CN114381465A - 优化的cpy4v2基因及其用途 - Google Patents
优化的cpy4v2基因及其用途 Download PDFInfo
- Publication number
- CN114381465A CN114381465A CN202111580873.0A CN202111580873A CN114381465A CN 114381465 A CN114381465 A CN 114381465A CN 202111580873 A CN202111580873 A CN 202111580873A CN 114381465 A CN114381465 A CN 114381465A
- Authority
- CN
- China
- Prior art keywords
- seq
- promoter
- nucleotide sequence
- polynucleotide
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 77
- 239000002773 nucleotide Substances 0.000 claims abstract description 93
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 93
- 101000896951 Homo sapiens Cytochrome P450 4V2 Proteins 0.000 claims abstract description 71
- 102100022028 Cytochrome P450 4V2 Human genes 0.000 claims abstract description 68
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 50
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 50
- 239000002157 polynucleotide Substances 0.000 claims abstract description 50
- 239000013604 expression vector Substances 0.000 claims abstract description 38
- 239000003814 drug Substances 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims description 75
- 239000013612 plasmid Substances 0.000 claims description 49
- 201000007795 Bietti crystalline corneoretinal dystrophy Diseases 0.000 claims description 44
- 208000008319 Bietti crystalline dystrophy Diseases 0.000 claims description 44
- 102000004169 proteins and genes Human genes 0.000 claims description 40
- 108020004414 DNA Proteins 0.000 claims description 39
- 239000002245 particle Substances 0.000 claims description 38
- 230000001105 regulatory effect Effects 0.000 claims description 36
- 239000013598 vector Substances 0.000 claims description 34
- 241000700605 Viruses Species 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 28
- 101150093909 Cyp4v2 gene Proteins 0.000 claims description 25
- 230000003612 virological effect Effects 0.000 claims description 24
- 241000702421 Dependoparvovirus Species 0.000 claims description 23
- 230000035772 mutation Effects 0.000 claims description 21
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 20
- 230000004663 cell proliferation Effects 0.000 claims description 16
- 239000003623 enhancer Substances 0.000 claims description 14
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 14
- 238000011282 treatment Methods 0.000 claims description 14
- 241000701022 Cytomegalovirus Species 0.000 claims description 12
- 241000282414 Homo sapiens Species 0.000 claims description 12
- 230000008021 deposition Effects 0.000 claims description 12
- 230000010076 replication Effects 0.000 claims description 12
- 239000013603 viral vector Substances 0.000 claims description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 10
- 230000001939 inductive effect Effects 0.000 claims description 10
- 241000701161 unidentified adenovirus Species 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 9
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 7
- 239000005090 green fluorescent protein Substances 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 7
- 229950010131 puromycin Drugs 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000001124 posttranscriptional effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000004098 Tetracycline Substances 0.000 claims description 5
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 229960002180 tetracycline Drugs 0.000 claims description 5
- 229930101283 tetracycline Natural products 0.000 claims description 5
- 235000019364 tetracycline Nutrition 0.000 claims description 5
- 150000003522 tetracyclines Chemical class 0.000 claims description 5
- 230000004544 DNA amplification Effects 0.000 claims description 4
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 claims description 4
- 241000713666 Lentivirus Species 0.000 claims description 4
- 108010047357 Luminescent Proteins Proteins 0.000 claims description 4
- 102000006830 Luminescent Proteins Human genes 0.000 claims description 4
- 241000700584 Simplexvirus Species 0.000 claims description 4
- 108010022394 Threonine synthase Proteins 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 230000010261 cell growth Effects 0.000 claims description 4
- 102000004419 dihydrofolate reductase Human genes 0.000 claims description 4
- 241001493065 dsRNA viruses Species 0.000 claims description 4
- 108091006047 fluorescent proteins Proteins 0.000 claims description 4
- 102000034287 fluorescent proteins Human genes 0.000 claims description 4
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 150000003431 steroids Chemical class 0.000 claims description 4
- 239000003053 toxin Substances 0.000 claims description 4
- 231100000765 toxin Toxicity 0.000 claims description 4
- 241001529453 unidentified herpesvirus Species 0.000 claims description 4
- 241001430294 unidentified retrovirus Species 0.000 claims description 4
- 241000271566 Aves Species 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- 206010064571 Gene mutation Diseases 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 241000125945 Protoparvovirus Species 0.000 claims description 3
- 230000002886 autophagic effect Effects 0.000 claims description 3
- 108010006025 bovine growth hormone Proteins 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000012212 insulator Substances 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 claims description 2
- FNQJDLTXOVEEFB-UHFFFAOYSA-N 1,2,3-benzothiadiazole Chemical compound C1=CC=C2SN=NC2=C1 FNQJDLTXOVEEFB-UHFFFAOYSA-N 0.000 claims description 2
- 239000005964 Acibenzolar-S-methyl Substances 0.000 claims description 2
- 241001136782 Alca Species 0.000 claims description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 2
- 241000710929 Alphavirus Species 0.000 claims description 2
- 241000193738 Bacillus anthracis Species 0.000 claims description 2
- 241001446316 Bohle iridovirus Species 0.000 claims description 2
- 241000178270 Canarypox virus Species 0.000 claims description 2
- 241000282465 Canis Species 0.000 claims description 2
- 241000713756 Caprine arthritis encephalitis virus Species 0.000 claims description 2
- 108020004638 Circular DNA Proteins 0.000 claims description 2
- 108091028075 Circular RNA Proteins 0.000 claims description 2
- 241000711573 Coronaviridae Species 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims description 2
- 241000450599 DNA viruses Species 0.000 claims description 2
- 102000016607 Diphtheria Toxin Human genes 0.000 claims description 2
- 108010053187 Diphtheria Toxin Proteins 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 claims description 2
- 241000713730 Equine infectious anemia virus Species 0.000 claims description 2
- 241000283073 Equus caballus Species 0.000 claims description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims description 2
- 239000005977 Ethylene Substances 0.000 claims description 2
- 108700024394 Exon Proteins 0.000 claims description 2
- 241000713800 Feline immunodeficiency virus Species 0.000 claims description 2
- 241000710831 Flavivirus Species 0.000 claims description 2
- 241000700662 Fowlpox virus Species 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 2
- 241000941423 Grom virus Species 0.000 claims description 2
