JP5815631B2 - 目的タンパク質の生産方法 - Google Patents
目的タンパク質の生産方法 Download PDFInfo
- Publication number
- JP5815631B2 JP5815631B2 JP2013207597A JP2013207597A JP5815631B2 JP 5815631 B2 JP5815631 B2 JP 5815631B2 JP 2013207597 A JP2013207597 A JP 2013207597A JP 2013207597 A JP2013207597 A JP 2013207597A JP 5815631 B2 JP5815631 B2 JP 5815631B2
- Authority
- JP
- Japan
- Prior art keywords
- gene
- antibody
- transgenic
- egg
- vector
- 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.)
- Expired - Lifetime
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 200
- 238000004519 manufacturing process Methods 0.000 title claims description 57
- 102000004169 proteins and genes Human genes 0.000 title claims description 49
- 230000009261 transgenic effect Effects 0.000 claims description 119
- 235000013601 eggs Nutrition 0.000 claims description 96
- 239000013598 vector Substances 0.000 claims description 95
- 241000287828 Gallus gallus Species 0.000 claims description 54
- 235000013330 chicken meat Nutrition 0.000 claims description 54
- 241000700605 Viruses Species 0.000 claims description 40
- 210000004369 blood Anatomy 0.000 claims description 35
- 239000008280 blood Substances 0.000 claims description 35
- 241001430294 unidentified retrovirus Species 0.000 claims description 34
- 238000011534 incubation Methods 0.000 claims description 33
- 210000001161 mammalian embryo Anatomy 0.000 claims description 29
- 230000001177 retroviral effect Effects 0.000 claims description 22
- 102000007469 Actins Human genes 0.000 claims description 6
- 108010085238 Actins Proteins 0.000 claims description 6
- 208000032839 leukemia Diseases 0.000 claims description 5
- 230000013011 mating Effects 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 94
- 239000012634 fragment Substances 0.000 description 91
- 239000013612 plasmid Substances 0.000 description 70
- 241000286209 Phasianidae Species 0.000 description 54
- 108010000912 Egg Proteins Proteins 0.000 description 50
- 102000002322 Egg Proteins Human genes 0.000 description 50
- 241000271566 Aves Species 0.000 description 49
- 238000000034 method Methods 0.000 description 36
- 108010005774 beta-Galactosidase Proteins 0.000 description 31
- 108700019146 Transgenes Proteins 0.000 description 30
- 239000000243 solution Substances 0.000 description 29
- 230000014509 gene expression Effects 0.000 description 26
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 25
- 210000000969 egg white Anatomy 0.000 description 25
- 235000014103 egg white Nutrition 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 24
- 210000002969 egg yolk Anatomy 0.000 description 23
- 235000018102 proteins Nutrition 0.000 description 22
- 108091034117 Oligonucleotide Proteins 0.000 description 21
- 102000005936 beta-Galactosidase Human genes 0.000 description 21
- 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 19
- 230000000694 effects Effects 0.000 description 18
- 235000013345 egg yolk Nutrition 0.000 description 18
- 210000002966 serum Anatomy 0.000 description 14
- 210000001172 blastoderm Anatomy 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 239000008363 phosphate buffer Substances 0.000 description 11
- 238000012546 transfer Methods 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 10
