 97924066 A EP97924066 A EP 97924066A EP 0910650 A1 EP0910650 A1 EP 0910650A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- glycoprotein
- virus
- plant
- rabies virus
- protein
- 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.)
- Withdrawn
Links
- 108010046722 Thrombospondin 1 Proteins 0.000 title claims abstract description 67
- 102100036034 Thrombospondin-1 Human genes 0.000 title claims abstract description 67
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 27
- 108090000288 Glycoproteins Proteins 0.000 title claims description 111
- 102000003886 Glycoproteins Human genes 0.000 title claims description 109
- 206010037742 Rabies Diseases 0.000 title claims description 54
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 109
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 83
- 241000711798 Rabies lyssavirus Species 0.000 claims abstract description 55
- 241000700605 Viruses Species 0.000 claims abstract description 48
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 36
- 108091026890 Coding region Proteins 0.000 claims abstract description 31
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 27
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 27
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 26
- 101710120037 Toxin CcdB Proteins 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 108010067390 Viral Proteins Proteins 0.000 claims abstract description 5
- 230000008929 regeneration Effects 0.000 claims abstract description 5
- 238000011069 regeneration method Methods 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 91
- 238000000034 method Methods 0.000 claims description 84
- 239000013598 vector Substances 0.000 claims description 32
- 230000001105 regulatory effect Effects 0.000 claims description 16
- 239000000284 extract Substances 0.000 claims description 13
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 150000004676 glycans Chemical class 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 238000013518 transcription Methods 0.000 claims description 11
- 230000035897 transcription Effects 0.000 claims description 11
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 10
- 229960005486 vaccine Drugs 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 239000003599 detergent Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 7
- 230000001681 protective effect Effects 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 230000000977 initiatory effect Effects 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- 230000003612 virological effect Effects 0.000 claims description 4
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 3
- 230000005030 transcription termination Effects 0.000 claims description 3
- 101900083372 Rabies virus Glycoprotein Proteins 0.000 claims description 2
- 230000005026 transcription initiation Effects 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 claims 3
- 108091006027 G proteins Proteins 0.000 claims 1
- 102000030782 GTP binding Human genes 0.000 claims 1
- 108091000058 GTP-Binding Proteins 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 239000002671 adjuvant Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 239000013612 plasmid Substances 0.000 description 143
- 241000196324 Embryophyta Species 0.000 description 78
- 239000012634 fragment Substances 0.000 description 76
- 102000012410 DNA Ligases Human genes 0.000 description 52
- 108010061982 DNA Ligases Proteins 0.000 description 52
- 210000004027 cell Anatomy 0.000 description 52
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 44
- 230000006862 enzymatic digestion Effects 0.000 description 38
- 239000000872 buffer Substances 0.000 description 35
- 239000002609 medium Substances 0.000 description 29
- 244000061176 Nicotiana tabacum Species 0.000 description 28
- 238000001962 electrophoresis Methods 0.000 description 28
- 241000894006 Bacteria Species 0.000 description 27
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 27
- 240000008042 Zea mays Species 0.000 description 26
- 108091008146 restriction endonucleases Proteins 0.000 description 26
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 25
- 230000009089 cytolysis Effects 0.000 description 25
- 239000011543 agarose gel Substances 0.000 description 24
- 239000012429 reaction media Substances 0.000 description 24
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 23
- 150000001413 amino acids Chemical class 0.000 description 21
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 20
- 238000012163 sequencing technique Methods 0.000 description 20
- 230000009466 transformation Effects 0.000 description 20
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 19
- 235000005822 corn Nutrition 0.000 description 19
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 17
- 238000012408 PCR amplification Methods 0.000 description 17
- 229960000723 ampicillin Drugs 0.000 description 16
- 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 16
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 15
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 15
- 206010020649 Hyperkeratosis Diseases 0.000 description 15
- 230000001476 alcoholic effect Effects 0.000 description 15
- 238000001556 precipitation Methods 0.000 description 15
- 238000010367 cloning Methods 0.000 description 14
- 108020004705 Codon Proteins 0.000 description 12
- 244000309466 calf Species 0.000 description 12
- 238000010276 construction Methods 0.000 description 12
- 239000000499 gel Substances 0.000 description 12
- 230000002068 genetic effect Effects 0.000 description 12
- 240000002791 Brassica napus Species 0.000 description 11
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 11
- 230000013595 glycosylation Effects 0.000 description 11
- 238000006206 glycosylation reaction Methods 0.000 description 11
- 210000000936 intestine Anatomy 0.000 description 11
- 239000001632 sodium acetate Substances 0.000 description 11
- 235000017281 sodium acetate Nutrition 0.000 description 11
- 239000004353 Polyethylene glycol 8000 Substances 0.000 description 10
- 239000004098 Tetracycline Substances 0.000 description 10
- 230000009471 action Effects 0.000 description 10
- 229940085678 polyethylene glycol 8000 Drugs 0.000 description 10
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 description 10
- 230000000717 retained effect Effects 0.000 description 10
- 229960002180 tetracycline Drugs 0.000 description 10
- 229930101283 tetracycline Natural products 0.000 description 10
- 235000019364 tetracycline Nutrition 0.000 description 10
- 150000003522 tetracyclines Chemical class 0.000 description 10
- 108091092724 Noncoding DNA Proteins 0.000 description 9
- 239000005546 dideoxynucleotide Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 8
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 8
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 7
- 102000053602 DNA Human genes 0.000 description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000701489 Cauliflower mosaic virus Species 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 235000009973 maize Nutrition 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 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 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 5
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 108010058731 nopaline synthase Proteins 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- HZZVJAQRINQKSD-UHFFFAOYSA-N Clavulanic acid Natural products OC(=O)C1C(=CCO)OC2CC(=O)N21 HZZVJAQRINQKSD-UHFFFAOYSA-N 0.000 description 4
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 4
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 4
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 4
- 108700005077 Viral Genes Proteins 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 101150055766 cat gene Proteins 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 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 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 210000004779 membrane envelope Anatomy 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 235000013311 vegetables Nutrition 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 3
- 241000219195 Arabidopsis thaliana Species 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- 235000011293 Brassica napus Nutrition 0.000 description 3
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 3
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 3
- LMKYZBGVKHTLTN-NKWVEPMBSA-N D-nopaline Chemical compound NC(=N)NCCC[C@@H](C(O)=O)N[C@@H](C(O)=O)CCC(O)=O LMKYZBGVKHTLTN-NKWVEPMBSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 229920001503 Glucan Polymers 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 3
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 229940098164 augmentin Drugs 0.000 description 3
- 108010021384 barley lectin Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 235000021186 dishes Nutrition 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 210000003934 vacuole Anatomy 0.000 description 3
- RVLOMLVNNBWRSR-KNIFDHDWSA-N (2s)-2-aminopropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound C[C@H](N)C(O)=O.NCCCC[C@H](N)C(O)=O RVLOMLVNNBWRSR-KNIFDHDWSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- UQBIAGWOJDEOMN-UHFFFAOYSA-N 2-O-(2-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranosyl)-D-mannopyranose Natural products OC1C(O)C(CO)OC(O)C1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(O)C(O)C(CO)O1 UQBIAGWOJDEOMN-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 240000001829 Catharanthus roseus Species 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 101710190853 Cruciferin Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 2
- 241000702463 Geminiviridae Species 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000725171 Mokola lyssavirus Species 0.000 description 2
- 230000004988 N-glycosylation Effects 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 244000088415 Raphanus sativus Species 0.000 description 2
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 102100021696 Syncytin-1 Human genes 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 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 2
- 239000008272 agar Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 239000012869 germination medium Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- 108010083942 mannopine synthase Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 108700003321 plant extensin Proteins 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000004382 potting Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 125000005629 sialic acid group Chemical group 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108700037654 Acyl carrier protein (ACP) Proteins 0.000 description 1
- 102000048456 Acyl carrier protein (ACP) Human genes 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 101710123228 Alkaline phosphatase D Proteins 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 1
- 235000007575 Calluna vulgaris Nutrition 0.000 description 1
- 241000871189 Chenopodiaceae Species 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 241000219130 Cucurbita pepo subsp. pepo Species 0.000 description 1
- 235000003954 Cucurbita pepo var melopepo Nutrition 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 241001520695 Duvenhage lyssavirus Species 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150082239 G gene Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 101710138460 Leaf protein Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 241000711828 Lyssavirus Species 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 101710175243 Major antigen Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 101000905770 Mokola virus Glycoprotein Proteins 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- 102100023315 N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase Human genes 0.000 description 1
- 108010056664 N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 101150005851 NOS gene Proteins 0.000 description 1
- 101710202365 Napin Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000026678 Paget disease of bone 6 Diseases 0.000 description 1
- 101710096342 Pathogenesis-related protein Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 1
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004264 Petrolatum Substances 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 108010047620 Phytohemagglutinins Proteins 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920005830 Polyurethane Foam Polymers 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241000711841 Rabies virus ERA Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000711931 Rhabdoviridae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000008406 SarachaNachtschatten Nutrition 0.000 description 1
- 241001515849 Satellite Viruses Species 0.000 description 1
- SBMNPABNWKXNBJ-BQBZGAKWSA-N Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CO SBMNPABNWKXNBJ-BQBZGAKWSA-N 0.000 description 1
- 235000004790 Solanum aculeatissimum Nutrition 0.000 description 1
- 235000008424 Solanum demissum Nutrition 0.000 description 1
- 235000018253 Solanum ferox Nutrition 0.000 description 1
- 235000000208 Solanum incanum Nutrition 0.000 description 1
- 235000013131 Solanum macrocarpon Nutrition 0.000 description 1
- 235000009869 Solanum phureja Nutrition 0.000 description 1
- 240000002307 Solanum ptychanthum Species 0.000 description 1
- 235000000341 Solanum ptychanthum Nutrition 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000017622 Solanum xanthocarpum Nutrition 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- ODMZDQKUHYGKKN-UHFFFAOYSA-N TCA C Natural products CC(CCCC(C)C1=CCC2(C)OC3=C(CC12)C(=O)C(O)CC3)C(=O)O ODMZDQKUHYGKKN-UHFFFAOYSA-N 0.000 description 1
- 235000019714 Triticale Nutrition 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- SHZGCJCMOBCMKK-SXUWKVJYSA-N alpha-L-fucose Chemical group C[C@@H]1O[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-SXUWKVJYSA-N 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin 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=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- LHJQIRIGXXHNLA-UHFFFAOYSA-N calcium peroxide Chemical compound [Ca+2].[O-][O-] LHJQIRIGXXHNLA-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 229960003324 clavulanic acid Drugs 0.000 description 1
- HZZVJAQRINQKSD-PBFISZAISA-N clavulanic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 HZZVJAQRINQKSD-PBFISZAISA-N 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008073 immune recognition Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 239000007003 mineral medium Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229940066842 petrolatum Drugs 0.000 description 1
- 235000019271 petrolatum Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000001885 phytohemagglutinin Effects 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011496 polyurethane foam Substances 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 229960003127 rabies vaccine Drugs 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 101150116497 sacm1l gene Proteins 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 241000228158 x Triticosecale Species 0.000 description 1
- -1 β-octyl Chemical group 0.000 description 1
Classifications
-
- 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)
- C12N15/8241—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a process for the production, by plant cells, of rabies glycoprotein G, or of a virus related to rabies.
- the invention also relates to the glycoprotein thus obtained and to its use as a medicament, in particular as a human or veterinary vaccine.
- the invention further relates to transformed plant cells, and transgenic plants, capable of producing the glycoprotein G of rabies.
- the rabies virus is part of the family Rhabdoviridae. genus Lyssavirus. It is an RNA virus- whose genome contains approximately 12,000 nucleotides, coding for five structural proteins: the nucleocapsid protein "N", the matrix proteins “Mi” and “M2” / glycoprotein from envelope "G” and the protein "L” encoding the viral polymerase.
- the viral envelope consists of a lipid bilayer containing spicules composed of a polymeric form of glycoprotein G. It is expressed in the form of a precursor having 524 amino acids, of which 19 constitute the N-terminal signal sequence hydrophobic. The N-terminal signal is cleaved to give rise to the mature glycoprotein of 505 amino acids.
- the molecular weight of mature monomeric rabies glycoprotein is 66 kDa.
- this form of glycoprotein is the so-called “insoluble” form, that is to say that it comprises a C-terminal transmembrane domain allowing the glycoprotein to bind in the viral envelope.
- the “soluble” form of glycoprotein has a molecular weight of around 60 kDa and is distinguished from the insoluble form by the absence of about 58 C-terminal amino acids. This form of glycoprotein, being devoid of transmembrane segment, can no longer associate with the viral envelope.
- Glycoprotein is the major antigen of the virus, responsible for the induction of neutralizing antibodies and protective immunity. Only the "insoluble" form is protective, although there are no antigenic determinants in the C-terminal (Dietzschold B. et al., 1983).
- rabies glycoprotein G has been widely studied and used in the development of subunit vaccines.
- the cDNAs of different strains of the rabies virus have been cloned (Anilionis A. et al., 1981; Yelverton et al., 1983) and expressed in different cellular hosts.
- Yelverton et al. (1983) described the expression of the rabies glycoprotein gene (CVS strain) in E. coli.
- the antigenic activity of the expression product is only 2 to 3% compared to the natural glycoprotein.
- Kieny et al. (1984) described the expression of glycoprotein G in cells of higher eukaryotes, infected with a vaccinia virus, recombinant and replicative.
- rabies glycoprotein (CVS strain) having a molecular weight of 63 and 65 kDa (Préhaud et al., 1989), and that of the Mokola virus, virus related to the rabies virus (Tordo N. and al., 1993), was obtained in insect cells infected with a recombinant baculovirus vector.
- the technical problem which the present invention proposes to solve is to produce, in a plant cell, the glycoprotein of the rabies virus, or of a virus related to the rabies virus, conforming to the “insoluble” form of the glycoprotein. natural, especially with regard to its molecular weight.
- the present inventors that the replacement of the endogenous N-terminal signal peptide of rabies glycoprotein, by a heterologous N-terminal signal peptide, makes it possible to obtain glycoproteins having a higher molecular weight than those obtained in plants before.
- the present inventors have therefore used, for the transformation of the plant cell, a chimeric coding sequence comprising the coding sequence of the rabies glycoprotein gene, in which the sequence of the natural N-terminal signal peptide has been replaced by a sequence coding for a heterologous N-terminal signal peptide.
- the present invention relates to a process for the production of the rabies virus glycoprotein G, or of a virus related to the rabies virus, characterized by: i) the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence, comprising on the one hand a sequence coding for said mature viral protein G, or for an analogous protein, and on the other hand, a sequence coding for an other N-terminal signal peptide as that naturally associated with the viral protein G, the introduction of the chimeric coding sequence allowing the expression in the cell of the protein G; ii) the multiplication, in the form of cell culture, of the transformed cells, or the regeneration of whole transgenic or chimeric plants from these cells; iii) optionally the extraction and purification of the glycoprotein G from the cells or plant tissues thus obtained.
- rabies virus or virus related to the rabies virus
- serotype i corresponds to the rabies virus itself and includes different strains of the rabies virus, for example CVS, HEP, ERA, PM. There is normally at least 90% homology in the amino acid sequence between the different strains of rabies.
- the other serotypes correspond to viruses related to the rabies virus (“rabies - related viruses”) and are exemplified by the “Lagos bat” virus (serotype 2); Mokola virus (serotype 3); Duvenhage virus (serotype 4). All rabies viruses are pathogenic to mammals, including humans, and give rise to rabies encephalitis.
- glycoprotein G means the glycoprotein constituting the spicules of the viral envelope. According to the invention, it is normally in monomeric form but can also exist in polymeric form, for example in homodimer. According to some authors, glycoprotein G is also called the “surface antigen” of the virus, as well as “haemagglutinin”, because of its ability to cause the agglutination of erythrocytes.
- the first step of the invention consists in the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence.
- a “chimeric coding sequence” means a nucleic acid sequence, coding for an amino acid chain, consisting of peptide fragments of different origins. In this case, it is a sequence comprising, on the one hand, a sequence encoding the mature viral glycoprotein G, that is to say devoid of the N-terminal signal naturally associated with the protein, and d 'somewhere else, a sequence coding for a heterologous N-terminal signal peptide to glycoprotein.
- sequence coding for mature glycoprotein G mention may be made of that of rabies glycoprotein, CVS strain, the nucleic acid and amino acid sequence of which have been described by Yelverton et al. (1983), or that of the ERA strain, the sequence of which has been described by Anilionis et al. (1981), and modified by Kieny et al. (1984).
- the mature protein lacks the 19 N-terminal amino acid sequence.
- Other sequences are described by N. Tordo et al., Virol., 1993, 194, 59-69.
- the nucleic acid coding for mature glycoprotein G is obtained by eliminating the part of the sequence which codes for the N-terminal signal peptide.
- the coding sequence can code for a protein “analogous” to glycoprotein G.
- Analogous proteins are proteins which normally have at least 90% homology with natural glycoprotein, and which also have functional and immunological homology with glycoprotein natural.
- sequence coding for the endogenous N-terminal signal peptide is therefore replaced by a heterologous signal.
- a sequence coding for a heterologous N-terminal signal peptide mention may be made of a plant signal peptide (or “pre-peptide”), or originating from a plant virus, or from a microorganism, by exempts yeast.
- N-terminal signal peptide is meant the peptide responsible for addressing the nascent protein in the endoplasmic reticulum.
- the addressing of proteins is based on the same principle as in animal cells. From chromosomal DNA, the gene is transcribed into messenger RNA, then translated into protein at the ribosome level. If the nascent protein has an N-terminal signal peptide or prepeptide, it enters the endoplasmic reticulum where a number of post-translational maturations take place, in particular the cleavage of the signal peptide, the N-glycosylations leading to polymannosidic glycans, and the formation of disulfide bridges.
- the sequence coding for the prepeptide responsible for addressing the protein in the endoplasmic reticulum, is part of the chimeric coding sequence. It is normally a hydrophobic N-terminal signal peptide having between 10 and 40 amino acids and being of animal or vegetable origin. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, plant extensin (pEXT), ⁇ -mating (factor, proteins plants involved in defense against microorganisms (PRla and PRS, "pathogenesis related proteins").
- the introduction into the plant cell of the nucleic acid molecule, comprising the chimeric coding sequence, is carried out so that expression of the sequence can be obtained.
- the chimeric coding sequence must be found, in the genome of the plant, under the control of transcriptional regulatory sequences recognized by the plant cell.
- regulatory sequences in particular a transcription initiation signal (promoter) and a transcription termination signal (comprising the polyadenylation signal), may be endogenous to the plant, in which case, the insertion of the chimeric coding sequence must be done by homologous recombination.
- the transforming nucleic acid comprises, on each end, a sequence homologous to the sequences which adjoin the desired site of insertion into the genome.
- the transcription regulatory sequences can be included in the nucleic acid molecule comprising the chimeric coding sequence.
- the nucleic acid molecule constitutes a chimeric gene which is also part of the invention.
- the regulatory sequences include one or more promoters of plant origin or from Agrobacterium tumefaciens or from a plant virus. It can be a constitutive promoter, for example 35S or double 35S of CaMV, NOS, OCS, or specific promoters of certain tissues such as grain, or specific of certain phases of plant development.
- promoters of the napin gene and of the acyl carrier protein (ACP) EP-A-0255378, as well as the promoters of the AT2S genes of Arabidopsis thaliana, ie ie the promoters PAT2S1, PAT2S2, PAT2S3 and PAT2S4 (Krebbers et al., Plant Physiol., 1988, vol. 87, pages 859-866).
- the cruciferin or phaseoline promoter the pGEA1 and pGEA6 promoters Arabidopsis promoters of the "EM, Early Methionine labeled protein" type gene which are highly expressed during the desiccation phases of the seed.
- the termination regulatory sequences are of plant, viral, or bacterial origin, for example 35S, NOS, etc.
- the chimeric coding sequence comprises, in addition to the N-terminal signal peptide part and the part coding for the mature envelope protein, other addressing signals, for example an endoplasmic retention signal or a vacuolar addressing signal.
- the endoplasmic retention signal consists of the peptides KDEL, SEKDEL or HKDEL. These signals are normally found at the C-terminus of the protein and remain on the mature protein. The presence of this signal on the proteins of the invention is advantageous for several reasons: on the one hand, the retention of the protein in the endoplasmic reticulum tends to increase the yields of recombinant proteins. On the other hand, the maturation of polymannosic glycosylation into complex glycans does not take place; the protein therefore retains polymannosic glycosylation, minimizing the risk of undesirable immunological reactions when the protein is administered to humans as a medicine.
- the glycoprotein therefore comprises the amino acid sequence of the protein G, a signal of the KDEL type, at least one glycan of the polymannosic type, and is free of glycans of the complex type.
- this type of protein can also be obtained by using plant mutants incapable of manufacturing N-acetyl glucosaminyl transferase (von Schaewen et al., Plant Physiol. 1993 102: 1109-1118), and therefore incapable of produce complex glycans.
- the protein of the invention can, in addition to the prepeptide, also include a vacuolar addressing signal or "propeptide".
- a vacuolar addressing signal or "propeptide” In the presence of such a signal, the protein is addressed to the vacuoles of the aqueous tissues, for example the leaves, as well as to the protein bodies of the reserve tissues, for example seeds, tubers and roots.
- the targeting of the protein to the protein bodies of the seed is particularly interesting because of the capacity of the seed to accumulate proteins, up to 40% of the proteins relative to the dry matter, in cellular organelles derived from vacuoles. , called protein bodies and because of the possibility of storing for several years the seeds containing the recombinant proteins in the dehydrated state.
- propeptide use may be made of a signal of animal or vegetable origin, plant signals being particularly preferred, for example proposporamine, or barley lectin.
- the propeptide can be N-terminal ("N-terminal targeting peptide” or NTTP), or C terminal (CTTP) or can consist of a sequence internal to the protein. Since the propeptide is normally cleaved upon entry of the protein into the vacuole, it is not present in the mature protein. It has been found that the use of the "propeptide", in combination with a prepeptide, is particularly advantageous, and gives rise to glycoproteins having a molecular weight in accordance with the natural molecule.
- a sequence coding for an agent allowing the selection of the transformed cells is introduced into the plant cell at the same time as the sequences coding for the glycoprotein.
- the gene coding for the selection agent can be a chimeric gene made up of regulatory sequences recognized by the plant in association with the coding sequence of the selection agent, for example NPT I, NPT II, dhfr, etc.
- the chimeric selection gene can be part of the same vector as that coding for glycoprotein. Alternatively, it can be carried by an independent vector and introduced by co-transformation.
- all known means for transforming the nuclear genome can be used, for example Agrobacterium, electroporation, fusion of protoplasts, bombardment with a particle gun, or penetration of DNA into cells such as pollen, microspore, seed and immature embryo, viral vectors such as Geminiviruses or satellite viruses.
- Agrobacterium tumefaciens and rhizogenes are the preferred means.
- the sequence of the invention is introduced into an appropriate vector with all the necessary regulatory sequences such as promoters, terminators, etc., as well as any sequence necessary for selecting the transformants.
- the transformed cells are selected by virtue of the presence of the gene coding for the selection agent. These cells are then subjected to a step of multiplication by cell culture, where they are regenerated into transgenic or chimeric plants.
- a biomass capable of producing glycoprotein G in large quantity.
- They can be cultures of plant cells in vitro, for example in a liquid medium.
- Different culture modes ("batch", “fed batch” or continuous) for this type of cells are currently being studied. Batch cultures are comparable to those carried out in an Erlenmeyer flask insofar as the medium is not renewed, the cells thus have only a limited quantity of nutritive elements.
- the "fed batch” culture corresponds to a "batch” culture with a programmed feeding in substrate.
- the cells are continuously supplied with nutritive medium. An equal volume of the biomass-medium mixture is removed in order to keep the volume of the reactor constant.
- biomass densities of around 10 to 30 g of dry weight per liter of culture can be obtained, for species such as Nicotiana tabacum, Vinca rosea and Catharanthus roseus.
- the cells of the invention can also be immobilized, which makes it possible to obtain a constant and prolonged production of the glycoprotein.
- immobilization method mention may be made of immobilization in alginate or agar beads, inside polyurethane foam, or even in hollow fibers.
- the cells of the invention can also be root cultures.
- the roots cultivated in vitro, in liquid medium, are called “Hairy roots", they are roots transformed by the bacterium Agrobacterium rhizo ⁇ enes.
- glycoprotein of the invention By culturing plant cells, it is possible to regenerate chimeric or transgenic plants from transformed explants, by using techniques known per se.
- the process of the invention may or may not include a step for recovering glycoprotein G.
- the process of the invention gives rise to plant cells, or to transgenic plants capable of produce glycoprotein G.
- Cells or plants are in themselves a source of the vaccinating protein and can be administered as such to animals or humans by ingestion.
- the glycoprotein G is recovered by extraction from the plant material, and optionally purified.
- the extraction step comprises the solubilization of the glycoprotein, associated with the cell membranes, with a detergent. More particularly, the plant material, for example, leaves, seeds, roots, etc., is ground in liquid nitrogen. The homogenate is then suspended in an appropriate buffer, added with a detergent, preferably nonionic.
- the detergent is used in concentrations of 0.1 to 2%, preferably 0.1 to 0.5%. It can for example be
- Triton X-100 Triton X-100, or NP40 or ⁇ -octyl glucopyranoside.
- the use of detergent releases glycoprotein from cell membranes, while retaining its integrity and immunogenic power.
- the homogenate thus obtained is centrifuged, the “solubilized” glycoprotein then being present in the supernatant. Normally, the supernatant is filtered.
- the plant extract corresponding to the supernatant can be used as such as a source of vaccinating protein, or can be subjected to one or more purification step (s).
- the detergent can be eliminated or reduced.
- the extract of plant material for example tobacco leaves or rapeseed
- the fraction corresponding to the retentate or to the dead volume is then subjected to an ion exchange chromatography on a QAE Sephadex A50 Pharmacia support equilibrated beforehand in ethylene diamine acetic acid buffer pH 8.1. After washing with this same pad, the glycoprotein is eluted with a 0.6 M sodium acetate solution (P. Atanasiu et al., 1976).
- the invention also relates to the glycoprotein G capable of being obtained by implementing the method of the invention.
- the invention relates to a glycoprotein characterized in that:
- glycoprotein 1 • invention is recognized by antibodies specific to the glycoprotein of rabies virus or related to rabies. They can be polyclonal or monoclonal antibodies against the virus, or against glycoprotein purified from the virus.
- the glycoprotein of the invention has a molecular weight of approximately 66 kDa.
- “approximately” means the variability resulting from measurement techniques. More particularly, the glycoprotein of the invention has a molecular weight of 66 kDa on average when several Western Blot tests are carried out, for example, at least 3 or 5 tests, under the same conditions.
- the molecular weight can be between 65 and 80 kDa, for example 66 to 67 kDa.
- the molecular weight is an apparent molecular weight, determined by electrophoresis on polyacrylamide gel under denaturing conditions, according to the technique of Laemmli et al. (1970).
- the polyacrylamide gel is a 10 or 12.5% gel, but lower concentrations can be used to obtain a clearer resolution of the areas between 60 and 65 kDa.
- the glycoprotein of the invention is highly insoluble.
- the term “insoluble” means that the molecule is not soluble in the exracellular medium, but is found associated with cell membranes. Indeed, it has been found that the presence of detergent, for example SDS, Triton X-100, is essential to extract and dissolve the glycoprotein.
- the “insoluble” nature of glycoprotein is linked to the presence of the C-terminal transmembrane domain. The presence of the C-terminal region, i.e., the region located approximately 40 to 60 amino acids from the carboxy end of the glycoprotein, is important to obtain a protective response when the glycoprotein is used as vaccine.
- the glycoprotein of the invention is indirectly insoluble by its association with cell membranes. In the absence of a detergent, it cannot be in the supernatant of cell cultures producing glycoprotein.
- the glycoprotein of the invention is glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues ⁇ -1,2, and / or a or several residues of fucose linked in ⁇ -1,3, and being free of sialic acid residues.
- glycosylation of glycoproteins produced in plant cells is different from those produced in animal cells. Although the glycosylation steps leading to the polymannosidic glycans are carried out identically in plants and in mammals, the maturation of the polymannosidic glycans into complex glycans is very different.
- the N-glycans of vegetable glycoproteins differ mainly from mammalian glycans by the absence of sialic acid, also known as N-acetylneuraminic acid, and the presence of a residue of ⁇ 1,2 xylose and a fucose residue linked in ⁇ 1,3 to the GlcNAc residue proximal to the "core" (Driouich et al., Regard sur la biochemimie, 1993, vol. 3, pages 33-42).
- Glycosylation plays an important role in the establishment of the spatial structure of the protein, in protection against proteolytic attacks and in the mechanisms of immune recognition.
- the protein of the invention is glycosylated at at least two natural N-glycosylation sites.
- the glycosylation is preferably at positions 247 and 319.
- the glycosylation is of mannosidic, polymannosidic or complex type, or a mixture of both.
- the mannosidic glucan (s) has the GlcNAc2-Manl structure.
- the polymannosidic glucan (s) has the structure GlcNAc2-Man2-9, for example GlcNAc2-Man9, GlcNAc2-Man8, GlcNAc2-Man6 or GlcNAc2-Man5, or GlcNAc2-Man3.
- the glucan (s) of complex type are biantennary and have a basic structure GlcNAc2-Man3 with which are optionally associated residues of xylose (Xyl), fucose (Fuc) and possibly galactose (Gai) or N-acetylglucosamine (GlcNAc ). They are normally free of sialic acid residues, this compound having not hitherto been detected in plant cells.
- the complex glycan is of the "phytohemagglutinin” (PHA) type consisting of a structure
- GlcNAc2Man3 whose ⁇ -linked mannose carries a residue of ⁇ 1,2-xylose and whose proximal GlcNAc carries a residue of ⁇ 1.3 fucose. This kind of structure is frequent for glycoproteins of vacuolar or extracellular localization.
- the 1,3-linked ⁇ -fucose residue may possibly be absent.
- the complex glycan can also be of the "Laccase” type composed of a basic structure GlcNAc2 (Fuc) Man3 (Xyl) associated with two side chains.
- Each side chain consists of a residue of ⁇ 1.2 GlcNAc to which is linked a residue of ⁇ 1.6 fucose and a residue of ⁇ 1.4 galactose.
- the glycoprotein of the invention can be glycosylated at the three natural sites or only at one or two of them.
- This type of glycosylation exists in identical form in plants and animals. Therefore, the appearance of an adverse immunological reaction is avoided.
- This type of glycosylation is obtained by the use of an N-terminal signal peptide in association with an endoplasmic retention signal.
- glycoproteins carrying exclusively glycans of the complex type for example two or three glycans of the PHA type: GlcNAc2 (Fuc) Man3 (Xyl).
- the carbohydrate part normally represents between 2 and 30%, for example 5 to 20% of the total mass of the glycoprotein of the invention, the natural molecule comprising 11% of glycosylation.
- composition of glycoprotein glycans can be determined by digestion of oligosaccharides with N-glycanase, as described by Tuchiya K. et al. (1992).
- the glycoprotein of the invention is capable of inducing the formation of neutralizing antibodies and protective immunity against the rabies virus or against a virus related to the rabies virus.
- glycoprotein The protective character of glycoprotein is demonstrated by the administration, to an animal, of the vaccine containing glycoprotein, followed by a virulent test with the rabies virus.
- the level of neutralizing antibodies produced in animals is determined by a test such as the RFFIT test ("Rapid Fluorescence Focus Inhibition Test" described by Smith et al. (1973).
- the glycoprotein of the invention may include one or more of the addressing signals described above.
- the only peptide signal left is the HKDEL-type endoplasmic retention, the other addressing signals being cleaved in the cell.
- Glycoprotein with an endoplasmic retention signal has a molecular weight greater than 66 kDa due to the presence of additional amino acids.
- the protein part of the glycoprotein of the invention may correspond to that of natural glycoprotein, or it may be a protein "analogous" to the natural protein.
- an "analogous" protein means a protein which has at least 90% homology with the reference molecule. It is for example a rabies glycoprotein of which certain amino acids have been replaced, deleted or inserted. The modifications preferably take place on the N-terminal side of the molecule.
- Analogous proteins are also insoluble, recognized by antibodies specific to rabies glycoprotein or to a virus related to the rabies virus and capable of inducing the formation of neutralizing antibodies.
- the invention also relates to the nucleic acids, RNA or DNA, used in the production of glycoprotein.
- the invention relates to a nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to the rabies virus, or for a similar protein , and on the other hand, a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned coding sequences.
- the regulatory sequences described above, as well as the sequences encoding the addressing signals are, if appropriate, also included in the nucleic acid molecule.
- the invention further relates to the vectors and plasmids used for the introduction of the nucleic acid into plant cells. They may be Ti plasmids of A ⁇ robacterium. or viral vectors such as Geminiviruses or CaMV.
- the invention also relates to transformed plant cells capable of producing the glycoprotein of the invention. These cells are in the form of cell culture, as indicated above, or are part of transgenic or chimeric plants.
- Angiosperms comprising monocots and dicots. More particularly, mention may be made of tobacco, species belonging to botanical families such as legumes (for example beans, peas, etc.), crucifers (for example cabbage, radishes, rapeseed, etc.) , nightshade (for example tomatoes, potatoes, etc.), cucurbits (for example melon, zucchini, cucumber), chenopodiaceae (for example vegetable beet), umbelliferae (for example carrots, celery, etc.). Mention may also be made of cereals such as wheat, corn, barley, triticale and rice, and oilseeds such as sunflower and soybeans.
- legumes for example beans, peas, etc.
- crucifers for example cabbage, radishes, rapeseed, etc.
- nightshade for example tomatoes, potatoes, etc.
- cucurbits for example melon, zucchini, cucumber
- chenopodiaceae for example vegetable beet
- the invention also relates to the seeds of transgenic plants capable of producing rabies glycoprotein G, as well as their progeny.
- the invention also relates to the use of glycoprotein G as a therapeutic product, in particular as a vaccine against infection by rabies, or by related viruses.
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising: one or more glycoprotein (s) according to the invention, or - cells according to the invention, or
- the pharmaceutical composition contains at least 1 ⁇ g of glycoprotein.
- it contains 1 ⁇ g to 1 mg, or preferably 10 ⁇ g to 200 ⁇ g, or preferably 25 to 100 mg, of purified glycoprotein, per dose, for example 200 or 300 ⁇ g.
- the glycoprotein is used in a form other than purified, for example in the form of an extract from the plant, or the plant itself, the quantity administered must be adjusted so that the dose of active principle reaches at least 100 ⁇ g.
- the excipient used in the composition is an excipient normally used for rabies vaccines, for example alumina gel.
- the vaccine is formulated for oral or parenteral administration. It can also take the form of a food product produced from the plant material of the invention.