- 208000031886 HIV Infections Diseases 0.000 claims description 2
- 102100032510 Heat shock protein HSP 90-beta Human genes 0.000 claims description 2
- 101000882584 Homo sapiens Estrogen receptor Proteins 0.000 claims description 2
- 101001016856 Homo sapiens Heat shock protein HSP 90-beta Proteins 0.000 claims description 2
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 claims description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims description 2
- 241000713340 Human immunodeficiency virus 2 Species 0.000 claims description 2
- 241000714192 Human spumaretrovirus Species 0.000 claims description 2
- 108091092195 Intron Proteins 0.000 claims description 2
- 101000839464 Leishmania braziliensis Heat shock 70 kDa protein Proteins 0.000 claims description 2
- 101000988090 Leishmania donovani Heat shock protein 83 Proteins 0.000 claims description 2
- 206010024229 Leprosy Diseases 0.000 claims description 2
- 108060001084 Luciferase Proteins 0.000 claims description 2
- 239000005089 Luciferase Substances 0.000 claims description 2
- 102000003792 Metallothionein Human genes 0.000 claims description 2
- 108090000157 Metallothionein Proteins 0.000 claims description 2
- 229930193140 Neomycin Natural products 0.000 claims description 2
- 241000714209 Norwalk virus Species 0.000 claims description 2
- 241000702244 Orthoreovirus Species 0.000 claims description 2
- 241000709664 Picornaviridae Species 0.000 claims description 2
- 206010037742 Rabies Diseases 0.000 claims description 2
- 241000711798 Rabies lyssavirus Species 0.000 claims description 2
- 101001023863 Rattus norvegicus Glucocorticoid receptor Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 206010039491 Sarcoma Diseases 0.000 claims description 2
- 108091023040 Transcription factor Proteins 0.000 claims description 2
- 102000040945 Transcription factor Human genes 0.000 claims description 2
- 241000700618 Vaccinia virus Species 0.000 claims description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 claims description 2
- 241001492404 Woodchuck hepatitis virus Species 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims description 2
- 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 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 208000004668 avian leukosis Diseases 0.000 claims description 2
- 229930189065 blasticidin Natural products 0.000 claims description 2
- 230000003210 demyelinating effect Effects 0.000 claims description 2
- 108010057988 ecdysone receptor Proteins 0.000 claims description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 claims description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 239000000865 liniment Substances 0.000 claims description 2
- 229960004927 neomycin Drugs 0.000 claims description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 2
- 108010054624 red fluorescent protein Proteins 0.000 claims description 2
- 230000032537 response to toxin Effects 0.000 claims description 2
- 150000004492 retinoid derivatives Chemical class 0.000 claims description 2
- 229960004889 salicylic acid Drugs 0.000 claims description 2
- 230000035939 shock Effects 0.000 claims description 2
- 102000004217 thyroid hormone receptors Human genes 0.000 claims description 2
- 108090000721 thyroid hormone receptors Proteins 0.000 claims description 2
- 241000701447 unidentified baculovirus Species 0.000 claims description 2
- 241000712461 unidentified influenza virus Species 0.000 claims description 2
- -1 cosmid Substances 0.000 claims 1
- 238000013518 transcription Methods 0.000 claims 1
- 230000035897 transcription Effects 0.000 claims 1
- 238000013519 translation Methods 0.000 claims 1
- 210000003583 retinal pigment epithelium Anatomy 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 238000001415 gene therapy Methods 0.000 abstract description 10
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000010473 stable expression Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 157
- 150000007523 nucleic acids Chemical class 0.000 description 53
- 102000039446 nucleic acids Human genes 0.000 description 38
- 108020004707 nucleic acids Proteins 0.000 description 38
- 230000015572 biosynthetic process Effects 0.000 description 27
- 238000003786 synthesis reaction Methods 0.000 description 27
- 239000000523 sample Substances 0.000 description 23
- 230000006870 function Effects 0.000 description 20
- 210000001508 eye Anatomy 0.000 description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 description 16
- 238000001514 detection method Methods 0.000 description 15
- 239000013607 AAV vector Substances 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 108020004705 Codon Proteins 0.000 description 13
- 208000015181 infectious disease Diseases 0.000 description 13
- 238000001262 western blot Methods 0.000 description 13
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 12
- 150000002632 lipids Chemical class 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 11
- 238000011002 quantification Methods 0.000 description 11
- 238000001890 transfection Methods 0.000 description 11
- 125000003275 alpha amino acid group Chemical group 0.000 description 10
- XDHNQDDQEHDUTM-UHFFFAOYSA-N bafliomycin A1 Natural products COC1C=CC=C(C)CC(C)C(O)C(C)C=C(C)C=C(OC)C(=O)OC1C(C)C(O)C(C)C1(O)OC(C(C)C)C(C)C(O)C1 XDHNQDDQEHDUTM-UHFFFAOYSA-N 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 230000002207 retinal effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 210000004957 autophagosome Anatomy 0.000 description 9
- 235000019197 fats Nutrition 0.000 description 9
- 210000001525 retina Anatomy 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 7
- 238000003306 harvesting Methods 0.000 description 7
- 238000012014 optical coherence tomography Methods 0.000 description 7
- 239000013608 rAAV vector Substances 0.000 description 7
- 101100329207 Homo sapiens CYP4V2 gene Proteins 0.000 description 6
- 229940114079 arachidonic acid Drugs 0.000 description 6
- 235000021342 arachidonic acid Nutrition 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 108091033409 CRISPR Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 239000012124 Opti-MEM Substances 0.000 description 5
- 230000004900 autophagic degradation Effects 0.000 description 5
- 210000005252 bulbus oculi Anatomy 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000010172 mouse model Methods 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 230000009385 viral infection Effects 0.000 description 5
- 201000004569 Blindness Diseases 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- XDHNQDDQEHDUTM-JQWOJBOSSA-N bafilomycin A1 Chemical compound CO[C@H]1\C=C\C=C(C)\C[C@H](C)[C@H](O)[C@H](C)\C=C(/C)\C=C(OC)\C(=O)O[C@@H]1[C@@H](C)[C@@H](O)[C@H](C)[C@]1(O)O[C@H](C(C)C)[C@@H](C)[C@H](O)C1 XDHNQDDQEHDUTM-JQWOJBOSSA-N 0.000 description 4
- XDHNQDDQEHDUTM-ZGOPVUMHSA-N bafilomycin A1 Natural products CO[C@H]1C=CC=C(C)C[C@H](C)[C@H](O)[C@H](C)C=C(C)C=C(OC)C(=O)O[C@@H]1[C@@H](C)[C@@H](O)[C@H](C)[C@]1(O)O[C@H](C(C)C)[C@@H](C)[C@H](O)C1 XDHNQDDQEHDUTM-ZGOPVUMHSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000004087 cornea Anatomy 0.000 description 4
- 210000000695 crystalline len Anatomy 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000003889 eye drop Substances 0.000 description 4