- 230000003321 amplification Effects 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 210000001082 somatic cell Anatomy 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 210000003278 egg shell Anatomy 0.000 description 7
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 6
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 6
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 241000714474 Rous sarcoma virus Species 0.000 description 6
- 210000004204 blood vessel Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 235000005956 Cosmos caudatus Nutrition 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 101150066002 GFP gene Proteins 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000012447 hatching Effects 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- 108700026220 vif Genes Proteins 0.000 description 5
- 239000013603 viral vector Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 4
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 230000004720 fertilization Effects 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000001638 lipofection Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 235000013372 meat Nutrition 0.000 description 4
- 210000004940 nucleus Anatomy 0.000 description 4
- 230000016087 ovulation Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 235000013594 poultry meat Nutrition 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101100391545 Mus musculus Fxyd3 gene Proteins 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 108010058846 Ovalbumin Proteins 0.000 description 3
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 101710120037 Toxin CcdB Proteins 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 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 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 101150066555 lacZ gene Proteins 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 229940092253 ovalbumin Drugs 0.000 description 3
- 230000017448 oviposition Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 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 2
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 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
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920001328 Polyvinylidene chloride Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 2
- 101150084279 TM gene Proteins 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 210000000991 chicken egg Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 229910003460 diamond Inorganic materials 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 238000010841 mRNA extraction Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 239000005033 polyvinylidene chloride Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000029058 respiratory gaseous exchange Effects 0.000 description 2
- 229920006298 saran Polymers 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241000271567 Struthioniformes Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 241000711975 Vesicular stomatitis virus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 1
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 101150031334 mvl gene Proteins 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 235000020030 perry Nutrition 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000010349 pulsation Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2806—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/30—Bird