- the invention also includes a method of immunizing mammals, including humans, against infection with this rabies virus, or with a virus related to the rabies virus, comprising administering a glycoprotein of the invention, or a plant material comprising this glycoprotein.
- Lane 1 Reference rabies glycoprotein G reconstruction in tobacco juice
- Lane 4 FFeeuuiilllleess of tobacco transformed with pBIOCllO;
- Lane 5 F Feeuuiilllleess of unprocessed tobacco
- Lane 7 MMaarrqqueueuurr cde molecular weight.
- Lane 1 Reference rabies glycoprotein G
- Lane 3 Reference rabies glycoprotein G reconstruction in corn callus juice
- Lane 1 Reconstruction of rabies glycoprotein G reference in tobacco leaf juice
- Lane 2 Unprocessed tobacco leaf
- Lane 3 Tobacco leaf transformed with pBIOCllO (PPS-RabG);
- Lane 4 Tobacco leaf transformed with pBI0C104 (PS-RabG);
- Lane 5 Molecular weight marker.
- rabies glycoprotein G required the following regulatory sequences:
- the constitutive double 35S promoter (pd35S) of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987). or the chimeric super-promoter (pSP; Ni et al., 1995). It consists of the fusion of the triple repetition of a transcriptional activating element of the promoter of the octopine synthase gene from Agrobacterium tumefaciens, a transcriptional activating element of the promoter of the mannopine synthase gene and the mannopine synthase promoter. Agrobacterium tumefaciens;
- polyA 35S terminator which corresponds to the 3 ′ non-coding region of the sequence of the double-stranded DNA virus circular of the cauliflower mosaic producing the 35S transcript (Franck et al., 1980).
- the plasmid pGA492 was doubly digested with Sac1 (restriction site of the polylinker) and by Seal (restriction site present in the sequence of the cat gene) and then subjected to the action of the enzyme T4 DNA polymerase (Biolabs) according to the manufacturer's recommendations.
- Ligation of the modified plasmid (20 ng) was carried out in a 10 ⁇ l reaction medium containing 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham); 2.5 U of T4 DNA ligase enzyme (Amersharo) at 14 ° C for 16 hours.
- the HindIII restriction site of the plasmid DNA of the retained clone was modified into an EcoRI restriction site using a phosphorylated HindIII-EcoRI adapter (Stratagene Cloning Systems).
- 500 ng of plasmid DNA of the retained clone were digested with HindIII, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations and coprecipitated in the presence of 1500 ng of DNA adapter HindIII-EcoRI, 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
- the ligation reaction mixture was digested with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, then ligated as described above.
- the resulting binary plasmid which only has the last 9 amino acids of the coding sequence for the cat gene and whose EcoRI site is unique, was called pBI0C4.
- the expression cassette consisting of the pd35S promoter and the polyA 35S terminator, was isolated from the plasmid pJIT163 ⁇ .
- the plasmid pJIT163 ⁇ derives from the plasmid pJIT163 which itself derives from the plasmid pJIT60 (Guerineau and Mullineaux, 1993).
- Plasmid pJIT163 has an ATG codon between the HindIII and SalI sites of the polylinker.
- the plasmid DNA pJIT163 was digested twice with HindIII and SalI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, subjected to the action of the enzyme Klenow (Biolabs) according to the manufacturer's recommendations, deproteinized by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) then 1 volume of chloroform: isoamyl alcohol ( 24: 1), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at - 80 ° C for 30 min.
- DH5 ⁇ previously made competent, have been transformed (Hanahan, 1983).
- the expression cassette consisting of the pd35S promoter and of the polyA 35S terminator (SacI-Xhol fragment) the plasmid DNA of the clone pJIT163 ⁇ retained was digested with SacI and Xhol.
- Sacl-Xhol fragment carrying the expression cassette, was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 of volume of 3M acetate. sodium pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, then subjected to the action of the enzyme Mung Bean Nuclease (Biolabs) according to the manufacturer's recommendations.
- This purified insert (200 ng) was cloned into the plasmid DNA of pBIOC4 (20 ng) digested with EcoRI, treated with the enzyme Mung Bean Nuclease and dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation reaction was carried out in 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the PvuII - Sali fragment subjected to the action of Klénow containing the pSP promoter was isolated from the plasmid pBISNl (Ni and al., 1995), purified by electrophoresis on 1% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pBIOC ⁇ l doubly digested with Kpnl and EcoRI subjected to the action of T4 DNA polymerase and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the plasmid pBIOC ⁇ l corresponds to pB10C21 whose Xbal site has been deleted. To do this, the plasmid pB10C21 was digested with Xbal then subjected to the action of Klenow and ligated by action of T4 DNA ligase.
- the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments carrying pSP described above in a 20 ⁇ l reaction medium in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ) and 2 ⁇ l of 50% polyethylene glycol 8000, and 5 ⁇ of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- T4 DNA ligase x 10 buffer Amersham
- 50% polyethylene glycol 8000 50% polyethylene glycol 8000
- 5 ⁇ of T4 DNA ligase enzyme Amersham
- Rabies glycoprotein G isolated from the ERA strain, is naturally synthesized in the form of a precursor.
- the mature protein RabG is made up of 505 amino acids. Its signal peptide is composed of 19 amino acids.
- the cDNA coding for the envelope glycoprotein of the rabies virus, strain ERA was amplified by PCR according to the usual methods, using the following two oligonucleotides:
- Oligo 1 (30 sea) 5 'AAA GGA TCC ATG GTT CCT CAG GCT CTC CTG 3'
- Oligo 2 (30 sea) 5 AAA CTG CAG TCA CAG TCT GGT CTC ACC CCC 3 '
- the 1.69 kb PCR fragment was digested with BamHI and Pst1 and then cloned into the vector pBlueScript previously digested with BamHI and Pstl, thus giving the plasmid pPBO10.
- the complementary DNA of the RabG precursor is contained in the plasmid pPBO10. It was used for the construction of the binary plasmids pBIOC104 and PBIOC105 containing the sequence coding for PS-RabG, pBIOCl07 and pBIOC108 containing the sequence coding for BL-RabG and pBIOCllO and pBIOClll containing the sequence coding for PPS-RabG where the sequence coding for RabG is preceded by the sequence coding for a signal peptide, PS and BL, and for an N-terminal prepropeptide (PPS i.e. a signal peptide followed by N-terminal vacuolar addressing sequences) of plant origin respectively.
- PPS N-terminal prepropeptide
- the PS and PPS sequences consisting respectively of 23 and 37 amino acids, are those of a reserve protein for tuberous sweetpotato roots: sporamine A (Murakami et al., 1986; Matsuoka and Nakamura, 1991).
- the BL sequence composed of 26 amino acids, is that of barley lectin (Lerner and Raikhel, 1989).
- cDNA RabG was modified at its 3 'end by PCR-directed mutagenesis using 2 oligodeoxynucleotides, 5' CTC AGG AGT TGA CTT GGG 3 '(containing the unique HincII site in the plasmid pPBO10) and 5' CCG GAT CCT CAC AGT CTG GTC TCA C 3 '(containing the unique BamHI site in the plasmid pPBO10).
- the PCR amplification of the HincII-BamHI fragment was carried out in 100 ⁇ l of reaction medium comprising 10 ⁇ l of Taq DNA polymerase x10 buffer (500 mM KC1, 100 mM Tris-HCl, pH9.0 and 1% Triton x100), 6 ⁇ l 25 mM MgCl2, 3 ⁇ l of 10 mM dNTP (dATP, dCTP, dGTP and dTTP), 100 ⁇ M of each of the 2 oligodeoxynucleotides described above, 5 ng of template DNA (vector pPBO10), 2.5 U of Taq DNA polymerase (Promega) and 2 drops of petrolatum oil.
- reaction medium comprising 10 ⁇ l of Taq DNA polymerase x10 buffer (500 mM KC1, 100 mM Tris-HCl, pH9.0 and 1% Triton x100), 6 ⁇ l 25 mM MgCl2, 3 ⁇ l of 10
- the DNA was denatured at 94 ° C for 5 min., Subjected to 30 cycles each consisting of 1 min. denaturation at 94 ° C, 1 min. hybridization at 44 ° C and 1 min. elongation at 72 ° C, then the elongation at 72 ° C was continued for 5 min.
- This PCR reaction was carried out in the "DNA Thermal Cycler" machine from PERKIN ELMER CETUS. The oil was removed by extraction with chloroform. Then, the DNA fragments of the reaction medium were precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
- T7 TM marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
- the resulting plasmid was called pBI ⁇ C102.
- the plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
- This sequence has been replaced by that coding for the signal peptide PS of 23 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG").
- the sequence "first PS-9 mature RabG codons" was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3' (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGA ATG GGC TGG ATT GGG CAG G 3' (containing the unique HindIII site in the plasmid pPBO10) by following the PCR amplification protocol described
- the hybridization temperature was 40 ° C.
- the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min ., washed with 70% ethanol, dried, then ligated with plasmi DNA pBIOClOl plate doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried.
- the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and -4.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
- the EcoRI fragment carrying the sequence PS-RabG was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBI0C21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation was carried out with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing PS-RabG, described above, in a 10 ⁇ l reaction medium in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- T4 DNA ligase x 10 buffer Amersham
- Amersham 2.5 U of T4 DNA ligase enzyme
- the nucleic acid sequence of the fragment coding for the recombinant protein PS-RabG was verified by sequencing using the T7 TM sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
- the plasmid of the binary vector pBIOC104 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
- pBIOClOS is similar to that of pBI0C104 except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82.
- the EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied.
- the plasmid pBIOC103 was digested twice with BglII and HindIII in order to suppress the sequence coding for the signal peptide PS and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
- This sequence has been replaced by that coding for the BL amino acid signal peptide of 26 amino acids (ATG AAG ATG ATG AGC ACC AGG GCC CTC GCT CTC GGC GCG GCC GCC GTC CTC GCC TTC GCG GCG GCG ACC GCG CAC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("BL-9 first codons of mature RabG").
- the sequence "BL-9 first codons of mature RabG” was amplified by PCR from the plasmid BLc3 (Lerner and Raikhel, 1989) using the 2 oligodeoxynucleotides, 5 'ggagatctgaattoaacaATG AAG ATG ATG AGC ACC AGG 3' (containing the unique BglII and additional EcoRI sites in the plasmid pBIOC102) and 5 'AGG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GTC CGC GGT CGC CGC G 3' (containing the unique HindIII site in the plasmid pBIOCl02) PCR amplification protocol described above.
- the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
- the sequences coding for BL and mature RabG were cloned maintaining their open reading phases (ie, so that they constitute a single open reading phase)
- the cleavage sequence between the BL and mature RabG sequences is Ala-Lys.
- the resulting plasmid was called pBIOClO ⁇ .
- the EcoRI fragment carrying the sequence BL-RabG was isolated by enzymatic digestion by EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme calf intestine alkaline phosphatase (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation was carried out with 100 ng of dephosphorylated pBI0C21 vector and 50 ng of DNA fragments containing BL-RabG, described above, in a 10 ⁇ l reaction medium in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- T4 DNA ligase x 10 buffer Amersham
- Amersham 2.5 U of T4 DNA ligase enzyme
- the nucleic acid sequence of the fragment coding for the recombinant protein BL-RabG was verified by sequencing using the T7 TM sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
- the plasmid DNA of the binary vector pBIOCl07 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
- pBIOClo ⁇ is similar to that of pBIOC107 except that the EcoRI fragment carrying the sequence BL-RabG was cloned at the Xbal site of pBIOC82.
- the EcoRI sites of the insert and Xbal of the vector were subject to the action of Klenow. The usual cloning methods were applied.
- the plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
- This sequence has been replaced by that coding for the PPS signal peptide of 37 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC AGG TTC AAT CCC ATC CGC CTC CCC ACC ACA CAC GAA CCC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG").
- the sequence "PPS-9 first codons of mature RabG” was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3 • (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GGG TTC GTG TGT GGT GGG GAG G 3' (containing the unique HindIII site in the plasmid pPBO10) following the PCR amplification protocol described above.
- the hybridization temperature was 40 ° C.
- the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 by volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, and then ligated to the plasmid DNA of pBIOClOl doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried.
- the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
- the EcoRI fragment carrying the PPS-RabG sequence was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- Ligation was carried out with 100 ng of pPHOC21 dephosphorylated vector and 50 ng of DNA fragments containing PPS-RabG, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 enzyme DNA ligase (Amersham) at 14 ° C for 16 hours.
- T4 DNA ligase x 10 buffer Amersham
- T4 enzyme DNA ligase Amersham
- the plasmid DNA of the binary vector pBIOC10O was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
- pBIOC111 is similar to that of pBIOC11O except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82.
- the EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied. II. CONSTRUCTION OF CHEMICAL GENES ENCODING THE RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND ALLOWING EXPRESSION IN CANNED SEEDS.
- rapeseed of the viral gene coding for rabies glycoprotein G required the following regulatory sequences:
- the transcription terminator sequence polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the transcript 35S (Franck et al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 • non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
- the plasmid pBI221-CRURSP is derived from pBI221 (marketed by Clontech) by replacing the 35S promoter with the pCRU promoter.
- the "EcoRI treated with Klenow - BamHI" fragment carrying the pCRU promoter was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pJIT163 (described in paragraph I.) digested with Kpnl, treated with T4 DNA Polymerase (Biolabs) according to the manufacturer's recommendations, then digested with BamHI, purified by electrophoresis on 0.8% agarose gel , electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments "EcoRI treated with Klenow - BamHI" described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of buffer T4 DNA ligase x 10 (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 -and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of XhoI-EcoRI DNA fragments described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
- the plasmid pBSII-pGEAID was obtained in two stages: - on the one hand, the Sacl-EcoRI fragment carrying tNOS (terminator of the nopaline synthase gene) of Agrobacterium tumefaciens, treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at EcoRV site of pBSIISK-t- marketed by Stratagene, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing tNOS described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham), of 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- T7 TM marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
- the resulting plasmid was called pBSII-tNOS.
- the ligation was carried out with 100 ng of the vector described above and 50 ng of DNA fragments described above in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the ligation was carried out with 20 ng of dephosphorylated pSCV1.2 and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the EcoRI-BamHI fragment treated with Klenow, containing the promoter pGEA6 was isolated from the plasmid pGUS2-pGEA6.
- the DNA fragment between the AccI site and the sequences upstream of the ATG of the GEA6 gene of the clone pGUS-2-pGEA6 was therefore amplified by PCR using the 2 oligonucleotides: 5 ′ AAGTACGGCCACTACCACG 3 • and 5 'CCCGGGGATCCTGGCTC 3'.
- the hybridization temperature has been adapted.
- the PCR amplified fragment was digested with AccI and BamHI, purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4, 8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
- the modified pBIOC21 plasmid was obtained by double digestion with Klenow-treated HindIII and Kpnl of pBIOC21 to delete the fragment carrying the pd35S promoter and replace it with the Kpnl-EcoRV fragment carrying the polylinker composed of the Kpnl-XhoI-SalI-ClaI sites -HindlII-BamHI-Smal-EcoRI- EcoRV from pBSIISK +.
- the ligation was carried out with 20 ng of dephosphorylated modified pBIOC21 and 200 ng of EcoRI-BamHI DNA fragments, described above, in a 20 ⁇ l reaction medium in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the expression cassette "pGEA6D - t35S” was isolated from pBIOC92.
- This expression cassette carried by the Kpnl-EcoRV fragment treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at the SmaI site of pSCV1.2 dephosphorylated by the enzyme alkaline phosphatase d ' calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
- the ligation was carried out with 20 ng of pSCV1.2 dephosphorylated and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the bacteria, Escherichia coli DH5cc, made competent beforehand, have been transformed (Hanahan, 1983).
- the plasmid pBIOC109 described previously in I.c, contains the EcoRI fragment carrying the sequence PPS-RabG.
- pd35S double constitutive promoter 35S of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987);
- the transcription terminator sequence polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the 35S transcript (Franck and al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
- the plasmid pBIOC11 ⁇ where the sequence coding for PS-RabG is placed under the control of pAR-IAR has been obtained by cloning of the EcoRI fragment carrying the sequence coding for PS-RabG at the "Ncol and Sali" sites of pBSII-pAR-IAR-tNOS.
- the EcoRI fragment carrying the sequence coding for PS-RabG was isolated from pBIOC103 by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried and then treated with the Klenow enzyme.
- the plasmid pBSII-pAR-IAR-tNOS was digested twice with Sali and Ncol, purified, treated with the enzyme Mung Bean Nuclease (Biolabs) and dephosphorylated by the enzyme alkaline phosphatase of calf (Boehringer Mannheim) .
- the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the sequence coding for RS-LGC, described above, in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase buffer.
- x 10 (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the plasmid pBSII-pAR-IAR-tNOS results from cloning at the sites "Eco0109I treated with Klenow and Kpnl" of pBSII-tNOS of the SnaBI-Kpnl fragment carrying the sequence corresponding to "pAR-IAR-start of the coding sequence for the gene gus "isolated from the plasmid pActl-F4.
- the ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l digested in the presence of 1 ⁇ l of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the bacteria, Escherichia coli DH5 ⁇ made previously competent, were transformed (Hanahan, 1983).
- the plasmid pBSII-tNOS was obtained by cloning at the dephosphorylated EcoRV site of pBSIISK + marketed by Stratagene, from the Sacl-EcoRI fragment carrying the tNOS sequence isolated from pBI121 marketed by Clontech by double enzymatic digestion with Sacl and EcoRV, subjected to purification by electrophoresis on 2% agarose gel, and treated with the T4 DNA polymerase enzyme.
- the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the tNOS sequence, described above, in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the plasmid pBIOC117 where the sequence coding for PS-RabG is placed under the control of pd35S was obtained by cloning at the "Kpnl and Xbal" sites of the plasmid pBSIISK + marketed by Stratagene, of the Kpnl-Xbal fragment carrying the sequence corresponding to "pd35S- PS-RabG "isolated from pBIOC103.
- the ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l digested in the presence of 1 ⁇ l of T4 buffer DNA ligase x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C. for 16 hours
- the plasmid DNA of the clones obtained selected from
- 50 ⁇ g / ml ampicillin was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
- the plasmid p ⁇ 63 results from the cloning of p ⁇ zein at the HindIII and Xbal sites of a plasmid pUC18 containing, between its HindIII and EcoRI sites, the expression cassette "p35S-gus-tNOS" of pBI221 marketed by Clontech. It allows an expression in the albumen of corn seeds.
- polyA NOS terminator which corresponds to the 3 'non-coding region of the nopaline synthase gene of the plasmid Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
- PS-RabG is placed under the control of p ⁇ zein, was obtained by cloning at the "Sacl sites treated with the enzyme T4 DNA polymerase and BamHI" of the plasmid p ⁇ 63, of the fragment "BamHI treated with Klenow - BglII” isolated from pBIOC103 .
- the ligation was carried out with 100 ng of the vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and of 2, 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
- the plasmid pBIOC119 results from the substitution of the BamHI-HincII fragment of pBIOC11 ⁇ by the BamHI-HincII fragment carrying the sequence coding for KDEL placed before the stop codon obtained by PCR amplification according to the methods described above.
- the 2 oligodeoxynucleotides used during this reaction were: 5 'GGT CTC AGG AGT TGA CTT GGG TCT CCC GAA CTG GG 3' (single HincII site) and 5 'CCC GGA TCC TCA TAG CTC ATC TTT CAG TCT GGT CTC ACC CCC ACT C 3 '(sequence coding for KDEL and unique BamHI site).
- the hybridization temperature was 50 ° C.
- the cloning was carried out as described previously.
- the tobacco plants used for the transformation experiments are cultivated in vitro on the basic medium of Murashige and Skoog (1962) supplemented with the vitamins of Gamborg et al. (1968, Sigma reference M0404), sucrose at 20g / L and agar (Merck) at 8g / L.
- the pH of the medium is adjusted to 5.8 with a potassium hydroxide solution before autoclaving at 120 ⁇ C for 20 min.
- Tobacco seedlings are transplanted by cutting internodes every 30 days on this MS20 multiplication medium.
- the transformation technique used is derived from that of Horsch et al. (1985).
- a preculture of Agrobacterium tumefaciens strain LBA4404 containing the binary plasmids is carried out for 48 h at 2 ⁇ ° C with stirring, in LB medium supplemented with appropriate antibiotics (rifampicin and tetracycline).
- the preculture is then diluted to 50th in the same medium and cultivated under the same conditions.
- the culture is centrifuged (10 min, 3000 rpm), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / L sucrose) and this suspension is diluted with 10eme.
- Explants of approximately 1 cm 2 are cut from the leaves of the seedlings described above. They are then placed in contact with the bacterial suspension for 1 h, then quickly dried on filter paper and placed on a coculture medium (solid MS30). .
- the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200mg / L), a bacteriostatic, augmentin (400mg / L) and the hormones necessary for bud induction (BAP, lmg / L and ANA, 0, lmg / L).
- the explants are subcultured on the same medium after 2 weeks of culture. After 2 new weeks, the buds are subcultured in Petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted into pots on the same medium, the concentration of kanamycin has been halved. It takes about 20 days to take root, at the end of which the seedlings can be cloned by cutting internodes or emerging in the greenhouse.
- the spring rapeseeds (Brassica napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% Domestos solution. After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per 7 cm diameter jars by 10 cm high, on Murashige and Skoog mineral medium (Sigma, reference M 5519) with 30 g / 1 of sucrose and solidified with 5 g / 1 of agargel. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 16h / ⁇ h and under a light intensity of the order of 80 ⁇ E m "" 2 S -1 . After 5 days of germination, the cotyledons are removed sterile by cutting each petiole about 1 mm above the cotyledon node.
- a culture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pBI0C114 is cultivated in a 50 ml Erlenmeyer flask for 36 h at 28 ° C. in 10 ml of YT bacterial medium supplemented with the antibiotics useful for the selection of the strain used.
- This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 rpm and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box.
- the severed end of the petiole is immersed for a few seconds in the agrobacteria solution thus prepared, then the petiole is pressed a few millimeters into the regeneration medium.
- This medium has the same basic composition as the germination medium with, in addition, 4 mg / 1 of benzylamino-purine, a phytohormone promoting the neoformation of buds. Twelve explants (cotyledon with petiole) are cultured in a 9 cm diameter petri dish (Greiner, reference 664102).
- the explants are subcultured in phytatray boxes (Sigma, reference P1552) containing the above medium supplemented with a selective agent: 45 mg / 1 of kanamycin sulfate (Sigma, reference K4000) and a bacteriostatics: mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 ammonium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1. Twice in succession, 3 weeks apart, the explants are transplanted sterile onto new medium under the same conditions.
- a selective agent 45 mg / 1 of kanamycin sulfate
- a bacteriostatics mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 ammonium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.
- the green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5 cm in diameter and 10 cm high containing a medium identical to the above but free of BAP. After 3 weeks of culture, the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium. After three to four weeks the roots are sufficiently developed to allow acclimatization of the seedling to the phytotron. Buds that are not green or rooted are removed. These seedlings are then transplanted into 7 cm side pots filled with potting soil (standard NF U44551: 40% brown peat, 30% sifted heather and 30% sand) saturated with water.
- standard NF U44551 40% brown peat, 30% sifted heather and 30% sand
- the seedlings are repotted in 12 cm diameter pots filled with the same soil enriched with late fertilizer (Osmocote, at a rate of 4g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at ⁇ ° C, with two daily waterings of 2 minutes.
- a greenhouse class S2
- the pods When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for the determination of biochemical activity.
- the selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes). Conditions The operating procedures are identical to those described at the beginning of this document except that the germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse.
- calluses are obtained from immature embryos of genotype HI, II or (A188 x B73) according to the method and on the media described by Armstrong (Malze Handbook, 1994, M. Freeling, V. Walbot Eds., Pages 665- 671). The calluses thus obtained are multiplied and maintained by successive subcultures every fortnight on the initiation medium.
- Seedlings are then regenerated from these calluses by modifying the hormonal and osmatic balance of the cells according to the method described by Vain et al. (Plant Cell Tissue and Organ Culture, 1969,! £: 143-151). These plants are then acclimatized in the greenhouse where they can be crossed or self-fertilized. b) Use of the particle gun for the genetic transformation of corn:
- the plasmids carrying the genes to be introduced are purified on a Qiagen® column, following the manufacturer's instructions. They are then precipitated on tungsten particles (M10) following the protocol described by Klein (Nature, 1987, 327: 70-73). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. Finer (Plant Cell Report, 1992, ⁇ : 323-328).
- the callus boxes thus bombarded are then sealed using Scellofrais® and then grown in the dark at 27 ° C.
- the first subculture takes place 24 hours later, then every fortnight for 3 months on medium identical to the initiation medium supplemented with a selective agent, the nature and concentration of which may vary depending on the gene used (see paragraph 3).
- the selective agents which can be used generally consist of active compounds of certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromycin, Kanamycin ).
- Calls are obtained after 3 months, or sometimes earlier, calluses whose growth is not inhibited by the selection agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene.
- the frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
- calluses are identified, individualized, amplified and then cultivated so as to regenerate seedlings (see paragraph a). In order to avoid any interference with untransformed cells, all of these operations are carried out on culture media containing the selective agent.
- the plants thus regenerated are acclimatized and then cultivated in a greenhouse where they can be crossed or self-fertilized.
- the homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 10,000 rpm.
- extraction is carried out at the rate of 100 mg of seeds for 4 ml of buffer.
- extraction is carried out at a rate of 0.5 g in 1 ml of buffer.
- Rabies glycoprotein G is demonstrated by "Western blots" (Renart and Sandoval, 1984) and assayed by an ELISA test.
- the proteins extracted according to the above protocol are denatured by heating at 95 ° C for 5 min, in the presence of 50 mM pH 6.8 Tris HCl buffer; SDS 4%; BSH 1%; Sucrose 20% and bromophenol blue 0.01%.
- the proteins are then separated by electrophoresis, on a polyacrylamide gel under denaturing conditions at 8%, 10% or 12.5%, according to the technique of Laemmli U.K. (1970) at the rate of 50 ⁇ g of total proteins per sample.
- the proteins are transferred to a nitrocellulose membrane.
- An anti-rabies G glycoprotein antibody obtained in sheep is used as a probe and the revelation is carried out by means of an anti-sheep IgG antibody margined with alkaline phosphatase.
- the tests were carried out on the transformed plants with the following constructs: pBIOC104, pBIOC10O, pBIOC114, pBIOC116.
- the control protein migrates in the form of 2 bands of apparent molecular weights 66 and 61 KDa.
- the lightest form corresponds to a protein, truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein.
- the following protocol was used: extraction of the glycoprotein: the samples of the leaves or seeds, taken on ice on the day of the test, are ground at a rate of 1 g for 4 ml of extraction buffer for the leaves and 100 mg for 4 ml of extraction buffer for the seeds.
- the amount of total protein is then determined by the Bradford method and all the extracts are brought to a concentration of 5 ⁇ g / ⁇ l. These extracts are diluted extemporaneously to 1/1000.
- the reference range (glycoprotein G: 0 to 20 mg / ml) is prepared in an extract of ground unprocessed plant, standardized and diluted to 1/1000 in the same way.
- the assay is then carried out by direct reading on a standard curve carried out under the same conditions, with purified glyco-protein G.
- glycoprotein G varies from 1.5 to 0.4 ⁇ g / ml for the different extracts tested.
- the homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 13000 g. Then, the supernatant is removed and filtered through Miracloth (CALBIOCHEM). A determination of the soluble proteins is then carried out by the method of Bradford (1976).
- Rabies glycoprotein G is demonstrated by immunodetection of the "Western blot” type (Renart and Sandoval, 1984).
- the proteins extracted according to the above protocol are denatured by heating at 95 ° C. for 5 min, in the presence of 50 mM Tris-HCl buffer, pH 6.8, 4% SDS, 1% BSH, 20% sucrose and bromophenol blue 0 , 01%.
- the proteins are then separated by electrophoresis on a polyacrylamide gel under denaturing conditions at 8% or 10%, according to the technique of Laemmli (1970) at the rate of 30 ⁇ g of soluble proteins per sample.
- the proteins are transferred to a PVDF (polyvinylidene difluoride) membrane.
- PVDF polyvinylidene difluoride
- the control reference rabies glycoprotein G is characterized by 2 bands of apparent molecular masses of 66 and 61 KDa.
- the form lighter corresponds to a protein truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein. No band corresponding to rabies glycoprotein G is detected in the unprocessed corn extracts.
- the modification of the cDNA encoding the rabies glycoprotein G was carried out according to the LbPCR technique by modifying the 5 '[fragment BglII (agatct) - Xhol (ctcgag)], central [fragment Xhol - Aatll (gacgtc)] and 3' [fragment Aatll - BamHI (ggatcc)]
- the LbPCR technique consists of an LCR ligation step (Barany F. PNAS USA 1991, 88, 189-193) to produce continuous single-stranded DNA using "sense" oligodeoxynucleotides and a PCR step to obtain double-stranded DNA.
- modified parts were verified by sequencing at using the Amersham Sequenase PCR product sequencing kit according to the manufacturer's recommendations Then, either the modified 5 'part, or the modified 5' and central parts, or the modified 5 ', central and 3' parts, were introduced into pBIOC109 by enzymatic digestions (restriction enzymes delimiting the fragments)
- the 5 ′ part was constituted using the following oligodeoxynucleotides
- the 5 ′ part amplified by PCR was doubly digested with BglII and Xhol, then cloned to replace the BglII - Xhol fragment of pBIOC109 according to the methods already described previously.
- the resulting plasmid was called pBIOC109-5'M This plasmid was used in genetic transformation of corn
- the central part was formed using the following oligodeoxynucleotides.
- the central part amplified by PCR was doubly digested with Xhol and Aatll, then cloned to replace the Xhol - Aatll fragment of pBIOC109-5'M according to the methods already described above.
- the resulting plasmid was called pBIOC109-5'CM. This plasmid was used in genetic transformation of corn.
- the 3 ′ part was constituted using the following oligodeoxynucleotides:
- the isolated fragments were ligated to the plasmid DNA of pBIOC21, pBIOC28, pBIOC82, pBIOC91 and pBIOC1 12 digested with EcoRI and dephosphorylated. The usual cloning methods were applied.
- the plasmid DNA of the resulting binary plasmids was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). Genetic transformation has been completed.
- the invention therefore particularly extends to methods, to glycoproteins and their uses, to nucleic acids, transformed plant cells, chimeric or transgenic plants, seeds of transgenic plants, monoclonal or polyclonal antibodies, pharmaceutical compositions and products.
- foodstuffs according to the invention characterized in that the coding sequence, allowing expression in the cell of the protein G, has been subjected to modifications to increase the expression, for example has been subjected to a "revegetation" according to example E above.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
This invention features a method for producing the glycoprotein G of the rabies virus, or a virus related to the rabies virus, characterised by: i) introducing, into a plant cell, a nucleic acid molecule comprising a chimeric coding sequence, comprising one coding sequence for the said mature viral protein G, or for an analogous protein, and a coding sequence for a N-terminal signal peptide other than the one naturally associated with the viral protein G, enabling the expression of the protein G in the cell; ii) multiplying, in the form of cell culture, transformed cells, or regeneration of whole transgenic or chimerical plants from these cells; iii) optionally extracting and purifying the glycoprotein G from the cells or plants thus obtained.
Description
PLANTES TRANSGENIQUES EXPRIMANT LA GLYCOPROTEINE G DE LA RAGE, ET GLYCOPROTEINES AINSI OBTENUES TRANSGENIC PLANTS EXPRESSING Rabies GLYCOPROTEIN G, AND GLYCOPROTEINS THUS OBTAINED
La présente invention concerne un procédé de production, par des cellules végétales, de la glycoproteine G de la rage, ou d'un virus apparenté à la rage. L'invention concerne également la glycoproteine ainsi obtenue et son utilisation en tant que médicament, notamment en tant que vaccin humain ou vétérinaire. L'invention vise en outre les cellules végétales transformées, et les plantes transgéniques, capables de produire la glycoproteine G de la rage.The present invention relates to a process for the production, by plant cells, of rabies glycoprotein G, or of a virus related to rabies. The invention also relates to the glycoprotein thus obtained and to its use as a medicament, in particular as a human or veterinary vaccine. The invention further relates to transformed plant cells, and transgenic plants, capable of producing the glycoprotein G of rabies.
Le virus de la rage fait partie de la famille des Rhabdoviridae. genre Lyssavirus. Il s'agit d'un virus à ARN- dont le génome comporte environ 12000 nucléotides, codant pour cinq protéines structurelles : la protéine de la nucléocapside « N », les protéines de matrice « Mi » et « M2 »/ la glycoproteine d'enveloppe « G » et la protéine « L » codant pour la polymérase virale.The rabies virus is part of the family Rhabdoviridae. genus Lyssavirus. It is an RNA virus- whose genome contains approximately 12,000 nucleotides, coding for five structural proteins: the nucleocapsid protein "N", the matrix proteins "Mi" and "M2" / glycoprotein from envelope "G" and the protein "L" encoding the viral polymerase.
L'enveloppe virale est constituée d'une bi-couche lipidique contenant des spicules composés d'une forme polymérique de la glycoproteine G. Elle est exprimée sous forme d'un précurseur ayant 524 acides aminés, dont 19 constituent la séquence signal N-terminal hydrophobe. Le signal N-terminal est clivé pour donner lieu à la glycoproteine mature de 505 acides aminés. Le poids moléculaire de la glycoproteine rabique monomérigue mature est de 66 kDa. cette forme de la glycoproteine est la forme dite « insoluble », c'est-à- dire qu'elle comprend un domaine C-terminal transmembranaire permettant à la glycoproteine de se fixer dans l'enveloppe virale. La forme « soluble » de la glycoproteine a un poids moléculaire d'environ 60 kDa et se distingue de la forme insoluble par l'absence
d'environ 58 acides aminés C-terminaux. Cette forme de la glycoproteine, étant dépourvue de segment transmembranaire, ne peut plus s'associer à l'enveloppe virale.The viral envelope consists of a lipid bilayer containing spicules composed of a polymeric form of glycoprotein G. It is expressed in the form of a precursor having 524 amino acids, of which 19 constitute the N-terminal signal sequence hydrophobic. The N-terminal signal is cleaved to give rise to the mature glycoprotein of 505 amino acids. The molecular weight of mature monomeric rabies glycoprotein is 66 kDa. this form of glycoprotein is the so-called “insoluble” form, that is to say that it comprises a C-terminal transmembrane domain allowing the glycoprotein to bind in the viral envelope. The “soluble” form of glycoprotein has a molecular weight of around 60 kDa and is distinguished from the insoluble form by the absence of about 58 C-terminal amino acids. This form of glycoprotein, being devoid of transmembrane segment, can no longer associate with the viral envelope.