- 235000009200 high fat diet Nutrition 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 3
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 3
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 3
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000005571 anion exchange chromatography Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940012356 eye drops Drugs 0.000 description 3
- 230000007849 functional defect Effects 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 3
- 210000001116 retinal neuron Anatomy 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 101100275556 Arabidopsis thaliana CYP19-3 gene Proteins 0.000 description 2
- 101150079826 CYP4 gene Proteins 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 241001135569 Human adenovirus 5 Species 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000006550 Mydriasis Diseases 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- YIPFBJGBRCJJJD-FHWLQOOXSA-N Pro-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@@H]3CCCN3 YIPFBJGBRCJJJD-FHWLQOOXSA-N 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 101100062195 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CPR4 gene Proteins 0.000 description 2
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 230000004908 autophagic flux Effects 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 239000003885 eye ointment Substances 0.000 description 2
- 230000004438 eyesight Effects 0.000 description 2
- 230000004129 fatty acid metabolism Effects 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 230000037433 frameshift Effects 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 102000056579 human CYP4V2 Human genes 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000006372 lipid accumulation Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 210000001747 pupil Anatomy 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- 230000004393 visual impairment Effects 0.000 description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- BFUUJUGQJUTPAF-UHFFFAOYSA-N 2-(3-amino-4-propoxybenzoyl)oxyethyl-diethylazanium;chloride Chemical compound [Cl-].CCCOC1=CC=C(C(=O)OCC[NH+](CC)CC)C=C1N BFUUJUGQJUTPAF-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 229940121819 ATPase inhibitor Drugs 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- PXKLCFFSVLKOJM-ACZMJKKPSA-N Ala-Asn-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PXKLCFFSVLKOJM-ACZMJKKPSA-N 0.000 description 1
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 description 1
- NJPMYXWVWQWCSR-ACZMJKKPSA-N Ala-Glu-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NJPMYXWVWQWCSR-ACZMJKKPSA-N 0.000 description 1
- IFKQPMZRDQZSHI-GHCJXIJMSA-N Ala-Ile-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O IFKQPMZRDQZSHI-GHCJXIJMSA-N 0.000 description 1
- LBYMZCVBOKYZNS-CIUDSAMLSA-N Ala-Leu-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O LBYMZCVBOKYZNS-CIUDSAMLSA-N 0.000 description 1
- ZBLQIYPCUWZSRZ-QEJZJMRPSA-N Ala-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 ZBLQIYPCUWZSRZ-QEJZJMRPSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- KGSJCPBERYUXCN-BPNCWPANSA-N Arg-Ala-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KGSJCPBERYUXCN-BPNCWPANSA-N 0.000 description 1
- VWVPYNGMOCSSGK-GUBZILKMSA-N Arg-Arg-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O VWVPYNGMOCSSGK-GUBZILKMSA-N 0.000 description 1
- RWWPBOUMKFBHAL-FXQIFTODSA-N Arg-Asn-Cys Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O RWWPBOUMKFBHAL-FXQIFTODSA-N 0.000 description 1
- SKTGPBFTMNLIHQ-KKUMJFAQSA-N Arg-Glu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SKTGPBFTMNLIHQ-KKUMJFAQSA-N 0.000 description 1
- 108010010777 Arg-Gly-Asp-Gly Proteins 0.000 description 1
- UPKMBGAAEZGHOC-RWMBFGLXSA-N Arg-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O UPKMBGAAEZGHOC-RWMBFGLXSA-N 0.000 description 1
- JCROZIFVIYMXHM-GUBZILKMSA-N Arg-Met-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N JCROZIFVIYMXHM-GUBZILKMSA-N 0.000 description 1
- HCAUEJAQCXVQQM-ACZMJKKPSA-N Asn-Glu-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HCAUEJAQCXVQQM-ACZMJKKPSA-N 0.000 description 1
- QYXNFROWLZPWPC-FXQIFTODSA-N Asn-Glu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QYXNFROWLZPWPC-FXQIFTODSA-N 0.000 description 1
- XFJKRRCWLTZIQA-XIRDDKMYSA-N Asn-Lys-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)N XFJKRRCWLTZIQA-XIRDDKMYSA-N 0.000 description 1
- NCXTYSVDWLAQGZ-ZKWXMUAHSA-N Asn-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O NCXTYSVDWLAQGZ-ZKWXMUAHSA-N 0.000 description 1
- WCFCYFDBMNFSPA-ACZMJKKPSA-N Asp-Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O WCFCYFDBMNFSPA-ACZMJKKPSA-N 0.000 description 1
- QXHVOUSPVAWEMX-ZLUOBGJFSA-N Asp-Asp-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXHVOUSPVAWEMX-ZLUOBGJFSA-N 0.000 description 1
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 1
- CRNKLABLTICXDV-GUBZILKMSA-N Asp-His-Glu Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N CRNKLABLTICXDV-GUBZILKMSA-N 0.000 description 1
- LDLZOAJRXXBVGF-GMOBBJLQSA-N Asp-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)O)N LDLZOAJRXXBVGF-GMOBBJLQSA-N 0.000 description 1
- UJGRZQYSNYTCAX-SRVKXCTJSA-N Asp-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UJGRZQYSNYTCAX-SRVKXCTJSA-N 0.000 description 1
- DPNWSMBUYCLEDG-CIUDSAMLSA-N Asp-Lys-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O DPNWSMBUYCLEDG-CIUDSAMLSA-N 0.000 description 1
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 1
- ZKAOJVJQGVUIIU-GUBZILKMSA-N Asp-Pro-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZKAOJVJQGVUIIU-GUBZILKMSA-N 0.000 description 1
- GXHDGYOXPNQCKM-XVSYOHENSA-N Asp-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O GXHDGYOXPNQCKM-XVSYOHENSA-N 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 229930003347 Atropine Natural products 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101150044789 Cap gene Proteins 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 208000008515 Choroidal sclerosis Diseases 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- CEZSLNCYQUFOSL-BQBZGAKWSA-N Cys-Arg-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O CEZSLNCYQUFOSL-BQBZGAKWSA-N 0.000 description 1
- 102000005297 Cytochrome P-450 CYP4A Human genes 0.000 description 1
- 108010081498 Cytochrome P-450 CYP4A Proteins 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000036893 GUCY2D-related dominant retinopathy Diseases 0.000 description 1
- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 description 1
- GURIQZQSTBBHRV-SRVKXCTJSA-N Gln-Lys-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GURIQZQSTBBHRV-SRVKXCTJSA-N 0.000 description 1
- OKARHJKJTKFQBM-ACZMJKKPSA-N Gln-Ser-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OKARHJKJTKFQBM-ACZMJKKPSA-N 0.000 description 1
- LTUVYLVIZHJCOQ-KKUMJFAQSA-N Glu-Arg-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LTUVYLVIZHJCOQ-KKUMJFAQSA-N 0.000 description 1
- NADWTMLCUDMDQI-ACZMJKKPSA-N Glu-Asp-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N NADWTMLCUDMDQI-ACZMJKKPSA-N 0.000 description 1
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 1