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/60—Vector systems having a special element relevant for transcription 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/90—Vector systems having a special element relevant for transcription from vertebrates avian
-
- 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
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
-
- 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
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
レトロウイルスベクターによって受精卵に導入された遺伝子が、発生後どの時期に不活性化されるのかを特定するため、本発明者らはβ−ガラクトシダーゼ発現遺伝子を指標とし、受精後の様々な時期にベクターを導入して発現量がどう変化するかを検討した。その結果、導入遺伝子の発現量は該遺伝子が胚発生のどの時期に導入されたかによって大きく変化してくることを発見し、本発明に至った。すなわち、導入遺伝子の不活性化は、放卵直後に導入された遺伝子に対して顕著であり、放卵から一定時間経過後に導入された遺伝子は、発現頻度が高いというものである。
本発明のG0トランスジェニックキメラ鳥類は、複製能欠失型レトロウイルスベクターによって外来性抗体遺伝子が導入された鳥類であって、導入遺伝子に由来する抗体を、血中、卵白中あるいは卵黄中に産生することを特徴とする。
キメラ抗体とは、2種以上の異なる遺伝形質から構成される抗体のことをいう。
医療用の組換え抗体には、低分子抗体と称される一群がある。免疫グロブリンIgGには直接抗原と結合する可変領域(Fv:Fragment of variable regeon)と呼ばれるVH、VLのヘテロ二量体からなるドメインがあり、このFvドメインはIgGの約5分の1の分子量でありながら、単独で充分な抗原結合能を持つ。VH、VLドメイン間を人工的にペプチドリンカーで結合したものが1本鎖抗体(scFv:single chain Fv)と呼ばれる低分子抗体で、VH、VL単独よりも安定性が向上することが知られていた。
遺伝子組み換えにより2種あるいはそれ以上のタンパクの一部を融合させた人工的な一群のタンパクは融合タンパクと称され、医薬品として実用化されているものに、TNF受容体に免疫グロブリンのFcを融合させたTNFR−FcやLFA3にFcを融合させたLFA3−Fcなどがある。これらはFcを融合させることで可溶化され、より強力な生理活性をもつように設計された人工タンパクである。
当該作製法の1つとして、鳥類受精卵を孵卵し、放卵直後の胚盤葉期を除くそれ以降の初期胚へ複製能欠失型レトロウイルスベクターを感染させ、その胚を孵化させる作製法が挙げられる。また、鳥類受精卵を孵卵し、孵卵開始から24時間以降の初期胚へ複製能欠失型レトロウイルスベクターを感染させ、その胚を孵化させる作製法も、本発明の作製法の1つとして挙げられる。
β−ガラクトシダーゼ発現ベクターコンストラクトpMSCVNΔAβは、以下のように作製した。
このように作製した複製能欠失型レトロウイルスベクターのベクターコンストラクトpMSCVNΔAβの構造を図1に示した。
実施例1で作製したベクターコンストラクトpMSCVNΔAβよりレトロウイルスベクターを調製するため、パッケージング細胞GP293(クロンテック社製)を直径100mmの培養ディッシュに5×106細胞植え、培養した。培地を新鮮なDMEM(ダルベッコ変法イーグル培地)に交換し、p−VSV−Gベクター(クロンテック社製)8μgとpMSCVNΔAβ8μgをリポフェクション法により前記GP293細胞に導入した。48時間後、ウイルス粒子を含む培養上清を回収し、0.45μm酢酸セルロースフィルター(アドバンテック社製)を通して、夾雑物を除去した。得られた溶液にポリブレン(シグマ社製)を10μg/mlとなるように加えウイルス液とした。
ウイルスタイター=細胞数×希釈率×発現割合(cfu/ml)
WE系統のウズラ受精卵(日本生物科学研究所)を使用した。この受精卵を自動転卵装置が内蔵された孵卵器(昭和フランキ社製P−008型)内で37.9℃、湿度65%環境に置いた時刻を孵卵開始時刻(0時間)とし、以後15分毎に90度転卵しながら孵卵を行った。
ニワトリ受精卵(日本生物科学研究所)を使用した。この受精卵を自動転卵装置が内蔵された孵卵器(昭和フランキ社製P−008型)内で37.9℃、湿度65%環境に置いた時刻を孵卵開始時刻(0時間)とし、以後15分毎に90度転卵しながら孵卵を行った。
孵卵開始から115時間後、胚を卵殻より取り出しPBS(リン酸緩衝液)にて洗浄し、胚をとり囲む膜を取り除いた。摘出した胚を細かくせん断し、0.8mlの反応バッファー(10mM KCl、1mM MgCl2、0.1% Triton X−100(和光純薬工業製)、5mM 2−メルカプトエタノール(和光純薬工業製)、2mM リン酸バッファー pH7.5)を加えて超音波破砕し、細胞液を得た。
実施例2で調製した1×108cfu/mlのウイルス液を、希釈溶媒(50mM Tris−HCl(pH7.8)、130mM NaCl、1mM EDTA溶液)で10倍、100倍、1000倍に3段階希釈し、1×107、1×106、1×105cfu/mlのタイター・ウイルス溶液とした。ウズラ受精卵を孵卵し、48時間後の発生初期心臓に、調製したウイルス液2μlをマイクロインジェクションした。同様にして48時間後の発生初期心臓に希釈溶媒のみを2μlインジェクションしたものを対照とした。
3種のサブクラスをもつヒト抗体(IgG1、2、3)(コスモバイオ社製)を混合したもの、および対応する3種の抗体フラグメント(Fab−1、Fab−2、Fab−3、Fc−1、Fc−2、Fc−3)(コスモバイオ社製)を100μg/mlとなるようPBSで希釈し、希釈液100μlをウズラ成鳥(3羽)またはニワトリ成鳥(3羽)の翼下静脈に注射した。
PBSで希釈した抗ヒトIgG抗体(コスモバイオ社製)をELISAプレートに100μg/well入れて、4℃で一晩静置した。PBS−0.05%Tween20溶液を200μlで各wellを3回洗浄した後、PBS−0.05%Tween20溶液−2%スキムミルクを150μl/well入れた。
抗CD2抗体発現用ベクターコンストラクトpMSCV/GΔAH、pMSCV/GΔAL及びpMSCV/GΔALIHは、以下のように作製した。
実施例2に準じて、ベクターコンストラクトpMSCV/GΔAH、pMSCV/GΔAL及びpMSCV/GΔALIHより3種類のレトロウイルスベクターを調製した。このレトロウイルスベクターのタイターを測定したところ、108cfu/ml〜109cfu/mlだった。