La glycoproteine constitue l'antigène majeur du virus, responsable de l'induction d'anticorps neutralisants et de l'immunité protectrice. Seule la forme « insoluble » est protectrice, bien qu'il n'y ait pas de déterminants antigéniques dans le C-terminal (Dietzschold B. et al., 1983).Glycoprotein is the major antigen of the virus, responsible for the induction of neutralizing antibodies and protective immunity. Only the "insoluble" form is protective, although there are no antigenic determinants in the C-terminal (Dietzschold B. et al., 1983).
Pour ces raisons, la glycoproteine G de la rage a été largement étudiée et utilisée dans le développement de vaccins sous-unités. Les cADN de différentes souches du virus de la rage ont été clones (Anilionis A. et al., 1981 ; Yelverton et al., 1983) et exprimées dans différents hôtes cellulaires.For these reasons, rabies glycoprotein G has been widely studied and used in the development of subunit vaccines. The cDNAs of different strains of the rabies virus have been cloned (Anilionis A. et al., 1981; Yelverton et al., 1983) and expressed in different cellular hosts.
Par exemple, Yelverton et al. (1983) ont décrit l'expression du gène de la glycoproteine rage (souche CVS) dans E. coli. L'activité antigénique du produit d'expression n'est que de 2 à 3% par rapport à la glycoproteine naturelle.For example, Yelverton et al. (1983) described the expression of the rabies glycoprotein gene (CVS strain) in E. coli. The antigenic activity of the expression product is only 2 to 3% compared to the natural glycoprotein.
Kieny et al. (1984) ont décrit l'expression de la glycoproteine G dans des cellules d'eucaryotes supérieures, infectées par un virus de la vaccine, recombinant et réplicatif.Kieny et al. (1984) described the expression of glycoprotein G in cells of higher eukaryotes, infected with a vaccinia virus, recombinant and replicative.
L'expression d'une glycoproteine de la rage (souche CVS) ayant un poids moléculaire de 63 et 65 kDa (Préhaud et al., 1989), et celle du virus Mokola, virus apparenté au virus de la rage (Tordo N. et al., 1993), a été obtenue dans des cellules d'insectes infectées par un vecteur baculovirus recombinant.The expression of a rabies glycoprotein (CVS strain) having a molecular weight of 63 and 65 kDa (Préhaud et al., 1989), and that of the Mokola virus, virus related to the rabies virus (Tordo N. and al., 1993), was obtained in insect cells infected with a recombinant baculovirus vector.
Parmi ces travaux, l'utilisation d'hôtes eucaryotes supérieurs a permis d'obtenir des glycoprotéines recombinantes capables de conférer une immunité protective in vivo, par voie orale ou par injection intramusculaire ou intrapéritonéale.
L'utilisation de plantes transgéniques pour la production de la glycoproteine G rabique a également été envisagée. En effet, la production de protéines vaccinantes dans les plantes présente des avantages économiques considérables car les plantes sont plus faciles à cultiver que les cellules animales. En outre, les risques de contamination par les virus ou par les prions sont éliminés lorsque les protéines sont produites dans les plantes.Among these works, the use of higher eukaryotic hosts made it possible to obtain recombinant glycoproteins capable of conferring protective immunity in vivo, by oral route or by intramuscular or intraperitoneal injection. The use of transgenic plants for the production of rabies glycoprotein G has also been envisaged. Indeed, the production of vaccinating proteins in plants has considerable economic advantages because plants are easier to cultivate than animal cells. In addition, the risk of contamination by viruses or prions is eliminated when proteins are produced in plants.
McGarvey et al. (1995) ont décrit la transformation de cotylédons de tomates par le cADN entier du gène de la glycoproteine G du virus rabique (souche ERA) . Des plantes transgéniques ont été régénérées. L'expression d'une protéine, reconnue par des anticorps anti-glycoprotéine rabique, et apparaissant comme deux bandes distinctes de 62 et 60 kDa sur gel d'électrophorèse, a été constatée dans les feuilles et dans les fruits des plantes transgéniques. La différence de poids moléculaire par rapport à la glycoproteine naturelle est probablement liée, selon les auteurs, à une modification post-traductionnelle de la protéine, notamment à un clivage protéolytique ou à une glycosylation particulière.McGarvey et al. (1995) described the transformation of cotyledons of tomatoes by the whole cDNA of the glycoprotein G gene of the rabies virus (strain ERA). Transgenic plants have been regenerated. The expression of a protein, recognized by anti-rabies glycoprotein antibodies, and appearing as two distinct bands of 62 and 60 kDa on electrophoresis gel, was observed in the leaves and in the fruits of transgenic plants. The difference in molecular weight compared to natural glycoprotein is probably linked, according to the authors, to a post-translational modification of the protein, in particular to a proteolytic cleavage or to a particular glycosylation.
Jusqu'à ce jour, la production, dans des plantes, de la glycoproteine rabique ayant un poids moléculaire comparable à celui de la glycoproteine naturelle, n'a pas été décrite.To date, the production in plants of rabies glycoprotein having a molecular weight comparable to that of natural glycoprotein has not been described.
Le problème technique que se propose de résoudre la présente invention est de produire, dans une cellule végétale, la glycoproteine du virus de la rage, ou d'un virus apparenté au virus de la rage, conforme à la forme « insoluble » de la glycoproteine naturelle, notamment en ce qui concerne son poids moléculaire.The technical problem which the present invention proposes to solve is to produce, in a plant cell, the glycoprotein of the rabies virus, or of a virus related to the rabies virus, conforming to the “insoluble” form of the glycoprotein. natural, especially with regard to its molecular weight.
De manière surprenante, il a été constaté par les présents inventeurs que le remplacement du peptide signal N-terminal endogène de la glycoproteine rabique,
par un peptide signal N-terminal héterologue, permet d'obtenir des glycoprotéines ayant un poids moléculaire plus élevé que celles obtenues dans des plantes auparavant. Les présents inventeurs ont donc mis en oeuvre pour la transformation de la cellule végétale, une séquence codante chimérique comportant la séquence codante du gène de la glycoproteine rabique, dans laquelle la séquence du peptide signal N-terminal naturel a été remplacée par une séquence codant pour un peptide signal N-terminal héterologue.Surprisingly, it has been found by the present inventors that the replacement of the endogenous N-terminal signal peptide of rabies glycoprotein, by a heterologous N-terminal signal peptide, makes it possible to obtain glycoproteins having a higher molecular weight than those obtained in plants before. The present inventors have therefore used, for the transformation of the plant cell, a chimeric coding sequence comprising the coding sequence of the rabies glycoprotein gene, in which the sequence of the natural N-terminal signal peptide has been replaced by a sequence coding for a heterologous N-terminal signal peptide.
Plus particulièrement, la présente invention concerne un procédé pour la production de la glycoproteine G du virus de la rage, ou d'un virus apparenté au virus de la rage, caractérisé par : i) l'introduction, dans une cellule végétale, d'une molécule d'acide nucléique comprenant une séquence codante chimérique, comportant d'une part une séquence codant pour ladite protéine G virale mature, ou pour une protéine analogue, et d'autre part, une séquence codant pour un peptide signal N-terminal autre que celui naturellement associé à la protéine G virale, 1'introduction de la séquence codante chimérique permettant l'expression dans la cellule de la protéine G ; ii) la multiplication, sous forme de culture cellulaire, des cellules transformées, ou la régénération de plantes entières transgéniques ou chimériques à partir de ces cellules ; iii) éventuellement l'extraction et la purification de la glycoproteine G à partir des cellules ou des tissus végétaux ainsi obtenus.More particularly, the present invention relates to a process for the production of the rabies virus glycoprotein G, or of a virus related to the rabies virus, characterized by: i) the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence, comprising on the one hand a sequence coding for said mature viral protein G, or for an analogous protein, and on the other hand, a sequence coding for an other N-terminal signal peptide as that naturally associated with the viral protein G, the introduction of the chimeric coding sequence allowing the expression in the cell of the protein G; ii) the multiplication, in the form of cell culture, of the transformed cells, or the regeneration of whole transgenic or chimeric plants from these cells; iii) optionally the extraction and purification of the glycoprotein G from the cells or plant tissues thus obtained.
Dans le contexte de l'invention, les termes « virus de la rage, ou virus apparenté au virus de la rage » signifient les virus des différents sérotypes du genre Lvssavirus (Bourhy et al., 1993). A l'heure actuelle, les Lvssavirus sont divisés en quatre
sérotypes distincts : le sérotype i correspond au virus de la rage proprement dit et comprend différentes souches du virus de la rage, par exemple CVS, HEP, ERA, PM. Il existe normalement au moins 90% d'homologie au niveau de la séquence en acides aminés, entre les différentes souches de la rage. Les autres sérotypes correspondent aux virus apparentés au virus de la rage (« rabies - related viruses ») et sont exemplifiés par le virus du « Lagos bat » (sérotype 2) ; le virus Mokola (sérotype 3) ; le virus Duvenhage (sérotype 4) . Tous les virus rabiques sont pathogènes pour les mammifères, y compris l'homme, et donnent lieu à une encéphalite rabique.In the context of the invention, the terms “rabies virus, or virus related to the rabies virus” mean the viruses of the various serotypes of the genus Lvssavirus (Bourhy et al., 1993). Lvssaviruses are currently divided into four distinct serotypes: serotype i corresponds to the rabies virus itself and includes different strains of the rabies virus, for example CVS, HEP, ERA, PM. There is normally at least 90% homology in the amino acid sequence between the different strains of rabies. The other serotypes correspond to viruses related to the rabies virus (“rabies - related viruses”) and are exemplified by the “Lagos bat” virus (serotype 2); Mokola virus (serotype 3); Duvenhage virus (serotype 4). All rabies viruses are pathogenic to mammals, including humans, and give rise to rabies encephalitis.
Dans le contexte de la présente invention, le terme « glycoproteine G » signifie la glycoproteine constituant les spicules de l'enveloppe virale. Selon l'invention, elle est normalement sous forme monomerique mais peut également exister sous forme polymerique, par exemple en homodimer. Selon certains auteurs, la glycoproteine G est également appelée « l'antigène de surface » du virus, ainsi que « l'haemagglutinine », en raison de sa capacité de provoquer l'agglutination des érythrocytes.In the context of the present invention, the term “glycoprotein G” means the glycoprotein constituting the spicules of the viral envelope. According to the invention, it is normally in monomeric form but can also exist in polymeric form, for example in homodimer. According to some authors, glycoprotein G is also called the “surface antigen” of the virus, as well as “haemagglutinin”, because of its ability to cause the agglutination of erythrocytes.
La première étape de l'invention consiste en l'introduction, dans une cellule végétale, d'une molécule d'acide nucléique comprenant une séquence codante chimérique.The first step of the invention consists in the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence.
Selon l'invention, une « séquence codante chimérique » signifie une séquence d'acide nucléique, codant pour un enchaînement d'acides aminés, constituée de fragments peptidiques d'origines différentes. En l'espèce, il s'agit d'une séquence comportant, d'une part une séquence codant pour la glycoproteine G virale mature, c'est-à-dire dépourvue du signal N-terminal naturellement associée à la protéine, et d'autre part,
une séquence codant pour un peptide signal N-terminal héterologue à la glycoproteine.According to the invention, a “chimeric coding sequence” means a nucleic acid sequence, coding for an amino acid chain, consisting of peptide fragments of different origins. In this case, it is a sequence comprising, on the one hand, a sequence encoding the mature viral glycoprotein G, that is to say devoid of the N-terminal signal naturally associated with the protein, and d 'somewhere else, a sequence coding for a heterologous N-terminal signal peptide to glycoprotein.
Comme exemple de séquence codant pour la glycoproteine G mature, l'on peut citer celle de la glycoproteine rabique, souche CVS dont la séquence en acide nucléique et en acide aminés ont été décrites par Yelverton et al. (1983) , ou encore celle de la souche ERA dont la séquence a été décrite par Anilionis et al. (1981), et modifiée par Kieny et al. (1984). La protéine mature est dépourvue de la séquence de 19 acides aminés N-terminaux. D'autres séquences sont décrites par N. Tordo et al., Virol., 1993, 194, 59-69.As an example of a sequence coding for mature glycoprotein G, mention may be made of that of rabies glycoprotein, CVS strain, the nucleic acid and amino acid sequence of which have been described by Yelverton et al. (1983), or that of the ERA strain, the sequence of which has been described by Anilionis et al. (1981), and modified by Kieny et al. (1984). The mature protein lacks the 19 N-terminal amino acid sequence. Other sequences are described by N. Tordo et al., Virol., 1993, 194, 59-69.
La séquence du gène de la glycoproteine G du virus Mokola a été décrite par Tordo et al. (1993) . La séquence de la protéine mature démarre à l'acide aminé 20.The sequence of the Mokola virus glycoprotein G gene has been described by Tordo et al. (1993). The mature protein sequence starts at amino acid 20.
L'acide nucléique codant pour la glycoproteine G mature est obtenu par l'élimination de la partie de la séquence qui code pour le peptide signal N-terminal.The nucleic acid coding for mature glycoprotein G is obtained by eliminating the part of the sequence which codes for the N-terminal signal peptide.
La séquence codante peut coder pour une protéine « analogue » à la glycoproteine G. Les protéines analogues sont des protéines qui présentent normalement au moins 90% d'homologie avec la glycoproteine naturelle, et qui présentent en outre une homologie fonctionnelle et immunolbgique avec la glycoproteine naturelle.The coding sequence can code for a protein “analogous” to glycoprotein G. Analogous proteins are proteins which normally have at least 90% homology with natural glycoprotein, and which also have functional and immunological homology with glycoprotein natural.
Selon l'invention, la séquence codant pour le peptide signal N-terminal endogène est donc remplacée par un signal héterologue. Comme exemple de séquence codant pour un peptide signal N-terminal héterologue, l'on peut citer un peptide signal (ou « pré-peptide ») végétal, ou provenant d'un virus de plante, ou d'un micro-organisme, par exempe la levure.According to the invention, the sequence coding for the endogenous N-terminal signal peptide is therefore replaced by a heterologous signal. As an example of a sequence coding for a heterologous N-terminal signal peptide, mention may be made of a plant signal peptide (or “pre-peptide”), or originating from a plant virus, or from a microorganism, by exempts yeast.
Par « peptide signal N-terminal », il faut comprendre le peptide responsable de l'addressage de la protéine naissante dans le réticulum endoplasmique.
Dans la cellule végétale, l'adressage des protéines est basé sur le même principe que dans les cellules animales. A partir de l'ADN chromosomique, le gène est transcrit en ARN messager, puis traduit en protéine au niveau des ribosomes. Si la protéine naissante possède un peptide signal N-terminal ou prépeptide, elle pénètre dans le réticulum endoplasmique où ont lieu un certain nombre de maturations post-traductionnelles, notamment le clivage du peptide signal, les N- glycosylations conduisant aux glycannes polymannosidiques, et la formation des ponts disulfures.By "N-terminal signal peptide" is meant the peptide responsible for addressing the nascent protein in the endoplasmic reticulum. In the plant cell, the addressing of proteins is based on the same principle as in animal cells. From chromosomal DNA, the gene is transcribed into messenger RNA, then translated into protein at the ribosome level. If the nascent protein has an N-terminal signal peptide or prepeptide, it enters the endoplasmic reticulum where a number of post-translational maturations take place, in particular the cleavage of the signal peptide, the N-glycosylations leading to polymannosidic glycans, and the formation of disulfide bridges.
Selon l'invention, la séquence codant pour le prépeptide, responsable de l'adressage de la protéine dans le réticulum endoplasmique, fait partie de la séquence codante chimérique. Il s'agit normalement d'un peptide signal N-terminal hydrophobe ayant entre 10 et 40 acides aminés et étant d'origine animale ou végétale. De préférence, il s'agit d'un prépeptide d'origine végétale, par exemple celui de la sporamine, de la lectine d'orge, de l'extensine végétale (pEXT) , de l'α-mating (factor, des protéines végétales impliquées dans la défense contre les micro-organismes (PRla et PRS, "pathogenesis related proteins").According to the invention, the sequence coding for the prepeptide, responsible for addressing the protein in the endoplasmic reticulum, is part of the chimeric coding sequence. It is normally a hydrophobic N-terminal signal peptide having between 10 and 40 amino acids and being of animal or vegetable origin. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, plant extensin (pEXT), α-mating (factor, proteins plants involved in defense against microorganisms (PRla and PRS, "pathogenesis related proteins").
L'introduction dans la cellule végétale de la molécule d'acide nucléique, comportant la séquence codante chimérique, est effectuée de manière à ce que l'expression de la séquence puisse être obtenue. En particulier, la séquence codante chimérique doit se trouver, dans le génome de la plante, sous le contrôle de séquences régulatrices de la transcription, reconnues par la cellule végétale.The introduction into the plant cell of the nucleic acid molecule, comprising the chimeric coding sequence, is carried out so that expression of the sequence can be obtained. In particular, the chimeric coding sequence must be found, in the genome of the plant, under the control of transcriptional regulatory sequences recognized by the plant cell.
Ces séquences régulatrices, notamment un signal d'initiation de transcription (promoteur) et un signal de terminaison de transcription (comprenant le signal de polyadénylation) , peuvent être endogènes à la
plante, auquel cas, l'insertion de la séquence codante chimérique doit se faire par recombinaison homologue. Dans ce cas, l'acide nucléique transformant comporte, sur chaque extrémité, une séquence homologue aux séquences qui jouxtent le site d'insertion souhaité dans le génome.These regulatory sequences, in particular a transcription initiation signal (promoter) and a transcription termination signal (comprising the polyadenylation signal), may be endogenous to the plant, in which case, the insertion of the chimeric coding sequence must be done by homologous recombination. In this case, the transforming nucleic acid comprises, on each end, a sequence homologous to the sequences which adjoin the desired site of insertion into the genome.
Alternativement, les séquences régulatrices de transcription peuvent être incluses dans la molécule d'acide nucléique comportant la séquence codante chimérique. Dans ce cas, la molécule d'acide nucléique constitue un gène chimérique qui fait également partie de l'invention. Les séquences régulatrices comprennent un ou plusieurs promoteurs d'origine végétale ou provenant d'Agrobacterium tumefaciens ou d'un virus de plante. Il peut s'agir d'un promoteur constitutif, par exemple le 35S ou le double 35S du CaMV, NOS, OCS, ou des promoteurs spécifiques de certains tissus comme le grain, ou spécifiques de certaines phases de développement de la plante.Alternatively, the transcription regulatory sequences can be included in the nucleic acid molecule comprising the chimeric coding sequence. In this case, the nucleic acid molecule constitutes a chimeric gene which is also part of the invention. The regulatory sequences include one or more promoters of plant origin or from Agrobacterium tumefaciens or from a plant virus. It can be a constitutive promoter, for example 35S or double 35S of CaMV, NOS, OCS, or specific promoters of certain tissues such as grain, or specific of certain phases of plant development.
Comme promoteurs spécifiques de graines, on peut citer le promoteur du gène de la napin et de l'acyl carrier protéine (ACP) (EP-A-0255378) , ainsi que les promoteurs des gènes AT2S d'Arabidopsis thaliana, c'est-à-dire les promoteurs PAT2S1, PAT2S2, PAT2S3 et PAT2S4 (Krebbers et al., Plant Physiol., 1988, vol. 87, pages 859-866). Il est particulièrement préféré d'utiliser le promoteur de la cruciférine ou de la phaséoline, les promoteurs pGEAl et pGEA6 promoteurs d'Arabidopsis de gène de type "EM, Early Methionine labelled protein" fortement exprimés au cours des phases de dessication de la graine.As specific seed promoters, mention may be made of the promoter of the napin gene and of the acyl carrier protein (ACP) (EP-A-0255378), as well as the promoters of the AT2S genes of Arabidopsis thaliana, ie ie the promoters PAT2S1, PAT2S2, PAT2S3 and PAT2S4 (Krebbers et al., Plant Physiol., 1988, vol. 87, pages 859-866). It is particularly preferred to use the cruciferin or phaseoline promoter, the pGEA1 and pGEA6 promoters Arabidopsis promoters of the "EM, Early Methionine labeled protein" type gene which are highly expressed during the desiccation phases of the seed.
Il peut être envisagé d'utiliser des "enhancers" pour améliorer l'efficacité d'expression.It can be envisaged to use "enhancers" to improve the efficiency of expression.
Les séquences régulatrices de terminaison sont d'origine végétale, virale, ou bactérienne, par exemple du 35S, NOS etc..
Selon une variante de l'invention, la séquence codante chimérique comprend, outre la partie peptide signal N-terminal et la partie codant pour la protéine d'enveloppe mature, d'autres signaux d'adressage, par exemple un signal de rétention endoplasmique ou un signal d'adressage vacuolaire.The termination regulatory sequences are of plant, viral, or bacterial origin, for example 35S, NOS, etc. According to a variant of the invention, the chimeric coding sequence comprises, in addition to the N-terminal signal peptide part and the part coding for the mature envelope protein, other addressing signals, for example an endoplasmic retention signal or a vacuolar addressing signal.
Le signal de rétention endoplasmique consiste en les peptides KDEL, SEKDEL ou HKDEL. Ces signaux se trouvent normalement à l'extrémité C-terminale de la protéine et subsistent sur la protéine mature. La présence de ce signal sur les protéines de l'invention est avantageuse pour plusieurs raisons : d'une part, la rétention de la protéine dans le réticulum endoplasmique a tendance à augmenter les rendements en protéines recombinantes. D'autre part, la maturation de la glycosylation polymannosique en glycannes complexes n'a pas lieu ; la protéine conserve donc la glycosylation polymannosique, minimisant le risque de réactions immunologiques indésirables lorsque la protéine sera administrée à l'homme comme médicament. Selon cette variante de l'invention, le glycoproteine comporte donc la séquence en acides aminés de la protéine G, un signal du type KDEL, au moins un glycanne de type polymannosique, et est exempt de glycannes de type complexe. Selon l'invention, ce type de protéine peut aussi être obtenu en utilisant des mutants de plantes incapables de fabriquer la N-acétyl glucosaminyl transférase (von Schaewen et al., Plant Physiol. 1993 102: 1109-1118), et donc incapables de produire des glycannes complexes.The endoplasmic retention signal consists of the peptides KDEL, SEKDEL or HKDEL. These signals are normally found at the C-terminus of the protein and remain on the mature protein. The presence of this signal on the proteins of the invention is advantageous for several reasons: on the one hand, the retention of the protein in the endoplasmic reticulum tends to increase the yields of recombinant proteins. On the other hand, the maturation of polymannosic glycosylation into complex glycans does not take place; the protein therefore retains polymannosic glycosylation, minimizing the risk of undesirable immunological reactions when the protein is administered to humans as a medicine. According to this variant of the invention, the glycoprotein therefore comprises the amino acid sequence of the protein G, a signal of the KDEL type, at least one glycan of the polymannosic type, and is free of glycans of the complex type. According to the invention, this type of protein can also be obtained by using plant mutants incapable of manufacturing N-acetyl glucosaminyl transferase (von Schaewen et al., Plant Physiol. 1993 102: 1109-1118), and therefore incapable of produce complex glycans.
La protéine de l'invention peut, outre le prépeptide, comporter aussi un signal d'adressage vacuolaire ou "propeptide". En présence d'un tel signal, la protéine est adressée aux vacuoles des tissus aqueux, par exemple les feuilles, ainsi qu'aux corps protéiques des tissus de réserve, par exemple
les graines, tubercules et racines. L'adressage de la protéine vers les corps protéiques de la graine est particulièrement intéressant en raison de la capacité de la graine à accumuler des protéines, jusqu'à 40% des protéines par rapport à la matière sèche, dans des organites cellulaires dérivés des vacuoles, appelés corps protéiques et en raison de la possibilité de stocker plusieurs années les graines contenant les protéines recombinantes à l'état déshydraté.The protein of the invention can, in addition to the prepeptide, also include a vacuolar addressing signal or "propeptide". In the presence of such a signal, the protein is addressed to the vacuoles of the aqueous tissues, for example the leaves, as well as to the protein bodies of the reserve tissues, for example seeds, tubers and roots. The targeting of the protein to the protein bodies of the seed is particularly interesting because of the capacity of the seed to accumulate proteins, up to 40% of the proteins relative to the dry matter, in cellular organelles derived from vacuoles. , called protein bodies and because of the possibility of storing for several years the seeds containing the recombinant proteins in the dehydrated state.
Comme propeptide, on peut utiliser un signal d'origine animale ou végétale, les signaux végétaux étant particulièrement préférés, par exemple la pro- sporamine, ou la lectine d'orge. Le propeptide peut être N-terminal ("N-terminal targeting peptide" ou NTTP) , ou C terminal (CTTP) ou peut consister en une séquence interne à la protéine. Dans la mesure où le propeptide est normalement clivé dès l'entrée de la protéine dans la vacuole, il n'est pas présent dans la protéine mature. Il a été constaté que l'utilisation du « propeptide », en association avec un prépeptide, est particulièrement avantageux, et donne lieu à des glycoprotéines ayant un poids moléculaire conforme à la molécule naturelle.As propeptide, use may be made of a signal of animal or vegetable origin, plant signals being particularly preferred, for example proposporamine, or barley lectin. The propeptide can be N-terminal ("N-terminal targeting peptide" or NTTP), or C terminal (CTTP) or can consist of a sequence internal to the protein. Since the propeptide is normally cleaved upon entry of the protein into the vacuole, it is not present in the mature protein. It has been found that the use of the "propeptide", in combination with a prepeptide, is particularly advantageous, and gives rise to glycoproteins having a molecular weight in accordance with the natural molecule.
De préférence, une séquence codant pour un agent permettant la sélection des cellules transformées est introduite dans la cellule végétale en même temps que les séquences codant pour la glycoproteine. Le gène codant pour l'agent de sélection peut être un gène chimérique constitué de séquences régulatrices reconnues par la plante en association avec la séquence codante de l'agent de sélection, par exemple NPT I, NPT II, dhfr, etc. Le gène chimérique de sélection peut faire partie du même vecteur que celui codant pour la glycoproteine. Alternativement, il peut être porté par un vecteur indépendant et introduit par co- transformation.
Pour introduire les différentes séquences hétérologues dans la cellule végétale, tous les moyens connus pour transformer le génome nucléaire peuvent être utilisés, par exemple Agrobacterium, électroporation, fusion de protoplastes, bombardement avec canon à particules, ou pénétration d'ADN dans des cellules comme le pollen, la microspore, la graine et l'embryon immature, vecteurs viraux tels que les Geminivirus ou les virus satellites. Agrobacterium tumefaciens et rhizogenes constituent le moyen préféré. Dans ce cas, la séquence de l'invention est introduite dans un vecteur approprié avec toutes les séquences régulatrices nécessaires tels que promoteurs, terminateurs, etc... ainsi que toute séquence nécessaire pour sélectionner les transformants.Preferably, a sequence coding for an agent allowing the selection of the transformed cells is introduced into the plant cell at the same time as the sequences coding for the glycoprotein. The gene coding for the selection agent can be a chimeric gene made up of regulatory sequences recognized by the plant in association with the coding sequence of the selection agent, for example NPT I, NPT II, dhfr, etc. The chimeric selection gene can be part of the same vector as that coding for glycoprotein. Alternatively, it can be carried by an independent vector and introduced by co-transformation. To introduce the different heterologous sequences into the plant cell, all known means for transforming the nuclear genome can be used, for example Agrobacterium, electroporation, fusion of protoplasts, bombardment with a particle gun, or penetration of DNA into cells such as pollen, microspore, seed and immature embryo, viral vectors such as Geminiviruses or satellite viruses. Agrobacterium tumefaciens and rhizogenes are the preferred means. In this case, the sequence of the invention is introduced into an appropriate vector with all the necessary regulatory sequences such as promoters, terminators, etc., as well as any sequence necessary for selecting the transformants.
Après l'étape de transformation des cellules végétales, les cellules transformées sont sélectionnées grâce à la présence du gène codant pour l'agent de sélection. Ces cellules sont ensuite soumises à une étape de multiplication par culture cellulaire, où elles sont régénérées en plantes transgéniques ou chimériques.After the stage of transformation of plant cells, the transformed cells are selected by virtue of the presence of the gene coding for the selection agent. These cells are then subjected to a step of multiplication by cell culture, where they are regenerated into transgenic or chimeric plants.
Lorsqu'il s'agit de multiplication par culture cellulaire in vitro, l'on obtient une biomasse capable de produire la glycoproteine G, en grande quantité. Il peut s'agir de cultures de cellules végétales in vitro, par exemple en milieu liquide. Différents modes de culture ("batch", "fed batch" ou en continu) pour ce type de cellules sont actuellement à l'étude. Les cultures en "batch" sont comparables à celles effectuées en erlenmeyer dans la mesure où le milieu n'est pas renouvelé, les cellules ne disposent ainsi que d'une quantité limitée d'éléments nutritifs. La culture en "fed batch" correspond quant à elle à une culture en "batch" avec une alimentation programmée en
substrat. Pour une culture en continu, les cellules sont alimentées en permanence avec du milieu nutritif. Un volume égal du mélange biomasse-milieu est ôté afin de maintenir le volume du réacteur constant. Les quantités de biomasse végétale envisageables avec des cultures en bioréacteurs sont variables selon l'espèce végétale, le mode de culture et le type de bioréacteur. Dans certaines conditions, des densités de biomasse d'environ 10 à 30g de poids sec par litre de culture peuvent être obtenus, pour des espèces comme Nicotiana tabacum, Vinca rosea et Catharanthus roseus.When it is a question of multiplication by cell culture in vitro, one obtains a biomass capable of producing glycoprotein G, in large quantity. They can be cultures of plant cells in vitro, for example in a liquid medium. Different culture modes ("batch", "fed batch" or continuous) for this type of cells are currently being studied. Batch cultures are comparable to those carried out in an Erlenmeyer flask insofar as the medium is not renewed, the cells thus have only a limited quantity of nutritive elements. The "fed batch" culture corresponds to a "batch" culture with a programmed feeding in substrate. For a continuous culture, the cells are continuously supplied with nutritive medium. An equal volume of the biomass-medium mixture is removed in order to keep the volume of the reactor constant. The quantities of plant biomass that can be envisaged with cultures in bioreactors vary according to the plant species, the culture method and the type of bioreactor. Under certain conditions, biomass densities of around 10 to 30 g of dry weight per liter of culture can be obtained, for species such as Nicotiana tabacum, Vinca rosea and Catharanthus roseus.
Les cellules de l'invention peuvent aussi être immobilisées, ce qui permet d'obtenir une production constante et prolongée de la glycoproteine. Comme méthode d'immobilisation, on peut citer l'immobilisation en billes d'alginate, d'agar, à l'intérieur de mousse de polyuréthane, ou bien encore dans des fibres creuses.The cells of the invention can also be immobilized, which makes it possible to obtain a constant and prolonged production of the glycoprotein. As immobilization method, mention may be made of immobilization in alginate or agar beads, inside polyurethane foam, or even in hollow fibers.
Les cellules de l'invention peuvent également être des cultures de racines. Les racines cultivées in vitro, en milieu liquide, sont nommées "Hairy roots", ce sont des racines transformées par la bactérie Agrobacterium rhizoσenes.The cells of the invention can also be root cultures. The roots cultivated in vitro, in liquid medium, are called "Hairy roots", they are roots transformed by the bacterium Agrobacterium rhizoσenes.
Au lieu de produire la glycoproteine de l'invention par culture de cellules végétales, on peut régénérer des plantes chimériques ou transgéniques à partir d'expiants transformés, en ayant recours à des techniques connues en soi.Instead of producing the glycoprotein of the invention by culturing plant cells, it is possible to regenerate chimeric or transgenic plants from transformed explants, by using techniques known per se.
Le procédé de l'invention peut ou non comprendre une étape de récupération de la glycoproteine G. En l'absence d'une étape de récupération, le procédé de l'invention donne lieu à des cellules végétales, ou à des plantes transgéniques capables de produire la glycoproteine G. Les cellules ou les plantes constituent en soi une source de la protéine vaccinante
et peuvent être administrées telles quelles à l'animal ou à l'homme par ingestion.The process of the invention may or may not include a step for recovering glycoprotein G. In the absence of a recovery step, the process of the invention gives rise to plant cells, or to transgenic plants capable of produce glycoprotein G. Cells or plants are in themselves a source of the vaccinating protein and can be administered as such to animals or humans by ingestion.
Selon une autre variante de l'invention, la glycoproteine G est récupérée par extraction de la matière végétale, et éventuellement purifiée.According to another variant of the invention, the glycoprotein G is recovered by extraction from the plant material, and optionally purified.
L'étape d'extraction comprend la solubilisation de la glycoproteine, associée aux membranes cellulaires, par un détergent. Plus particulièrement, la matière végétale, par exemple, feuilles, graines, racines etc., est broyée dans l'azote liquide. L'homogénat est alors suspendu dans un tampon approprié, additionné d'un détergent, de préférence non-ionique. Le détergent est employé à des concentrations de 0.1 à 2%, de préférence 0.1 à 0.5%. Il peut s'agir par exemple deThe extraction step comprises the solubilization of the glycoprotein, associated with the cell membranes, with a detergent. More particularly, the plant material, for example, leaves, seeds, roots, etc., is ground in liquid nitrogen. The homogenate is then suspended in an appropriate buffer, added with a detergent, preferably nonionic. The detergent is used in concentrations of 0.1 to 2%, preferably 0.1 to 0.5%. It can for example be
Triton X-100, ou NP40 ou β-octyl glucopyranoside. L'emploi du détergent libère la glycoproteine des membranes cellulaires, tout en conservant son intégrité et son pouvoir immunogène. L'homogénat ainsi obtenu est centrifugé, la glycoproteine « solubilisée » étant alors présente dans le surnageant. Normalement, le surnageant est filtré.Triton X-100, or NP40 or β-octyl glucopyranoside. The use of detergent releases glycoprotein from cell membranes, while retaining its integrity and immunogenic power. The homogenate thus obtained is centrifuged, the “solubilized” glycoprotein then being present in the supernatant. Normally, the supernatant is filtered.