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 description 1
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 description 1
- GGJOGFJIPPGNRK-JSGCOSHPSA-N Glu-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)N)C(O)=O)=CNC2=C1 GGJOGFJIPPGNRK-JSGCOSHPSA-N 0.000 description 1
- QLPYYTDOUQNJGQ-AVGNSLFASA-N Glu-His-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N QLPYYTDOUQNJGQ-AVGNSLFASA-N 0.000 description 1
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- LKOAAMXDJGEYMS-ZPFDUUQYSA-N Glu-Met-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LKOAAMXDJGEYMS-ZPFDUUQYSA-N 0.000 description 1
- ALMBZBOCGSVSAI-ACZMJKKPSA-N Glu-Ser-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ALMBZBOCGSVSAI-ACZMJKKPSA-N 0.000 description 1
- HZISRJBYZAODRV-XQXXSGGOSA-N Glu-Thr-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O HZISRJBYZAODRV-XQXXSGGOSA-N 0.000 description 1
- HBMRTXJZQDVRFT-DZKIICNBSA-N Glu-Tyr-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HBMRTXJZQDVRFT-DZKIICNBSA-N 0.000 description 1
- KIEICAOUSNYOLM-NRPADANISA-N Glu-Val-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KIEICAOUSNYOLM-NRPADANISA-N 0.000 description 1
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 1
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 1
- DWUKOTKSTDWGAE-BQBZGAKWSA-N Gly-Asn-Arg Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DWUKOTKSTDWGAE-BQBZGAKWSA-N 0.000 description 1
- LXXANCRPFBSSKS-IUCAKERBSA-N Gly-Gln-Leu Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LXXANCRPFBSSKS-IUCAKERBSA-N 0.000 description 1
- FQKKPCWTZZEDIC-XPUUQOCRSA-N Gly-His-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 FQKKPCWTZZEDIC-XPUUQOCRSA-N 0.000 description 1
- VAXIVIPMCTYSHI-YUMQZZPRSA-N Gly-His-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN VAXIVIPMCTYSHI-YUMQZZPRSA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 1
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 1
- IALQAMYQJBZNSK-WHFBIAKZSA-N Gly-Ser-Asn Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O IALQAMYQJBZNSK-WHFBIAKZSA-N 0.000 description 1
- GWNIGUKSRJBIHX-STQMWFEESA-N Gly-Tyr-Arg Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)CN)O GWNIGUKSRJBIHX-STQMWFEESA-N 0.000 description 1
- VJJSDSNFXCWCEJ-DJFWLOJKSA-N His-Ile-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O VJJSDSNFXCWCEJ-DJFWLOJKSA-N 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101001078886 Homo sapiens Retinaldehyde-binding protein 1 Proteins 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 1
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 1
- IGJWJGIHUFQANP-LAEOZQHASA-N Ile-Gly-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N IGJWJGIHUFQANP-LAEOZQHASA-N 0.000 description 1
- AFERFBZLVUFWRA-HTFCKZLJSA-N Ile-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(=O)O)N AFERFBZLVUFWRA-HTFCKZLJSA-N 0.000 description 1
- SJLVSMMIFYTSGY-GRLWGSQLSA-N Ile-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SJLVSMMIFYTSGY-GRLWGSQLSA-N 0.000 description 1
- DBXXASNNDTXOLU-MXAVVETBSA-N Ile-Leu-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N DBXXASNNDTXOLU-MXAVVETBSA-N 0.000 description 1
- WVUDHMBJNBWZBU-XUXIUFHCSA-N Ile-Lys-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)O)N WVUDHMBJNBWZBU-XUXIUFHCSA-N 0.000 description 1
- XVUAQNRNFMVWBR-BLMTYFJBSA-N Ile-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N XVUAQNRNFMVWBR-BLMTYFJBSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- FJUKMPUELVROGK-IHRRRGAJSA-N Leu-Arg-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N FJUKMPUELVROGK-IHRRRGAJSA-N 0.000 description 1
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 1
- CUXRXAIAVYLVFD-ULQDDVLXSA-N Leu-Arg-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CUXRXAIAVYLVFD-ULQDDVLXSA-N 0.000 description 1
- DLFAACQHIRSQGG-CIUDSAMLSA-N Leu-Asp-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O DLFAACQHIRSQGG-CIUDSAMLSA-N 0.000 description 1
- DLCOFDAHNMMQPP-SRVKXCTJSA-N Leu-Asp-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DLCOFDAHNMMQPP-SRVKXCTJSA-N 0.000 description 1
- WMTOVWLLDGQGCV-GUBZILKMSA-N Leu-Glu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N WMTOVWLLDGQGCV-GUBZILKMSA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- VZBIUJURDLFFOE-IHRRRGAJSA-N Leu-His-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VZBIUJURDLFFOE-IHRRRGAJSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- FOBUGKUBUJOWAD-IHPCNDPISA-N Leu-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FOBUGKUBUJOWAD-IHPCNDPISA-N 0.000 description 1
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 1
- KPYAOIVPJKPIOU-KKUMJFAQSA-N Leu-Lys-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O KPYAOIVPJKPIOU-KKUMJFAQSA-N 0.000 description 1
- IBSGMIPRBMPMHE-IHRRRGAJSA-N Leu-Met-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O IBSGMIPRBMPMHE-IHRRRGAJSA-N 0.000 description 1
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 1
- ADJWHHZETYAAAX-SRVKXCTJSA-N Leu-Ser-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ADJWHHZETYAAAX-SRVKXCTJSA-N 0.000 description 1
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 1
- WUHBLPVELFTPQK-KKUMJFAQSA-N Leu-Tyr-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O WUHBLPVELFTPQK-KKUMJFAQSA-N 0.000 description 1
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 1
- 208000034800 Leukoencephalopathies Diseases 0.000 description 1
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- CLBGMWIYPYAZPR-AVGNSLFASA-N Lys-Arg-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O CLBGMWIYPYAZPR-AVGNSLFASA-N 0.000 description 1
- YVMQJGWLHRWMDF-MNXVOIDGSA-N Lys-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N YVMQJGWLHRWMDF-MNXVOIDGSA-N 0.000 description 1
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 1
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 1
- VSTNAUBHKQPVJX-IHRRRGAJSA-N Lys-Met-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O VSTNAUBHKQPVJX-IHRRRGAJSA-N 0.000 description 1
- ALEVUGKHINJNIF-QEJZJMRPSA-N Lys-Phe-Ala Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ALEVUGKHINJNIF-QEJZJMRPSA-N 0.000 description 1
- LNMKRJJLEFASGA-BZSNNMDCSA-N Lys-Phe-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LNMKRJJLEFASGA-BZSNNMDCSA-N 0.000 description 1
- MGKFCQFVPKOWOL-CIUDSAMLSA-N Lys-Ser-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N MGKFCQFVPKOWOL-CIUDSAMLSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- GAELMDJMQDUDLJ-BQBZGAKWSA-N Met-Ala-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O GAELMDJMQDUDLJ-BQBZGAKWSA-N 0.000 description 1
- DCHHUGLTVLJYKA-FXQIFTODSA-N Met-Asn-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O DCHHUGLTVLJYKA-FXQIFTODSA-N 0.000 description 1
- JACAKCWAOHKQBV-UWVGGRQHSA-N Met-Gly-Lys Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN JACAKCWAOHKQBV-UWVGGRQHSA-N 0.000 description 1
- FBLBCGLSRXBANI-KKUMJFAQSA-N Met-Phe-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FBLBCGLSRXBANI-KKUMJFAQSA-N 0.000 description 1
- NLDXSXDCNZIQCN-ULQDDVLXSA-N Met-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCSC)CC1=CC=CC=C1 NLDXSXDCNZIQCN-ULQDDVLXSA-N 0.000 description 1
- YLDSJJOGQNEQJK-AVGNSLFASA-N Met-Pro-Leu Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O YLDSJJOGQNEQJK-AVGNSLFASA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010020004 Microtubule-Associated Proteins Proteins 0.000 description 1