実施例10により孵化させたG0トランスジェニックキメラウズラを1ヶ月間飼育して雛を成長させた。30日後および60日後、成長したG0トランスジェニックウズラの翼下静脈より採血を行い、血液サンプルを得た。得られた血液を15,000rpmで10分間遠心し、上清として得られる血清から抗CD2抗体量の測定を行った。
孵化から1.5ヵ月後、産卵を始めた雌性トランスジェニックウズラより採卵を行い、実施例7に準じて調製した卵白、卵黄中の抗CD2抗体量を、実施例8に準じてELISA法により定量した。
実験例1、実験例2の定量結果を示す。
pMSCV/GΔAH、pMSCV/GΔALから調製したベクター(3〜4×108cfu/ml)を同時に感染させたG0トランスジェニックキメラウズラ(個体識別番号#1113)は、卵黄中に0.6μg/ml、卵白中に0.5μg/mlの抗CD2抗体を発現した。
pMSCV/GΔALIHより調製したベクター(5×108cfu/ml)を単独で導入したG0トランスジェニックキメラウズラ(#4202)は、血清中に5.2μg/mlの抗CD2抗体を発現した。
ScFv−Fc抗体発現ベクターコンストラクトpMSCV/scFv−Fcは、以下のように作製した。
実施例2に準じて、ベクターコンストラクトpMSCV/GΔAscFv−Fcよりレトロウイルスベクターを調製した。このレトロウイルスベクターのタイターを測定したところ、108cfu/ml〜109cfu/mlだった。
実施例13により誕生したG0トランスジェニックキメラウズラを1ヶ月間飼育して雛を成長させた。30日後および60日後、成長したG0トランスジェニックキメラウズラ(個体識別番号#3303、#3306、#3310、#3311、#3313)の翼下静脈より採血を行い、血液サンプルを得た。得られた血液を15,000rpmで10分間遠心し、上清として得られる血清からscFv−Fc抗体量の測定を行った。
孵化から1.5ヵ月後、産卵を始めた雌性トランスジェニックウズラ(個体識別番号#3310)より採卵を行い、実施例7に準じて調製した卵白、卵黄中のscFv−Fc抗体量を、実施例8に準じてELISA法により定量した。
実施例13で作製したG0トランスジェニックキメラウズラ血清1mlから実施例10に準じて、硫安沈殿とプロテインGカラムによりscFv−Fcを精製した。精製したscFv−FcをSDS−PAGEで解析し、その結果を図16に示した。未処理のレーンより、scFv−Fcの分子量約120kDaが示された。還元処理したscFv−Fcの分子量は未処理の場合のほぼ半分(約60kDa)になっていることから、G0トランスジェニックキメラウズラにより産生されたscFv−FcはS−S結合による2量体を形成していることがわかった。これらは、S−S結合に関与するシステイン残基をFc部に有するscFv−Fcの構造的特徴に符合し、G0トランスジェニックキメラウズラにより産生されたscFv−Fcは正しい構造を保持していることが示唆された。
実施例9に準じてTNFR−Fc発現ベクターコンストラクトを作製し、実施例2に準じてレトロウイルスベクターを作製した。このレトロウイルスベクターのタイターは、1.7×107cfu/mlだった。
Claims (7)
- ニワトリ受精卵を孵卵し、孵卵開始から50時間以降、かつ60時間以前の初期胚の心臓内へ目的タンパク質をコードする塩基配列を含む複製能欠失型レトロウイルスベクターを感染させ、その胚を孵化させて得られるG0トランスジェニックニワトリの血中及び/又は卵中から目的タンパク質を回収することを特徴とする、目的タンパク質の生産方法。
- 目的タンパク質がヒトモノクローナル抗体、キメラ抗体、及びscFv−Fc抗体からなる群から選択される抗体である請求項1に記載の目的タンパク質の生産方法。
- 複製能欠失型レトロウイルスベクターのタイターが1×107cfu/ml以上である請求項1又は2に記載の目的タンパク質の生産方法。
- レトロウイルスベクターがモロニー・ミューリン・ロイケミア・ウイルス由来ベクターである請求項1〜3のいずれかに記載の目的タンパク質の生産方法。
- 目的タンパク質をコードする塩基配列がβ−アクチンプロモーターにより制御されている請求項1〜4のいずれかに記載の目的タンパク質の生産方法。
- レトロウイルスベクターがVSV−Gシュードタイプである請求項1〜5のいずれかに記載の目的タンパク質の生産方法。
- さらに、G0トランスジェニックニワトリを配偶型の同種ニワトリと交配させ、その卵を孵化させたトランスジェニックニワトリの血中及び/又は卵中から、目的タンパク質を回収する、請求項1〜6のいずれかに記載の目的タンパク質の生産方法。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013207597A JP5815631B2 (ja) | 2002-08-13 | 2013-10-02 | 目的タンパク質の生産方法 |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002236089 | 2002-08-13 | ||
JP2002236089 | 2002-08-13 | ||
JP2013207597A JP5815631B2 (ja) | 2002-08-13 | 2013-10-02 | 目的タンパク質の生産方法 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2011040380A Division JP5473963B2 (ja) | 2002-08-13 | 2011-02-25 | レトロウイルスベクターによる遺伝子導入鳥類での遺伝子発現法およびそれによって得られる遺伝子導入鳥類 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2013255521A JP2013255521A (ja) | 2013-12-26 |
JP5815631B2 true JP5815631B2 (ja) | 2015-11-17 |
Family
ID=31884397
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2004528857A Expired - Lifetime JP5468719B2 (ja) | 2002-08-13 | 2003-08-11 | レトロウイルスベクターによる遺伝子導入鳥類での遺伝子発現法およびそれによって得られる遺伝子導入烏類 |
JP2011040380A Expired - Lifetime JP5473963B2 (ja) | 2002-08-13 | 2011-02-25 | レトロウイルスベクターによる遺伝子導入鳥類での遺伝子発現法およびそれによって得られる遺伝子導入鳥類 |
JP2013207597A Expired - Lifetime JP5815631B2 (ja) | 2002-08-13 | 2013-10-02 | 目的タンパク質の生産方法 |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2004528857A Expired - Lifetime JP5468719B2 (ja) | 2002-08-13 | 2003-08-11 | レトロウイルスベクターによる遺伝子導入鳥類での遺伝子発現法およびそれによって得られる遺伝子導入烏類 |