L'extrait végétal correspondant au surnageant peut être utilisé tel quel comme source de protéine vaccinante, ou peut être soumis à une ou plusieurs étape(s) de purification. Eventuellement, le détergent peut être éliminé ou réduit. De préférence, l'extrait de matière végétale, par exemple feuilles de tabac ou graines de colza, est dialyse contre un tampon éthylène diamine acide acétique pH 8.1, ou est soumis à une gel filtration sur support Séphadex G25 Pharmacia. La fraction correspondant au rétentat ou au volume mort est ensuite soumise à une chromatographie d'échange d'ion sur support QAE Séphadex A50 Pharmacia équilibré préalablement en tampon éthylène diamine acide acétique pH 8.1. Après lavage avec ce même tampon, la
glycoproteine est éluée par une solution 0.6 M acétate de sodium (P. Atanasiu et al., 1976).The plant extract corresponding to the supernatant can be used as such as a source of vaccinating protein, or can be subjected to one or more purification step (s). Optionally, the detergent can be eliminated or reduced. Preferably, the extract of plant material, for example tobacco leaves or rapeseed, is dialyzed against an ethylene diamine acetic acid buffer pH 8.1, or is subjected to a filtration gel on a Sephadex G25 Pharmacia support. The fraction corresponding to the retentate or to the dead volume is then subjected to an ion exchange chromatography on a QAE Sephadex A50 Pharmacia support equilibrated beforehand in ethylene diamine acetic acid buffer pH 8.1. After washing with this same pad, the glycoprotein is eluted with a 0.6 M sodium acetate solution (P. Atanasiu et al., 1976).
D'autres techniques de purification sont décrites dans l'art, par exemple chromatographie d'affinité utilisant un anticorps monoclonal purifié couplé à un support Sépharose CL-4B activé au bromure de cyanogène (B. Dietzschold et al., 1983) ; chromatographie de gel perméation sur support Sépharose CL-4B (G. Coslett et al., 1980) ; technique d'électrofocalisation (J. Cox et al., 1980).Other purification techniques are described in the art, for example affinity chromatography using a purified monoclonal antibody coupled to a Sepharose CL-4B support activated with cyanogen bromide (B. Dietzschold et al., 1983); gel permeation chromatography on Sepharose CL-4B support (G. Coslett et al., 1980); electrofocusing technique (J. Cox et al., 1980).
L'invention vise également la glycoproteine G susceptible d'être obtenue par la mise en oeuvre du procédé de l'invention.The invention also relates to the glycoprotein G capable of being obtained by implementing the method of the invention.
Plus particulièrement, l'invention vise une glycoproteine caractérisée en ce que :More particularly, the invention relates to a glycoprotein characterized in that:
- elle est reconnue par des anticorps spécifiques à la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage ; elle a un poids moléculaire de 66 kDa approximativement ;- it is recognized by antibodies specific to the glycoprotein G of the rabies virus or of a virus related to the rabies virus; it has a molecular weight of approximately 66 kDa;
- elle est insoluble ;- it is insoluble;
- elle est N-glycosylée par au moins un glycanne de type polymannosidique, et/ou par au moins un glycanne de type complexe comportant au sein de sa structure un ou plusieurs résidus de xylose β-1,2, et/ou un ou plusieurs résidus de fucose lié en α-1,3, et étant dépourvu de résidus d'acide sialique.- It is N-glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues β-1,2, and / or one or more residues of fucose linked in α-1,3, and being free of residues of sialic acid.
Les propriétés antigéniques de la glycoproteine de l'invention sont, au moins en partie, celles de la glycoproteine correspondante naturelle. En effet, la glycoproteine de 1•invention est reconnue par des anticorps spécifiques à la glycoproteine de la rage ou d'un virus apparenté à la rage. Il peut s'agir d'anticorps polyclonaux ou monoclonaux dirigés contre
le virus, ou contre la glycoproteine purifiée à partir du virus.The antigenic properties of the glycoprotein of the invention are, at least in part, those of the corresponding natural glycoprotein. Indeed, the glycoprotein 1 • invention is recognized by antibodies specific to the glycoprotein of rabies virus or related to rabies. They can be polyclonal or monoclonal antibodies against the virus, or against glycoprotein purified from the virus.
La glycoproteine de l'invention a un poids moléculaire de 66 kDa approximativement. Dans le contexte de la présente invention, « approximativement » signifie la variabilité résultant des techniques de mesure. Plus particulièrement, la glycoproteine de l'invention a un poids moléculaire de 66 kDa en moyenne lorsque plusieurs essais de Western Blot sont effectués, par exemple, au moins 3 ou 5 essais, dans les mêmes conditions. Par exemple le poids moléculaire peut être entre 65 et 80 kDa, par exemple 66 à 67 kDa. Le poids moléculaire est un poids moléculaire apparent, déterminé par électrophorèse sur gel de polyacrylamide en conditions dénaturantes, selon la technique de Laemmli et al. (1970) . De préférence, le gel de polyacrylamide est un gel à 10 ou 12.5%, mais des concentrations moins élevées peuvent être utilisées pour obtenir une résolution plus nette des zones entre 60 et 65 kDa.The glycoprotein of the invention has a molecular weight of approximately 66 kDa. In the context of the present invention, "approximately" means the variability resulting from measurement techniques. More particularly, the glycoprotein of the invention has a molecular weight of 66 kDa on average when several Western Blot tests are carried out, for example, at least 3 or 5 tests, under the same conditions. For example, the molecular weight can be between 65 and 80 kDa, for example 66 to 67 kDa. The molecular weight is an apparent molecular weight, determined by electrophoresis on polyacrylamide gel under denaturing conditions, according to the technique of Laemmli et al. (1970). Preferably, the polyacrylamide gel is a 10 or 12.5% gel, but lower concentrations can be used to obtain a clearer resolution of the areas between 60 and 65 kDa.
La glycoproteine de l' invention est fortement insoluble. Dans le contexte de l'invention, le terme « insoluble » signifie que la molécule n'est pas soluble dans le milieu exracellulaire, mais se trouve associée aux membranes cellulaires. En effet, il a été constaté que la présence de détergent, par exemple SDS, Triton X-100, est indispensable pour extraire et solubiliser la glycoproteine. La nature « insoluble » de la glycoproteine est liée à la présence du domaine transmembranaire C-terminal. La présence de la région C-terminale, c'est-à-dire la région se trouvant environ 40 à 60 acides aminés de l'extrémité carboxy de la glycoproteine, est importante pour obtenir une réponse protectrice lorsque la glycoproteine est utilisée en tant que vaccin.
La glycoproteine de l'invention se montre insoluble indirectement par son association aux membranes cellulaires. En l'absence de détergent, elle ne peut pas se trouver dans le surnageant de cultures de cellules produisant la glycoproteine.The glycoprotein of the invention is highly insoluble. In the context of the invention, the term “insoluble” means that the molecule is not soluble in the exracellular medium, but is found associated with cell membranes. Indeed, it has been found that the presence of detergent, for example SDS, Triton X-100, is essential to extract and dissolve the glycoprotein. The “insoluble” nature of glycoprotein is linked to the presence of the C-terminal transmembrane domain. The presence of the C-terminal region, i.e., the region located approximately 40 to 60 amino acids from the carboxy end of the glycoprotein, is important to obtain a protective response when the glycoprotein is used as vaccine. The glycoprotein of the invention is indirectly insoluble by its association with cell membranes. In the absence of a detergent, it cannot be in the supernatant of cell cultures producing glycoprotein.
La glycoproteine de l'invention est glycosylée par au moins un glycanne de type polymannosidique, et/ou par au moins un glycanne de type complexe comportant au sein de sa structure un ou plusieurs résidus de xylose β-1,2, et/ou un ou plusieurs résidus de fucose lié en α-1,3, et étant dépourvu de résidus d'acide sialique.The glycoprotein of the invention is glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues β-1,2, and / or a or several residues of fucose linked in α-1,3, and being free of sialic acid residues.
La glycosylation de glycoprotéines produites dans des cellules végétales est différente de celles produites dans des cellules animales. Bien que les étapes de glycosylation conduisant aux glycannes polymannosidiques soient réalisées de manière identique chez les plantes et chez les mammifères, la maturation des glycannes polymannosidiques en glycannes complexes est très différente. Les N- glycannes des glycoprotéines végétales diffèrent principalement des glycannes de mammifères par l'absence d'acide sialique, également connu sous le nom d'acide N-acétylneuraminique, et la présence d'un résidu de β 1,2 xylose et d'un résidu de fucose lié en α 1,3 au résidu de GlcNAc proximal du "core" (Driouich et al., Regard sur la biochimie, 1993, vol. 3, pages 33-42) .The glycosylation of glycoproteins produced in plant cells is different from those produced in animal cells. Although the glycosylation steps leading to the polymannosidic glycans are carried out identically in plants and in mammals, the maturation of the polymannosidic glycans into complex glycans is very different. The N-glycans of vegetable glycoproteins differ mainly from mammalian glycans by the absence of sialic acid, also known as N-acetylneuraminic acid, and the presence of a residue of β 1,2 xylose and a fucose residue linked in α 1,3 to the GlcNAc residue proximal to the "core" (Driouich et al., Regard sur la biochemimie, 1993, vol. 3, pages 33-42).
La glycosylation joue un rôle important dans la mise en place de la structure spatiale de la protéine, dans la protection contre les attaques protéolytiques et dans les mécanismes de reconnaissance immunitaire.Glycosylation plays an important role in the establishment of the spatial structure of the protein, in protection against proteolytic attacks and in the mechanisms of immune recognition.
Selon une variante préférée, la protéine de l'invention est glycosylée à au moins deux sites naturels de N-glycosylation. Par exemple, pour la
souche ERA, la glycosylation est de préférence aux positions 247 et 319. La glycosylation est de type mannosidique, polymannosidique ou de type complexe, ou un mélange des deux.According to a preferred variant, the protein of the invention is glycosylated at at least two natural N-glycosylation sites. For example, for the strain ERA, the glycosylation is preferably at positions 247 and 319. The glycosylation is of mannosidic, polymannosidic or complex type, or a mixture of both.
Le ou les glucanne(s) de type mannosidique ont la structure GlcNAc2-Manl. Le ou les glucanne(s) polymannosidique(s) ont la structure GlcNAc2-Man2-9, par exemple GlcNAc2-Man9, GlcNAc2-Man8, GlcNAc2-Man6 ou GlcNAc2-Man5, ou GlcNAc2-Man3.The mannosidic glucan (s) has the GlcNAc2-Manl structure. The polymannosidic glucan (s) has the structure GlcNAc2-Man2-9, for example GlcNAc2-Man9, GlcNAc2-Man8, GlcNAc2-Man6 or GlcNAc2-Man5, or GlcNAc2-Man3.
Le ou les glucanne(s) de type complexe sont biantennés et ont une structure de base GlcNAc2-Man3 à laquelle sont éventuellement associés des résidus de xylose (Xyl) , fucose (Fuc) et éventuellement galactose (Gai) ou N-acétylglucosamine (GlcNAc) . Ils sont normalement exempts de résidus d'acide sialique, ce composé n'ayant pas jusqu'alors été mis en évidence dans les cellules végétales.The glucan (s) of complex type are biantennary and have a basic structure GlcNAc2-Man3 with which are optionally associated residues of xylose (Xyl), fucose (Fuc) and possibly galactose (Gai) or N-acetylglucosamine (GlcNAc ). They are normally free of sialic acid residues, this compound having not hitherto been detected in plant cells.
De préférence, le glycanne complexe est de type "phytohémagglutinine" (PHA) constitué d'une structurePreferably, the complex glycan is of the "phytohemagglutinin" (PHA) type consisting of a structure
"core" GlcNAc2Man3 dont le mannose lié en β porte un résidu de β 1,2-xylose et dont le GlcNAc proximal porte un résidu d'α 1,3 fucose. Ce genre de structure est fréquent pour les glycoprotéines de localisation vacuolaire ou extracellulaire. Le résidu de fucose α 1,3-lié peut éventuellement être absent."core" GlcNAc2Man3 whose β-linked mannose carries a residue of β 1,2-xylose and whose proximal GlcNAc carries a residue of α 1.3 fucose. This kind of structure is frequent for glycoproteins of vacuolar or extracellular localization. The 1,3-linked α-fucose residue may possibly be absent.
Le glycanne complexe peut aussi être du type "Laccase" composé d'une structure de base GlcNAc2 (Fuc) Man3 (Xyl) associée à deux chaînes latérales. Chaque chaîne latérale est constituée d'un résidu de β 1,2 GlcNAc auquel est lié un résidu d'α 1,6 fucose et un résidu de β 1,4 galactose.The complex glycan can also be of the "Laccase" type composed of a basic structure GlcNAc2 (Fuc) Man3 (Xyl) associated with two side chains. Each side chain consists of a residue of β 1.2 GlcNAc to which is linked a residue of α 1.6 fucose and a residue of β 1.4 galactose.
La glycoproteine de l'invention peut être glycosylée aux trois sites naturels ou seulement à l'un ou deux d'entre eux. Parmi les variantes
préférées, l'on peut citer des glycoprotéines portant exclusivement des glycannes polymannosidiques. Ce type de glycosylation existe sous forme identique chez la plante et chez l'animal. Par conséquent, l'apparition de réaction immunologique indésirable est évitée. Ce type de glycosylation est obtenue par l'utilisation d'un peptide signal N-terminal en association avec un signal de rétention endoplasmique.The glycoprotein of the invention can be glycosylated at the three natural sites or only at one or two of them. Among the variants preferred, mention may be made of glycoproteins carrying exclusively polymannosidic glycans. This type of glycosylation exists in identical form in plants and animals. Therefore, the appearance of an adverse immunological reaction is avoided. This type of glycosylation is obtained by the use of an N-terminal signal peptide in association with an endoplasmic retention signal.
On peut aussi citer les glycoprotéines portant exclusivement des glycannes de type complexe, par exemple deux ou trois glycannes de type PHA : GlcNAc2(Fuc)Man3(Xyl) .Mention may also be made of glycoproteins carrying exclusively glycans of the complex type, for example two or three glycans of the PHA type: GlcNAc2 (Fuc) Man3 (Xyl).
La partie glucidique représente, normalement, entre 2 et 30%, par exemple 5 à 20% de la masse totale de la glycoproteine de l'invention, la molécule naturelle comportant 11% de glycosylation.The carbohydrate part normally represents between 2 and 30%, for example 5 to 20% of the total mass of the glycoprotein of the invention, the natural molecule comprising 11% of glycosylation.
La composition des glycannes de la glycoproteine peut être déterminée par digestion des oligosaccharides par N-glycanase, telle que décrite par Tuchiya K. et al. (1992) .The composition of glycoprotein glycans can be determined by digestion of oligosaccharides with N-glycanase, as described by Tuchiya K. et al. (1992).
La glycoproteine de l'invention est capable d'induire la formation d'anticorps neutralisants et une immunité protectrice contre le virus de la rage ou contre un virus apparenté au virus de la rage.The glycoprotein of the invention is capable of inducing the formation of neutralizing antibodies and protective immunity against the rabies virus or against a virus related to the rabies virus.
Le caractère protecteur de la glycoproteine est mis en évidence par l'administration, à un animal, du vaccin contenant la glycoproteine, suivie par une épreuve virulente avec le virus rabique. Le taux d'anticorps neutralisants produit chez l'animal est déterminé par un test tel que le test RFFIT (« Rapid Fluorescence Focus Inhibition Test » décrit par Smith et al. (1973) .The protective character of glycoprotein is demonstrated by the administration, to an animal, of the vaccine containing glycoprotein, followed by a virulent test with the rabies virus. The level of neutralizing antibodies produced in animals is determined by a test such as the RFFIT test ("Rapid Fluorescence Focus Inhibition Test" described by Smith et al. (1973).
La glycoproteine de l'invention peut comporter un ou plusieurs des signaux d'adressage décrits ci- dessus. Lorsqu'il s'agit de la glycoproteine mature, le seul signal peptidique restant est le signal de
rétention endoplasmique de type HKDEL, les autres signaux d'adressage étant clivés dans la cellule. La glycoproteine comportant un signal de rétention endoplasmique a un poids moléculaire supérieur à 66 kDa en raison de la présence des acides aminés supplémentaires.The glycoprotein of the invention may include one or more of the addressing signals described above. When it comes to mature glycoprotein, the only peptide signal left is the HKDEL-type endoplasmic retention, the other addressing signals being cleaved in the cell. Glycoprotein with an endoplasmic retention signal has a molecular weight greater than 66 kDa due to the presence of additional amino acids.
La partie protéique de la glycoproteine de 1•invention peut correspondre à celle de la glycoproteine naturelle, ou il peut s'agir d'une protéine « analogue » à la protéine naturelle. Dans le contexte de l'invention, une protéine « analogue » signifie une protéine qui présente au moins 90% d'homologie avec la molécule de référence. Il s'agit par exemple d'une glycoproteine rabique dont certains acides aminés ont été remplacés, délétéε ou insérés. Les modifications ont lieu de préférence du côté N- terminal de la molécule. Les protéines analogues sont également insolubles, reconnues par des anticorps spécifiques à la glycoproteine de la rage ou à un virus apparenté au virus de la rage et capable d'induire la formation d'anticorps neutralisants.The protein part of the glycoprotein of the invention may correspond to that of natural glycoprotein, or it may be a protein "analogous" to the natural protein. In the context of the invention, an "analogous" protein means a protein which has at least 90% homology with the reference molecule. It is for example a rabies glycoprotein of which certain amino acids have been replaced, deleted or inserted. The modifications preferably take place on the N-terminal side of the molecule. Analogous proteins are also insoluble, recognized by antibodies specific to rabies glycoprotein or to a virus related to the rabies virus and capable of inducing the formation of neutralizing antibodies.
L'invention vise également les acides nucléiques, ARN ou ADN, mis en oeuvre dans la production de la glycoproteine. En particulier, l'invention vise un acide nucléique comportant une séquence codante chimérique comprenant d'une part une séquence codant pour la protéine G mature du virus de la rage ou d'un virus apparenté au virus de la rage, ou pour une protéine analogue, et d'autre part, une séquence codant pour un peptide signal N-terminal autre que celui naturellement associé au virus, ou les séquences complémentaires aux sus-dites séquences codantes.The invention also relates to the nucleic acids, RNA or DNA, used in the production of glycoprotein. In particular, the invention relates to a nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to the rabies virus, or for a similar protein , and on the other hand, a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned coding sequences.
Les séquences régulatrices décrites ci-dessus, ainsi que les séquences codant pour les signaux d'adressage sont, le cas échéant, également compris dans la molécule d'acide nucléique.
L'invention concerne en outre les vecteurs et plasmides employés pour 1•introduction de l'acide nucléique dans les cellules végétales. Il peut s'agir de plasmides Ti d'Aσrobacterium. ou de vecteurs viraux tels que les Geminivirus ou le CaMV.The regulatory sequences described above, as well as the sequences encoding the addressing signals are, if appropriate, also included in the nucleic acid molecule. The invention further relates to the vectors and plasmids used for the introduction of the nucleic acid into plant cells. They may be Ti plasmids of Aσrobacterium. or viral vectors such as Geminiviruses or CaMV.
L'invention vise également les cellules végétales transformées et capable de produire la glycoproteine de l'invention. Ces cellules sont sous forme de culture cellulaire, comme indiqué plus haut, ou font partie de plantes transgéniques ou chimériques.The invention also relates to transformed plant cells capable of producing the glycoprotein of the invention. These cells are in the form of cell culture, as indicated above, or are part of transgenic or chimeric plants.
Comme plantes appropriées, on peut citer les Angiospermes comprenant les monocotylédones et les dicotylédons. Plus particulièrement, on peut citer le tabac, les espèces appartenant aux familles botaniques telles que les légumineuses (par exemple les haricots, pois, etc...), les crucifères (par exemple les choux, radis, le colza etc...), les solanacées (par exemple les tomates, pommes de terre, etc...), les cucurbitacées (par exemple le melon, courgette, concombre) , les chénopodiacées (par exemple la betterave potagère) , les ombellifères (par exemple les carottes, céleris, etc...). On peut également citer les céréales telles que le blé, le maïs, l'orge, le triticale et le riz, et les oléagineux tels que le tournesol et le soja.As suitable plants, mention may be made of Angiosperms comprising monocots and dicots. More particularly, mention may be made of tobacco, species belonging to botanical families such as legumes (for example beans, peas, etc.), crucifers (for example cabbage, radishes, rapeseed, etc.) , nightshade (for example tomatoes, potatoes, etc.), cucurbits (for example melon, zucchini, cucumber), chenopodiaceae (for example vegetable beet), umbelliferae (for example carrots, celery, etc.). Mention may also be made of cereals such as wheat, corn, barley, triticale and rice, and oilseeds such as sunflower and soybeans.
L'invention concerne également les graines des plantes transgéniques capables de produire la glycoproteine G rabique, ainsi que leurs descendances.The invention also relates to the seeds of transgenic plants capable of producing rabies glycoprotein G, as well as their progeny.
L'invention vise également l'utilisation de la glycoproteine G comme produit thérapeutique, notamment comme vaccin contre l'infection par la rage, ou par des virus apparentés.The invention also relates to the use of glycoprotein G as a therapeutic product, in particular as a vaccine against infection by rabies, or by related viruses.
En particulier, l'invention vise une composition pharmaceutique comprenant : une ou plusieurs glycoproteine(s) selon l'invention, ou
- des cellules selon l'invention, ouIn particular, the invention relates to a pharmaceutical composition comprising: one or more glycoprotein (s) according to the invention, or - cells according to the invention, or
- toute ou partie d'une plante selon l'invention, en association avec un excipient acceptable du point de vue physiologique.- All or part of a plant according to the invention, in combination with an excipient which is acceptable from the physiological point of view.
La composition pharmaceutique contient au moins 1 μg de glycoproteine. Par exemple, elle contient 1 μg à 1 mg, ou de préférence de 10 μg à 200 μg, ou de préférence de 25 à 100 mg, de la glycoproteine purifiée, par dose, par exemple 200 ou 300 μg. Si la glycoproteine est employée sous une forme autre que purifiée, par exemple sous forme d'extrait de la plante, ou la plante elle-même, la quantité administrée doit être adaptée afin que la dose de principe actif atteigne au moins 100 μg.The pharmaceutical composition contains at least 1 μg of glycoprotein. For example, it contains 1 μg to 1 mg, or preferably 10 μg to 200 μg, or preferably 25 to 100 mg, of purified glycoprotein, per dose, for example 200 or 300 μg. If the glycoprotein is used in a form other than purified, for example in the form of an extract from the plant, or the plant itself, the quantity administered must be adjusted so that the dose of active principle reaches at least 100 μg.
L'excipient utilisé dans la composition est un excipient normalement utilisé pour les vaccins rabiques, par exemple le gel d'alumine.The excipient used in the composition is an excipient normally used for rabies vaccines, for example alumina gel.
Normalement, le vaccin est formulé pour une administration par voie orale, ou parentérale. Il peut également prendre la forme d'un produit alimentaire élaboré à partir de la matière végétale de 1'invention.Normally, the vaccine is formulated for oral or parenteral administration. It can also take the form of a food product produced from the plant material of the invention.
L'invention comprend également une méthode d'immuniser des mammifères, y compris l'homme, contre l'infection par ce virus de la rage, ou par un virus apparenté au virus de la rage, comprenant l'administration d'une glycoproteine de l'invention, ou d'une matière végétale comprenant cette glycoproteine.The invention also includes a method of immunizing mammals, including humans, against infection with this rabies virus, or with a virus related to the rabies virus, comprising administering a glycoprotein of the invention, or a plant material comprising this glycoprotein.
Différents aspects de l'invention sont illustrés dans les figures.Different aspects of the invention are illustrated in the figures.
Figure 1 :Figure 1 :
Western Blot de protéines de feuilles de tabac transformées avec le cDNA de la glycoproteine G rabique (construit pBIOCHO ≈ PPS-RabG)Western blot of tobacco leaf proteins transformed with the cDNA of rabies glycoprotein G (constructed pBIOCHO ≈ PPS-RabG)
Gel à 12% :
Piste 1 Feuilles de tabac non transformées ;12% gel: Lane 1 Unprocessed tobacco leaves;
Piste 2 Feuilles de tabac transformées avec pBIOCllO ;Lane 2 Tobacco leaves transformed with pBIOCllO;
Piste 3 Glycoproteine G rabique de référence ;Track 3 Reference rabies glycoprotein G;
Piste 4 Marqueur de poids moléculaire.Lane 4 Molecular weight marker.
Figure 2 :Figure 2:
Western Blot de protéines de feuilles de tabac transformées avec pBIOCllO (PPS-RabG) et de graines de colza transformées avec pBIOC114 (PPS-RabG) .Western blot of proteins from tobacco leaves transformed with pBIOC11O (PPS-RabG) and rapeseed transformed with pBIOC114 (PPS-RabG).
Gel à 10% :10% gel:
Piste 1 : Reconstruction glycoproteine G rabique de référence dans du jus de tabac ;Lane 1: Reference rabies glycoprotein G reconstruction in tobacco juice;
Piste 2 : Graines de colza transformées avec pBIOC114 ;Lane 2: Rapeseed transformed with pBIOC114;
Piste 3 : Graines deLane 3: Seeds of
Piste 4 : : FFeeuuiilllleess de tabac transformées avec pBIOCllO ;Lane 4:: FFeeuuiilllleess of tobacco transformed with pBIOCllO;
Piste 5 : F Feeuuiilllleess cde tabac non transformées ;Lane 5: F Feeuuiilllleess of unprocessed tobacco;
Piste 6 : GGllyyccoopprroottéeine G rabique de référence ;Track 6: Rabid GGllyyccoopprroottéeine G reference;
Piste 7 : MMaarrqquueeuurr cde poids moléculaire.Lane 7: MMaarrqqueueuurr cde molecular weight.
Figure 3 ••Figure 3 ••
Western Blot de protéines de cals de maïs transformés avec pBI0C116 (PS-RabG) .Western blot of corn calli proteins transformed with pBI0C116 (PS-RabG).
Gel à 10% :10% gel:
Piste 1 : Glycoproteine G rabique de référence ;Lane 1: Reference rabies glycoprotein G;
Piste 2 : Marqueur de poids moléculaire ;Lane 2: Molecular weight marker;
Piste 3 : Reconstruction glycoproteine G rabique de référence dans dujus de cals de maïs ;Lane 3: Reference rabies glycoprotein G reconstruction in corn callus juice;
Piste Cals de mais transformés avec PBI0C116 ;Track Corn callus processed with PBI0C116;
Piste 5 Cals de maïs non transformés.Track 5 Unprocessed corn calluses.
Figure 4 :Figure 4:
Gel à 8% :8% gel:
Piste 1 : Reconstruction glycoproteine G rabique de référence dans du jus de feuille de tabac ;Lane 1: Reconstruction of rabies glycoprotein G reference in tobacco leaf juice;
Piste 2 : Feuille de tabac non transformé ;
Piste 3 : Feuille de tabac transformé avec pBIOCllO (PPS-RabG) ;Lane 2: Unprocessed tobacco leaf; Lane 3: Tobacco leaf transformed with pBIOCllO (PPS-RabG);
Piste 4 : Feuille de tabac transformé avec pBI0C104 (PS-RabG) ;Lane 4: Tobacco leaf transformed with pBI0C104 (PS-RabG);
Piste 5 : Marqueur de poids moléculaire.Lane 5: Molecular weight marker.
EXEMPLESEXAMPLES
A. EXEMPLES DE CONSTRUCTIONS MOLECULAIRESA. EXAMPLES OF MOLECULAR CONSTRUCTIONS
I. CONSTRUCTION DE GENES CHIMERIQUES CODANT FOUR LA PROTEINE RECOMBINANTE DE LA GLYCOPROTEINE G RABIQUE ET PERMETTANT UNE EXPRESSION DANS LES FEUILLES ET LES GRAINES DE TABAC.I. CONSTRUCTION OF CHEMICAL GENES ENCODING THE RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND ALLOWING EXPRESSION IN TOBACCO LEAVES AND SEEDS.
L'expression dans les feuilles de tabac du gène viral codant pour la glycoproteine G rabique (RabG) a nécessité les séquences régulatrices suivantes :Expression in tobacco leaves of the viral gene encoding rabies glycoprotein G (RabG) required the following regulatory sequences:
1. un promoteur constitutif :1. a constituent promoter:
- soit le promoteur constitutif double 35S (pd35S) du CaMV (virus de la mosaïque du chou-fleur) . Il correspond à une duplication des séquences activant la transcription situées en amont de l'élément TATA du promoteur 35S naturel (Kay et al., 1987). soit le promoteur chimérique super-promoteur (pSP ; Ni et al., 1995). Il est constitué de la fusion de la triple répétition d'un élément activateur transcriptionnel du promoteur du gène de l'octopine synthase d'Agrobacterium tumefaciens, d'un élément activateur transcriptionnel du promoteur du gène de mannopine synthase et du promoteur mannopine synthase d'Agrobacterium tumefaciens ;- or the constitutive double 35S promoter (pd35S) of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987). or the chimeric super-promoter (pSP; Ni et al., 1995). It consists of the fusion of the triple repetition of a transcriptional activating element of the promoter of the octopine synthase gene from Agrobacterium tumefaciens, a transcriptional activating element of the promoter of the mannopine synthase gene and the mannopine synthase promoter. Agrobacterium tumefaciens;
2. la séquence terminatrice de transcription, terminateur polyA 35S, qui correspond à la région en 3' non codante de la séquence du virus à ADN bicaténaire
circulaire de la mosaïque du chou-fleur produisant le transcrit 35S (Franck et al., 1980).2. the transcription terminator sequence, polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the double-stranded DNA virus circular of the cauliflower mosaic producing the 35S transcript (Franck et al., 1980).
Les constructions des différents plasmides via l'utilisation de techniques d'ADN recombinant (Sambrook et al., 1989) dérivent de pBIOC4. Ce plasmide binaire dérive de pGA492 (An, 1986) qui contient entre les bordures droite et gauche, issues du plasmide pTiT37 d'Agrobacterium tumefaciens, sur son ADN de transfert, les séquences suivantes :The constructions of the various plasmids via the use of recombinant DNA techniques (Sambrook et al., 1989) derive from pBIOC4. This binary plasmid derives from pGA492 (An, 1986) which contains, between the right and left borders, derived from the plasmid pTiT37 from Agrobacterium tumefaciens, on its transfer DNA, the following sequences:
- le promoteur constitutif du gène nos codant pour la nopaline synthase (Depicker et al., 1982), la séquence codante du gène nptll codant pour la néomycine phosphotransferase II (Berg et Berg, 1983) délétée de la région des 8 premiers acides aminés dont le codon initiateur méthionine ATG et fusionnée à la séquence des 14 premiers acides aminés de la séquence codante du gène nos (Depicker et al., 1982), la séquence codante du gène nos dépourvue de la région des 14 premiers acides aminés, le terminateur nos (Depicker et al. , 1982), un polylinker (HindlII-Xbal-SacI-Hpal-Kpnl-Clal- BglII) précédant le gène cat codant pour la chloramphénicol acétyltransférase (Close et Rodriguez, 1982) et les séquences terminatrices du gène 6 du plasmide pTiA6 d'Agrobacterium tumefaciens (Liu et al., 1993).- the promoter constituting the gene nos coding for nopaline synthase (Depicker et al., 1982), the coding sequence of the nptll gene coding for neomycin phosphotransferase II (Berg and Berg, 1983) deleted from the region of the first 8 amino acids, the initiator codon methionine ATG and fused to the sequence of the first 14 amino acids of the coding sequence of the nos gene (Depicker et al., 1982), the coding sequence of the gene nos devoid of the region of the first 14 amino acids, the terminator nos (Depicker et al., 1982), a polylinker (HindlII-Xbal-SacI-Hpal-Kpnl-Clal- BglII) preceding the cat gene coding for chloramphenicol acetyltransferase (Close and Rodriguez, 1982) and the terminator sequences of gene 6 of plasmid pTiA6 from Agrobacterium tumefaciens (Liu et al., 1993).
Pour éliminer la quasi-totalité de la séquence codante du gène cat, le plasmide pGA492 a été doublement digéré par Sacl (site de restriction du polylinker) et par Seal (site de restriction présent dans la séquence du gène cat) puis soumis à l'action de l'enzyme T4 DNA polymérase (Biolabs) selon les recommandations du fabricant. La ligation du plasmide modifié (20 ng) a été réalisée dans un milieu reactionnel de 10 μl contenant 1 μl de tampon T4 DNA ligase x 10 (Amersham) ; 2,5 U d'enzyme T4 DNA ligase (Amersharo) à 14°C pendant 16 heures.
Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction.To eliminate almost all of the coding sequence for the cat gene, the plasmid pGA492 was doubly digested with Sac1 (restriction site of the polylinker) and by Seal (restriction site present in the sequence of the cat gene) and then subjected to the action of the enzyme T4 DNA polymerase (Biolabs) according to the manufacturer's recommendations. Ligation of the modified plasmid (20 ng) was carried out in a 10 μl reaction medium containing 1 μl of T4 DNA ligase x 10 buffer (Amersham); 2.5 U of T4 DNA ligase enzyme (Amersharo) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
Puis, le site de restriction HindIII de l'ADN plasmidique du clone retenu a été modifié en un site de restriction EcoRI à l'aide d'un adaptateur HindIII - EcoRI phosphorylé (Stratagene Cloning Systems) . Pour réaliser cette modification, 500 ng d'ADN plasmidique du clone retenu ont été digérés par HindIII, déphosphorylés par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant et coprécipités en présence de 1500 ng d'ADN adaptateur HindIII-EcoRI, 1/10 volume d'acétate de sodium 3M pH4,8 et 2,5 volumes d'éthanol absolu à -80°C pendant 30 min. Après centrifugation à 12000g pendant 30 min., l'ADN précipité a été lavé à l'éthanol 70%, séché, repris dans 8 μl d'eau, porté à 65°C pendant 10 min., puis ligué en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Après inactivation de la T4 DNA ligase à 65°C pendant 10 min., le mélange reactionnel de ligation a été digéré par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), précipité en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugé à 12000g pendant 30 min., lavé à l'éthanol 70%, séché, puis, ligué comme décrit ci-dessus.Then, the HindIII restriction site of the plasmid DNA of the retained clone was modified into an EcoRI restriction site using a phosphorylated HindIII-EcoRI adapter (Stratagene Cloning Systems). To carry out this modification, 500 ng of plasmid DNA of the retained clone were digested with HindIII, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations and coprecipitated in the presence of 1500 ng of DNA adapter HindIII-EcoRI, 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min. After centrifugation at 12000 g for 30 min., The precipitated DNA was washed with 70% ethanol, dried, taken up in 8 μl of water, brought to 65 ° C. for 10 min., Then ligated in the presence of 1 μl. of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. After inactivation of the T4 DNA ligase at 65 ° C. for 10 min., The ligation reaction mixture was digested with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, then ligated as described above.
Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées
(Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par HindIII et EcoRI notamment. Le plasmide binaire résultant, qui ne possède plus que les 9 derniers acides aminés de la séquences codante du gène cat et dont le site EcoRI est unique, a été appelé pBI0C4.The bacteria, Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with HindIII and EcoRI in particular. The resulting binary plasmid, which only has the last 9 amino acids of the coding sequence for the cat gene and whose EcoRI site is unique, was called pBI0C4.