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 101100501720 Mus musculus Erg gene Proteins 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 208000022873 Ocular disease Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- LZDIENNKWVXJMX-JYJNAYRXSA-N Phe-Arg-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC1=CC=CC=C1 LZDIENNKWVXJMX-JYJNAYRXSA-N 0.000 description 1
- UUWCIPUVJJIEEP-SRVKXCTJSA-N Phe-Asn-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N UUWCIPUVJJIEEP-SRVKXCTJSA-N 0.000 description 1
- IILUKIJNFMUBNF-IHRRRGAJSA-N Phe-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O IILUKIJNFMUBNF-IHRRRGAJSA-N 0.000 description 1
- IDUCUXTUHHIQIP-SOUVJXGZSA-N Phe-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O IDUCUXTUHHIQIP-SOUVJXGZSA-N 0.000 description 1
- FINLZXKJWTYYLC-ACRUOGEOSA-N Phe-His-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FINLZXKJWTYYLC-ACRUOGEOSA-N 0.000 description 1
- DSXPMZMSJHOKKK-HJOGWXRNSA-N Phe-Phe-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O DSXPMZMSJHOKKK-HJOGWXRNSA-N 0.000 description 1
- RVEVENLSADZUMS-IHRRRGAJSA-N Phe-Pro-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RVEVENLSADZUMS-IHRRRGAJSA-N 0.000 description 1
- QARPMYDMYVLFMW-KKUMJFAQSA-N Phe-Pro-Glu Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=CC=C1 QARPMYDMYVLFMW-KKUMJFAQSA-N 0.000 description 1
- YCEWAVIRWNGGSS-NQCBNZPSSA-N Phe-Trp-Ile Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C1=CC=CC=C1 YCEWAVIRWNGGSS-NQCBNZPSSA-N 0.000 description 1
- 241000233805 Phoenix Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 1
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 1
- FRKBNXCFJBPJOL-GUBZILKMSA-N Pro-Glu-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FRKBNXCFJBPJOL-GUBZILKMSA-N 0.000 description 1
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 1
- FKVNLUZHSFCNGY-RVMXOQNASA-N Pro-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 FKVNLUZHSFCNGY-RVMXOQNASA-N 0.000 description 1
- SUENWIFTSTWUKD-AVGNSLFASA-N Pro-Leu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SUENWIFTSTWUKD-AVGNSLFASA-N 0.000 description 1
- DLZBBDSPTJBOOD-BPNCWPANSA-N Pro-Tyr-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O DLZBBDSPTJBOOD-BPNCWPANSA-N 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 238000001190 Q-PCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 102100028001 Retinaldehyde-binding protein 1 Human genes 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 1
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 1
- BRKHVZNDAOMAHX-BIIVOSGPSA-N Ser-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N BRKHVZNDAOMAHX-BIIVOSGPSA-N 0.000 description 1
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- FUMGHWDRRFCKEP-CIUDSAMLSA-N Ser-Leu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O FUMGHWDRRFCKEP-CIUDSAMLSA-N 0.000 description 1
- GJFYFGOEWLDQGW-GUBZILKMSA-N Ser-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GJFYFGOEWLDQGW-GUBZILKMSA-N 0.000 description 1
- HDBOEVPDIDDEPC-CIUDSAMLSA-N Ser-Lys-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O HDBOEVPDIDDEPC-CIUDSAMLSA-N 0.000 description 1
- AXOHAHIUJHCLQR-IHRRRGAJSA-N Ser-Met-Tyr Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CO)N AXOHAHIUJHCLQR-IHRRRGAJSA-N 0.000 description 1
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- VGYBYGQXZJDZJU-XQXXSGGOSA-N Thr-Glu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VGYBYGQXZJDZJU-XQXXSGGOSA-N 0.000 description 1
- UDQBCBUXAQIZAK-GLLZPBPUSA-N Thr-Glu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UDQBCBUXAQIZAK-GLLZPBPUSA-N 0.000 description 1
- ADPHPKGWVDHWML-PPCPHDFISA-N Thr-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N ADPHPKGWVDHWML-PPCPHDFISA-N 0.000 description 1
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- BGDKAVGWHJFAGW-UHFFFAOYSA-N Tropicamide Chemical compound C=1C=CC=CC=1C(CO)C(=O)N(CC)CC1=CC=NC=C1 BGDKAVGWHJFAGW-UHFFFAOYSA-N 0.000 description 1
- PTAWAMWPRFTACW-SZMVWBNQSA-N Trp-Gln-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PTAWAMWPRFTACW-SZMVWBNQSA-N 0.000 description 1
- BOBZBMOTRORUPT-XIRDDKMYSA-N Trp-Ser-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O)=CNC2=C1 BOBZBMOTRORUPT-XIRDDKMYSA-N 0.000 description 1
- DVLHKUWLNKDINO-PMVMPFDFSA-N Trp-Tyr-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DVLHKUWLNKDINO-PMVMPFDFSA-N 0.000 description 1
- XQMGDVVKFRLQKH-BBRMVZONSA-N Trp-Val-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O)=CNC2=C1 XQMGDVVKFRLQKH-BBRMVZONSA-N 0.000 description 1
- KSVMDJJCYKIXTK-IGNZVWTISA-N Tyr-Ala-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 KSVMDJJCYKIXTK-IGNZVWTISA-N 0.000 description 1
- WDIJBEWLXLQQKD-ULQDDVLXSA-N Tyr-Arg-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O WDIJBEWLXLQQKD-ULQDDVLXSA-N 0.000 description 1
- KHCSOLAHNLOXJR-BZSNNMDCSA-N Tyr-Leu-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHCSOLAHNLOXJR-BZSNNMDCSA-N 0.000 description 1
- 102000011731 Vacuolar Proton-Translocating ATPases Human genes 0.000 description 1
- 108010037026 Vacuolar Proton-Translocating ATPases Proteins 0.000 description 1
- GBESYURLQOYWLU-LAEOZQHASA-N Val-Glu-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N GBESYURLQOYWLU-LAEOZQHASA-N 0.000 description 1
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 1
- VHRLUTIMTDOVCG-PEDHHIEDSA-N Val-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](C(C)C)N VHRLUTIMTDOVCG-PEDHHIEDSA-N 0.000 description 1
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 1
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 1
- VENKIVFKIPGEJN-NHCYSSNCSA-N Val-Met-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N VENKIVFKIPGEJN-NHCYSSNCSA-N 0.000 description 1
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 1
- WANVRBAZGSICCP-SRVKXCTJSA-N Val-Pro-Met Chemical compound CSCC[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C)C(O)=O WANVRBAZGSICCP-SRVKXCTJSA-N 0.000 description 1
- QIVPZSWBBHRNBA-JYJNAYRXSA-N Val-Pro-Phe Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O QIVPZSWBBHRNBA-JYJNAYRXSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000362 adenosine triphosphatase inhibitor Substances 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 229960000396 atropine Drugs 0.000 description 1
- RKUNBYITZUJHSG-SPUOUPEWSA-N atropine Chemical compound O([C@H]1C[C@H]2CC[C@@H](C1)N2C)C(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-SPUOUPEWSA-N 0.000 description 1
- 230000005033 autophagosome formation Effects 0.000 description 1
- 208000025341 autosomal recessive disease Diseases 0.000 description 1
- 101150091634 bcd gene Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 229960001631 carbomer Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000004456 color vision Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- HQPMKSGTIOYHJT-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2-diol Chemical compound OCCO.CC(O)CO HQPMKSGTIOYHJT-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229940020947 fluorescein sodium Drugs 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 238000002695 general anesthesia Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 108010091871 leucylmethionine Proteins 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 229960003376 levofloxacin Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 208000018769 loss of vision Diseases 0.000 description 1