JP2011040380A Expired - Lifetime JP5473963B2 (ja) | 2002-08-13 | 2011-02-25 | レトロウイルスベクターによる遺伝子導入鳥類での遺伝子発現法およびそれによって得られる遺伝子導入鳥類 |
Country Status (14)
Country | Link |
---|---|
US (1) | US20060143725A1 (ja) |
EP (1) | EP1548114B8 (ja) |
JP (3) | JP5468719B2 (ja) |
KR (1) | KR20050067138A (ja) |
CN (1) | CN1674779A (ja) |
AU (1) | AU2003254929A1 (ja) |
BR (1) | BR0313441A (ja) |
CA (1) | CA2492927A1 (ja) |
ES (1) | ES2394792T3 (ja) |
HR (1) | HRP20050237A2 (ja) |
IL (1) | IL166395A0 (ja) |
MX (1) | MXPA05001331A (ja) |
RU (1) | RU2005106867A (ja) |
WO (1) | WO2004016081A1 (ja) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060191026A1 (en) * | 2005-02-18 | 2006-08-24 | Origen Therapeutics, Inc. | Tissue specific expression of antibodies in chickens |
BR0313441A (pt) * | 2002-08-13 | 2005-07-12 | Kaneka Corp | Método para expressar gene em aves transgênicas usando vetor retroviral, e as aves transgênicas assim obtidas |
US20070214511A1 (en) * | 2004-01-08 | 2007-09-13 | Kaneka Corporation | Transgenic Bird And Method Of Constructing The Same |
JP4995574B2 (ja) * | 2004-09-28 | 2012-08-08 | 株式会社カネカ | 遺伝子導入鳥類作製法 |
JP2006271266A (ja) * | 2005-03-29 | 2006-10-12 | Univ Nagoya | 高発現および効率的遺伝子導入鳥類 |
WO2006112147A1 (ja) * | 2005-03-30 | 2006-10-26 | Kaneka Corporation | エリスロポエチンを産生するトランスジェニック鳥類およびその作製法 |
US7524626B2 (en) | 2005-10-05 | 2009-04-28 | Synageva Biopharma Corp. | Rapid production of high titer virus |
WO2007046439A1 (ja) * | 2005-10-18 | 2007-04-26 | National Institute Of Agrobiological Sciences | 抗体を産生するトランスジェニックカイコとその製造方法 |
WO2008018562A1 (fr) * | 2006-08-10 | 2008-02-14 | Kaneka Corporation | Production d'une protéine utile telle qu'un anticorps ayant une chaîne glucidique modifiée dans le jaune d'oeuf d'un poulet transgénique |
EP2126071A4 (en) | 2007-01-26 | 2010-12-08 | Synageva Biopharma Corp | TRANSGENEXPRESSION IN BIRDS |
US8431770B2 (en) * | 2008-01-07 | 2013-04-30 | Synageva Biopharma Corp. | Method of producing sialytransferase-modified proteins |
CN102036683A (zh) * | 2008-05-20 | 2011-04-27 | 株式会社钟化 | 细胞毒性组合物 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0424044A1 (en) * | 1989-10-16 | 1991-04-24 | Merck & Co. Inc. | Transgenic fowl expressing bovine growth hormone |
JPH0928235A (ja) * | 1995-07-17 | 1997-02-04 | Hoechst Japan Ltd | 骨減少症モデルトランスジェニック動物 |
EP1023442B1 (en) * | 1997-10-16 | 2010-11-24 | University Of Georgia Research Foundation, Inc. | Transgenic birds and protein production |
ATE469970T1 (de) * | 1999-06-04 | 2010-06-15 | Nippon Inst For Biolog Science | Plasmidvektor |
WO2001043540A2 (en) * | 1999-12-17 | 2001-06-21 | Oregon Health And Science University | Methods for producing transgenic animals |
JP2002176880A (ja) * | 2000-12-12 | 2002-06-25 | Kanegafuchi Chem Ind Co Ltd | 効率的な遺伝子導入鳥類の作製法及びそれによって得られる遺伝子導入鳥類 |
US20020108132A1 (en) * | 2001-02-02 | 2002-08-08 | Avigenics Inc. | Production of a monoclonal antibody by a transgenic chicken |
EP1438401B1 (en) * | 2001-09-18 | 2011-06-15 | Synageva BioPharma Corp. | Production of a transgenic avian by cytoplasmic injection |
BR0313441A (pt) * | 2002-08-13 | 2005-07-12 | Kaneka Corp | Método para expressar gene em aves transgênicas usando vetor retroviral, e as aves transgênicas assim obtidas |