La cassette d'expression, constituée du promoteur pd35S et du terminateur polyA 35S, a été isolée à partir du plasmide pJIT163Δ. Le plasmide pJIT163Δ dérive du plasmide pJIT163 qui dérive lui-même du plasmide pJIT60 (Guerineau et Mullineaux, 1993) . Le plasmide pJIT163 possède un codon ATG entre les sites HindIII et Sali du polylinker. Pour supprimer cet ATG et obtenir le plasmide pJIT163Δ, l'ADN plasmidique pJIT163 a été digéré doublement par HindIII et Sali, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), précipité en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugé à 12000g pendant 30 min., lavé à l'éthanol 70%, séché, soumis à l'action de l'enzyme Klenow (Biolabs) selon les recommandations du fabricant, déprotéinisé par extraction avec 1 volume de phénol:chloroforme:alcool isoamylique (25:24:1) puis 1 volume de chloroforme:alcool isoamylique (24:1), précipité en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à - 80°C pendant 30 min. , centrifugé à 12000g pendant 30 min., lavé à l'éthanol 70%, séché, et enfin, ligué en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coliThe expression cassette, consisting of the pd35S promoter and the polyA 35S terminator, was isolated from the plasmid pJIT163Δ. The plasmid pJIT163Δ derives from the plasmid pJIT163 which itself derives from the plasmid pJIT60 (Guerineau and Mullineaux, 1993). Plasmid pJIT163 has an ATG codon between the HindIII and SalI sites of the polylinker. To remove this ATG and obtain the plasmid pJIT163Δ, the plasmid DNA pJIT163 was digested twice with HindIII and SalI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, subjected to the action of the enzyme Klenow (Biolabs) according to the manufacturer's recommendations, deproteinized by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) then 1 volume of chloroform: isoamyl alcohol ( 24: 1), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at - 80 ° C for 30 min. , centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, and finally, ligated in the presence of 1 μl of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme ( Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli
DH5α rendues préalablement compétentes, ont été
transformées (Hanahan, 1983). L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Pour isoler la cassette d'expression constituée du promoteur pd35S et du terminateur polyA 35S (fragment Sacl-Xhol) , l'ADN plasmidique du clone pJIT163Δ retenu a été digéré par Sacl et Xhol. Le fragment Sacl-Xhol, portant la cassette d'expression, a été purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989) , précipité en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°c pendant 30 min., centrifugé à 12000g pendant 30 min., lavé à l'éthanol 70%, séché, puis soumis à l'action de l'enzyme Mung Bean Nuclease (Biolabs) selon les recommandations du fabricant. Cet insert purifié (200 ng) a été clone dans l'ADN plasmidique de pBIOC4 (20 ng) digéré par EcoRI, traité par l'enzyme Mung Bean Nuclease et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La réaction de ligation a été effectuée dans 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983). L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBI0C21.
Pour obtenir un plasmide binaire similaire à pBI0C21 mais dont le promoteur pd35S a été remplacé par le promoteur pSP, le fragment PvuII - Sali soumis à l'action de la Klénow contenant le promoteur pSP, a été isolé à partir du plasmide pBISNl (Ni et al., 1995), purifié par électrophorèse sur gel d'agarose 1%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché et ligué à l'ADN plasmidique de pBIOCδl doublement digéré par Kpnl et EcoRI soumis à l'action de la T4 DNA polymérase et déphosphorylé par l'enzyme phosphatase alcaline d•intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. Le plasmide pBIOCδl correspond à pB10C21 dont le site Xbal a été délété. Pour ce faire, le plasmide pB10C21 a été digéré par Xbal puis soumis à l'action de la Klenow et ligué par action de la T4 DNA ligase.DH5α previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. To isolate the expression cassette consisting of the pd35S promoter and of the polyA 35S terminator (SacI-Xhol fragment), the plasmid DNA of the clone pJIT163Δ retained was digested with SacI and Xhol. The Sacl-Xhol fragment, carrying the expression cassette, was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 of volume of 3M acetate. sodium pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, then subjected to the action of the enzyme Mung Bean Nuclease (Biolabs) according to the manufacturer's recommendations. This purified insert (200 ng) was cloned into the plasmid DNA of pBIOC4 (20 ng) digested with EcoRI, treated with the enzyme Mung Bean Nuclease and dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation reaction was carried out in 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBI0C21. To obtain a binary plasmid similar to pBI0C21 but whose pd35S promoter has been replaced by the pSP promoter, the PvuII - Sali fragment subjected to the action of Klénow containing the pSP promoter, was isolated from the plasmid pBISNl (Ni and al., 1995), purified by electrophoresis on 1% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pBIOCδl doubly digested with Kpnl and EcoRI subjected to the action of T4 DNA polymerase and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The plasmid pBIOCδl corresponds to pB10C21 whose Xbal site has been deleted. To do this, the plasmid pB10C21 was digested with Xbal then subjected to the action of Klenow and ligated by action of T4 DNA ligase.
La ligation a été réalisée avec 20 ng du vecteur déphosphorylé décrit ci-dessus et 200 ng de fragments d'ADN portant pSP décrits ci-dessus dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) et 2 μl de 50% polyéthylène glycol 8000, et de 5 ϋ d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,The ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments carrying pSP described above in a 20 μl reaction medium in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham ) and 2 μl of 50% polyethylene glycol 8000, and 5 ϋ of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, • 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC82.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, • 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC82.
La glycoproteine G rabique (RabG) , isolée à partir de la souche ERA, est naturellement synthétisée sous forme d'un précurseur. La protéine mature RabG est
constituée de 505 acides aminés. Son peptide signal est composé de 19 acides aminés.Rabies glycoprotein G (RabG), isolated from the ERA strain, is naturally synthesized in the form of a precursor. The mature protein RabG is made up of 505 amino acids. Its signal peptide is composed of 19 amino acids.
Le cDNA codant pour la glycoproteine d'enveloppe du virus de la rage, souche ERA (A. Anilionis et al., Nature, 1981, 294. 275-278 et M.P. Kieny et al., Nature, 1984, 312. 163-166) a été amplifié par PCR selon les procédés usuels, à l'aide des deux oligonucléotides suivants :The cDNA coding for the envelope glycoprotein of the rabies virus, strain ERA (A. Anilionis et al., Nature, 1981, 294. 275-278 and MP Kieny et al., Nature, 1984, 312. 163-166 ) was amplified by PCR according to the usual methods, using the following two oligonucleotides:
Oligo 1 (30 mer) 5' AAA GGA TCC ATG GTT CCT CAG GCT CTC CTG 3'Oligo 1 (30 sea) 5 'AAA GGA TCC ATG GTT CCT CAG GCT CTC CTG 3'
Oligo 2 (30 mer) 5» AAA CTG CAG TCA CAG TCT GGT CTC ACC CCC 3'Oligo 2 (30 sea) 5 ” AAA CTG CAG TCA CAG TCT GGT CTC ACC CCC 3 '
Le fragment PCR de 1.69 kb a été digéré par BamHI et Pstl pui clone dans le vecteur pBlueScript préalablement digéré par BamHI et Pstl, donnant ainsi le plasmide pPBOlO.The 1.69 kb PCR fragment was digested with BamHI and Pst1 and then cloned into the vector pBlueScript previously digested with BamHI and Pstl, thus giving the plasmid pPBO10.
L'ADN complémentaire du précurseur de RabG est contenu dans le plasmide pPBOlO. Il a été utilisé pour la construction des plasmides binaires pBIOC104 et PBIOC105 contenant la séquence codant pour PS-RabG, pBIOCl07 et pBIOC108 contenant la séquence codant pour BL-RabG et pBIOCllO et pBIOClll contenant la séquence codant pour PPS-RabG où la séquence codant pour RabG est précédée de la séquence codant pour un peptide signal, PS et BL, et pour un prépropeptide N-terminal (PPS c'est-à-dire un peptide signal suivi des séquences N-terminales d'adressage vacuolaire) d'origine végétale respectivement. Les séquences PS et PPS, constituées respectivement de 23 et 37 acides aminés, sont celles d'une protéine de réserve des racines tubérisées de patate douce : la sporamine A (Murakami et al., 1986 ; Matsuoka et Nakamura, 1991) . La séquence BL, composée de 26 acides aminés, est celle de la lectine d'orge (Lerner et Raikhel, 1989) .The complementary DNA of the RabG precursor is contained in the plasmid pPBO10. It was used for the construction of the binary plasmids pBIOC104 and PBIOC105 containing the sequence coding for PS-RabG, pBIOCl07 and pBIOC108 containing the sequence coding for BL-RabG and pBIOCllO and pBIOClll containing the sequence coding for PPS-RabG where the sequence coding for RabG is preceded by the sequence coding for a signal peptide, PS and BL, and for an N-terminal prepropeptide (PPS i.e. a signal peptide followed by N-terminal vacuolar addressing sequences) of plant origin respectively. The PS and PPS sequences, consisting respectively of 23 and 37 amino acids, are those of a reserve protein for tuberous sweetpotato roots: sporamine A (Murakami et al., 1986; Matsuoka and Nakamura, 1991). The BL sequence, composed of 26 amino acids, is that of barley lectin (Lerner and Raikhel, 1989).
Afin d'obtenir une séquence codant pour la glycoproteine G rabique ne possédant que le premier
codon stop, le cDNA RabG a été modifié en son extrémité 3' par mutagenèse dirigée par PCR en utilisant 2 oligodésoxynucléotides, 5' CTC AGG AGT TGA CTT GGG 3' (contenant le site HincII unique dans le plasmide pPBOlO) et 5' CCG GAT CCT CAC AGT CTG GTC TCA C 3' (contenant le site BamHI unique dans le plasmide pPBOlO) .In order to obtain a sequence coding for rabies glycoprotein G having only the first stop codon, cDNA RabG was modified at its 3 'end by PCR-directed mutagenesis using 2 oligodeoxynucleotides, 5' CTC AGG AGT TGA CTT GGG 3 '(containing the unique HincII site in the plasmid pPBO10) and 5' CCG GAT CCT CAC AGT CTG GTC TCA C 3 '(containing the unique BamHI site in the plasmid pPBO10).
L'amplification PCR du fragment HincII-BamHI a été réalisée dans 100 μl de milieu reactionnel comprenant 10 μl de tampon Taq DNA polymérase xlO (500 mM KC1, 100 mM Tris-HCl, pH9,0 et 1% Triton xlOO) , 6 μl de 25 mM MgCl2, 3 μl de 10 mM dNTP (dATP, dCTP, dGTP et dTTP) , 100 pM de chacun des 2 oligodésoxynucléotides décrits ci-dessus, 5 ng d'ADN matrice (vecteur pPBOlO) , 2,5 U de Taq DNA polymérase (Promega) et 2 gouttes d'huile de vaseline. L'ADN a été dénaturé à 94°C pendant 5 min., soumis à 30 cycles constitués chacun de 1 min. de dénaturation à 94°C, 1 min. d'hybridation à 44°C et de 1 min. d'élongation à 72°C, puis l'élongation à 72°C a été poursuivie pendant 5 min. Cette réaction PCR a été réalisée dans la machine "DNA Thermal Cycler" de PERKIN ELMER CETUS. L'huile a été éliminée par extraction au chloroforme. Puis, les fragments d'ADN du milieu reactionnel ont été précipités en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min. , centrifugés à 12000g pendant 30 min., lavés à l'éthanol 70%, séchés et digérés par les 2 enzymes de restriction HincII et BamHI. Les fragments d'ADN digérés issus de l'amplification par PCR ont été purifiés par électrophorèse sur gel d'agarose 2%, électroélués (Sambrook et al., 1989), précipités en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugés à 12000g pendant 30 min. , lavés à
l'éthanol 70%, séchés, puis ligués à l'ADN plasmidique de pPBOlO doublement digéré par HincII et BamHI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué, soumis à la précipitation alcoolique, séché. La ligation a été réalisée avec 100 ng du vecteur et 50 ng de fragments d'ADN digérés issus de l'amplification PCR, décrits ci-dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à l'aide du kit de séquençageThe PCR amplification of the HincII-BamHI fragment was carried out in 100 μl of reaction medium comprising 10 μl of Taq DNA polymerase x10 buffer (500 mM KC1, 100 mM Tris-HCl, pH9.0 and 1% Triton x100), 6 μl 25 mM MgCl2, 3 μl of 10 mM dNTP (dATP, dCTP, dGTP and dTTP), 100 μM of each of the 2 oligodeoxynucleotides described above, 5 ng of template DNA (vector pPBO10), 2.5 U of Taq DNA polymerase (Promega) and 2 drops of petrolatum oil. The DNA was denatured at 94 ° C for 5 min., Subjected to 30 cycles each consisting of 1 min. denaturation at 94 ° C, 1 min. hybridization at 44 ° C and 1 min. elongation at 72 ° C, then the elongation at 72 ° C was continued for 5 min. This PCR reaction was carried out in the "DNA Thermal Cycler" machine from PERKIN ELMER CETUS. The oil was removed by extraction with chloroform. Then, the DNA fragments of the reaction medium were precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min. , centrifuged at 12000g for 30 min., washed with 70% ethanol, dried and digested with the 2 restriction enzymes HincII and BamHI. The digested DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 of volume of 3M sodium acetate. pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min. , washed at ethanol 70%, dried, then ligated to the plasmid DNA of pPBO10 doubly digested with HincII and BamHI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected were checked by sequencing using the sequencing kit
T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Le plasmide résultant a été appelé pBIθC102.T7 ™ marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The resulting plasmid was called pBIθC102.
a. CONSTRUCTION DES PLASMIDES BINAIRES pBIOC104 ET PBIOC105 CONTENANT PS-RabG.at. CONSTRUCTION OF BINARY PLASMIDS pBIOC104 AND PBIOC105 CONTAINING PS-RabG.
Le plasmide pBIOC102 a été digéré doublement par BglII et HindIII afin de supprimer la séquence codant pour le peptide signal naturel de la glycoproteine G rabique et les 9 premiers acides aminés de la protéine RabG mature (KFPIYTIPDKL) . Cette séquence a été remplacée par celle codant pour le peptide signal PS de 23 acides aminés (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC) fusionnée à celle codant pour les 9 premiers codons de la protéine RabG mature ("PS-9 premiers codons de RabG mature") . La séquence "PS-9 premiers
codons de RabG mature" a été amplifiée par PCR à partir du plasmide pMAT103 (Matuoka et Nakamura, 1991) à l'aide des 2 oligodésoxynucléotides, 5' cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3' (contenant les sites BglII unique et EcoRI supplémentaire dans le plasmide pPBOlO) et 5' GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGA ATG GGC TGG ATT GGG CAG G 3' (contenant le site HindIII unique dans le plasmide pPBOlO) en suivant le protocole d'amplification PCR décrit précédemment. La température d'hybridation a été de 40 °C. Après double digestion enzymatique par BglII et HindIII, les fragments d'ADN issus de l'amplification PCR ont été purifiés par électrophorèse sur gel d'agarose 2%, électroélués (Sambrook et al., 1989), précipités en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugés à 12000g pendant 30 min., lavés à l'éthanol 70%, séchés, puis ligués à l'ADN plasmidique de pBIOClOl doublement digéré par BglII et HindIII, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché. La ligation a été réalisée avec 100 ng du vecteur et 50 ng de fragments d'ADN digérés issus de l'amplification PCR, décrits ci- dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et -de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendantThe plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL). This sequence has been replaced by that coding for the signal peptide PS of 23 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG"). The sequence "first PS-9 mature RabG codons "was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3' (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGA ATG GGC TGG ATT GGG CAG G 3' (containing the unique HindIII site in the plasmid pPBO10) by following the PCR amplification protocol described The hybridization temperature was 40 ° C. After double enzymatic digestion with BglII and HindIII, the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min ., washed with 70% ethanol, dried, then ligated with plasmi DNA pBIOClOl plate doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer. (Amersham) and -4.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à l'aide du kit de
séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Les séquences codant pour PS et RabG mature ont été clonées en maintenant leurs phases de lecture ouverte (c'est-à-dire, de telle sorte qu'elles constituent une phase ouverte de lecture unique) . La séquence de clivage entre les séquences PS et RabG mature est Ser- Lys. Le plasmide résultant a été appelé pBIOC103.16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones retained were verified by sequencing using the T7 ™ sequencing marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). Sequences encoding PS and mature RabG were cloned by maintaining their open reading phases (i.e., so that they constitute a single open reading phase). The cleavage sequence between the PS and mature RabG sequences is Ser-Lys. The resulting plasmid was called pBIOC103.
A partir de pBIOC103, le fragment EcoRI portant la séquence PS-RabG a été isolé par digestion enzymatique par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché, et ligué à l'ADN plasmidique de pBI0C21 digéré au site EcoRI et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 100 ng de vecteur pBIOC21 déphosphorylé et 50 ng de fragments d'ADN contenant PS-RabG, décrits ci-dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,From pBIOC103, the EcoRI fragment carrying the sequence PS-RabG was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBI0C21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing PS-RabG, described above, in a 10 μl reaction medium in the presence of 1 μl of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983). L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le clone résultant a été appelé pBIθC104. La séquence nucléique du fragment codant pour la protéine recombinante PS-RabG a été vérifiée par séquençage à l'aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). L'ADN
plasmidique du vecteur binaire pBIOC104 a été introduit par transformation directe dans la souche LBA4404 d'Agrobacterium tumefaciens selon le procédé de Holsters et al. (1978) . La validité du clone retenu a été vérifiée par digestion enzymatique de l'ADN plasmidique introduit.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was called pBIθC104. The nucleic acid sequence of the fragment coding for the recombinant protein PS-RabG was verified by sequencing using the T7 ™ sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). DNA The plasmid of the binary vector pBIOC104 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
L'obtention de pBIOClOS est similaire à celle de pBI0C104 excepté que le fragment EcoRI portant la séquence PS-RabG a été clone au site Xbal de pBIOC82. Les sites EcoRI de l'insert et Xbal du vecteur ont été soumis à l'action de la Klenow. Les méthodes usuelles de clonages ont été appliquées.Obtaining pBIOClOS is similar to that of pBI0C104 except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82. The EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied.
b. CONSTRUCTION DES PLASMIDES BINAIRES pBIOC107 ET pBIOC108 CONTENANT BL-RabG.b. CONSTRUCTION OF BINARY PLASMIDS pBIOC107 AND pBIOC108 CONTAINING BL-RabG.
Le plasmide pBIOC103 a été digéré doublement par BglII et HindIII afin de supprimer la séquence codant pour le peptide signal PS et les 9 premiers acides aminés de la protéine RabG mature (KFPIYTIPDKL) . Cette séquence a été remplacée par celle codant pour le peptide signal BL de 26 acides aminés (ATG AAG ATG ATG AGC ACC AGG GCC CTC GCT CTC GGC GCG GCC GCC GTC CTC GCC TTC GCG GCG GCG ACC GCG CAC GCC) fusionnée à celle codant pour les 9 premiers codons de la protéine RabG mature ("BL-9 premiers codons de RabG mature") . La séquence "BL-9 premiers codons de RabG mature" a été amplifiée par PCR à partir du plasmide BLc3 (Lerner et Raikhel, 1989) à l'aide des 2 oligodésoxynucléotides, 5' ggagatctgaattoaacaATG AAG ATG ATG AGC ACC AGG 3 ' (contenant les sites BglII unique et EcoRI supplémentaire dans le plasmide pBIOC102) et 5' AGG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GTC CGC GGT CGC CGC G 3' (contenant le site HindIII unique dans le plasmide pBIOCl02) en suivant le protocole d'amplification PCR décrit précédemment. La température
d'hybridation a été de 65°C. Après double digestion enzymatique par BglII et HindIII, les fragments d'ADN issus de l'amplification PCR ont été purifiés par électrophorèse sur gel d'agarose 2%, électroélués (Sambrook et al., 1989), précipités en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugés à 12000g pendant 30 min., lavés à l'éthanol 70%, séchés, puis ligués à l'ADN plasmidique de pBIOC102 doublement digéré par BglII et HindIII, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché. La ligation a été réalisée avec 100 ng du vecteur et 50 ng de fragments d'ADN digérés issus de l'amplification PCR, décrits ci- dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendantThe plasmid pBIOC103 was digested twice with BglII and HindIII in order to suppress the sequence coding for the signal peptide PS and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL). This sequence has been replaced by that coding for the BL amino acid signal peptide of 26 amino acids (ATG AAG ATG ATG AGC ACC AGG GCC CTC GCT CTC GGC GCG GCC GCC GTC CTC GCC TTC GCG GCG GCG ACC GCG CAC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("BL-9 first codons of mature RabG"). The sequence "BL-9 first codons of mature RabG" was amplified by PCR from the plasmid BLc3 (Lerner and Raikhel, 1989) using the 2 oligodeoxynucleotides, 5 'ggagatctgaattoaacaATG AAG ATG ATG AGC ACC AGG 3' (containing the unique BglII and additional EcoRI sites in the plasmid pBIOC102) and 5 'AGG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GTC CGC GGT CGC CGC G 3' (containing the unique HindIII site in the plasmid pBIOCl02) PCR amplification protocol described above. Temperature hybridization was 65 ° C. After double enzymatic digestion with BglII and HindIII, the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 by volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, then ligated with plasmid DNA from pBIOC102 doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à 1"aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Les séquences codant pour BL et RabG mature ont été clonées en maintenant leurs phases de lecture ouverte (c'est-à-dire, de telle sorte qu'elles constituent une phase ouverte de lecture unique) . La séquence de clivage entre les séquences BL et RabG mature est Ala- Lys. Le plasmide résultant a été appelé pBIOClOβ.16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones retained were verified by 1 "sequencing using the T7 ™ sequencing kit marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The sequences coding for BL and mature RabG were cloned maintaining their open reading phases (ie, so that they constitute a single open reading phase) The cleavage sequence between the BL and mature RabG sequences is Ala-Lys. The resulting plasmid was called pBIOClOβ.
A partir de pBIOC106, le fragment EcoRI portant la séquence BL-RabG a été isolé par digestion enzymatique
par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché, et ligué à l'ADN plasmidique de pBIOC21 digéré au site EcoRI et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 100 ng de vecteur pBI0C21 déphosphorylé et 50 ng de fragments d'ADN contenant BL-RabG, décrits ci-dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,From pBIOC106, the EcoRI fragment carrying the sequence BL-RabG was isolated by enzymatic digestion by EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme calf intestine alkaline phosphatase (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 100 ng of dephosphorylated pBI0C21 vector and 50 ng of DNA fragments containing BL-RabG, described above, in a 10 μl reaction medium in the presence of 1 μl of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le clone résultant a été appelé pBIOC107. La séquence nucléique du fragment codant pour la protéine recombinante BL-RabG a été vérifiée par séquençage à l'aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). L'ADN plasmidique du vecteur binaire pBIOCl07 a été introduit par transformation directe dans la souche LBA4404 d'Agrobacterium tumefaciens selon le procédé de Holsters et al. (1978) . La validité du clone retenu a été vérifiée par digestion enzymatique de l'ADN plasmidique introduit.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was named pBIOC107. The nucleic acid sequence of the fragment coding for the recombinant protein BL-RabG was verified by sequencing using the T7 ™ sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The plasmid DNA of the binary vector pBIOCl07 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
L'obtention de pBIOCloβ est similaire à celle de pBIOC107 excepté que le fragment EcoRI portant la séquence BL-RabG a été clone au site Xbal de pBIOC82. Les sites EcoRI de l'insert et Xbal du vecteur ont été
soumis à l'action de la Klenow. Les méthodes usuelles de clonages ont été appliquées.Obtaining pBIOCloβ is similar to that of pBIOC107 except that the EcoRI fragment carrying the sequence BL-RabG was cloned at the Xbal site of pBIOC82. The EcoRI sites of the insert and Xbal of the vector were subject to the action of Klenow. The usual cloning methods were applied.
C. CONSTRUCTION DES PLASMIDES BINAIRES pBIOCllO ET pBIOClll CONTENANT PPS-RabG.C. CONSTRUCTION OF PBIOCllO AND pBIOClll BINARY PLASMIDS CONTAINING PPS-RabG.
Le plasmide pBIOC102 a été digéré doublement par BglII et HindIII afin de supprimer la séquence codant pour le peptide signal naturel de la glycoproteine G rabique et les 9 premiers acides aminés de la protéine RabG mature (KFPIYTIPDKL) . Cette séquence a été remplacée par celle codant pour le peptide signal PPS de 37 acides aminés (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC AGG TTC AAT CCC ATC CGC CTC CCC ACC ACA CAC GAA CCC GCC) fusionnée à celle codant pour les 9 premiers codons de la protéine RabG mature ("PS-9 premiers codons de RabG mature") . La séquence "PPS-9 premiers codons de RabG mature" a été amplifiée par PCR à partir du plasmide pMAT103 (Matuoka et Nakamura, 1991) à l'aide des 2 oligodésoxynucléotides, 5' cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3• (contenant les sites BglII unique et EcoRI supplémentaire dans le plasmide pPBOlO) et 5' GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GGG TTC GTG TGT GGT GGG GAG G 3' (contenant le site HindIII unique dans le plasmide pPBOlO) en suivant le protocole d'amplification PCR décrit précédemment. La température d'hybridation a été de 40°C. Après double digestion enzymatique par BglII et HindIII, les fragments d'ADN issus de l'amplification PCR ont été purifiés par électrophorèse sur gel d'agarose 2%, électroélués (Sambrook et al., 1989), précipités en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à -80°C pendant 30 min., centrifugés à 12000g pendant 30 min., lavés à l'éthanol
70%, séchés, puis ligués à l'ADN plasmidique de pBIOClOl doublement digéré par BglII et HindIII, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché. La ligation a été réalisée avec 100 ng du vecteur et 50 ng de fragments d'ADN digérés issus de l'amplification PCR, décrits ci- dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendantThe plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL). This sequence has been replaced by that coding for the PPS signal peptide of 37 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC AGG TTC AAT CCC ATC CGC CTC CCC ACC ACA CAC GAA CCC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG"). The sequence "PPS-9 first codons of mature RabG" was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3 • (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GGG TTC GTG TGT GGT GGG GAG G 3' (containing the unique HindIII site in the plasmid pPBO10) following the PCR amplification protocol described above. The hybridization temperature was 40 ° C. After double enzymatic digestion with BglII and HindIII, the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 by volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, and then ligated to the plasmid DNA of pBIOClOl doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à l'aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Les séquences codant pour PPS et RabG mature ont été clonées en maintenant leurs phases de lecture ouverte (c'est-à-dire, de telle sorte qu'elles constituent une phase ouverte de lecture unique) . La séquence de clivage entre les séquences PPS et RabG mature est Ala- Lys. Le plasmide résultant a été appelé pBIOC109.16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected were verified by sequencing using the T7 ™ sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). Sequences encoding PPS and mature RabG were cloned by maintaining their open reading phases (i.e., so that they constitute a single open reading phase). The cleavage sequence between the PPS and mature RabG sequences is Ala-Lys. The resulting plasmid was called pBIOC109.
A partir de pBIθC109, le fragment EcoRI portant la séquence PPS-RabG a été isolé par digestion enzymatique par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché, et ligué à l'ADN plasmidique de pBIOC21 digéré au site EcoRI et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 100 ng de vecteur pBIOC21 déphosphorylé
et 50 ng de fragments d'ADN contenant PPS-RabG, décrits ci-dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,From pBIθC109, the EcoRI fragment carrying the PPS-RabG sequence was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. Ligation was carried out with 100 ng of pPHOC21 dephosphorylated vector and 50 ng of DNA fragments containing PPS-RabG, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 enzyme DNA ligase (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le clone résultant a été appelé pBIOCllO. La séquence nucléique du fragment codant pour la protéine recombinante PPS-RabG a été vérifiée par séquençage à l'aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). L'ADN plasmidique du vecteur binaire pBIOCllO a été introduit par transformation directe dans la souche LBA4404 d'Agrobacterium tumefaciens selon le procédé de Holsters et al. (1978) . La validité du clone retenu a été vérifiée par digestion enzymatique de l'ADN plasmidique introduit.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was named pBIOC10O. The nucleic acid sequence of the fragment coding for the recombinant protein PPS-RabG was verified by sequencing using the T7 ™ sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The plasmid DNA of the binary vector pBIOC10O was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
L'obtention de pBIOClll est similaire à celle de pBIOCllO excepté que le fragment EcoRI portant la séquence PS-RabG a été clone au site Xbal de pBIOC82. Les sites EcoRI de l'insert et Xbal du vecteur ont été soumis à l'action de la Klenow. Les méthodes usuelles de clonages ont été appliquées.
II. CONSTRUCTION DE GENES CHIMERIQUES CODANT POUR LA PROTEINE RECOMBINANTE DE LA GLYCOPROTEINE G RABIQUE ET PERMETTANT UNE EXPRESSION DANS LES GRAINES DE COLZA.Obtaining pBIOC111 is similar to that of pBIOC11O except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82. The EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied. II. CONSTRUCTION OF CHEMICAL GENES ENCODING THE RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND ALLOWING EXPRESSION IN CANNED SEEDS.
L'expression dans les graines de colza du gène viral codant pour la glycoproteine G rabique (RabG) a nécessité les séquences régulatrices suivantes :The expression in rapeseed of the viral gene coding for rabies glycoprotein G (RabG) required the following regulatory sequences:
1. un des promoteurs décrits ci-dessous :1. one of the promoters described below:
- le promoteur pCRU correspondant à la région 5' non codante du gène de la protéine de réserve de graines, la CRUCIFERINE A de radis (Depigny-This et al. , 1992) , et permettant une expression spécifique dans les graines;- the pCRU promoter corresponding to the 5 ′ non-coding region of the gene for the seed reserve protein, radish CRUCIFERIN A (Depigny-This et al., 1992), and allowing specific expression in the seeds;
- le promoteur pGEAl correspondant à la région 5' non codante du gène de la protéine de réserve de graines, GEAI d'Arabidopsis thaliana (Gaubier et al., 1993) , et permettant une expression spécifique dans les graines ;- the pGEAl promoter corresponding to the 5 ′ non-coding region of the gene for the seed reserve protein, GEAI of Arabidopsis thaliana (Gaubier et al., 1993), and allowing specific expression in the seeds;
- le promoteur pGEA6 correspondant à la région 5• non codante du gène de la protéine de réserve de graines, GEA6 d'Arabidopsis thaliana (Gaubier et al., 1993) , et permettant une expression spécifique dans les graines ;- the pGEA6 promoter corresponding to the non-coding region 5 • of the gene for the seed reserve protein, GEA6 from Arabidopsis thaliana (Gaubier et al., 1993), and allowing specific expression in the seeds;
2. une des séquences terminatrices décrites ci- dessous : la séquence terminatrice de transcription, terminateur polyA 35S, qui correspond à la région en 3' non codante de la séquence du virus à ADN bicaténaire circulaire de la mosaïque du chou-fleur produisant le transcrit 35S (Franck et al., 1980) ; la séquence terminatrice de transcription, terminateur polyA NOS, qui correspond à la région en 3• non codante du gène de la nopaline synthase du plasmide Ti d'Agrobacterium tumefaciens souche à nopaline (Depicker et al., 1982).
Pour obtenir un plasmide binaire similaire à pBI0C21 mais dont le promoteur pd35S a été remplacé par le promoteur pCRU, le fragment "EcoRI traité à la Klenow - BamHI", contenant le promoteur pCRU, a été isolé à partir du plasmide pBI221-CRURSP. Le plasmide pBI221-CRURSP dérive de pBI221 (commercialisé par Clontech) par remplacement du promoteur 35S par le promoteur pCRU.2. one of the terminator sequences described below: the transcription terminator sequence, polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the transcript 35S (Franck et al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 • non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982). To obtain a binary plasmid similar to pBI0C21 but whose promoter pd35S has been replaced by the promoter pCRU, the fragment "EcoRI treated with Klenow - BamHI", containing the promoter pCRU, was isolated from the plasmid pBI221-CRURSP. The plasmid pBI221-CRURSP is derived from pBI221 (marketed by Clontech) by replacing the 35S promoter with the pCRU promoter.
Le fragment "EcoRI traité à la Klenow - BamHI" portant le promoteur pCRU a été purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché et ligué à l'ADN plasmidique de pJIT163 (décrit dans le paragraphe I.) digéré par Kpnl, traité à la T4 DNA Polymérase (Biolabs) selon les recommandations du fabricant, puis digéré par BamHI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 20 ng du vecteur déphosphorylé décrit ci-dessus et 200 ng de fragments d'ADN "EcoRI traité à la Klenow - BamHI" décrits ci-dessus dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 -et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983). L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC27.
La cassette d'expression, constituée du promoteur pCRU et du terminateur polyA35S a été isolée à partir de pBI0C27 par digestion totale Xhol suivie d'une digestion partielle EcoRI. Elle a été purifiée par électrophorèse sur gel d'agarose 0,8%, électroéluée (Sambrook et al., 1989), soumise à la précipitation alcoolique, séchée, traitée à la Klenow (Biolabs) selon les recommandations du fabricant et liguée à l'ADN plasmidique de pBIOC4 au site EcoRI traité à la Klenow et déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 20 ng du vecteur déphosphorylé décrit ci- dessus et 200 ng de fragments d'ADN XhoI-EcoRI décrits ci-dessus dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendantThe "EcoRI treated with Klenow - BamHI" fragment carrying the pCRU promoter was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pJIT163 (described in paragraph I.) digested with Kpnl, treated with T4 DNA Polymerase (Biolabs) according to the manufacturer's recommendations, then digested with BamHI, purified by electrophoresis on 0.8% agarose gel , electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments "EcoRI treated with Klenow - BamHI" described above in a reaction medium of 20 μl in the presence of 2 μl of buffer T4 DNA ligase x 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 -and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC27. The expression cassette, consisting of the pCRU promoter and the polyA35S terminator, was isolated from pBI0C27 by total digestion Xhol followed by partial digestion EcoRI. It was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried, treated with Klenow (Biolabs) according to the manufacturer's recommendations and ligated to l Plasmid DNA of pBIOC4 at the EcoRI site treated with Klenow and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of XhoI-EcoRI DNA fragments described above in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer ( Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC28.16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC28.
Pour obtenir le plasmide binaire pBIOC91, 'la cassette d'expression "pGEAlD - tNOS", isolée à partir de pBSII-pGEAlD, a été introduite dans le plasmide binaire pSCV1.2 lui-même obtenu par clonage du fragment HindIII portant la cassette d'expression "p35S - nptll - tNOS" décrite par Fromm et al. (1986) au site HindIII de pSCVl construit par Edwards G.A. en 1990 en suivant les procédés usuels de clonage.To obtain the binary plasmid pBIOC91, "the expression cassette" pGEAlD - tNOS ", isolated from pBSII-pGEAlD, was introduced into the binary plasmid pSCV1.2 itself obtained by cloning of the HindIII fragment carrying the cassette expression "p35S - nptll - tNOS" described by Fromm et al. (1986) at the HindIII site of pSCV1 constructed by Edwards GA in 1990 following the usual cloning methods.