- 231100000864 loss of vision Toxicity 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108010022588 methionyl-lysyl-proline Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000005043 peripheral vision Effects 0.000 description 1
- 108010082795 phenylalanyl-arginyl-arginine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 150000008298 phosphoramidates Chemical group 0.000 description 1
- 210000000608 photoreceptor cell Anatomy 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000013326 plasmid cotransfection Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 229960001371 proparacaine hydrochloride Drugs 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 210000003786 sclera Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000013334 tissue model Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 229960004791 tropicamide Drugs 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
- C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- 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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
-
- 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
- C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Ophthalmology & Optometry (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
Abstract
本公开涉及一种优化的CPY4V2基因及其用途,所述基因涉及一种编码CYP4V2蛋白的多核苷酸,其包含与SEQ ID NO 5、SEQ ID NO 6、SEQ ID NO 7、SEQ ID NO 8或SEQ ID NO 9所示核苷酸序列具有90%或以上同一性的核苷酸序列。本公开还涉及包含所述基因的表达载体,所述表达载体可以实现在眼睛的视网膜色素上皮层中高效,持久且稳定的表达能有效地降低用于治疗BCD的基因治疗药物的剂量和可能的副作用,提高治疗效果。
Description
技术领域
本公开属于基因治疗领域,具体涉及优化CPY4V2基因表达用于针对Bietti结晶性营养不良(BCD)的基因治疗。
背景技术
Bietti结晶性营养不良(Bietti crystalline dystrophy,BCD,人类孟德尔遗传病OMIM210370)是一种罕见的CYP4V2基因的双等位基因突变的遗传性常染色体隐性疾病。1937年意大利医生G.B.Bietti首次描述该疾病。其特征在于在视网膜的后极存在许多细小闪光的黄白色晶体样沉积物,视网膜色素上皮(retinal pigment epithelium,RPE)萎缩、色素凝块及脉络膜硬化。该疾病的进展最终导致视力下降,夜盲症,视野丧失和色觉受损。该疾病的发作可发生于青少年时期至三十岁,但也可发生于三十岁以后。随着疾病的进展,周围视力,中枢视力或两者的降低最终导致大多数患者的法律失明(Garcia-Garcia,Martinez-Rubio et al.(2019)."Current perspectives in Bietti crystallinedystrophy."Clin Ophthalmol 13:1379-1399.)。
BCD是由CYP4V2基因突变引起的。该基因位于人类4号染色体的长臂上,编码细胞色素P450(家族4、亚家族IV、多肽2)的一个重要的525个氨基酸的蛋白质成员,参与脂肪酸代谢(Li,Jiao et al.(2004)."Bietti crystalline corneoretinal dystrophy iscaused by mutations in the novel gene CYP4V2."Am J Hum Genet 74(5):817-826)。CYP4V2蛋白存在于视网膜和角膜的上皮细胞中,该酶定位于内质网。该酶对中链饱和脂肪酸具有典型的CYP4的ω-羟化酶活性。CYP4V2是视网膜细胞中唯一以显着水平存在的CYP4,它可能是视网膜细胞中多不饱和脂肪酸代谢的重要贡献者。导致CYP4V2催化功能缺陷的基因突变阻断眼部脂质的降解,随后在BCD病人眼中形成积累(Hata,Ikeda et al.(2018)."Reduction of lipid accumulation rescues Bietti's crystalline dystrophyphenotypes."Proc Natl Acad Sci U S A115(15):3936-3941;Zhang,Yan et al.(2020)."PSCs Reveal PUFA-Provoked Mitochondrial Stress as a Central NodePotentiating RPE Degeneration in Bietti's Crystalline Dystrophy."Mol Ther 28(12):2642-2661)。在有些患者的还发现有全身性的脂质包裹体(Lai,Chu et al.(2010)."Alterations in serum fatty acid concentrations and desaturase activities inBietti crystalline dystrophy unaffected by CYP4V2 genotypes."InvestOphthalmol Vis Sci 51(2):1092-1097)。
BCD估计每67,000人中就有1人发生。它在东亚血统的人群中更为常见,尤其是中国和日本背景的人群。估计中国有21,000名患者,美国约有5,000名患者。BCD可能确诊不足,因为其症状与其他逐渐损害视网膜的眼部疾病的症状相似。目前,没有可用于BCD的治疗方法(Ng,Lai et al.(2016)."Genetics of Bietti Crystalline Dystrophy."AsiaPac J Ophthalmol(Phila)5(4):245-252;Wang,Chen et al.(2021)."New compoundheterozygous CYP4V2 mutations in bietti crystalline corneoretinal dystrophy."Gene 790:145698.)。
BCD的基因治疗策略正在开发中。在高脂肪喂养(HFD)诱导的BCD小鼠模型中,腺相关病毒(AAV)载体携带的人类CYP4V2基因通过视网膜下腔注射在RPE层表达,有效地恢复了一些生理和功能缺陷,概念验证了基因治疗BCD疾病的有效性(Qu,Wu et al.(2020)."Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murinemodel using gene therapy."Gene Ther 27(7-8):370-382)。
目前,AAV介导的CYP4V2基因治疗在多家制药公司处于研究和开发的早期阶段。到目前为止,还没有报告证明能够实现CYP4V2在体内充分和持续表达以达到有效的治疗效果。
发明内容
为了解决现有技术中存在的问题,本公开的目的在于提供一种优化基因表达的病毒载体,以在眼睛的视网膜RPE层中高效持久且稳定地表达人CYP4V2并用于治疗BCD。
在一方面,本公开提供了一种编码CYP4V2蛋白的多核苷酸,其包含与SEQ ID NO5、SEQ ID NO 6、SEQ ID NO 7、SEQ ID NO 8或SEQ ID NO 9所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。
在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 5、SEQ ID NO 6、SEQ IDNO 7、SEQ ID NO 8或SEQ ID NO 9所示。
在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 5所示。
在另一方面,本公开提供了一种表达盒,其包含所述的多核苷酸以及与所述多核苷酸可操作连接的启动子。
在另一方面,本公开提供了一种包含前述方面的多核苷酸的载体,其包含所述的多核苷酸或所述的表达盒。
在本公开的一些实施方案中,所述编码CYP4V2蛋白的多核苷酸与所述表达控制元件可操作地连接。
在本公开的一些实施方案中,所述表达控制元件选自转录/翻译控制信号、复制起点、启动子、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和/或蛋白质的稳定性的元件中的一种或多种。
在另一方面,本公开提供了包含所述表达载体的细胞。
在另一方面,本公开提供了一种病毒颗粒,其包含所述的表达载体。
在另一方面,本公开提供了一种治疗BCD的药物组合物,其包含所述的多核苷酸、所述的表达盒、所述的表达载体和/或所述的病毒颗粒,以及药学上可接受的载体,其中,所述的药物组合物表达野生型或密码子优化的CYP4V2蛋白。
在另一方面,本公开提供了所述的多核苷酸、所述的表达盒、所述的表达载体、所述的病毒颗粒和/或所述的药物组合物在制备治疗Bietti结晶性营养不良(BCD)中的用途。
在另一方面,本公开提供了一种治疗Bietti结晶性营养不良(BCD)的方法,所述方法包括向受试者施用有效量的所述的多核苷酸、所述的表达盒、所述的表达载体、所述的病毒颗粒和/或所述的药物组合物。
在另一方面,本公开提供了一种构建带有CYP4V2突变的BCD细胞模型的方法,其包括以下步骤:
(1)设计针对CYP4V2基因的sgRNA;
(2)将步骤(1)获得的所述sgRNA构建至Cas9-sgRNA载体,获得Cas9-sgRNA质粒;
(3)将步骤(2)获得的所述Cas9-sgRNA质粒导入细胞,获得的具有CYP4V2基因突变的细胞。
附图说明
图1为本公开的重组AAV(rAAV)载体CYP4V2表达盒的结构示意图。
图2示出了在由质粒转染的HEK293细胞中密码子优化的CYP4V2 opt基因的体外表达。其中,(A)CYP4V2蛋白质Western印迹的代表性图像;(B)CYP4V2蛋白质Western印迹的定量,以检测HEK293细胞中CYP4V2和GFP(质粒转染对照)的表达水平,其中WT或opt基因和GFP用CAG-CYP4V2-WT/opt和CMV-EGFP双质粒表达。等量的质粒转染后2天收获细胞裂解物。通过SDS-PAGE分离等量的总蛋白,然后进行免疫印迹。CYP4V2的表达水平用GFP标准化,并计算为与WT表达水平的比率。
图3示出了在AAV2转染的ARPE-19细胞中密码子优化的CYP4V2的体外表达。其中,(A)CYP4V2蛋白质Western印迹的代表性图像;(B)CYP4V2蛋白质Western印迹的定量,以检测ARPE-19细胞中CYP4V2和GAPDH(内部对照)的表达水平,其中WT或opt基因或EGFP对照用等量的AAV2病毒颗粒(MOI=20000)介导。病毒颗粒侵染后2天收获细胞裂解物。通过SDS-PAGE分离等量的总蛋白,然后进行免疫印迹。CYP4V2的表达水平标准化为GAPDH,并计算为与WT表达水平的比率(和WT表达水平比较:*,p<0.05;**,p<0.01;n=3,student检验)。
图4-6示出了AAV2-CYP4V2在BCD细胞模型中的体外药效。
图4示出了HEK293 CYP4V2突变细胞模型的建立和表型鉴定。CYP4V2突变的HEK293细胞表现出有缺陷的细胞表型。其中,(A)使用CRIPSR/Cas9方法生成CYP4V2外显子5突变的示意图。(B)HEK293外显子5突变细胞14号克隆(5-C14)中CYP4V2基因靶向位点的Sanger测序结果。(C)HEK293 WT和突变5-C14细胞在6天的培养过程中细胞增殖率的评估(和WT比较:**,p<0.01;***,p<0.005;student检验;n=4)。(D-E)HEK293WT和突变细胞中在加入或没有加入巴弗洛霉素A1(bafilomycin-A1)(Baf,100nM,2h)处理的情况下自噬标记物LC3B-I/II的评估。(D)蛋白质印迹的代表性图像和(E)LC3B-II蛋白质水平的量化,其被标准化为GAPDH并计算为与WT细胞未加入Baf处理的情况(NT)的比率(*,p<0.05;ns:无显著差别;student检验;n=3)。
图5示出了CYP4V2突变的ARPE-19细胞模型的建立、表型鉴定和AAV2-CYP4V2 WT和优化基因opt18对ARPE-19突变细胞模型表型的影响。使用CRISPR/Cas9方法生成CYP4V2外显子5突变。其中,(A)ARPE-19细胞中CYP4V2基因靶向位点的Sanger测序。(B)WT和突变5-C13细胞在在6天的培养时间过程中评估细胞增殖率。***,P<0.01;student检验;n=6。(C)细胞脂质沉积。WT、突变5-C13细胞、AAV2-CYP4V2 WT或opt18以MOI 20000侵染5-C13细胞。细胞在花生四烯酸(AA)处理后的脂质和DAPI染色。激光共聚焦显微镜的代表性图像。比例尺,25微米。(D)细胞内脂肪颗粒沉积的量化。AAV2-CYP4V WT和opt18侵染突变细胞后脂肪颗粒沉积明显减少。opt18对脂肪颗粒沉积减少的效应优于WT(***,p<0.005;****,p<0.001;n=50,student检验)。(E)WT和突变细胞5-C13的电子显微照片,显示自噬泡(黑色箭头)和空泡(白色箭头)。比例尺,500纳米。
图6示出了AAV2-CYP4V2 WT和优化基因对HEK293 CYP4V2突变细胞模型表型的影响。(A)AAV2-CYP4V2WT或opt18或EGFP对照以MOI 20000侵染细胞2天后CYP4V2蛋白表达水平的Western印迹检测。(B)侵染后,6天时间过程中细胞增殖率的评估。(C)在第6天,用AAV2-CYP4V2 opt18或EGFP对照以MOI 5000或20000侵染的HEK293突变细胞中的细胞增殖。(D)在第6天,野生型HEK293细胞和用AAV2-CYP4V2 opt18及EGFP对照以MOI 20000侵染的HEK293突变细胞的细胞增殖比较(*,p<0.05;****,p<0.001;n=6,student检验)。(E-F)用AAV2-CYP4V2 WT opt18及EGFP对照以MOI 20000侵染的HEK293突变细胞中在加入或没有加入Baf处理的情况下自噬标记物LC3B-I/II的评估。(E)蛋白质印迹的代表性图像和(F)LC3B-II蛋白质水平的量化,其被标准化为GAPDH并计算为EGFP NT情况的比率(*,p<0.05;**,p<0.01;ns:无显著差别;student检验;n=3)。