US20060259997A1 (en) * | 2003-08-29 | 2006-11-16 | Shinji Iijima | Method of constructing transgenic bird using lentivirus vector and transgenic bird obtained thereby |
-
2003
- 2003-08-11 BR BR0313441-5A patent/BR0313441A/pt not_active Application Discontinuation
- 2003-08-11 RU RU2005106867/13A patent/RU2005106867A/ru not_active Application Discontinuation
- 2003-08-11 MX MXPA05001331A patent/MXPA05001331A/es unknown
- 2003-08-11 EP EP03788072A patent/EP1548114B8/en not_active Expired - Lifetime
- 2003-08-11 JP JP2004528857A patent/JP5468719B2/ja not_active Expired - Lifetime
- 2003-08-11 CA CA002492927A patent/CA2492927A1/en not_active Abandoned
- 2003-08-11 ES ES03788072T patent/ES2394792T3/es not_active Expired - Lifetime
- 2003-08-11 WO PCT/JP2003/010198 patent/WO2004016081A1/ja active Application Filing
- 2003-08-11 AU AU2003254929A patent/AU2003254929A1/en not_active Abandoned
- 2003-08-11 KR KR1020057002420A patent/KR20050067138A/ko not_active Application Discontinuation
- 2003-08-11 CN CNA038192020A patent/CN1674779A/zh active Pending
- 2003-08-11 US US10/523,191 patent/US20060143725A1/en not_active Abandoned
-
2005
- 2005-01-19 IL IL16639505A patent/IL166395A0/xx unknown
- 2005-03-11 HR HR20050237A patent/HRP20050237A2/hr not_active Application Discontinuation
-
2011
- 2011-02-25 JP JP2011040380A patent/JP5473963B2/ja not_active Expired - Lifetime
-
2013
- 2013-10-02 JP JP2013207597A patent/JP5815631B2/ja not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
IL166395A0 (en) | 2006-01-15 |
US20060143725A1 (en) | 2006-06-29 |
JPWO2004016081A1 (ja) | 2005-12-02 |
JP5468719B2 (ja) | 2014-04-09 |
BR0313441A (pt) | 2005-07-12 |
EP1548114B1 (en) | 2012-09-26 |
MXPA05001331A (es) | 2005-10-06 |
EP1548114A1 (en) | 2005-06-29 |
EP1548114B8 (en) | 2012-10-31 |
EP1548114A4 (en) | 2008-05-14 |
ES2394792T3 (es) | 2013-02-05 |
CA2492927A1 (en) | 2004-02-26 |
AU2003254929A1 (en) | 2004-03-03 |
RU2005106867A (ru) | 2006-01-20 |
KR20050067138A (ko) | 2005-06-30 |
WO2004016081A1 (ja) | 2004-02-26 |
JP2011147447A (ja) | 2011-08-04 |
CN1674779A (zh) | 2005-09-28 |
HRP20050237A2 (en) | 2006-06-30 |
JP2013255521A (ja) | 2013-12-26 |
JP5473963B2 (ja) | 2014-04-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5815631B2 (ja) | 目的タンパク質の生産方法 | |
US20190017021A1 (en) | Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals | |
US10370641B2 (en) | Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals | |
JPWO2006112147A1 (ja) | エリスロポエチンを産生するトランスジェニック鳥類およびその作製法 | |
JP2009082033A (ja) | 完全ヒト型抗体生産法 | |
EP1712126B1 (en) | Method of constructing a transgenic bird. | |
EP1672076A1 (en) | Method of constructing transgenic bird using lentivirus vector and transgenic bird obtained thereby | |
JP4995574B2 (ja) | 遺伝子導入鳥類作製法 | |
WO2009142186A1 (ja) | 細胞障害性組成物 | |
JP4747329B2 (ja) | トランスジェニック鳥類及びその作製法、並びにタンパク質の生産法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20131015 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150113 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150305 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20150901 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150924 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5815631 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313117 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
EXPY | Cancellation because of completion of term |