Le plasmide pBSII-pGEAID a été obtenu en deux étapes :
- d'une part, le fragment Sacl-EcoRI portant tNOS (terminateur du gène de la nopaline synthase) d'Agrobacterium tumefaciens, traité à l'enzyme T4 DNA polymérase (Biolabs) selon les recommandations du fabricant et purifié, a été clone au site EcoRV de pBSIISK-t- commercialisé par Stratagene, déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 20 ng du vecteur déphosphorylé et 200 ng de fragments d'ADN contenant tNOS décrits ci-dessus dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à l'aide du kit de séquençageThe plasmid pBSII-pGEAID was obtained in two stages: - on the one hand, the Sacl-EcoRI fragment carrying tNOS (terminator of the nopaline synthase gene) of Agrobacterium tumefaciens, treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at EcoRV site of pBSIISK-t- marketed by Stratagene, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing tNOS described above in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham), of 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected were checked by sequencing using the sequencing kit
T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Le plasmide résultant a été appelé pBSII-tNOS.T7 ™ marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The resulting plasmid was called pBSII-tNOS.
- d'autre part, le fragment portant pGEAlD digéré doublement par HindIII traité à l'enzyme Klenow et BamHI, purifié, a été clone aux sites "Xbal traité à la Klenow et BamHI" du plasmide pBSII-tNOS. La ligation a été réalisée avec 100 ng du vecteur décrit ci-dessus et 50 ng de fragments d'ADN décrits ci- dessus dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant
16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBSII-pGEAID.- On the other hand, the fragment carrying pGEAlD digested doubly with HindIII treated with the enzyme Klenow and BamHI, purified, was cloned at the sites "Xbal treated with Klenow and BamHI" of the plasmid pBSII-tNOS. The ligation was carried out with 100 ng of the vector described above and 50 ng of DNA fragments described above in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBSII-pGEAID.
Puis, la cassette d'expression "pGEAlD - tNOS" portée par le fragment Xbal-HindIII traité à la Klenow, a été clonée au site Smal de pSCV1.2 déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La ligation a été réalisée avec 20 ng de pSCV1.2 déphosphorylé et 200 ng de fragments portant la cassette d'expression "pGEAlD - tNOS", dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC91.Then, the expression cassette "pGEAlD - tNOS" carried by the Xbal-HindIII fragment treated with Klenow, was cloned at the SmaI site of pSCV1.2 dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of dephosphorylated pSCV1.2 and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer ( Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC91.
Pour obtenir le plasmide binaire pBIOC92 similaire à pBI0C21 mais dont le promoteur pd35S a été remplacé par le promoteur pGEA6D, le fragment EcoRI - BamHI traité à la Klenow, contenant le promoteur pGEA6, a été isolé à partir du plasmide pGUS2-pGEA6. Le clone pGUS-2-pGEA6 dérivant de pBI221 par remplacement du p35S par le promoteur pGEA6. Il comporte 2 ATG en phase : ATG du gène GEA6 (Em6) et
ATG du gène gus. L'ATG du gène GEA6 a été détruit. Le fragment d'ADN compris entre le site AccI et les séquences en amont de l'ATG du gène GEA6 du clone pGUS-2-pGEA6 a donc été amplifié par PCR à l'aide des 2 oligonucléotides : 5' AAGTACGGCCACTACCACG 3 • et 5' CCCGGGGATCCTGGCTC 3'. La température d'hybridation a été adaptée.To obtain the binary plasmid pBIOC92 similar to pBI0C21 but whose promoter pd35S has been replaced by the promoter pGEA6D, the EcoRI-BamHI fragment treated with Klenow, containing the promoter pGEA6, was isolated from the plasmid pGUS2-pGEA6. The pGUS-2-pGEA6 clone deriving from pBI221 by replacing p35S with the pGEA6 promoter. It has 2 ATGs in phase: ATG of the GEA6 gene (Em6) and ATG of the gus gene. The ATA of the GEA6 gene has been destroyed. The DNA fragment between the AccI site and the sequences upstream of the ATG of the GEA6 gene of the clone pGUS-2-pGEA6 was therefore amplified by PCR using the 2 oligonucleotides: 5 ′ AAGTACGGCCACTACCACG 3 • and 5 'CCCGGGGATCCTGGCTC 3'. The hybridization temperature has been adapted.
Le fragment amplifié par PCR a été digéré par AccI et BamHI, purifié par électrophorèse sur gel d'agarose 2%, électroélué (Sambrook et al., 1989), précipité en présence de 1/10 de volume de 3M acétate de sodium pH4,8 et de 2,5 volumes d'éthanol absolu à - 80°C pendant 30 min. , centrifugé à 12000g pendant 30 min., lavé à l'éthanol 70%, séché, puis ligué à l'ADN plasmidique de pGUS-2-GEA6 doublement digéré par AccI et BamHI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché. La ligation a été réalisée avec 100 ng du vecteur décrit ci-dessus et 50 ng de fragments d'ADN digérés issus de l'amplification PCR décrits ci-dessus dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,The PCR amplified fragment was digested with AccI and BamHI, purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4, 8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min. , centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, then ligated to the plasmid DNA of pGUS-2-GEA6 doubly digested with AccI and BamHI, purified by electrophoresis on 0.8 agarose gel %, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng of the vector described above and 50 ng of digested DNA fragments resulting from the PCR amplification described above in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase buffer. x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sûr 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Certains des clones retenus ont été vérifiés par séquençage à l'aide du kit de séquençage T7™ commercialisé par Pharmacia selon la méthode des didésoxynucléotides (Sanger et al., 1977). Le clone résultant a été appelé pGUS-2-pGEA6D.
Le fragment EcoRI - BamHI portant le promoteur pGEA6D isolé à partir de pGUS-2-pGEA6D, traité à la Klenow selon les recommandations du fabricant (Biolabs) , a été purifié par électrophorèse sur gel d'agarose 0,8%, électroélué (Sambrook et al., 1989), soumis à la précipitation alcoolique, séché et ligué au site Xhol traité par l'enzyme Klenow de l'ADN plasmidique de pBIOC21 modifié. Le plasmide pBIOC21 modifié a été obtenu par double digestion par HindIII traité à la Klenow et Kpnl de pBIOC21 pour déléter le fragment portant le promoteur pd35S et le remplacer par le fragment Kpnl-EcoRV portant le polylinker composé des sites Kpnl-XhoI-SalI-Clal-HindlII-BamHI-Smal-EcoRI- EcoRV de pBSIISK+.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected from 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected were verified by sequencing using the T7 ™ sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977). The resulting clone was named pGUS-2-pGEA6D. The EcoRI - BamHI fragment carrying the pGEA6D promoter isolated from pGUS-2-pGEA6D, treated with Klenow according to the manufacturer's recommendations (Biolabs), was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the Xhol site treated with the Klenow enzyme of the modified pBIOC21 plasmid DNA. The modified pBIOC21 plasmid was obtained by double digestion with Klenow-treated HindIII and Kpnl of pBIOC21 to delete the fragment carrying the pd35S promoter and replace it with the Kpnl-EcoRV fragment carrying the polylinker composed of the Kpnl-XhoI-SalI-ClaI sites -HindlII-BamHI-Smal-EcoRI- EcoRV from pBSIISK +.
La ligation a été réalisée avec 20 ng de pBIOC21 modifié déphosphorylé et 200 ng de fragments d'ADN EcoRI-BamHI, décrits ci-dessus, dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 12 μg/ml tétracycline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC92.The ligation was carried out with 20 ng of dephosphorylated modified pBIOC21 and 200 ng of EcoRI-BamHI DNA fragments, described above, in a 20 μl reaction medium in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham ), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 μg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC92.
Puis, la cassette d'expression "pGEA6D - t35S" a été isolée à partir de pBIOC92. Cette cassette d'expression portée par le fragment Kpnl-EcoRV traité à l'enzyme T4 DNA polymérase (Biolabs) selon les recommandations du fabricant et purifié, a été clonée au site Smal de pSCV1.2 déphosphorylé par l'enzyme phosphatase alcaline d'intestin de veau (Boehringer Mannheim) selon les recommandations du fabricant. La
ligation a été réalisée avec 20 ng de pSCV1.2 déphosphorylé et 200 ng de fragments portant la cassette d'expression "pGEAlD - tNOS", dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5cc rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIθC112.Then, the expression cassette "pGEA6D - t35S" was isolated from pBIOC92. This expression cassette carried by the Kpnl-EcoRV fragment treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at the SmaI site of pSCV1.2 dephosphorylated by the enzyme alkaline phosphatase d ' calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of pSCV1.2 dephosphorylated and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham ), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5cc, made competent beforehand, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIθC112.
CONSTRUCTION DES PLASMIDES BINAIRES pBIOC113, pBI0C114 ET pBIOCHS CONTENANT PPS-RABG SOUS CONTROLE DE PROMOTEURS PERMETTANT UNE EXPRESSION DANS LES SEMENCES.CONSTRUCTION OF BINARY PLASMIDS pBIOC113, pBI0C114 AND pBIOCHS CONTAINING PPS-RABG UNDER CONTROL OF PROMOTERS ALLOWING EXPRESSION IN SEEDS.
Le plasmide pBIOC109, décrit précédemment en I.c, contient le fragment EcoRI portant la séquence PPS-RabG.The plasmid pBIOC109, described previously in I.c, contains the EcoRI fragment carrying the sequence PPS-RabG.
Ce fragment a été isolé par digestion enzymatique par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué, soumis à la précipitation alcoolique, séché et ligué à pBIOC28, pBI0C91 et PBI0C112 digéré par EcoRI et déphosphorylé pour résulter en pBI0C113, pBI0CH4 et pBIOCHS respectivement.
III. CONSTRUCTION DE GENES CHIMERIQUES CODANT POUR LA PROTEINE RECOMBINANTE DE LA GLYCOPROTEINE G RABIQUE ET PERMETTANT UNE EXPRESSION DANS LES SEMENCES DE MAIS.This fragment was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried and ligated to pBIOC28, pBI0C91 and PBI0C112 digested with EcoRI and dephosphorylated to result in pBI0C113 , pBI0CH4 and pBIOCHS respectively. III. CONSTRUCTION OF CHEMICAL GENES ENCODING THE RECOMBINANT PROTEIN OF GLYCOPROTEIN G RABIC AND ALLOWING EXPRESSION IN MAIZE SEEDS.
a. CONSTRUCTION DES PLASMIDES pBIOCllβ ET PBIOC117 CONTENANT RS-LGC ET PERMETTANT UNE EXPRESSION CONSTITUTIVE DANS LES SEMENCES DE MAIS.at. CONSTRUCTION OF PLASMIDES pBIOCllβ AND PBIOC117 CONTAINING RS-LGC AND ALLOWING CONSTITUTIVE EXPRESSION IN MAIZE SEEDS.
L'expression constitutive dans les semences de maïs du gène viral codant pour la glycoproteine G rabique (RabG) a nécessité les séquences régulatrices suivantes :The constitutive expression in maize seeds of the viral gene coding for rabies glycoprotein G (RabG) required the following regulatory sequences:
1. un des deux promoteurs permettant une expression constitutive :1. one of the two promoters allowing a constitutive expression:
- promoteur actine de riz suivi de l'intron actine de riz (pAR-IAR) contenu dans le plasmide pActl-F4 décrit par McElroy et al. (1991) ;- rice actin promoter followed by the rice actin intron (pAR-IAR) contained in the plasmid pActl-F4 described by McElroy et al. (1991);
- promoteur constitutif double 35S (pd35S) du CaMV (virus de la mosaïque du chou-fleur) . Il correspond à une duplication des séquences activant la transcription situées en amont de l'élément TATA du promoteur 35S naturel (Kay et al., 1987);- double constitutive promoter 35S (pd35S) of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987);
2. un des deux terminateurs : la séquence terminatrice de transcription, terminateur polyA 35S, qui correspond à la région en 3' non codante de la séquence du virus à ADN bicaténaire circulaire de la mosaïque du chou-fleur produisant le transcrit 35S (Franck et al., 1980) ; la séquence terminatrice de transcription, terminateur polyA NOS, qui correspond à la région en 3' non codante du gène de la nopaline synthase du plasmide Ti d'Agrobacterium tumefaciens souche à nopaline (Depicker et al. , 1982) .2. one of the two terminators: the transcription terminator sequence, polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the 35S transcript (Franck and al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
Le plasmide pBIOCllβ où la séquence codant pour PS-RabG est placée sous contrôle de pAR-IAR a été
obtenu par clonage du fragment EcoRI portant la séquence codant pour PS-RabG aux sites "Ncol et Sali" de pBSII-pAR-IAR-tNOS.The plasmid pBIOC11β where the sequence coding for PS-RabG is placed under the control of pAR-IAR has been obtained by cloning of the EcoRI fragment carrying the sequence coding for PS-RabG at the "Ncol and Sali" sites of pBSII-pAR-IAR-tNOS.
Le fragment EcoRI portant la séquence codant pour PS-RabG a été isolée à partir de pBIOC103 par digestion enzymatique par EcoRI, purifié par électrophorèse sur gel d'agarose 0,8%, électroélué, soumis à la précipitation alcoolique, séché, puis traité par l'enzyme Klenow. Le plasmide pBSII-pAR-IAR- tNOS a été digéré doublement par Sali et Ncol, purifié, traité à l'enzyme Mung Bean Nuclease (Biolabs) et déphosphorylé par l'enzyme phosphatase alcaline de veau (Boehringer Mannheim) selon les recommandations des fabricants. La ligation a été réalisée avec 20 ng du vecteur déphosphorylé et 200 ng de fragments d'ADN contenant la séquence codant pour RS-LGC, décrits ci-dessus, dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le plasmide résultant a été appelé pBIOC116.The EcoRI fragment carrying the sequence coding for PS-RabG was isolated from pBIOC103 by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried and then treated with the Klenow enzyme. The plasmid pBSII-pAR-IAR-tNOS was digested twice with Sali and Ncol, purified, treated with the enzyme Mung Bean Nuclease (Biolabs) and dephosphorylated by the enzyme alkaline phosphatase of calf (Boehringer Mannheim) . The ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the sequence coding for RS-LGC, described above, in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase buffer. x 10 (Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pBIOC116.
Le plasmide pBSII-pAR-IAR-tNOS résulte du clonage aux sites "Eco0109I traité à la Klenow et Kpnl" de pBSII-tNOS du fragment SnaBI-Kpnl portant la séquence correspondant à "pAR-IAR-début de la séquence codante pour le gène gus" isolé à partir du plasmide pActl-F4. La ligation a été réalisée avec 100 ng de vecteur et 50 ng de fragments d'ADN, décrits ci-dessus, dans un milieu reactionnel de 10 μl digérés en présence de 1
μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983). L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction.The plasmid pBSII-pAR-IAR-tNOS results from cloning at the sites "Eco0109I treated with Klenow and Kpnl" of pBSII-tNOS of the SnaBI-Kpnl fragment carrying the sequence corresponding to "pAR-IAR-start of the coding sequence for the gene gus "isolated from the plasmid pActl-F4. The ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 μl digested in the presence of 1 μl of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
Le plasmide pBSII-tNOS a été obtenu par clonage au site EcoRV déphosphorylé de pBSIISK+ commercialisé par Stratagene, du fragment Sacl-EcoRI portant la séquence tNOS isolé à partir de pBI121 commercialisé par Clontech par double digestion enzymatique par Sacl et EcoRV, soumis à une purification par électrophorèse sur gel d'agarose à 2%, et traité à l'enzyme T4 DNA polymérase. La ligation a été réalisée avec 20 ng du vecteur déphosphorylé et 200 ng de fragments d'ADN contenant la séquence tNOS, décrits ci-dessus, dans un milieu reactionnel de 20 μl en présence de 2 μl de tampon T4 DNA ligase x 10 (Amersham) , de 2 μl de 50% polyéthylène glycol 8000 et de 5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction.The plasmid pBSII-tNOS was obtained by cloning at the dephosphorylated EcoRV site of pBSIISK + marketed by Stratagene, from the Sacl-EcoRI fragment carrying the tNOS sequence isolated from pBI121 marketed by Clontech by double enzymatic digestion with Sacl and EcoRV, subjected to purification by electrophoresis on 2% agarose gel, and treated with the T4 DNA polymerase enzyme. The ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the tNOS sequence, described above, in a reaction medium of 20 μl in the presence of 2 μl of T4 DNA ligase x 10 buffer (Amersham ), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
Le plasmide pBIOC117 où la séquence codant pour PS-RabG est placée sous contrôle de pd35S a été obtenu par clonage aux sites "Kpnl et Xbal" du plasmide pBSIISK+ commercialisé par Stratagene, du fragment Kpnl-Xbal portant la séquence correspondant à "pd35S-
PS-RabG" isolé à partir de pBIOC103. La ligation a été réalisée avec 100 ng de vecteur et 50 ng de fragments d'ADN, décrits ci-dessus, dans un milieu reactionnel de 10 μl digérés en présence de l μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries, Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) .The plasmid pBIOC117 where the sequence coding for PS-RabG is placed under the control of pd35S was obtained by cloning at the "Kpnl and Xbal" sites of the plasmid pBSIISK + marketed by Stratagene, of the Kpnl-Xbal fragment carrying the sequence corresponding to "pd35S- PS-RabG "isolated from pBIOC103. The ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 μl digested in the presence of 1 μl of T4 buffer DNA ligase x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C. for 16 hours The bacteria, Escherichia coli DH5α made previously competent, were transformed (Hanahan, 1983).
L'ADN plasmidique des clones obtenus, sélectionnés surThe plasmid DNA of the clones obtained, selected from
50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction.50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
b. CONSTRUCTION DES PLASMIDES pBIOCllβ ET PBI0C119 CONTENANT PS-RabG ET PS-RabG-KDEL RESPECTIVEMENT ET PERMETTANT UNE EXPRESSION DANS L'ALBUMEN DES SEMENCES DE MAIS.b. CONSTRUCTION OF PLASMIDES pBIOCllβ AND PBI0C119 CONTAINING PS-RabG AND PS-RabG-KDEL RESPECTIVELY AND ALLOWING EXPRESSION IN THE CORN SEED ALBUMEN.
L'expression dans l'albumen des semences de maïs du gène viral codant pour la glycoproteine G rabique (RabG) a nécessité les séquences régulatrices suivantesThe expression in the albumen of maize seeds of the viral gene coding for rabies glycoprotein G (RabG) required the following regulatory sequences
1. le promoteur du gène de γzéine de maïs (pγzéine) contenu dans le plasmide pγ63 (Reina et al., 1990,1. the promoter of the corn γzein gene (pγzein) contained in the plasmid pγ63 (Reina et al., 1990,
N.A.R. , JJL, 6426) . Le plasmide pγ63 résulte du clonage de pγzéine aux sites HindIII et Xbal d'un plasmide pUC18 renfermant, entre ses sites HindIII et EcoRI, la cassette d'expression "p35S-gus-tNOS" de pBI221 commercialisé par Clontech. Il permet une expression dans l'albumen des semences de maïs.N.A.R. , JJL, 6426). The plasmid pγ63 results from the cloning of pγzein at the HindIII and Xbal sites of a plasmid pUC18 containing, between its HindIII and EcoRI sites, the expression cassette "p35S-gus-tNOS" of pBI221 marketed by Clontech. It allows an expression in the albumen of corn seeds.
2. la séquence terminatrice de transcription, terminateur polyA NOS, qui correspond à la région en 3' non codante du gène de la nopaline synthase du plasmide
Ti d'Agrobacterium tumefaciens souche à nopaline (Depicker et al., 1982).2. the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'non-coding region of the nopaline synthase gene of the plasmid Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
Le plasmide pBIOCllβ où la séquence codant pourThe plasmid pBIOCllβ where the sequence coding for
PS-RabG est placée sous contrôle du pγzéine, a été obtenu par clonage aux sites "Sacl traité par l'enzyme T4 DNA polymérase et BamHI" du plasmide pγ63, du fragment "BamHI traité à la Klenow - BglII" isolé à partir de pBIOC103. La ligation a été réalisée avec 100 ng du vecteur et 50 ng de fragments d'ADN, décrits ci-dessus, dans un milieu reactionnel de 10 μl en présence de 1 μl de tampon T4 DNA ligase x 10 (Amersham) et de 2,5 U d'enzyme T4 DNA ligase (Amersham) à 14°C pendant 16 heures. Les bactéries,PS-RabG is placed under the control of pγzein, was obtained by cloning at the "Sacl sites treated with the enzyme T4 DNA polymerase and BamHI" of the plasmid pγ63, of the fragment "BamHI treated with Klenow - BglII" isolated from pBIOC103 . The ligation was carried out with 100 ng of the vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 μl in the presence of 1 μl of T4 DNA ligase x 10 buffer (Amersham) and of 2, 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria,
Escherichia coli DH5α rendues préalablement compétentes, ont été transformées (Hanahan, 1983) . L'ADN plasmidique des clones obtenus, sélectionnés sur 50 μg/ml ampicilline, a été extrait selon la méthode de la lyse alcaline (Birnboim et Doly, 1979) et analysé par digestion enzymatique par des enzymes de restriction. Le clone résultant a été appelé pBIOCllδ.Escherichia coli DH5α made previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 μg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting clone was named pBIOCllδ.
Le plasmide pBIOC119 résulte de la substitution du fragment BamHI-HincII de pBIOCllδ par le fragment BamHI-HincII portant la séquence codant pour KDEL placée avant le codon stop obtenu par amplification PCR selon les procédés décrits ci-dessus. Les 2 oligodésoxynucléotides utilisés lors de cette réaction ont été : 5' GGT CTC AGG AGT TGA CTT GGG TCT CCC GAA CTG GG 3' (site HincII unique) et 5' CCC GGA TCC TCA TAG CTC ATC TTT CAG TCT GGT CTC ACC CCC ACT C 3' (séquence codant pour KDEL et site BamHI unique) . La température d'hybridation a été de 50°C. Le clonage a été effectué comme décrit précédemment.
B. EXEMPLES DE TRANSFORMATION GENETIQUE DU TABAC, DU COLZA ET DU MAISThe plasmid pBIOC119 results from the substitution of the BamHI-HincII fragment of pBIOC11δ by the BamHI-HincII fragment carrying the sequence coding for KDEL placed before the stop codon obtained by PCR amplification according to the methods described above. The 2 oligodeoxynucleotides used during this reaction were: 5 'GGT CTC AGG AGT TGA CTT GGG TCT CCC GAA CTG GG 3' (single HincII site) and 5 'CCC GGA TCC TCA TAG CTC ATC TTT CAG TCT GGT CTC ACC CCC ACT C 3 '(sequence coding for KDEL and unique BamHI site). The hybridization temperature was 50 ° C. The cloning was carried out as described previously. B. EXAMPLES OF GENETIC TRANSFORMATION OF TOBACCO, RUSSIA AND CORN
I. OBTENTION DE PLANTES DE TABAC TRANSGENIQUESI. OBTAINING TRANSGENIC TOBACCO PLANTS
Les plantes de tabac utilisées pour les expériences de transformation (Nicotiana tabacum var. Xanthi NC et PDB6) sont cultivées in vitro sur le milieu de base de Murashige et Skoog (1962) additionné des vitamines de Gamborg et al. (1968, Sigma référence M0404) , de saccharose à 20g/L et d'agar (Merck) à 8g/L. Le pH du milieu est ajusté à 5,8 avec une solution de potasse avant autoclavage à 120βC pendant 20 min. Les plantules de tabac sont repiquées par bouture des entre-noeuds tous les 30 jours sur ce milieu de multiplication MS20.The tobacco plants used for the transformation experiments (Nicotiana tabacum var. Xanthi NC and PDB6) are cultivated in vitro on the basic medium of Murashige and Skoog (1962) supplemented with the vitamins of Gamborg et al. (1968, Sigma reference M0404), sucrose at 20g / L and agar (Merck) at 8g / L. The pH of the medium is adjusted to 5.8 with a potassium hydroxide solution before autoclaving at 120 β C for 20 min. Tobacco seedlings are transplanted by cutting internodes every 30 days on this MS20 multiplication medium.
Toutes les cultures in vitro sont réalisées en enceinte climatisée, dans les conditions définiées ci- dessous :All in vitro cultures are carried out in an air-conditioned enclosure, under the conditions defined below:
Intensité lumineuse de SOμE.m^.S"1; photopériode de 16h;Light intensity of SOμE.m ^ .S "1; photoperiod of 16h;
- Thermopériode de 26°C le jour, 24°C la nuit.- Thermoperiod of 26 ° C during the day, 24 ° C at night.
La technique de transformation utilisée est dérivée de celle de Horsch et al. (1985).The transformation technique used is derived from that of Horsch et al. (1985).
Une préculture d'Agrobacterium tumefaciens souche LBA4404 contenant les plasmides binaires, est réalisée durant 48h à 2δ°C sous agitation, dans du milieu LB additionné des antibiotiques adéquats (rifampicine et tétracycline) . La préculture est ensuite diluée au 50ème dans le même milieu et cultivée dans les mêmes conditions. Après une nuit, la culture est centrifugée (10 min, 3000 rpm) , les bactéries sont reprises dans un volume équivalent de milieu MS30 (30g/L saccharose) liquide et cette suspension est diluée au lOème.
Des explants d'environ 1cm2 sont découpés à partir des feuilles des plantules décrites ci- dessus.Ils sont ensuite mis au contact de la suspension bactérienne pendant lh, puis séchés rapidement sur papier filtre et placés sur un milieu de coculture (MS30 solide) .A preculture of Agrobacterium tumefaciens strain LBA4404 containing the binary plasmids is carried out for 48 h at 2δ ° C with stirring, in LB medium supplemented with appropriate antibiotics (rifampicin and tetracycline). The preculture is then diluted to 50th in the same medium and cultivated under the same conditions. After one night, the culture is centrifuged (10 min, 3000 rpm), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / L sucrose) and this suspension is diluted with 10eme. Explants of approximately 1 cm 2 are cut from the leaves of the seedlings described above. They are then placed in contact with the bacterial suspension for 1 h, then quickly dried on filter paper and placed on a coculture medium (solid MS30). .
Après 2 jours, les explants sont transférés en boîtes de Pétri sur le milieu de régénération MS30, contenant un agent sélectif, la kanamycine (200mg/L) , un bactériostatique, l'augmentin (400mg/L) et les hormones nécessaires à l'induction de bourgeons (BAP, lmg/L et ANA, 0,lmg/L). Un repiquage des explants est effectué sur le même milieu après 2 semaines de culture. Après 2 nouvelles semaines, les bourgeons sont repiqués en boîtes de Pétri sur le milieu de développement composé du milieu MS20 additionné de kanamycine et d'augmentin. Après 15 jours, les bourgeons sont repiqués en pots sur le même milieu dont la concentration en kanamycine a été diminuée de moitié.L'enracinement prend environ 20 jours, au terme desquels les plantules peuvent être clonées par bouture d'entre-noeuds ou sorties en serre.After 2 days, the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200mg / L), a bacteriostatic, augmentin (400mg / L) and the hormones necessary for bud induction (BAP, lmg / L and ANA, 0, lmg / L). The explants are subcultured on the same medium after 2 weeks of culture. After 2 new weeks, the buds are subcultured in Petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted into pots on the same medium, the concentration of kanamycin has been halved. It takes about 20 days to take root, at the end of which the seedlings can be cloned by cutting internodes or emerging in the greenhouse.
II. OBTENTION DE PLANTES DE COLZA TRANSGENIQUESII. OBTAINING TRANSGENIC COLZA PLANTS
Les graines de colza de printemps (Brassica napus cv WESTAR ou lignées Limagrain) sont désinfectées pendant 40 minutes dans une solution de Domestos à 15%. Après 4 rinçages à l'eau stérile, les graines sont mises à germer, à raison de 20 graines par pots de 7 cm de diamètre sur 10cm de haut, sur du milieu minéral de Murashige et Skoog (Sigma, référence M 5519) avec 30 g/1 de saccharose et solidifié avec de l'agargel à 5 g/1. Ces pots sont placés dans une chambre de culture à 26°C avec une photopériode de 16h/δh et sous une intensité lumineuse de l'ordre de
80 μE m""2 S-1. Après 5 jours de germination, les cotylédons sont prélevés stérilement en coupant chaque pétiole environ 1 mm au dessus du noeud cotylédonaire.The spring rapeseeds (Brassica napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% Domestos solution. After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per 7 cm diameter jars by 10 cm high, on Murashige and Skoog mineral medium (Sigma, reference M 5519) with 30 g / 1 of sucrose and solidified with 5 g / 1 of agargel. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 16h / δh and under a light intensity of the order of 80 μE m "" 2 S -1 . After 5 days of germination, the cotyledons are removed sterile by cutting each petiole about 1 mm above the cotyledon node.
Parallèlement une culture d'Agrobacterium tumefaciens souche LBA4404, contenant le plasmide pBI0C114, est cultivée en erlen meyer de 50 ml, pendant 36h à 28°C dans 10 ml de milieu bactérien YT complémenté avec les antibiotiques utiles à la sélection de la souche utilisée. Cette préculture sert à ensemencer à 1% une nouvelle culture bactérienne effectuée dans les mêmes conditions. Au bout de 14h la culture est centrifugée 15 min à 3000 rpm et les bactéries sont reprises dans un volume équivalent de milieu de germination liquide. Cette suspension est distribuée dans des boîtes de pétri de 5 cm de diamètre à raison de 5 ml/ boite.In parallel, a culture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pBI0C114, is cultivated in a 50 ml Erlenmeyer flask for 36 h at 28 ° C. in 10 ml of YT bacterial medium supplemented with the antibiotics useful for the selection of the strain used. This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 rpm and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box.
L'extrémité sectionnée du pétiole est immergée quelques secondes dans la solution d'agrobactéries ainsi préparée, puis le pétiole est enfoncé de quelques millimètres dans le milieu de régénération. Ce milieu a la même composition de base que le milieu de germination avec en plus 4 mg/1 de benzyl-amino- purine, phytohormone favorisant la néoformation de bourgeons. Douze explants (cotylédon avec pétiole) sont mis en culture par boîte de pétri de 9 cm de diamètre (Greiner, référence 664102) .The severed end of the petiole is immersed for a few seconds in the agrobacteria solution thus prepared, then the petiole is pressed a few millimeters into the regeneration medium. This medium has the same basic composition as the germination medium with, in addition, 4 mg / 1 of benzylamino-purine, a phytohormone promoting the neoformation of buds. Twelve explants (cotyledon with petiole) are cultured in a 9 cm diameter petri dish (Greiner, reference 664102).
Après 2 jours de coculture dans les mêmes conditions environnementales que les germinations, les explants sont repiqués dans des boites phytatray (Sigma, référence P1552) contenant le milieu précèdent complémenté avec un agent sélectif: 45 mg/1 du sulfate de kanamycine (Sigma, référence K4000) et un bactériostatigue: mélange de 1/6 (en poids) de sel de potassium d'acide clavulanique et de 5/6 sel d'ammonium d'amoxicilline (Augmentin injectable) à raison de 600 mg/1.
Deux fois de suite, à 3 semaines d'intervalle, les explants sont repiqués stérilement sur du milieu neuf dans les mêmes conditions.After 2 days of coculture under the same environmental conditions as germination, the explants are subcultured in phytatray boxes (Sigma, reference P1552) containing the above medium supplemented with a selective agent: 45 mg / 1 of kanamycin sulfate (Sigma, reference K4000) and a bacteriostatics: mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 ammonium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1. Twice in succession, 3 weeks apart, the explants are transplanted sterile onto new medium under the same conditions.
Les bourgeons verts apparus à la fin du deuxième ou du troisième repiquage sont séparés de l'expiant et mis en culture individuellement dans des pots transparents de 5 cm de diamètre et de 10 cm de haut contenant un milieu identique au précèdent mais dépourvu de BAP. Après 3 semaines de culture la tige du bourgeon transformé est sectionnée et le bourgeon est repiqué dans un pot de milieu frais. Au bout de trois à quatre semaines les racines sont assez développées pour permettre l'acclimatation de la plantule au phytotron. Les bourgeons qui ne sont pas verts ou enracinés sont éliminés. Ces plantules sont alors transplantées dans des godets de 7 cm de coté remplis de terreau (norme NF U44551: 40 % tourbe brune, 30 % bruyère tamisée et 30 % sable) saturé en eau. Après deux semaines d'acclimatation en phytotron (température 21°C, photopériode I6h/8h et 64 % d'humidité relative), les plantules sont rempotées dans des pots de 12 cm de diamètre remplis du même terreau enrichi en engrais retard (Osmocote, à raison de 4g/1 de terreau) puis transportées en serre (classe S2) régulée à lδ°C, avec deux arrosages quotidien de 2 minutes à l'eau.The green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5 cm in diameter and 10 cm high containing a medium identical to the above but free of BAP. After 3 weeks of culture, the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium. After three to four weeks the roots are sufficiently developed to allow acclimatization of the seedling to the phytotron. Buds that are not green or rooted are removed. These seedlings are then transplanted into 7 cm side pots filled with potting soil (standard NF U44551: 40% brown peat, 30% sifted heather and 30% sand) saturated with water. After two weeks of acclimatization in a phytotron (temperature 21 ° C, photoperiod I6h / 8h and 64% relative humidity), the seedlings are repotted in 12 cm diameter pots filled with the same soil enriched with late fertilizer (Osmocote, at a rate of 4g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at δ ° C, with two daily waterings of 2 minutes.
Dès l'apparition des fleurs celles-ci sont ensachées (Crispac, référence SM 57Oy 300 mm * 700 mm) de façon à empêcher la fécondation croisée.As soon as the flowers appear, these are bagged (Crispac, reference SM 57Oy 300 mm * 700 mm) so as to prevent cross-fertilization.
Lorsque les siliques sont arrivées à maturité, elles sont récoltées, séchées, puis battues. Les graines obtenues servent au dosage de l'activité biochimique. La sélection de la descendance transgénique se fait par germination sur un milieu contenant du sulfate de kanamycine à raison de 100 à 150 mg/1 (selon les génotypes) . Les conditions
opératoires sont identiques à celles décrites au début de ce document à ceci près que les germinations sont effectuées en tubes de verre avec une seule graine par tube. Seules les plantules développant des racines secondaires les trois premières semaines sont acclimatées en phytotron avant d'être passées en serre.When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for the determination of biochemical activity. The selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes). Conditions The operating procedures are identical to those described at the beginning of this document except that the germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse.