图7示出了通过视网膜下注射的密码子优化的AAV-CYP4V2在小鼠眼中的体内表达。(A)AAV2-CYP4V2野生型和优化基因在小鼠眼部的表达。野生型小鼠眼球通过视网膜下腔注射入1μl 5×1012vg/ml的AAV2-CYP4V2 WT或opt重组病毒颗粒。4周后,取眼部组织,分离视网膜,匀浆裂解。通过SDS-PAGE分离等量的总蛋白,然后进行免疫印迹。(A)CYP4V2蛋白质Western印迹的代表性图像;(B)CYP4V2蛋白质Western印迹的定量,以检测CYP4V2和GAPDH(内部对照)的表达水平,CYP4V2的表达水平标准化为GAPDH,并计算为与WT表达水平的比率(和WT表达水平比较:*,p<0.05;student检验;n=6-9)。Std:纯化的重组人CYP4V2蛋白标准样品。NC:未注射的小鼠眼球阴性对照。
具体实施方式
在本公开中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时,为了更好地理解本公开,下面提供相关术语的定义和解释。
如本文使用本文提及“约”某个值或参数包括(和描述)针对值或参数本身的实施方案。例如,提及“约X”的描述包括“X”的描述。
如本文使用,除非另外表明,单数形式的冠词“一种”、“一个”和“所述”包括复数指代物。
如本文使用的“载体”指包含待递送入宿主细胞(体外或体内)的核酸的重组质粒或病毒。
如本文使用的术语“多核苷酸”或“核酸”指任何长度的聚合形式的核苷酸,其为核糖核苷酸或脱氧核糖核苷酸。因此,该术语包括但不限于单、双或多链DNA或RNA、基因组DNA、cDNA、DNA-RNA杂交物或包含嘌呤和嘧啶碱基或其他天然、化学或生化修饰的、非天然或衍生的核苷酸碱基的聚合物。核酸的骨架可包含糖和磷酸基团(如通常在RNA或DNA中所发现),或修饰或取代的糖或磷酸基团。可替换地,核酸的骨架可包含合成的亚单元如氨基磷酸的聚合物且因此可以是寡脱氧核苷氨基磷酸(P-NH2)或混合的氨基磷酸酯-磷酸二酯低聚物。此外,双链核酸可从化学合成(通过在适当的条件下合成互补链并退火该链,或使用DNA聚合酶以适当的引物从头开始合成互补链)的单链多核苷酸产物获得。
“重组病毒载体”指包含一种或多种异源序列(即非病毒来源的核酸序列)的重组多核苷酸载体。在重组AAV载体的情况中,重组核酸侧翼为至少一个,优选两个反向末端重复序列(ITR)。
“重组AAV载体(rAAV载体)”指含有一个或多个异源序列(即非AAV来源的核酸序列)的多核苷酸载体,其侧翼为至少一个,优选两个AAV反向末端重复序列(ITR)。当存在于已经使用适当的辅助病毒(或表达适当的辅助功能)感染并表达AAV rep和cap基因产物(即AAV Rep和Cap蛋白)的宿主细胞中时,该rAAV载体可复制并包装入感染的病毒颗粒。当rAAV载体并入较大多核苷酸(例如,在染色体中或在用于克隆或转染的另一载体如质粒中)时,可将rAAV载体称为“原载体(pro-vector)”,其在AAV包装功能和适当的辅助功能存在下可通过复制和壳体化“援救”。rAAV载体可以是多种形式中的任一种,包括但不限于质粒、线性人工染色体,其可与脂质体复合、包封在脂质体内,且在实施方案中,包封在病毒颗粒特别是AAV颗粒中。可将rAAV载体包装入AAV病毒衣壳以生成“重组腺伴随病毒颗粒(rAAV颗粒)”。AAV辅助功能(即允许AAV由宿主细胞复制和包装的功能)可以多种形式中的任一种提供,包括但不限于帮助AAV复制和包装的辅助病毒或辅助病毒基因。其他AAV辅助功能为本领域已知。
“rAAV病毒”或“rAAV病毒颗粒”指由至少一种AAV衣壳蛋白和壳体化的rAAV载体基因组组成的病毒颗粒。
“异源”意为源自与其所比较的或其所导入或并入的实体的其余部分在基因型上不同的实体。例如,由基因工程技术引入不同细胞类型的核酸是异源核酸(且当表达时,可编码异源多肽)。相似地,并入病毒载体的细胞序列(例如基因或其部分)相对于载体是异源核苷酸序列。
如指代病毒滴度中使用,术语“基因组颗粒(gp)”、“基因组等价物”或“基因组拷贝”指包含重组AAV DNA基因组的病毒粒数目,不论其感染性或功能性。在特定载体制备中基因组颗粒的数目可通过如本文实施例或例如Clark等人(1999)Hum.Gene Ther.,10:1031-1039;Veldwijk等人(2002)Mol.Ther.,6:272-278中所述的方法测量。
如指代病毒滴度中使用,术语“感染单位(iu)”、“感染颗粒”或“复制单位”指如通过感染中心测定所测量的具有感染和复制能力的重组AAV载体颗粒的数目,也称为复制中心测定,如例如McLaughlin等人(1988)J.Virol.,62:1963-1973中所述。
如指代病毒滴度中使用,术语“转导单位(tu)”指导致功能性转基因产物产生的感染性重组AAV载体颗粒的数目,如在功能性测定中所测量,如本文实施例或例如Xiao等人(1997)Exp.Neurobiol.,144:113-124中;或Fisher等人(1996)J.Virol.,70:520-532(LFU测定)中所述。
“反向末端重复”或“ITR”序列是本领域熟知的术语且指代在病毒基因组的末端发现的相对短的序列,其处于相反方向。
“AAV反向末端重复(ITR)”序列是本领域熟知的术语,其为在天然单链AAV基因组两末端存在的约145个核苷酸的序列。ITR的最外面125个核苷酸可以两种可替换的方向中的任一种存在,导致不同AAV基因组间和单AAV基因组的两末端间的异质性。最外面125个核苷酸还包含自身互补的数个较短区(称为A、A'、B、B'、C、C'和D区),允许在该ITR部分内发生链间碱基配对。
对于AAV的“辅助病毒”指允许AAV(为有缺陷的细小病毒)通过宿主细胞复制并包装的病毒。已识别多种这样的辅助病毒,包括腺病毒、泡疹病毒和痘类病毒如牛痘。腺病毒涵盖多个不同的亚类,尽管亚类C的腺病毒5型(Ad5)是最常用的。已知人、非人哺乳动物和鸟类来源的多种腺病毒且可从保藏机构如ATCC获得。也可从保藏机构如ATCC获得的泡疹病毒家族包括例如单纯泡疹病毒(herpes simplex viruses,HSV)、EB病毒(Epstein-Barrviruses,EBV)、巨细胞病毒(cytomegaloviruses,CMV)和伪狂犬病病毒(pseudorabiesviruses,PRV)。
相对于参照多肽或核酸序列的“百分比(%)序列同一性”定义为对齐序列并引进缺口后(若需要,以实现最大百分比序列同一性,且不将任何保守取代考虑为序列同一性的部分),候选序列中与参照多肽或核酸序列中的氨基酸残基或核苷酸相同的氨基酸残基或核苷酸的百分比。出于确定百分比氨基酸或核酸序列同一性的目的的比对可以多种本领域内的方式实现,例如使用公共可获得的计算机软件程序,例如Current Protocols inMolecular Biology(Ausubel et al.,eds.,1987),Supp.30,section 7.7.18,表7.7.1中所述的那些,且包括BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。优选的比对软件是ALIGN Plus(Scientific and Educational Software,Pennsylvania)。本领域的技术人员可确定用于测量比对的适当的参数,包括任何在比较的序列全长上需要实现最大比对的算法。就本文而言,给定氨基酸序列A对、与或针对给定氨基酸序列B的%氨基酸序列同一性(其可替换地可称为具有或包含对、与或针对给定氨基酸序列B具有一定%氨基酸序列同一性的氨基酸序列A)如下计算:100乘以分数X/Y,其中X是在A和B的程序比对中通过序列比对程序评分为相同匹配的氨基酸残基的数目,且其中Y是B中氨基酸残基的总数。可以理解的是其中氨基酸序列A的长度与氨基酸序列B的长度不等,A对B的%氨基酸序列同一性将不等于B对A的%氨基酸序列同一性。就本文而言,给定核酸序列C对、与或针对给定核酸序列D的%核酸序列同一性(其可替换地可称为对、与或针对给定核酸序列D具有或包含一定%核酸序列同一性的给定核酸序列C)如下计算:100乘以分数W/Z,其中W是在C和D的程序比对中通过序列比对程序评分为相同匹配的核苷酸的数目,且其中Z是D中核苷酸的总数。可以理解的是其中核酸序列C的长度不等于核酸序列D的长度,C对D的%核酸序列同一性将不等于D对C的%核酸序列同一性。
“有效量”是足以影响有益的或预期的结果,包括临床结果(例如改善症状、实现临床终点等)的量。有效量可以一次或多次施用。就疾病状态而言,有效量是足以改善、稳定或延迟疾病进展的量。例如,有效量的rAAV颗粒表达预期量的异源核酸如治疗性多肽或治疗性核酸。
“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于驯养动物(例如,牛、绵羊(sheep)、猫、狗和马)、灵长类动物(例如,人和非人灵长类动物如猴)、兔和啮齿类动物(例如小鼠和大鼠)。在一些实施方案中,个体或受试者是人。
如本文使用的“治疗”是用于获得有益或预期的临床结果的方式。就本公开而言,有益或预期的临床结果包括但不限于症状改善、疾病程度的减少、疾病的稳定化(例如未恶化)状态、疾病扩散(例如转移)的防止、疾病进展的延迟或减缓、疾病状态的改善或缓和以及缓解(无论部分或全部),无论是可检测的或不可检测的。“治疗”还可意为与未接受治疗的预期生存率相比延长生存率。
在一方面,本公开提供了一种编码CYP4V2蛋白的多核苷酸,其包含与SEQ ID NO5、SEQ ID NO 6、SEQ ID NO 7、SEQ ID NO 8或SEQ ID NO 9所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。
在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 5、SEQ ID NO 6、SEQ IDNO 7、SEQ ID NO 8或SEQ ID NO 9所示。
在另一方面,本公开提供了一种表达盒,其包含所述的多核苷酸以及与所述多核苷酸可操作连接的启动子。
在另一方面,本公开提供了一种表达载体,其包含所述的多核苷酸或所述的表达盒。
在本公开的一些实施方案中,所述编码CYP4V2蛋白的多核苷酸与所述表达控制元件可操作地连接。
在本公开的一些实施方案中,所述表达控制元件选自转录/翻译控制信号、复制起点、启动子、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和/或蛋白质的稳定性的元件中的一种或多种。
在本公开的一些实施方案中,所述表达载体包含复制起点;优选地,所述复制起点序列选自f1噬菌体ori、RK2oriV、pUC ori以及pSC101ori。
在本公开的一些实施方案中,所述表达载体还包含5’ITR;优选地,所述5’ITR包含与SEQ ID NO 1所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 1所示。
在本公开的一些实施方案中,所述表达载体还包含3’ITR;优选地,所述3’ITR包含与SEQ ID NO 11所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 11所示。
在本公开的一些实施方案中,所述表达载体还包含增强子;优选地,所述增强子为CMV增强子;更优选地,所述增强子包含与SEQ ID NO 2所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 2所示。
在本公开的一些实施方案中,所述表达载体还包含启动子。在本公开的一些实施方案中,所述启动子为特异性或非特异性启动子。在本公开的一些实施方案中,所述启动子选自CBA启动子、CMV启动子、SV40启动子、hPGK启动子和TRE3GS启动子。在本公开的一些实施方案中,所述启动子为CBA启动子,优选地,所述CBA启动子包含与SEQ ID NO 3所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 3所示。在本公开的一些实施方案中,所述启动子是可诱导启动子优选地,所述可诱导系统包含四环素调控启动子、醇调控启动子、类固醇调控启动子、金属调控启动子、致病调控启动子、温度/热诱导型启动子和光调控启动子、IPTG诱导型系统中的一种或多种。在本公开的一些实施方案中,所述四环素调控启动子选自Tet on启动子、Tet off的启动子和Tet Activator启动子。在本公开的一些实施方案中,所述醇调控启动子选自醇脱氢酶I(alcA)基因启动子、响应于醇反式激活蛋白(AlcR)的启动子。在本公开的一些实施方案中,所述类固醇调控启动子选自大鼠糖皮质激素受体启动子、人雌激素受体启动子、蛾蜕皮激素受体启动子,类视黄醇启动子和甲状腺受体超家族启动子。在本公开的一些实施方案中,所述金属调控启动子选自酵母、小鼠和人的金属硫蛋白启动子。在本公开的一些实施方案中,所述致病调控启动子选自由水杨酸调控启动子、乙烯调控启动子和苯并噻二唑调控(BTH)启动子。在本公开的一些实施方案中,所述温度/热诱导型启动子选自HSP-70启动、HSP-90启动和大豆热激启动子。在本公开的一些实施方案中,所述光调控启动子是植物细胞的光应答型启动子。
在本公开的一些实施方案中,所述表达载体还包含外显子和内含子。在本公开的一些实施方案中,所述外显子和内含子为鸡β-actin基因的第一个外显子和第一个内含子。在本公开的一些实施方案中,所述外显子和内含子包含与SEQ ID NO 4所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 4所示。
在本公开的一些实施方案中,所述表达载体还包含polyA信号。在本公开的一些实施方案中,所述polyA信号为牛生长激素poly A(BGH poly A)、短poly A、SV40 polyA、和/或人β珠蛋白poly A。在本公开的一些实施方案中,所述polyA信号包含与SEQ ID NO 10所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 10所示。
在本公开的一些实施方案中,所述表达载体包含与SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15或SEQ ID NO 16所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列。在本公开的一些实施方案中,所述多核苷酸如SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15或SEQ ID NO 16所示。
在本公开的一些实施方案中,所述载体包含转录后调节元件。在本公开的一些实施方案中,所述转录后调节元件为土拨鼠肝炎病毒转录后调节元件(WPRE)。
在本公开的一些实施方案中,所述载体还包含编码标志物基因。在本公开的一些实施方案中,所述标志物选自抗生素抗性蛋白质、毒素抗性蛋白质、有色的或荧光的或发光的蛋白质和介导增强的细胞生长和/或基因扩增的蛋白质中的一种或多种。
在本公开的一些实施方案中,所述抗生素选自氨苄青霉素、新霉素、G418、嘌呤霉素和杀稻瘟素。
在本公开的一些实施方案中,所述毒素选自炭疽毒素和白喉毒素。
在本公开的一些实施方案中,所述有色的或荧光的或发光的蛋白质选自绿色荧光蛋白、增强型绿色荧光蛋白、红色荧光蛋白和萤光素酶。在本公开的一些实施方案中,所述介导增强的细胞生长和/或基因扩增的蛋白质为二氢叶酸还原酶(DHFR)。
在本公开的一些实施方案中,所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。
在本公开的一些实施方案中,所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603和pCMV。
在本公开的一些实施方案中,所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如,腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如,流感病毒)、弹状病毒(例如,狂犬病和水疱性口炎病毒)、副粘病毒(例如,麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒,和双链DNA病毒,所述双链DNA病毒包括腺病毒、疱疹病毒(例如,单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如,牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒。
在本公开的一些实施方案中,所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒和泡沫病毒。