III. OBTENTION DE PLANTES DE MAIS TRANSGENIQUESIII. OBTAINING TRANSGENIC CORN PLANTS
a) Obtention et utilisation de cal de maïs comme cible pour la transformation génétique :a) Obtaining and using corn callus as a target for genetic transformation:
La transformation génétique du maïs, quelle que soit la méthode employée (électroporation, Agrobacterium. microfibres, canon à particules) , requiert généralement l'utilisation de cellules indifférenciées en divisions rapides ayant conservé une aptitude à la régénération de plantes entières. Ce type de cellules compose le cal friable embryogène (dit de type II) de maïs.The genetic transformation of maize, whatever the method used (electroporation, Agrobacterium. Microfibers, particle cannon), generally requires the use of undifferentiated cells in rapid divisions which have retained an ability to regenerate whole plants. This type of cell makes up the embryogenic friable callus (called type II) of corn.
Ces cals sont obtenus à partir d'embryons immatures de génotype HI, II ou (A188 x B73) selon la méthode et sur les milieux décrits par Armstrong (Malze Handbook, 1994, M. Freeling, V. Walbot Eds. , pages 665-671) . Les cals ainsi obtenus sont multipliés et maintenus par repiquages successifs tous les quinze jours sur le milieu d'initiation.These calluses are obtained from immature embryos of genotype HI, II or (A188 x B73) according to the method and on the media described by Armstrong (Malze Handbook, 1994, M. Freeling, V. Walbot Eds., Pages 665- 671). The calluses thus obtained are multiplied and maintained by successive subcultures every fortnight on the initiation medium.
Des plantules sont ensuite régénérées à partir de ces cals en modifiant l'équilibre hormonal et osmatique des cellules selon la méthode décrite par Vain et al. (Plant Cell Tissue and Organ Culture, 1969, !£ : 143-151). Ces plantes sont ensuite acclimatées en serre où elles peuvent être croisées ou autofécondées.
b) Utilisation du canon à particules pour la transformation génétigue du maïs :Seedlings are then regenerated from these calluses by modifying the hormonal and osmatic balance of the cells according to the method described by Vain et al. (Plant Cell Tissue and Organ Culture, 1969,! £: 143-151). These plants are then acclimatized in the greenhouse where they can be crossed or self-fertilized. b) Use of the particle gun for the genetic transformation of corn:
Le paragraphe précédent décrit l'obtention et la régénération des lignées cellulaires nécessaires à la transformation ; on décrit ici une méthode de transformation génétique conduisant à 1•intégration stable des gènes modifiés dans le génome de la plante. Cette méthode repose sur l'utilisation d'un canon à particules identique à celui décrit par J. Finer (Plant Cell Report, 1992, H : 323-326) ; les cellules cibles sont des fragments de cals décrits dans le paragraphe 1. Ces fragments d'une surface de 10 à 20 mm 2 ont ete disposés, 4 heures avant bombardement, à raison de 16 fragments par boîte au centre d'une boite de pétri contenant un milieu de culture identique au milieu d'initiation, additionné de 0.2M de mannitol +The previous paragraph describes obtaining and regenerating the cell lines necessary for transformation; we describe here a method of genetic transformation leading to 1 • stable integration of modified genes in the genome of the plant. This method is based on the use of a particle gun identical to that described by J. Finer (Plant Cell Report, 1992, H: 323-326); the target cells are fragments of calluses described in paragraph 1. These fragments with an area of 10 to 20 mm 2 were placed, 4 hours before bombardment, at the rate of 16 fragments per dish in the center of a petri dish containing a culture medium identical to the initiation medium, supplemented with 0.2M mannitol +
0.2 M de sorbitol. Les plasmides portant les gènes à introduire sont purifiés sur colonne Qiagen®, en suivant les instructions du fabricant. Ils sont ensuite précipités sur des particules de tungsten (M10) en suivant le protocole décrit par Klein (Nature, 1987, 327 :70-73). Les particules ainsi enrobées sont projetées vers les cellules cibles à l'aide du canon et selon le protocole décrit par J. Finer (Plant Cell Report, 1992, ϋ : 323-328).0.2 M sorbitol. The plasmids carrying the genes to be introduced are purified on a Qiagen® column, following the manufacturer's instructions. They are then precipitated on tungsten particles (M10) following the protocol described by Klein (Nature, 1987, 327: 70-73). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. Finer (Plant Cell Report, 1992, ϋ: 323-328).
Les boites de cals ainsi bombardées sont ensuite scellées à l'aide de Scellofrais® puis cultivées à l'obscurité à 27°C. Le premier repiquage a lieu 24 heures après, puis tous les quinze jours pendant 3 mois sur milieu identique au milieu d'initiation additionné d'un agent sélectif dont la nature et la concentration peuvent varier selon le gène utilisé (voir paragraphe 3). Les agents sélectifs utilisables consistent généralement en composés actifs de certains
herbicides (Basta®, Round up®) ou certains antibiotiques (Hygromycine, Kanamycine... )The callus boxes thus bombarded are then sealed using Scellofrais® and then grown in the dark at 27 ° C. The first subculture takes place 24 hours later, then every fortnight for 3 months on medium identical to the initiation medium supplemented with a selective agent, the nature and concentration of which may vary depending on the gene used (see paragraph 3). The selective agents which can be used generally consist of active compounds of certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromycin, Kanamycin ...)
On obtient après 3 mois, ou parfois plus tôt, des cals dont la croissance n'est pas inhibée par l'agent sélection, habituellement et majoritairement composés de cellules résultant de la division d'une cellule ayant intégré dans son patrimoine génétique une ou plusieurs copies du gène de sélection. La fréquence d'obtention de tels cals est d'environ 0.8 cal par boite bombardée.Calls are obtained after 3 months, or sometimes earlier, calluses whose growth is not inhibited by the selection agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene. The frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
Ces cals sont identifiés, individualisés, amplifiés puis cultivés de façon à régénérer des plantules (cf. paragraphe a). Afin d'éviter toute interférence avec des cellules non transformées, toutes ces opérations sont menées sur des milieux de culture contenant l'agent sélectif.These calluses are identified, individualized, amplified and then cultivated so as to regenerate seedlings (see paragraph a). In order to avoid any interference with untransformed cells, all of these operations are carried out on culture media containing the selective agent.
Les plantes ainsi régénérées sont acclimatées puis cultivées en serre où elles peuvent être croisées ou autofécondées.The plants thus regenerated are acclimatized and then cultivated in a greenhouse where they can be crossed or self-fertilized.
C. MISE EN EVIDENCE DE LA GLYCOPROTEINE G RABIQUEC. EVIDENCE OF G RABIC GLYCOPROTEIN
I. ANALYSE DE L'EXPRESSION DE LA GLYCOPROTEINE G RABIQUE DANS LES FEUILLES , LES GRAINES DE TABAC ET LES GRAINES DE COLZA.I. ANALYSIS OF THE EXPRESSION OF G RABIC GLYCOPROTEIN IN LEAVES, TOBACCO SEEDS AND CANNED SEEDS.
Le protocole d'extraction de la glycoproteine G de la rage à partir des feuilles est le suivant:The protocol for extracting G glycoprotein from rabies from the leaves is as follows:
- 1 g de feuilles (poids frais) de tabac est broyé dans l'azote liquide puis suspendu, à 4°C, dans 4 ml de tampon Tris-HCl 100 mM pH 7.5, additionné d'EDTA 1 mM, Triton X100 0.2% et NaCl 250 mM.- 1 g of tobacco leaves (fresh weight) is ground in liquid nitrogen and then suspended, at 4 ° C, in 4 ml of 100 mM Tris-HCl buffer pH 7.5, supplemented with 1 mM EDTA, 0.2% Triton X100 and 250 mM NaCl.
L'homogénat ainsi obtenu est immédiatement centrifugé à 4°C pendant 10 min à 10 000 rpm.The homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 10,000 rpm.
Pour les graines de tabac ou de colza, l'extraction est réalisée à raison de 100 mg de
graines pour 4 ml de tampon. Pour les cals de maïs, l'extraction est effectuée à raison de 0.5 g dans 1 ml de tampon.For tobacco or rapeseed seeds, extraction is carried out at the rate of 100 mg of seeds for 4 ml of buffer. For corn calluses, the extraction is carried out at a rate of 0.5 g in 1 ml of buffer.
Apres centrifugation, le surnageant est récupéré puis filtré sur MIRACLOTH (CALBIOCHEM) . Un dosage des protéines solubles est alors réalisé par la méhode de Bradford (1976) .After centrifugation, the supernatant is recovered and then filtered through MIRACLOTH (CALBIOCHEM). A determination of the soluble proteins is then carried out by the method of Bradford (1976).
La glycoproteine G rabique est mise en évidence par immunodétection de type "Western" ("Western blots") (Renart et Sandoval, 1984) et dosée par un test ELISA.Rabies glycoprotein G is demonstrated by "Western blots" (Renart and Sandoval, 1984) and assayed by an ELISA test.
II. IMMUNODETECTION PAR WESTERN BLOT DE LA GLYCOPROTEINE G RABIQUE EXPRIMEE DANS LES FEUILLES DE TABAC, DANS LES GRAINES DE TABAC ET DE COLZA TRANSGENIQUES ET DANS LES CALS DE MAIS TRANSFORMES.II. WESTERN BLOT IMMUNODETECTION OF G RABIC GLYCOPROTEIN G EXPRESSED IN TOBACCO LEAVES, IN TRANSGENIC TOBACCO AND CANNED SEEDS AND IN PROCESSED CORN.
Les protéines extraites selon le protocole ci- dessus sont dénaturées par chauffage à 95°C pendant 5 min, en présence de tampon Tris HC1 50 mM ph 6.8; SDS 4%; BSH 1%; Saccharose 20% et bleu de bromophénol 0.01%.The proteins extracted according to the above protocol are denatured by heating at 95 ° C for 5 min, in the presence of 50 mM pH 6.8 Tris HCl buffer; SDS 4%; BSH 1%; Sucrose 20% and bromophenol blue 0.01%.
Les protéines sont ensuites séparées par électrophorèse, sur un gel de polyacrylamide en conditions dénaturantes à 8%, 10% ou 12.5%, selon la technique de Laemmli U.K. (1970) à raison de 50 ug de protéines totales par échantillon.The proteins are then separated by electrophoresis, on a polyacrylamide gel under denaturing conditions at 8%, 10% or 12.5%, according to the technique of Laemmli U.K. (1970) at the rate of 50 μg of total proteins per sample.
Après migration les protéines sont transférées sur une membrane de nitrocellulose. Un anticorps anti- glycoprotéine G de la rage, obtenu chez le mouton est utilisé comme sonde et la révélation est effectuée au moyen d'un anticorps anti-IgG de mouton margué à la phosphatase alcaline.After migration, the proteins are transferred to a nitrocellulose membrane. An anti-rabies G glycoprotein antibody obtained in sheep is used as a probe and the revelation is carried out by means of an anti-sheep IgG antibody margined with alkaline phosphatase.
Les essais ont été réalisés sur les plantes transformées avec les construits suivants : pBIOC104, pBIOCllO, pBIOC114, pBIOC116.
La protéine témoin migre sous la forme de 2 bandes de poids moléculaires apparents 66 et 61 KDa. La forme la plus légère correspond à une protéine, tronquée au niveau de l'extrémité C terminale (Wunner and al, 1983), plus soluble que la protéine complète.The tests were carried out on the transformed plants with the following constructs: pBIOC104, pBIOC10O, pBIOC114, pBIOC116. The control protein migrates in the form of 2 bands of apparent molecular weights 66 and 61 KDa. The lightest form corresponds to a protein, truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein.
L'analyse des extraits de feuilles et de graines de tabac, ainsi que dans les graines de colza et les cals de maïs, révèle une bande de poids moléculaire d'environ 66 KDa, (Fig 1, 2, 3 et 4) ce qui correspond à celui de la glycoproteine G du virus.Analysis of extracts from tobacco leaves and seeds, as well as in rapeseeds and corn calluses, reveals a band of molecular weight of approximately 66 KDa, (Fig 1, 2, 3 and 4) which corresponds to that of the glycoprotein G of the virus.
Aucune bande correspondant à la glycoproteine G rabique n'est détectée dans les extraits protéiques de feuilles et de graines de tabac et de colza non transformées.No band corresponding to rabies glycoprotein G is detected in the protein extracts of leaves and seeds of tobacco and rapeseed unprocessed.
III. DOSAGE PAR TEST ELISA DE LA GLYCOPROTEINE G RABIQUE EXPRIMEE DANS LES FEUILLES DE TABAC ET LES GRAINES DE TABAC ET DE COLZA TRANSGENIQUES.III. ASSAY BY ELISA TEST OF G RABIC GLYCOPROTEIN EXPRESSED IN TOBACCO LEAVES AND TRANSGENIC TOBACCO AND CANNED SEEDS.
Pour le dosage de la glycoproteine G exprimée dans les feuilles et dans les graines de tabac, le protocole suivant a été mis en oeuvre : extraction de la glycoproteine : les échantillons de feuilles ou de graines, prélevés sur glace le jour du test, sont broyés à raison de lg pour 4ml de tampon d'extraction pour les feuilles et lOOmg pour 4ml de tampon d'extraction pour les graines.For the determination of the glycoprotein G expressed in the leaves and in the tobacco seeds, the following protocol was used: extraction of the glycoprotein: the samples of the leaves or seeds, taken on ice on the day of the test, are ground at a rate of 1 g for 4 ml of extraction buffer for the leaves and 100 mg for 4 ml of extraction buffer for the seeds.
La quantité de protéines totales est alors déterminée par la méthode de Bradford et tous les extraits sont ramenés à une concentration de 5 μg/μl. Ces extraits sont dilués extemporairement au 1/1000. La gamme de référence (glycoproteine G : 0 à 20 mg/ml) est préparée dans un extrait de plante non transformée broyé, standardisé et dilué au 1/1000 de la même façon.The amount of total protein is then determined by the Bradford method and all the extracts are brought to a concentration of 5 μg / μl. These extracts are diluted extemporaneously to 1/1000. The reference range (glycoprotein G: 0 to 20 mg / ml) is prepared in an extract of ground unprocessed plant, standardized and diluted to 1/1000 in the same way.
Graines : Technique ELISA sandwich
La glycoproteine de la rage, après avoir été retenue par un anticorps monoclonal anti-glycoprotéine G de la rage adsorbé sur une plaque de polychlorure de vinyl, est mise en contact avec un anticorps polyclonal anti-glycoprotéine G de la rage. Celui-ci est révélé à l'aide d'IgG, marqués à la peroxydase ou à la phosphatase alcaline et dirigés contre le fragment F(ab')2 de l'anticorps polyclonal précédent.Seeds: ELISA sandwich technique The rabies glycoprotein, after having been retained by a monoclonal anti-rabies glycoprotein G antibody adsorbed on a polyvinyl chloride plate, is brought into contact with a polyclonal anti-rabies glycoprotein G antibody. This is revealed using IgG, labeled with peroxidase or alkaline phosphatase and directed against the F (ab ') 2 fragment of the preceding polyclonal antibody.
Le dosage s'effectue alors par lecture directe sur une courbe étalon réalisée dans les mêmes conditions, avec de la glyco-protéine G purifiée.The assay is then carried out by direct reading on a standard curve carried out under the same conditions, with purified glyco-protein G.
Feuilles : Technique ELISA directeSheets: Direct ELISA technique
Chacun de ces échantillons est absorbé sur la plaque (150 μl/puits) pendant 2 heures à 37°C, puis marqué, sans étape de saturation préalable, par un anticorps polyclonal de cheval purifié (100 μl/puits, 1 heure, température ambiante) , et enfin, révélé par un anticorps marqué à la phosphatase alcaline (SIGMA réf. A6063), dirigé contre le précédent et dilué au 1/1000 (100 μl/puits, 30 minutes, température ambiante) . Entre chaque étape, les lavages sont effectués avec du PBS additionné de Tween 20 à 0.1% (P/V) . Les résultats sont ramenés à une quantité de glycoproteine par mg de protéines totales.Each of these samples is absorbed on the plate (150 μl / well) for 2 hours at 37 ° C., then labeled, without prior saturation step, with a purified polyclonal horse antibody (100 μl / well, 1 hour, room temperature ), and finally, revealed by an antibody labeled with alkaline phosphatase (SIGMA ref. A6063), directed against the previous one and diluted to 1/1000 (100 μl / well, 30 minutes, room temperature). Between each step, the washes are carried out with PBS added with 0.1% Tween 20 (W / V). The results are reduced to an amount of glycoprotein per mg of total protein.
La quantité de glycoproteine G varie de 1.5 à 0.4 μg/ml pour les différents extraits testés.
The amount of glycoprotein G varies from 1.5 to 0.4 μg / ml for the different extracts tested.
D. MISE EN EVIDENCE DE LA GLYCOPROTEINE G RABIQUE DANS LES GRAINS DE MAIS.D. EVIDENCE OF G RABIC GLYCOPROTEIN IN CORN GRAINS.
Le protocole d'extraction de la glycoproteine G de la rage à partir de grains de maïs est le suivant :The protocol for extracting G glycoprotein from rabies from corn kernels is as follows:
- 200 mg de grains sont broyés dans l'azote liquide, puis suspendu dans 1ml de tampon Tris-HCl 100 mM pH 7,5, additionné d' EDTA ImM, Triton X100 0,2% et NaCl 250 mM .- 200 mg of grains are ground in liquid nitrogen, then suspended in 1 ml of 100 mM Tris-HCl buffer pH 7.5, supplemented with EDTA ImM, 0.2% Triton X100 and 250 mM NaCl.
L'homogénat ainsi obtenu est immédiatement centrifugé à 4 °C pendant 10 min à 13000 g. Puis, le surnageant est prélevé et filtré sur Miracloth (CALBIOCHEM) . Un dosage des protéines solubles est alors réalisé par la méthode de Bradford (1976) .The homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 13000 g. Then, the supernatant is removed and filtered through Miracloth (CALBIOCHEM). A determination of the soluble proteins is then carried out by the method of Bradford (1976).
La glycoproteine G rabique est mise en évidence par immunodétection de type "Western blot" (Renart et Sandoval , 1984) .Rabies glycoprotein G is demonstrated by immunodetection of the "Western blot" type (Renart and Sandoval, 1984).
Les protéines extraites selon le protocole ci-dessus sont dénaturées par chauffage à 95°C pendant 5 min ,en présence de tampon Tris-HCl 50mM pH 6,8,SDS 4%, BSH 1%, Saccharose 20% et bleu de bromophénol 0,01%.The proteins extracted according to the above protocol are denatured by heating at 95 ° C. for 5 min, in the presence of 50 mM Tris-HCl buffer, pH 6.8, 4% SDS, 1% BSH, 20% sucrose and bromophenol blue 0 , 01%.
Les protéines sont ensuite séparées par électrophorèse sur un gel de polyacrylamide en conditions dénaturantes à 8% ou 10%, selon la technique de Laemmli (1970) à raison de 30μg de protéines solubles par échantillon.The proteins are then separated by electrophoresis on a polyacrylamide gel under denaturing conditions at 8% or 10%, according to the technique of Laemmli (1970) at the rate of 30 μg of soluble proteins per sample.
Après migration, les protéines sont transférées sur une membrane PVDF (polyvinylidene difluoride). Un anti-sérum polyclonal anti- glycoprotéine G rabique, obtenu chez le mouton, est utilisé comme sonde et la révélation est effectuée au moyen d'un anticorps anti-IgG de mouton marqué à la phosphatase alcaline .After migration, the proteins are transferred to a PVDF (polyvinylidene difluoride) membrane. A rabies polyclonal anti-glycoprotein G anti-serum, obtained in sheep, is used as probe and the revelation is carried out using an anti-sheep IgG antibody labeled with alkaline phosphatase.
La glycoproteine G rabique de référence témoin est caractérisée par 2 bandes de masses moléculaires apparentes de 66 et 61 KDa. La forme la
plus légère correspond à une protéine tronquée au niveau de l'extrémité C terminale (Wunner et al, 1983), plus soluble que la protéine complète. Aucune bande correspondant à la glycoproteine G rabique n'est détectée dans les extraits de maïs non transformés.The control reference rabies glycoprotein G is characterized by 2 bands of apparent molecular masses of 66 and 61 KDa. The form lighter corresponds to a protein truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein. No band corresponding to rabies glycoprotein G is detected in the unprocessed corn extracts.
L'analyse des extraits de grains de maïs transformés avec pBIOCllδ met en évidence des bandes spécifiques caractéristiques de la glycoproteine G. Une des bandes est de masse moléculaire d'environ 66 KDa.Analysis of the extracts of corn kernels transformed with pBIOCllδ reveals specific bands characteristic of glycoprotein G. One of the bands has a molecular mass of approximately 66 KDa.
E. VEGETALISATION DE LA SEQUENCE CODANT LA GLYCOPROTEINE G RABIQUEE. VEGETATION OF THE SEQUENCE ENCODING G RABIC GLYCOPROTEIN
I. MODIFICATION DE LA SEQUENCE CODANT LA GLYCOPROTEINE G RABIQUE : CREATION DE SEQUENCES SYNTHETIQUES UTILISABLES EN TRANSFORMATION GENETIQUE DU MAÏS.I. MODIFICATION OF THE SEQUENCE CODING G RABIC GLYCOPROTEIN: CREATION OF SYNTHETIC SEQUENCES FOR USE IN GENETIC TRANSFORMATION OF CORN.
Pour augmenter le taux d'expression, la séquence codant la glycoproteine G rabique a été soumise à plusieurs modifications :To increase the level of expression, the sequence coding for rabies glycoprotein G has been subjected to several modifications:
- utilisation du code génétique des plantes (Murray E.E. Nucleic Acids Res., 1989, 17, 477)- use of plant genetic code (Murray E.E. Nucleic Acids Res., 1989, 17, 477)
- modification du contenu en GC- modification of content in GC
- suppression des introns cryptiques- removal of cryptic introns
- suppression des signaux de polyadénylation internes- suppression of internal polyadenylation signals
- suppression des séquences déstabilisant TARN- removal of TARN destabilizing sequences
- suppression de signaux de terminaison de la Polymérase II- suppression of Polymerase II termination signals
La modification du cDNA codant la glycoproteine G rabique a été réalisée selon la technique LbPCR en modifiant les parties 5' [fragment BglII (agatct) - Xhol (ctcgag)], centrale [fragment Xhol - Aatll (gacgtc)] et 3' [fragment Aatll - BamHI (ggatcc)] La technique LbPCR consiste en une étape de ligation LCR (Barany F.
P N A S USA 1991, 88, 189-193) pour produire un ADN simple brin continu à l'aide des oligodésoxynucléotides "sens" et d'une étape de PCR pour obtenir l'ADN double brin Chacune des parties modifiées a été vérifiée par séquençage à l'aide du kit "Sequenase PCR product sequencing" d'Amersham selon les recommandations du fabricant Puis, soit la partie 5' modifiée, soit les parties 5' et centrale modifiées, soit les parties 5', centrale et 3' modifiées, ont été introduites dans pBIOC109 par digestions enzymatiques (enzymes de restriction délimitant les fragments)The modification of the cDNA encoding the rabies glycoprotein G was carried out according to the LbPCR technique by modifying the 5 '[fragment BglII (agatct) - Xhol (ctcgag)], central [fragment Xhol - Aatll (gacgtc)] and 3' [fragment Aatll - BamHI (ggatcc)] The LbPCR technique consists of an LCR ligation step (Barany F. PNAS USA 1991, 88, 189-193) to produce continuous single-stranded DNA using "sense" oligodeoxynucleotides and a PCR step to obtain double-stranded DNA. Each of the modified parts was verified by sequencing at using the Amersham Sequenase PCR product sequencing kit according to the manufacturer's recommendations Then, either the modified 5 'part, or the modified 5' and central parts, or the modified 5 ', central and 3' parts, were were introduced into pBIOC109 by enzymatic digestions (restriction enzymes delimiting the fragments)
La partie 5' a été constituée à l'aide des oligodésoxynucléotides suivantsThe 5 ′ part was constituted using the following oligodeoxynucleotides
- oligodésoxynucléotides "sens" pour former l'ADN simple brin continu 1 cgagatctgaattcaacaATGAAAGCCTTCACACTCGCTCTCTTCTTAGCTCTTTC CCTCTATCT- "sense" oligodeoxynucleotides to form continuous single-stranded DNA 1 cgagatctgaattcaacaATGAAAGCCTTCACACTCGCTCTCTTCTTAGCTCTTTC CCTCTATCT
2 CCTGCCCAATCCAGCCcattccaggttcaatcccatccgcctccccaccacacacgaacccgccAA2 CCTGCCCAATCCAGCCcattccaggttcaatcccatccgcctccccaccacacacgaacccgccAA
3 gTTCCCaATcTACACcATCCCAGACaAGCTcGGcCCaTGGAGCCCaATcGAC3 gTTCCCaATcTACACcATCCCAGACaAGCTcGGcCCaTGGAGCCCaATcGAC
ATcCAcCACCTCAATcCAcCACCTCA
44
GCTGCCCAAACAAcTTGGTcGTcGAGGACGAgGGcTGCACCAACCTcTCcG GcTTCTCCTACATGGCTGCCCAAACAAcTTGGTcGTcGAGGACGAgGGcTGCACCAACCTcTCcG GcTTCTCCTACATG
55
GAgCTcAAgGTcGGcTACATCTTgGCCATcAAgATGAACGGcTTCACcTGCAGAgCTcAAgGTcGGcTACATCTTgGCCATcAAgATGAACGGcTTCACcTGCA
CcGGCGTcGTcAC
6 cGAGGCcGAgACCTACACcAACTTCGTcGGcTAcGTCACcACCACcTTCAAgCcGGCGTcGTcAC 6 cGAGGCcGAgACCTACACcAACTTCGTcGGcTAcGTCACcACCACcTTCAAg
AGgAAGCAcTTCCGAGgAAGCAcTTCCG
7 gCCAACcCCAGAcGCcTGcAGgGCCGCcTACAACTGGAAGATGGCCGGcG7 gCCAACcCCAGAcGCcTGcAGgGCCGCcTACAACTGGAAGATGGCCGGcG
ACCCaAGgTAcGAgACCCaAGgTAcGAg
88
GAGTCcCTcCACAAcCCaTACCCaGACTACCGCTGGCTcCGcACcGTcAAgAGAGTCcCTcCACAAcCCaTACCCaGACTACCGCTGGCTcCGcACcGTcAAgA
CCACCAAGGAGTCCCACCAAGGAGTC
9 cCTCGTcATCATcTCcCCAAGcGTcGCcGAcTTGGACCCATAcGACAGgTCC9 cCTCGTcATCATcTCcCCAAGcGTcGCcGAcTTGGACCCATAcGACAGgTCC
CTcCACTCGAGGGTCTTCTcCACTCGAGGGTCTT
- oligodésoxynucléotides "guides" pour relier les oligodésoxynucléotides "sens"- "guide" oligodeoxynucleotides to connect the "sense" oligodeoxynucleotides
1 CTGGATTGGGCAGGagatagagggaaagagctaag1 CTGGATTGGGCAGGagatagagggaaagagctaag
2 GATgGTGTAGATTGGGAACttggcgggttcg2 GATgGTGTAGATTGGGAACttggcgggttcg
3 AGTTGTTTGGGCAGCtgaggtggtggatgtc3 AGTTGTTTGGGCAGCtgaggtggtggatgtc
4 GCCGACCTTGAGCTCcatgtaggagaagccg4 GCCGACCTTGAGCTCcatgtaggagaagccg
5 GGTCTCGGCCTCGgtgacgacgccg5 GGTCTCGGCCTCGgtgacgacgccg
6 GTCTGGGGTTGGCcggaagtgcttcctct6 GTCTGGGGTTGGCcggaagtgcttcctct
7 GGTTGTGGAGGGACTCctcgtaccttgggtcg7 GGTTGTGGAGGGACTCctcgtaccttgggtcg
8 GGAGATGATGACGAGGgactccttggtggtctt8 GGAGATGATGACGAGGgactccttggtggtctt
- oligodésoxynucléotides pour réaliser l'amplification PCR de l'ADN simple brin obtenu
cgagatctgaattcaacaATGAAAGCC et- oligodeoxynucleotides to carry out PCR amplification of the single-stranded DNA obtained cgagatctgaattcaacaATGAAAGCC and
AAGACCCTCGAGTGGAGGgaccAAGACCCTCGAGTGGAGGgacc
La partie 5' amplifiée par PCR a été doublement digérée par BglII et Xhol, puis, clonée en remplacement du fragment BglII - Xhol de pBIOC109 selon les procédés déjà décrits précédemment Le plasmide résultant a été appelé pBIOC109-5'M Ce plasmide a été utilisé en transformation génétique du maisThe 5 ′ part amplified by PCR was doubly digested with BglII and Xhol, then cloned to replace the BglII - Xhol fragment of pBIOC109 according to the methods already described previously. The resulting plasmid was called pBIOC109-5'M This plasmid was used in genetic transformation of corn
La partie centrale a été constituée à l'aide des oligodésoxynucléotides suivants .The central part was formed using the following oligodeoxynucleotides.
- oligodésoxynucléotides "sens" : 1- "sense" oligodeoxynucleotides: 1
CCCaAGCGGcAAGTGCTCcGGcGTcGCcGTcTCcTCcACCTACTGCTCCACc AACCACGAcCCCaAGCGGcAAGTGCTCcGGcGTcGCcGTcTCcTCcACCTACTGCTCCACc AACCACGAc
22
TACACCATcTGGATGCCaGAGAAcCCaAGgCTcGGcATGTCcTGcGACATcTTACACCATcTGGATGCCaGAGAAcCCaAGgCTcGGcATGTCcTGcGACATcT
TcACCAAcATcACCAAcA
3.3.
GcAGgGGcAAGAGgGCcTCCAAgGGcAGcGAGACcTGCGGCTTcGTcGAcGGcAGgGGcAAGAGgGCcTCCAAgGGcAGcGAGACcTGCGGCTTcGTcGAcG
AgAGgGGCCTAgAGgGGCCT
4. cTAcAAGTCcTTgAAgGGcGCcTGCAAgCTCAAGTTgTGcGGcGTcCTcGGcC TcAGgCTc4. cTAcAAGTCcTTgAAgGGcGCcTGCAAgCTCAAGTTgTGcGGcGTcCTcGGcC TcAGgCTc
55
ATGGAcGGcACcTGGGTCGCcATGCAAACcTCcAAcGAgACCAAgTGGTGC CCaCCaGAcC
6.ATGGAcGGcACcTGGGTCGCcATGCAAACcTCcAAcGAgACCAAgTGGTGC CCaCCaGAcC 6.
AaTTGGTcAACCTcCACGACTTcCGCTCcGACGAgATcGAGCACCTcGTcGT cGAGGAGTTAaTTGGTcAACCTcCACGACTTcCGCTCcGACGAgATcGAGCACCTcGTcGT cGAGGAGTT
7.7.
GGTCAGGAAGAGgGAGGAGTGcCTcGAcG€cCTtGAGTCCATCATGACcAGGTCAGGAAGAGgGAGGAGTGcCTcGAcG € cCTtGAGTCCATCATGACcA
CCAAGTCcGTcACCAAGTCcGTcA
- oligodésoxynucléotides "guides" : 1 GCATCCAGATGGTGTAgtcgtggttggtgga 2. CTCTTGCCCCTGCtgttggtgaagatgtcg- "guide" oligodeoxynucleotides: 1 GCATCCAGATGGTGTAgtcgtggttggtgga 2. CTCTTGCCCCTGCtgttggtgaagatgtcg
3 CCCTTCAAGGACTTGTAGaggcccctctcgt3 CCCTTCAAGGACTTGTAGaggcccctctcgt
4 CAGGTGCCGTCCATgagcctgaggccg4 CAGGTGCCGTCCATgagcctgaggccg
5. GTGGAGGTTGACCAATTggtctggtgggcac 6 CTCCCTCTTCCTGACCaactcctcgacgacg5. GTGGAGGTTGACCAATTggtctggtgggcac 6 CTCCCTCTTCCTGACCaactcctcgacgacg
- oligodésoxynucléotides pour réaliser l'amplification PCR : tccgctcgagCCCAAGCGGCAAGTGCTCCGgcg et actgacgtcTGACGGACTTGGTGGTCATGATGGACtcaagggcgtcgag- oligodeoxynucleotides to carry out PCR amplification: tccgctcgagCCCAAGCGGCAAGTGCTCCGgcg and actgacgtcTGACGGACTTGGTGGTCATGATGGACtcaagggcgtcgag
La partie centrale amplifiée par PCR a été doublement digérée par Xhol et Aatll, puis, clonée en remplacement du fragment Xhol - Aatll de pBIOC109-5'M selon les procédés déjà décrits précédemment. Le plasmide résultant a été appelé pBIOC109-5'CM. Ce plasmide a été utilisé en transformation génétique du maïs.The central part amplified by PCR was doubly digested with Xhol and Aatll, then cloned to replace the Xhol - Aatll fragment of pBIOC109-5'M according to the methods already described above. The resulting plasmid was called pBIOC109-5'CM. This plasmid was used in genetic transformation of corn.
La partie 3' a été constituée à l'aide des oligodésoxynucléotides suivants :The 3 ′ part was constituted using the following oligodeoxynucleotides:
- oligodésoxynucléotides "sens" :
1- "sense" oligodeoxynucleotides: 1
GcTTCAGACGTCTCAGcCAcTTgAGgAAgCTcGTCCCaGGcTTcGGcAAgGC cTAcACCATcTTCAACAGcTTCAGACGTCTCAGcCAcTTgAGgAAgCTcGTCCCaGGcTTcGGcAAgGC cTAcACCATcTTCAACA
22
AGACCTTGATGGAgGCCGAcGCcCACTACAAGTCcGTCAGgACcTGGAAcAGACCTTGATGGAgGCCGAcGCcCACTACAAGTCcGTCAGgACcTGGAAc
GAGATCCTCCCaTCcGAGATCCTCCCaTCc
33
AAgGGcTGcTTgAGgGTcGGcGGcAGGTGcCAcCCaCAcGTcAACGGcGTcTT cTTCAAcGGcATAAgGGcTGcTTgAGgGTcGGcGGcAGGTGcCAcCCaCAcGTcAACGGcGTcTT cTTCAAcGGcAT
4 cATcTTgGGcCCaGACGGCAAcGTCTTgATCCCAGAGATGCAATCcTCCCT4 cATcTTgGGcCCaGACGGCAAcGTCTTgATCCCAGAGATGCAATCcTCCCT
CCTCCAaCAACAcATCCTCCAaCAACAcAT
55
GGAGTTGTTGGAgTCCTCcGTcATCCCaCTcGTcCACCCaCTcGCcGACCCaGGAGTTGTTGGAgTCCTCcGTcATCCCaCTcGTcCACCCaCTcGCcGACCCa
TCcACCGTcTTCTCcACCGTcTTC
66
AAGGACGGcGACGAGGCcGAGGAcTTcGTcGAgGTcCACCTcCCaGATGTcAAGGACGGcGACGAGGCcGAGGAcTTcGTcGAgGTcCACCTcCCaGATGTc
CACAAcCAaGTCTCACAAcCAaGTCT
77
CcGGcGTcGACTTGGGcCTCCCaAACTGGGGcAAGTAcGTcTTgCTcAGcGC cGGcGCCCTcACCcGGcGTcGACTTGGGcCTCCCaAACTGGGGcAAGTAcGTcTTgCTcAGcGC cGGcGCCCTcAC
8 cGCCTTGATGTTGATcATcTTCCTcATGACcTGcTGcAGgAGgGTCAAcCGc TCcGAgCCaACc
98 cGCCTTGATGTTGATcATcTTCCTcATGACcTGcTGcAGgAGgGTCAAcCGc TCcGAgCCaACc 9
CAACACAAcCTCAGgGGcACcGGcAGGGAGGTcTCcGTCACcCCaCAAAGCCAACACAAcCTCAGgGGcACcGGcAGGGAGGTcTCcGTCACcCCaCAAAGC
GGcAAGATCATcTGGcAAGATCATcT
10 CcTCcTGGGAgTCcCACAAGAGcGGcGGcGAGACCAGgCTcTGAggatccgggg aattctgtga10 CcTCcTGGGAgTCcCACAAGAGcGGcGGcGAGACCAGgCTcTGAggatccgggg aattctgtga
- oligodésoxynucléotides "guides"- "guide" oligodeoxynucleotides
1 GCCTCCATCAAGGTCTtgttgaagatggtgtagg1 GCCTCCATCAAGGTCTtgttgaagatggtgtagg
2 CTCAAGCAGCCCTTggatgggaggatctc2 CTCAAGCAGCCCTTggatgggaggatctc
3 TGGGCCCAAGATGatgccgttgaagaagac3 TGGGCCCAAGATGatgccgttgaagaagac
4 AGGACTCCAACAACTCCatgtgttgttggaggag4 AGGACTCCAACAACTCCatgtgttgttggaggag
5 CGTCGCCGTCCTTgaagacggtggatgg5 CGTCGCCGTCCTTgaagacggtggatgg
6 AGTCGACGCCGGagacttggttgtggacat6 AGTCGACGCCGGagacttggttgtggacat
7 GATCAACATCAAGGCGgtgagggcgccg7 GATCAACATCAAGGCGgtgagggcgccg
8 CCCTGAGGTTGTGTTGggttggctcggag8 CCCTGAGGTTGTGTTGggttggctcggag
9 GGACTCCCAGGAGGagatgatcttgccgct9 GGACTCCCAGGAGGagatgatcttgccgct
- oligodésoxynucléotides pour réaliser l'amplification PCR GCTTCAGACGTCTCAGCCACTTga et TCACAGAATTCCCCGGATCCtc- oligodeoxynucleotides to carry out PCR amplification GCTTCAGACGTCTCAGCCACTTga and TCACAGAATTCCCCGGATCCtc
La partie 3' amplifiée par PCR a été doublement digérée par Aatll et BamHI, puis, clonée en remplacement du fragment Aatll - BamHI de pBIOC109-5'CM selon les procédés déjà décrits précédemment Le plasmide résultant a été appelé pBIOC109-5'C3'M Ce plasmide a été utilisé en transformation génétique du mais
II. CONSTRUCTION DES PLASMIDES BINAIRES CONTENANT LES DIFFERENTES SEQUENCES MODIFIEES DU cDNA CODANT LA GLYCOPROTEINE G RABIQUE UTILISABLES EN TRANSGENESE VIA AGROBACTERIUM TUMEFACIENSThe 3 ′ part amplified by PCR was doubly digested with Aatll and BamHI, then cloned to replace the Aatll - BamHI fragment of pBIOC109-5'CM according to the methods already described previously. The resulting plasmid was called pBIOC109-5'C3 ' M This plasmid was used in genetic transformation of maize II. CONSTRUCTION OF BINARY PLASMIDS CONTAINING THE DIFFERENT MODIFIED SEQUENCES OF cDNA ENCODING GLABIC GLYCOPROTEIN FOR USE IN TRANSGENESIA VIA AGROBACTERIUM TUMEFACIENS
Les fragments EcoRI portant les différentes séquences modifiées du cDNA codant la glycoproteine G rabique ont été isolés à partir des plasmides pBIOC109-5'M, pBIOC109-5'CM et pBIOC109-5'C3'M. Les procédés utilisés ont déjà été décrits précédemmentThe EcoRI fragments carrying the different modified sequences of the cDNA coding for rabies glycoprotein G were isolated from the plasmids pBIOC109-5'M, pBIOC109-5'CM and pBIOC109-5'C3'M. The processes used have already been described previously
Les fragments isolés ont été ligués à l'ADN plasmidique de pBIOC21, pBIOC28, pBIOC82, pBIOC91 et pBIOC1 12 digéré par EcoRI et déphosphorylé Les méthodes usuelles de clonage ont été appliquées. L'ADN plasmidique des plasmides binaires résultants a été introduit par transformation directe dans la souche LBA4404 d'Agrobacterium tumefaciens selon le procédé de Holsters et al. (1978). La transformation génétique a été réalisée.The isolated fragments were ligated to the plasmid DNA of pBIOC21, pBIOC28, pBIOC82, pBIOC91 and pBIOC1 12 digested with EcoRI and dephosphorylated. The usual cloning methods were applied. The plasmid DNA of the resulting binary plasmids was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). Genetic transformation has been completed.
L'invention s'étend donc particulièrement aux procédés, aux glycoprotéines et leurs utilisations, aux acides nucléiques, aux cellules végétales transformées, aux plantes chimériques ou transgéniques, aux graines de plantes transgénique, aux anticorps monoclonaux ou polyclonaux, aux compositions pharmaceutiques et aux produits alimentaires selon l'invention caractérisés en ce que la séquence codante, permettant l'expression dans la cellule de la protéine G, a été soumise à des modifications pour augmenter l'expression, par exemple a été soumis à une "végétalisation" selon l'exemple E ci-dessus.
REFERENCESThe invention therefore particularly extends to methods, to glycoproteins and their uses, to nucleic acids, transformed plant cells, chimeric or transgenic plants, seeds of transgenic plants, monoclonal or polyclonal antibodies, pharmaceutical compositions and products. foodstuffs according to the invention characterized in that the coding sequence, allowing expression in the cell of the protein G, has been subjected to modifications to increase the expression, for example has been subjected to a "revegetation" according to example E above. REFERENCES
Kay R. , Chan A., Daly M., McPherson J. , 1987, Science, 236 : 1299 ;Kay R., Chan A., Daly M., McPherson J., 1987, Science, 236: 1299;
Franck A., Guilley H., Jonard G., Richards K. , Hirth L., 1980, Cell, 21 : 285 ;Franck A., Guilley H., Jonard G., Richards K., Hirth L., 1980, Cell, 21: 285;
An G., 1986, Plant Physiol., 81 : 86 ;An G., 1986, Plant Physiol., 81: 86;
Sambrook J. , Fritsch E. F., Maniatis T., 1989, Molecular Cloning. A laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press ;Sambrook J., Fritsch E. F., Maniatis T., 1989, Molecular Cloning. A laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press;
Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H.M. , 1982, J. Mol. Appl. Genêt., i : 561 ;Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H.M., 1982, J. Mol. Appl. Genet., I: 561;
Berg D. E. , Berg C. M., 1983, Biotechnology, 1 : 417 ;Berg D. E., Berg C. M., 1983, Biotechnology, 1: 417;
Close, Rodriguez, 1982, Gène, 2_0 : 305 ;Close, Rodriguez, 1982, Gene, 2_0: 305;
Liu C-N. , Steck T. R. , Habeck L. L. , Meyer J. A., Gelvin S. B., 1993, Mol. Plant Microbe Interactions, 6. : 144 ;Liu C-N. , Steck T. R., Habeck L. L., Meyer J. A., Gelvin S. B., 1993, Mol. Plant Microbe Interactions, 6.: 144;
Hanahan D. , 1983, J. Mol. Biol., 166 : 557 ;Hanahan D., 1983, J. Mol. Biol., 166: 557;
Birnboim H. C. , Doly J. , 1979, Nucl. Acids Res., 2 : 1513 ;Birnboim H. C., Doly J., 1979, Nucl. Acids Res., 2: 1513;
Matsuoka K., Nakamura K. , 1991, Proc. Natl. Acad. Sci. , 8_8_ : 834 ;Matsuoka K., Nakamura K., 1991, Proc. Natl. Acad. Sci. , 8_8_: 834;
Guerineau F., Mullineaux P., 1993, Plant Molecular Biology LABFAX, Croy R. R. D. (ed) , BIOS Scientific Publishers, Blackwell Scientific Publications ;Guerineau F., Mullineaux P., 1993, Plant Molecular Biology LABFAX, Croy R. R. D. (ed), BIOS Scientific Publishers, Blackwell Scientific Publications;
Murakami S., Hattori T., Nakamura K. , 1986, Plant Mol. Biol. , 2 : 343 ;Murakami S., Hattori T., Nakamura K., 1986, Plant Mol. Biol. , 2: 343;
Sanger F., Nicklen S., Coulson A. R. , 1977, Proc. Natl. Acad. Sci., 74. Î 5463 ;Sanger F., Nicklen S., Coulson A. R., 1977, Proc. Natl. Acad. Sci., 74. Î 5463;
Holster M., de Waele D., Depicker A., Messens E. , van Montagu M., Schell J. , 1978, Mol. Gen. Genêt., 163 : 181 ;Holster M., de Waele D., Depicker A., Messens E., van Montagu M., Schell J., 1978, Mol. Gen. Genet., 163: 181;
Depigny-This D. , Raynal M. , Aspart L. , Delseny M. , Grellet F., 1992, Plant Mol. Biol., 2J) : 467 ;Depigny-This D., Raynal M., Aspart L., Delseny M., Grellet F., 1992, Plant Mol. Biol., 2J): 467;
Ni M., Cui D., Einstein J. , Narasimhulu S., Vergara C. E. , Gelvin S.B., 1995, Plant J. , 1_ : 661 ;
Lerner D. R. , Raikhel N. V., 1989, Plant Physiol., 91 : 124 ;Ni M., Cui D., Einstein J., Narasimhulu S., Vergara CE, Gelvin SB, 1995, Plant J., 1_: 661; Lerner DR, Raikhel NV, 1989, Plant Physiol., 91: 124;
Wunner W. H., Dietzschold B. , Curtis P. J;, Wiktor T. J., 1983, J. Gen. Virol., M : 1649 ;Wunner W. H., Dietzschold B., Curtis P. J;, Wiktor T. J., 1983, J. Gen. Virol., M: 1649;
Bradford M., 1976, Anal. Biochem. , 22 : 248 ;Bradford M., 1976, Anal. Biochem. , 22: 248;
Renart J., Sandoval I. V;, 1984, Meth. Enzymol., 104 : 455 ;Renart J., Sandoval I. V;, 1984, Meth. Enzymol., 104: 455;
Laemmli U. K., 1970, Nature, 227 : 680 ;Laemmli U. K., 1970, Nature, 227: 680;
Kieny M. P., Lathe R. , Drillien R. , Spehner D. , Skory S., Schmitt D. , Wiktor T., Koprowski H., Lecocg J. P., 1984, Nature, 3_12 : 163 ;Kieny M. P., Lathe R., Drillien R., Spehner D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocg J. P., 1984, Nature, 3_12: 163;
Anilionis A., Wunner W. H., Curtis P. J. , 1981, Nature, 221, 275-278 ;Anilionis A., Wunner W. H., Curtis P. J., 1981, Nature, 221, 275-278;
Yelverton E. , Norton S., Obijeski J. F., Goeddel D. V., 1983, Science, 219. 614-620 ;Yelverton E., Norton S., Obijeski J. F., Goeddel D. V., 1983, Science, 219. 614-620;
Prehaud C. et al., 1989, Virology, 121, 390-399 ;Prehaud C. et al., 1989, Virology, 121, 390-399;
Tordo N., Bourhy H., Sather S., Ollo R. , 1993, Virology, 194. 59-69 ;Tordo N., Bourhy H., Sather S., Ollo R., 1993, Virology, 194. 59-69;
McGarvey P. B. et al., 1995, Bio/Technology, 13., 1484- 1487 ;McGarvey P. B. et al., 1995, Bio / Technology, 13., 1484-1487;
Bourhy H. et al., 1993, Virology, 194. 70-81 ;Bourhy H. et al., 1993, Virology, 194. 70-81;
Dietzschold B. et al., 1983, Virology, 124. 330-337 ;Dietzschold B. et al., 1983, Virology, 124. 330-337;
Coslett G. et al., 1980, J. Gen. Virol., 4_9_, 161-190 ;Coslett G. et al., 1980, J. Gen. Virol., 4_9_, 161-190;
Cox J. et al., 1980, Infection and Immunity, 3_0, 572- 577 ;Cox J. et al., 1980, Infection and Immunity, 30, 572-577;
Atanasiu et al., 1976, Ann. Microbiol., 127B. 257-267Atanasiu et al., 1976, Ann. Microbiol., 127B. 257-267
Tuchiya et al., 1992, Virus Research, 21, 1-13 ;Tuchiya et al., 1992, Virus Research, 21, 1-13;
Smith at al., « Laboratory Techniques in Rabies »,Smith at al., "Laboratory Techniques in Rabies",
W.H.O. Monograph Séries n° 23 (M. Kaplan and H.W.H.O. Monograph Series No. 23 (M. Kaplan and H.
Koprowski, Eds) , 3rd éd., pages 354-357, Geneva, Switzeland.
Koprowski, Eds), 3rd ed., Pages 354-357, Geneva, Switzeland.
Claims
REVENDICATIONS
1 - Procédé pour la production de la glycoproteine G du virus de la rage, ou d'un virus apparenté au virus de la rage, caractérisé par : i) l'introduction, dans une cellule végétale, d'une molécule d'acide nucléique comprenant une séquence codante chimérique, comportant d'une part une séquence codant pour ladite protéine G virale mature, ou pour une protéine analogue, et d'autre part, une séquence codant pour un peptide signal N-terminal autre que celui naturellement associé à la protéine G virale, 1•introduction de la séquence codante chimérique permettant l'expression dans la cellule de la protéine G ; ii) la multiplication, sous forme de culture cellulaire, des cellules transformées, ou la régénération de plantes entières transgéniques ou chimériques à partir de ces cellules ; iii) éventuellement l'extraction et la purification de la glycoproteine G à partir des cellules ou des tissus végétaux ainsi obtenus.1 - Process for the production of the rabies virus glycoprotein G, or of a virus related to the rabies virus, characterized by: i) the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence, comprising on the one hand a sequence coding for said mature viral G protein, or for an analogous protein, and on the other hand, a sequence coding for an N-terminal signal peptide other than that naturally associated with viral protein G, 1 • introduction of the chimeric coding sequence allowing expression in the cell of protein G; ii) the multiplication, in the form of cell culture, of the transformed cells, or the regeneration of whole transgenic or chimeric plants from these cells; iii) optionally the extraction and purification of the glycoprotein G from the cells or plant tissues thus obtained.
2 - Procédé selon la revendication 1 caractérisé en ce que la séquence codant pour le peptide signal N- terminal code pour un peptide signal d'origine végétale.2 - Method according to claim 1 characterized in that the sequence coding for the signal peptide N-terminal code for a signal peptide of plant origin.
3 - Procédé selon la revendication 1 ou *.2 caractérisé en ce que l'acide nucléique introduit dans la cellule comprend en outre des séquences régulatrices de la transcription reconnues par la cellule végétale.3 - Method according to claim 1 or * .2 characterized in that the nucleic acid introduced into the cell further comprises regulatory sequences of transcription recognized by the plant cell.
4 - Procédé selon la revendication 3 caractérisé en ce que les séquences régulatrices comprennent au moins un signal d'initiation de transcription et un signal de terminaison de transcription.
5 - Procédé selon la revendication 3 caractérisé en ce que les signaux d'initiation et de terminaison de transcription sont d'origine végétale, ou proviennent d'un virus végétal, ou d'Aσrobacterium.4 - Method according to claim 3 characterized in that the regulatory sequences comprise at least one transcription initiation signal and a transcription termination signal. 5 - Process according to claim 3 characterized in that the initiation and transcription termination signals are of plant origin, or come from a plant virus, or from Aσrobacterium.
6 - Procédé selon l'une quelconque des revendications précédentes caractérisé en ce que la séquence codante chimérique comprend en outre une séquence codant pour un signal de rétention endoplasmique d'origine végétale, ou pour un signal d•adressage vacuolaire.6 - Process according to any one of the preceding claims, characterized in that the chimeric coding sequence further comprises a sequence coding for an endoplasmic retention signal of plant origin, or for a vacuolar addressing signal.
7 - Procédé selon l'une quelconque des revendications précédentes caractérisé en ce qu'une séquence codant pour un agent permettant la sélection des cellules transformées est également introduite dans la cellule végétale.7 - Process according to any one of the preceding claims, characterized in that a sequence coding for an agent allowing the selection of the transformed cells is also introduced into the plant cell.
8 - Procédé selon l'une quelconque des revendications précédentes caractérisé en ce que l'étape d'extraction de la glycoproteine G comprend l'utilisation d'un ou plusieurs détergent(s) , de préférence non-ionique(s) .8 - Process according to any one of the preceding claims, characterized in that the step of extracting the glycoprotein G comprises the use of one or more detergent (s), preferably nonionic (s).
9 - Glycoproteine, caractérisée en ce que :9 - Glycoprotein, characterized in that:
- elle est reconnue par des anticorps spécifiques à la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage ;- it is recognized by antibodies specific to the glycoprotein G of the rabies virus or of a virus related to the rabies virus;
- elle a un poids moléculaire de 66 kDa approximativement ;- It has a molecular weight of approximately 66 kDa;
- elle est insoluble ;- it is insoluble;
- elle est N-glycosylée par au moins un glycanne de type polymannosidique, et/ou par au moins un glycanne de type complexe comportant au sein de sa structure un ou plusieurs résidus de xylose β-1,2, et/ou un ou plusieurs résidus de fucose lié en α-1,3, et étant dépourvu de résidus d'acide sialigue.
10 - Glycoproteine selon la revendication 9 caractérisée en ce qu'elle comprend en outre un signal de rétention endoplasmique végétale.- It is N-glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues β-1,2, and / or one or more residues of fucose bound in α-1,3, and being free of residues of sialigue acid. 10 - Glycoprotein according to claim 9 characterized in that it further comprises a plant endoplasmic retention signal.
11 - Glycoproteine selon l'une quelconque des revendications 9 ou 10 caractérisée en ce qu'elle est capable d'induire la formation d'anticorps protecteurs contre le virus de la rage ou contre un virus apparenté au virus de la rage.11 - Glycoprotein according to any one of claims 9 or 10 characterized in that it is capable of inducing the formation of protective antibodies against the rabies virus or against a virus related to the rabies virus.
12 - Glycoproteine selon l'une quelconque des revendications 9 à 11 caractérisée en ce qu'il s'agit de la glycoproteine G du virus rabique, ayant un poids moléculaire d'environ 66 kDa.12 - Glycoprotein according to any one of claims 9 to 11 characterized in that it is glycoprotein G of the rabies virus, having a molecular weight of approximately 66 kDa.
13 - Glycoproteine selon la revendication 9 caractérisée en ce qu'il s'agit d'une analogue de la glycoproteine G du virus rabique présentant une homologie d'environ 90% à 95% avec la protéine G du virus rabique.13 - Glycoprotein according to claim 9 characterized in that it is an analog of glycoprotein G of the rabies virus having a homology of about 90% to 95% with the protein G of the rabies virus.
14 - Glycoproteine susceptible d'être produite par le procédé selon l'une quelconque des revendications l à 8.14 - Glycoprotein capable of being produced by the process according to any one of claims 1 to 8.
15 - Acide nucléique comportant une séquence codante chimérique comprenant d'une part une séquence codant pour la protéine G mature du virus de la rage ou d'un virus apparenté au virus de la rage, ou pour une protéine analogue, et d'autre part, une séquence codant pour un peptide signal N-terminal autre que celui naturellement associé au virus, ou les séquences complémentaires aux sus-dites séquences codantes.15 - Nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to the rabies virus, or for a similar protein, and on the other hand , a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned coding sequences.
16 - Acide nucléique selon la revendication 15 caractérisé en ce que la séquence codant pour le peptide signal N-terminal code pour un peptide signal d'origine végétale.16 - Nucleic acid according to claim 15 characterized in that the sequence coding for the N-terminal signal peptide codes for a signal peptide of plant origin.
17 - Acide nucléique selon la revendication 16 caractérisé en ce qu'il s'agit d'ADN.17 - Nucleic acid according to claim 16 characterized in that it is DNA.
18 - Acide nucléique selon la revendication 16 caractérisé en ce qu'il s'agit d'ARN.
19 - Acide nucléique selon l'une quelconque des revendications 16 à 18, caractérisé en ce qu'il comporte en outre des séquences régulatrices de transcription reconnues par une cellule végétale.18 - Nucleic acid according to claim 16 characterized in that it is RNA. 19 - Nucleic acid according to any one of claims 16 to 18, characterized in that it also comprises regulatory transcription sequences recognized by a plant cell.
20 - Vecteur comprenant un acide nucléique selon les revendications 16 à 19.20 - Vector comprising a nucleic acid according to claims 16 to 19.
21 - Cellules végétales transformées de manière stable par un acide nucléique selon l'une quelconque des revendications 16 à 19 et capable de produire la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage, ou une glycoproteine analogue.21 - Plant cells transformed in a stable manner with a nucleic acid according to any one of claims 16 to 19 and capable of producing the glycoprotein G of the rabies virus or of a virus related to the rabies virus, or a glycoprotein analog thereof .
22 - Cellules végétales selon la revendication 21 caractérisées en ce qu'il s'agit d'une culture de cellules végétales, par exemple en milieu liquide ou immobilisées.22 - Plant cells according to claim 21 characterized in that it is a culture of plant cells, for example in liquid or immobilized medium.
23 - Cellules végétales selon la revendication 21 caractérisées en ce qu'il s'agit de cellules faisant partie d'une plante transformée entière.23 - Plant cells according to claim 21 characterized in that they are cells forming part of a whole transformed plant.
24 - Plante chimérique ou transgénique capable de produire la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage, ou une glycoproteine analogue, caractérisée en ce qu'elle comporte des cellules selon la revendication 21.24 - Chimeric or transgenic plant capable of producing the glycoprotein G of the rabies virus or of a virus related to the rabies virus, or an analogous glycoprotein, characterized in that it comprises cells according to claim 21.
25 - Graines de plante transgénique selon la revendication 24.25 - Seeds of transgenic plant according to claim 24.
26 - Anticorps monoclonaux ou polyclonaux capable de reconnaître spécifiquement la glycoproteine selon les revendications 9 à 14.26 - Monoclonal or polyclonal antibodies capable of specifically recognizing the glycoprotein according to claims 9 to 14.
27 - Composition pharmaceutique comprenant :27 - Pharmaceutical composition comprising:
- une ou plusieurs glycoproteine(s) selon l'une quelconque des revendications 9 à 14, ou- one or more glycoprotein (s) according to any one of claims 9 to 14, or
- des cellules selon l'une des revendications 21 à 23, ou- cells according to one of claims 21 to 23, or
- toute ou partie d'une plante selon la revendication 24,
en association avec un excipient acceptable du point de vue physiologique.- all or part of a plant according to claim 24, in combination with a physiologically acceptable excipient.
28 - Composition pharmaceutique selon la revendication 27 caractérisé en ce qu'il s'agit d'un vaccin.28 - Pharmaceutical composition according to claim 27 characterized in that it is a vaccine.
29 - Composition pharmaceutique selon la revendication 28 caractérisée en ce qu'il s'agit d'un vaccin pour administration par voie orale ou injectable, éventuellement en association avec un ou plusieurs adjuvant(s) .29 - Pharmaceutical composition according to claim 28 characterized in that it is a vaccine for administration by oral or injectable route, optionally in combination with one or more adjuvant (s).
30 - Glycoproteine selon l'une quelconque des revendications 9 à 14 pour une utilisation en thérapie et notamment en tant que vaccin humain ou vétérinaire contre une infection par le virus de la rage, ou par un virus apparenté au virus de la rage.30 - Glycoprotein according to any one of claims 9 to 14 for use in therapy and in particular as a human or veterinary vaccine against infection by the rabies virus, or by a virus related to the rabies virus.
31 - Cellules végétales selon l'une quelconque des revendications 21 à 23 pour une utilisation en thérapie et notamment en tant que vaccin humain ou vétérinaire contre une infection par le virus de la rage, ou par un virus apparenté au virus de la rage.31 - Plant cells according to any one of claims 21 to 23 for use in therapy and in particular as a human or veterinary vaccine against infection by the rabies virus, or by a virus related to the rabies virus.
32 - Plante transgénique ou chimérique selon la revendication 24, ou extrait d'une plante selon la revendication 24, pour une utilisation en thérapie et notamment en tant que vaccin humain ou vétérinaire contre une infection par le virus de la rage, ou par un virus apparenté au virus de la rage.32 - Transgenic or chimeric plant according to claim 24, or extract of a plant according to claim 24, for use in therapy and in particular as a human or veterinary vaccine against infection by the rabies virus, or by a virus related to rabies virus.
33 - Utilisation d'une glycoproteine selon l'une quelconque des revendications 9 à 14, des cellules selon l'une quelconque des revendications 21 à 23, des plantes selon la revendication 24, ou d'un extrait de ladite plante, pour la préparation d'un médicament contre le virus de la rage, ou un virus apparenté au virus de la rage.33 - Use of a glycoprotein according to any one of claims 9 to 14, cells according to any one of claims 21 to 23, plants according to claim 24, or an extract of said plant, for the preparation a rabies virus medication, or a virus related to the rabies virus.
34 - Produit alimentaire caractérisé en ce qu'il comprend une glycoproteine qui a les caractéristiques suivantes :
- elle est reconnue par des anticorps spécifiques à la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage ; elle a un poids moléculaire de 66 kDa approximativement ;34 - Food product characterized in that it comprises a glycoprotein which has the following characteristics: - it is recognized by antibodies specific to the glycoprotein G of the rabies virus or of a virus related to the rabies virus; it has a molecular weight of approximately 66 kDa;
- elle est insoluble ;- it is insoluble;
- elle est N-glycosylée par au moins un glycanne de type polymannosidique, et/ou par au moins un glycanne de type complexe comportant au sein de sa structure un ou plusieurs résidus de xylose β-1,2, et/ou un ou plusieurs résidus de fucose lié en α-1,3, et étant dépourvu de résidus d'acide sialique.- It is N-glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues β-1,2, and / or one or more residues of fucose linked in α-1,3, and being free of residues of sialic acid.
35 - Produit alimentaire caractérisé en ce qu'il comprend des cellules végétales transformées de manière stable par un acide nucléique, ledit acide nucléique comportant une séquence codante chimérique comprenant d'une part une séquence codant pour la protéine G mature du virus de la rage ou d'un virus apparenté au virus de la rage, ou pour une protéine analogue, et d'autre part, une séquence codant pour un peptide signal N-terminal autre que celui naturellement associé au virus, ou les séquences complémentaires aux sus-dites séquences codantes.35 - Food product characterized in that it comprises plant cells transformed in a stable manner with a nucleic acid, said nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to the rabies virus, or for a protein analogous, and on the other hand, a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned sequences coding.
36 - Produit alimentaire caractérisé en ce qu'il comprend une plante transgénique ou chimérique, ou un extrait d'une plante transgénique ou chimérique, ladite plante étant capable de produire la glycoproteine G du virus de la rage ou d'un virus apparenté au virus de la rage, ou une glycoproteine analogue, les cellules de la plante étant transformées par un acide nucléique comportant une séquence codante chimérique comprenant d'une part une séquence codant pour la protéine G mature du virus de la rage ou d'un virus apparenté au virus de la rage, ou pour une protéine analogue, et d'autre part, une séquence
codant pour un peptide signal N-terminal autre que celui naturellement associé au virus, ou les séquences complémentaires aux sus-dites séquences codantes.
36 - Food product characterized in that it comprises a transgenic or chimeric plant, or an extract of a transgenic or chimeric plant, said plant being capable of producing the glycoprotein G of the rabies virus or of a virus related to the virus rabies, or a glycoprotein analog, the cells of the plant being transformed by a nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to rabies virus, or for a protein analog, and secondly, a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned coding sequences.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9606029 | 1996-05-09 | ||
FR9606029A FR2748480B1 (en) | 1996-05-09 | 1996-05-09 | TRANSGENIC PLANTS EXPRESSING Rabies GLYCOPROTEIN G, AND GLYCOPROTEINS THUS OBTAINED |
PCT/FR1997/000827 WO1997043428A1 (en) | 1996-05-09 | 1997-05-07 | Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtained |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0910650A1 true EP0910650A1 (en) | 1999-04-28 |
Family
ID=9492145
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97924066A Withdrawn EP0910650A1 (en) | 1996-05-09 | 1997-05-07 | Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtained |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0910650A1 (en) |
AR (1) | AR013817A1 (en) |
AU (1) | AU2965797A (en) |
CA (1) | CA2254115A1 (en) |
FR (1) | FR2748480B1 (en) |
WO (1) | WO1997043428A1 (en) |
ZA (1) | ZA974037B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000068392A1 (en) * | 1999-05-11 | 2000-11-16 | The Board Of Trustees Of The University Of Illinois | Plant-derived antigens against respiratory syncytial virus |
WO2006016380A2 (en) * | 2004-08-13 | 2006-02-16 | Council Of Scientific And Industrial Research | A chimeric g protein based rabies vaccine |
WO2008063982A2 (en) * | 2006-11-13 | 2008-05-29 | Procell Corp | High mannose glycoprotein epitopes |
CA2839009C (en) * | 2011-06-13 | 2015-10-20 | Medicago Inc. | Rabies virus like particle production in plants |
CN109111507B (en) * | 2017-08-22 | 2021-12-24 | 浙江派迪畅科技发展有限公司 | Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1985001516A1 (en) * | 1983-10-03 | 1985-04-11 | Transgene S.A. | Vectors for the expression of an antigenic protein of rabies in eucaryotic cells and application thereof to the preparation of a vaccine |
NL8901932A (en) * | 1989-07-26 | 1991-02-18 | Mogen Int | PRODUCTION OF heterologous PROTEINS IN PLANTS OR PLANTS. |
EP0519011A1 (en) * | 1990-03-05 | 1992-12-23 | The Upjohn Company | Protein expression via seed specific regulatory sequences |
ES2235150T3 (en) * | 1990-06-15 | 2005-07-01 | Syngenta Participations Ag | NEW SIGNAL SEQUENCES. |
US5612487A (en) * | 1991-08-26 | 1997-03-18 | Edible Vaccines, Inc. | Anti-viral vaccines expressed in plants |
DE69533964T2 (en) * | 1994-10-24 | 2006-03-30 | The Texas A & M University System, College Station | ORAL IMMUNIZATION THROUGH THE USE OF TRANSGENIC PLANTS |
JPH08269092A (en) * | 1995-03-31 | 1996-10-15 | Chemo Sero Therapeut Res Inst | Rabies vaccine produced by recombinant gene in escherichia coli |
WO1996040229A1 (en) * | 1995-06-07 | 1996-12-19 | Thomas Jefferson University | Vaccines synthesized by transgenic plants |
-
1996
- 1996-05-09 FR FR9606029A patent/FR2748480B1/en not_active Expired - Fee Related
-
1997
- 1997-05-07 EP EP97924066A patent/EP0910650A1/en not_active Withdrawn
- 1997-05-07 AU AU29657/97A patent/AU2965797A/en not_active Abandoned
- 1997-05-07 WO PCT/FR1997/000827 patent/WO1997043428A1/en not_active Application Discontinuation
- 1997-05-07 CA CA002254115A patent/CA2254115A1/en not_active Abandoned
- 1997-05-09 ZA ZA9704037A patent/ZA974037B/en unknown
- 1997-09-05 AR ARP970101956A patent/AR013817A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9743428A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1997043428A1 (en) | 1997-11-20 |
FR2748480B1 (en) | 1998-09-04 |
AU2965797A (en) | 1997-12-05 |
ZA974037B (en) | 1998-01-08 |
CA2254115A1 (en) | 1997-11-20 |
AR013817A1 (en) | 2001-01-31 |
FR2748480A1 (en) | 1997-11-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6034298A (en) | Vaccines expressed in plants | |
US6136320A (en) | Vaccines expressed in plants | |
US6551820B1 (en) | Expression of immunogenic hepatitis B surface antigens in transgenic plants | |
US7504560B2 (en) | Vaccines expressed in plants | |
US5686079A (en) | Oral immunization by transgenic plants | |
EP2374892B1 (en) | Expression of viral proteins in plants | |
KR101963792B1 (en) | Rabies virus like particle production in plants | |
EP0517833B2 (en) | Regeneration and genetic transformation of sugar beet | |
EP0412912B1 (en) | Transgenic plants of the species cucumis melo | |
WO2005032457A2 (en) | Vectors and methods for immunization against norwalk virus using transgenic plants | |
FR2774379A1 (en) | PROCESS FOR THE PRODUCTION OF ALPHA 1-ANTITRYPSIN AND ITS VARIANTS BY PLANT CELLS, AND PRODUCTS CONTAINING THE ALPHA-ANTITRYPSIN OBTAINED THEREBY | |
EP0822988B1 (en) | Recombinant preduodenal lipases and polypeptide derivatives produced by plants, processes for obtaining them and their uses | |
WO1997043428A1 (en) | Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtained | |
FR2913694A1 (en) | EXPRESSION REGULATORY ELEMENTS | |
US20030138456A1 (en) | Vaccines expressed in plants | |
FR2754827A1 (en) | RECOMBINANT PANCREATIC LIPASES AND / OR COLIPASES AND DERIVED POLYPEPTIDES PRODUCED BY PLANTS, METHODS OF MAKING THEM AND USES THEREOF | |
US20020006411A1 (en) | Vaccines expressed in plants | |
Devine | Expression Of Heterologous Proteins In Transgenic Tobacco Chloroplasts To Produce A Biopharmaceutical And Biopolymer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19981130 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: MERIAL Owner name: MERISTEM THERAPEUTICS |
|
17Q | First examination report despatched |
Effective date: 20010611 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20021224 |