在本公开的一些实施方案中,所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV和绵羊脱髓鞘性脑白质炎慢病毒。
在本公开的一些实施方案中,所述表达载体为腺相关病毒。
在本公开的一些实施方案中,所述腺相关病毒选自AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV 9型、AAV 10型、禽AAV、牛AAV、犬AAV、马AAV和绵羊AAV等。
在本公开的一些实施方案中,所述表达载体还包含缩短的嵌合内含子和Kozak起始序列。
在本公开的一些实施方案中,所述缩短的嵌合内含子如SEQ ID NO 4所示。
在本公开的一些实施方案中,所述Kozak起始序列如SEQ ID NO 17所示。
在另一方面,本公开提供了一种病毒颗粒,其包含所述的表达载体。
在另一方面,本公开提供了一种治疗BCD的药物组合物,其包含所述的多核苷酸、所述的表达盒、任一所述的表达载体和/或所述的病毒颗粒,以及药学上可接受的载体,其中,所述的药物组合物表达野生型或密码子优化的CYP4V2蛋白。
在另一方面,本公开提供了所述的多核苷酸、所述的表达盒、所述的表达载体、所述的病毒颗粒和/或所述的药物组合物在制备治疗Bietti结晶性营养不良(BCD)中的用途。
在另一方面,本公开提供了一种治疗Bietti结晶性营养不良(BCD)的方法,所述方法包括向受试者施用有效量的所述的多核苷酸、所述的表达盒、所述的表达载体、所述的病毒颗粒和/或所述的药物组合物。
在另一方面,本公开提供了一种构建带有CYP4V2突变的BCD细胞模型的方法,其包括以下步骤:
(1)设计针对CYP4V2基因的sgRNA;
(2)将步骤(1)获得的所述sgRNA构建至Cas9-sgRNA载体,获得Cas9-sgRNA质粒;
(3)将步骤(3)获得的所述Cas9-sgRNA质粒导入细胞,获得具有CYP4V2基因突变的细胞。
在本公开的一些实施方案中,所述方法还包括步骤(4)筛选具有CYP4V2基因突变的细胞。
在本公开的一些实施方案中,所述方法还包括步骤(5)对所述CYP4V2基因突变细胞进行鉴定。
在本公开的一些实施方案中,所述鉴定选自细胞增殖鉴定、自噬变化鉴定及脂肪颗粒沉积鉴定。
在本公开的一些实施方案中,所述sgRNA选自SEQ ID NO 18-21中的一种或多种。
本公开提供腺相关病毒载体表达密码子优化的CPY4V2基因以实现在眼睛的视网膜色素上皮层中高效,持久且稳定的表达。本公开能有效地降低用于治疗BCD的基因治疗药物的剂量和可能的副作用,提高治疗效果。
为了达到清楚和简洁描述的目的,本文中作为相同的或分开的一些实施方案的一部分来描述特征,然而,将要理解的是,本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
下面,参考具体实施例更详细地描述本公开,然而,实施例仅用于说明目的,对于本公开不具有限制作用。
实验材料和方法:
CYP4V2表达盒和AAV载体
在本研究中使用的所有DNA序列由金斯瑞合成。密码子优化使用GenSmart codonoptimization tool加以改进进行。AAV载体介导的CYP4V2表达盒如图1所示。表达盒序列克隆至穿梭质粒载体获得含AAV载体介导的表达CYP4V2目的基因的穿梭质粒。
AAV载体生产及纯化方法
AAV载体的生产采用三质粒系统,即用含CYP4V2目的基因的穿梭质粒、带AAV载体repcap基因的pRepCap质粒和辅助质粒pHelper,以PEI为转染试剂,共同转染HEK293细胞,重组包装出AAV病毒载体。转染后48-72小时进行收获,收获液经纯化后获得一定纯度的重组AAV病毒载体。纯化方法如下:
首先对收获液进行预处理:充分裂解HEK293细胞,释放出细胞内的AAV病毒载体,同时加入核酸酶对游离的核酸进行消化,消化结束后,采用深层过滤除去大分子杂质和细胞碎片,深层过滤后滤液用二次过滤得到澄清液用于亲和上样。
亲和层析利用配基与蛋白的特异性吸附捕获收获液中的AAV病毒载体,并除去大部分工艺相关杂质,达到浓缩与除杂的效果。将收集的洗脱液混匀,并用中和缓冲液中和,保存在无菌储液瓶中作为阴离子层析上样液。
阴离子层析利用不同组分的等电点差异分离实心与空壳AAV病毒,同时继续去除残留杂质,洗脱液收集在新的无菌储液瓶中,再经超滤浓缩的方法置换缓冲液为制剂稳定的缓冲液,同时病毒滴度浓缩至约5×1012vg/mL,最后除菌过滤分装后备用。
AAV载体储用物的滴度定量
在AAV病毒纯化完成之后,需要对病毒的含量进行测定,基因组滴度是表征AAV物理滴度最经典的标准。通过针对rAAV的基因组序列设计引物探针,然后进行Q-PCR检测是基因组滴度检测最通用的方法。
考虑到本公开中针对ORF编码框进行了密码子优化,涉及到多个载体结构的筛选,为了保证不同载体结构之间定量的稳定性和准确性,选择载体中的共有序列设计引物探针。CMV增强子序列为CAG启动子的一部分,在进行密码子优化过程中,这一部分为不同载体结构的共有部分,因此针对该序列设计引物探针。
在基因组滴度检测过程中,首先要进行标准曲线的建立,阳性标准质粒使用样品稀释液稀释至2×107、2×106、2×105、2×104、2×103、2×102拷贝/μl,作为标准曲线模板,标准曲线需要控制其线性和扩增效率,一般要求R2>0.99,扩增效率在90%-110%之间。之后采用预处理完成的rAAV样品稀释后进行QPCR检测,确保样品检测Ct值在标准曲线范围内,根据样品的Ct值代入到标准曲线中进行rAAV样品基因组滴度的计算,对产品的含量进行标识。
体外细胞质粒转染实验
提前一天消化处理HEK293细胞,按7.0*105细胞/孔接种细胞至6孔板中。过夜培养后进行质粒转染实验。按如下用量进行质粒与转染试剂混合:在一个1.5ml离心管中加入125μl opti-MEM培养基(Gibco,31985-070),2μgCYP4V2-opt/WT表达质粒,200ng CMV-EGFP质粒和5μl P3000,混匀,获得质粒管;在另一个1.5ml离心管中加入125μl opti-MEM培养基和5μl Lipo 3000(Thermo,L3000015),混匀,然后加入至质粒管中,室温静置孵育15min。将混合液缓慢滴加至6孔板HEK293细胞中,继续置于CO2恒温培养箱中培养48h。
体外细胞病毒侵染实验
提前一天消化处理ARPE-19细胞,按3.0*105细胞/孔接种细胞至6孔板中。过夜培养24h后进行AAV2病毒侵染实验。取一个孔的细胞消化后计数,根据20000MOI感染参数计算所需病毒Vg数,用Opti-MEM培养基将所需病毒量稀释至1ml。接着将接种过夜的6孔板中细胞培养基吸干净,加入1ml已稀释好的病毒液,继续置于CO2恒温培养箱中培养;4h后补加1ml DMEM完全培养基(DMEM+10%FBS+1%双抗培养基(Hyclone,SV30010)),置于CO2恒温培养箱中培养48h。
带有CYP4V2突变的BCD细胞模型的构建
采用CRISPR-Cas9方法构建带有CYP4V2突变的BCD细胞模型,具体方法如下:提前一天消化处理HEK293或ARPE-19细胞,按7.0*105细胞/孔接种细胞至6孔板中;24h后进行质粒转染实验,质粒样品稀释准备:在1.5ml离心管中加入125μl opti-MEM培养基,2μg Cas9-sgRNA质粒(addgene,#58766)和5μl P3000(Exon7-sgRNA1-HDR修复构建补加3μg CYP4V2-ssDNA),混匀;Lipo 3000(Thermo,L3000015)稀释准备:在1.5ml离心管中加入125μl opti-MEM培养基和5μl Lipo 3000,混匀后加入至质粒样品稀释管中,混匀并室温孵育15min。将混合液缓慢滴加至6孔板待转染细胞中,轻轻摇晃混匀,置于CO2恒温培养箱中培养。转染48h后荧光拍照确认转染效率,消化6孔板中细胞,加入含5μg/ml嘌呤霉素(Puromycin)的DMEM完全培养基加压培养,期间每天更换培养基,培养基为含5μg/ml嘌呤霉素的DMEM-Fμll培养基;培养72h后,弃掉6孔板中细胞培养基,加入不含嘌呤霉素的DMEM培养基培养。培养48h后,消化细胞并计数,按1个细胞/孔分选细胞至96孔板中(含200μlDMEM+20%FBS+1%双抗培养基)培养。培养2~3周后,观察到克隆斑长大,将克隆斑按96孔板→24孔板→6孔板逐步扩大培养。6孔板细胞长满后,取部分细胞提取细胞基因组DNA(天根,DP304-03),PCR扩增,切胶回收后Sanger测序验证,使用SeqMan软件分析单克隆细胞基因型。剩余部分细胞根据实验需要扩大培养冻存。
Western印迹分析
在接种培养细胞的6孔板的每个孔内加入200μl含蛋白酶抑制剂的RIPA裂解液(碧云天生物技术有限公司,P0013B),冰上充分裂解细胞5min。收集细胞裂解液于1.5ml离心管中,冰上放置30min,4℃,15000g离心20min,取上清。将蛋白样品用PBS稀释10倍,充分混匀。用BCA蛋白定量试剂盒(PierceTM BCA蛋白检测试剂盒(Thermo Fisher,23225)进行蛋白定量。在蛋白样品中加入5×SDS上样缓冲液,充分混匀。100℃煮沸5min。SDS-PAGE电泳分离30μg蛋白样品后转PVDF膜。用5%脱脂牛奶(PBS配制)室温封闭1h后,抗体孵育,ECL检测。Bio-Rad ChemiDocTM Touch Imaging System进行成像分析。
细胞增殖分析
将CYP4V2突变的细胞与野生型细胞消化,计数。将调整好细胞密度的细胞悬液充分混匀,接种至96孔板内,每个细胞设四个复孔。在体外药效实验中,种板24h之后,进行病毒侵染。从种板后48h开始进行CCK-8检测分析,检测的天数分别为Day2,Day3,Day4,Day5,Day6。检测当天,计算总共需要分析的孔的数量配置适当量的含有CCK-8试剂的DMEM培养基。CCK-8试剂(同仁化学所,CK04)与DMEM培养基的量按照1:10的比例配制,每孔加入100μl含有CCK-8试剂的DMEM培养基,37℃细胞培养箱孵育1.5h后,用多功能酶标仪(BioTek,SYNERGY/LX)在450nm处读板。用Graphpad Prism软件对数据进行统计分析。
自噬体功能检测
野生型和突变细胞及病毒侵染的细胞,分别用含有100nM Bafilomycin-A1(Baf)或者不含有药物的培基处理2h,提取细胞蛋白,用WB检测分析LC3B-Ⅱ/GAPDH的比值,以此确定细胞内自噬流是否通畅。
脂质沉积染色
细胞用160μM花生四烯酸(AA)连续处理4天,用4%多聚甲醛室温固定细胞20min。用3μM BODIPY 493/503,37℃染细胞20min。用5μg/ml DAPI衬染10min。激光共聚焦显微镜拍照。每一个实验组,选取50个细胞,用Image pro plus软件分析细胞内脂质沉积颗粒面积和细胞内单位面积内脂质沉积颗粒的数目。
小鼠视网膜下腔注射
小鼠全身麻醉后充分散瞳,在眼科专用手术显微镜直视下,在角巩膜缘外侧用301/2gauge的一次性尖针头穿刺巩膜,避免伤及虹膜和晶状体,然后用带33gauge平针头的微量进样器沿穿刺口进入,针头绕过晶体后到达玻璃体,然后逐渐进针至神经视网膜层和视网膜色素上皮(RPE)层之间的潜在视网膜腔隙并缓慢推注,注射量为1μl。注射载体混悬液中添加0.1%荧光素钠染料(安全浓度),方便观察注射是否成功,及视网膜脱离范围。注射过程中,眼表滴2.5%羟丙基甲基纤维素以便于随时观察眼底情况。手术显微镜下如清楚看到眼底局部视网膜呈圆形隆起且视网膜隆起下方可见绿色,证明注射成功。一定时间后小泡消失,视网膜局部隆起变平。术中如不能看到视网膜隆起及其下方的绿色或看到视网膜有大出血等并发症,则另选小鼠重新注射。术毕涂1%阿托品眼膏和四环素可的松眼膏,以后每隔1天重复一次,共三次,以减少炎症反应、防止感染。
小鼠眼球视网膜样品提取
取小鼠眼部组织,去除角膜和晶状体,加入100μl ATL溶液(QIAGEN,19076),匀浆2min。DNA样品提取:吸取10μl匀浆液,加入10μl ATL溶液和2μl蛋白酶K(QIAGEN,19133)混匀,加入20μl AL溶液(QIAGEN,19075)混匀后56℃孵育10min,制备获得DNA检测样品;蛋白提取:吸取剩余50μl匀浆液,加入5μl 10*RIPA裂解液(CST)和蛋白酶抑制剂(碧云天)混匀,冰上裂解30min;4℃,15000g,离心30min,收集上清,蛋白定量后进行WB检测。
载体基因组拷贝数分析
取5μl DNA检测样品,加入45μl样品稀释液(1ml无RNA酶的水中加5μl PluronicF68(Gibco,24040032)和2μl tRNA(Ambion,AM7119);混匀后吸取5μl稀释液,加入45μl样品稀释液混匀,得到100倍稀释的待测DNA样品;以线性化并绝对定量的质粒(pAAV-CMV-EGFP)作为标准品进行标准曲线的绘制。按如下反应体系配制qPCR反应液:Taqman PCR Mix,上游引物(10μM),下游引物(10μM),探针(10μM),DNA模板。按如下反应条件进行qPCR反应:50℃,2min;95℃,10min;95℃,15s,60℃,30s 40个循环;37℃,2s。反应结束后,根据标准品Ct值制作标准曲线,线性回归计算每个待测样品的Vg数。
小鼠OCT检测
光学相干断层扫描(OCT)检查:将麻醉好的动物置于升降台上,使用复方托吡卡胺滴眼液常规散瞳,并用盐酸丙美卡因滴眼液进行表麻,摆好眼位,待瞳孔散大后将医用卡波姆滴眼液凝胶涂于待测眼角膜上,将眼科超显微成像系统光源调整聚焦,调整镜头焦点至视网膜,而后对视网膜进行光学相干断层扫描(OCT)。每只实验动物双眼均进行拍照。实验完成后用生理盐水清洗双眼,并用左氧氟沙星滴眼液滴眼预防感染。
小鼠ERG检测
将待检测小鼠放置于暗室内饲养3天,到达实验时间点,将小鼠在暗室内进行视网膜电图(ERG)的波形采集,将小鼠麻醉和散瞳后,0.25%羟丙基甲基纤维素溶液点双眼保护角膜,将小鼠放置于有加热垫的操作台上,在小鼠双眼之间的中央处皮肤表皮下插入红色金属丝电极,黑色接地电极插于小鼠尾部表皮下,采用全视网膜眼电图Ganzfeld ERG系统(Micron IV,Phoenix Research Laboratories,Inc)进行ERG图像采集,利用该系统内软件自动分析,得到每个刺激强度a波和b波的平均振幅用于统计分析。视网膜电图的波形可以反映出视网膜神经元传导功能,所测得的波形主要由负向的a波和正向的b波组成,其中a波所反映的是视网膜一级神经元即光感受器细胞的功能,而b波所反应的是视网膜二级神经元即双极细胞的功能。
实施例
实施例1:腺病毒载体的构建和分离纯化
1.1腺病毒载体的构建
CYP4V2表达盒的结构如图1所示。所述CYP4V2表达盒从5’至3’端依次包含5’ITR、CMV增强子、CBA启动子、CBA外显子1&内含子1、Kozak序列、目的基因:野生型人源CYP4V2基因hCYP4V2 WT或优化的CYP4V2基因hCYP4V2 opt、BGH polyA)和3’ITR。其中:
5’ITR的核苷酸序列如SEQ ID NO 1所示;
CMV增强子为巨细胞病毒(CMV)增强子元件,其核苷酸序列如SEQ ID NO 2所示;
CBA启动子为鸡β-肌动蛋白基因启动子,其核苷酸序列如SEQ ID NO 3所示;
CBA外显子1&内含子1为鸡β-actin基因的第一个外显子和第一个内含子,其核苷酸序列如SEQ ID NO 4所示;
hCYP4V2WT基因源自野生型人源CYP4V2基因(GeneID:285440),其基因序列如SEQID NO 9所示,其编码的野生型hCYP4V2WT蛋白的NCBI登记号为NP_997235.3。
优化的CYP4V2基因hCYP4V2 opt为编码野生型hCYP4V2WT蛋白的密码子优化的基因opt18、opt7、opt8和opt6,其核苷酸序列分别为SEQ ID NO 5、SEQ ID NO 6、SEQ ID NO 7和SEQ ID NO 8。
BGH polyA为牛生长激素聚腺苷酸化信号,其核苷酸序列如SEQ ID NO 10所示。
3’ITR的核苷酸序列如SEQ ID NO 11所示。
Kozak序列被插入到CYP4V2 cDNA序列之前,其序列如SEQ ID NO 17。
包含野生型人源CYP4V2基因hCYP4V2WT的表达盒构建为质粒pAAV2-CYP4V2 WT。
包含优化的CYP4V2基因的表达盒构建为质粒pAAV2-CYP4V2 opt。其中,SEQ ID NO12示出了包含密码子优化基因opt6的表达盒的核苷酸序列,SEQ ID NO 13示出了包含密码子优化基因opt7的表达盒的核苷酸序列,SEQ ID NO 14示出了包含密码子优化基因opt8的表达盒的核苷酸序列。SEQ ID NO 15示出了包含密码子优化基因opt18的表达盒的核苷酸序列。SEQ ID NO 16示出了包含CYP4V2 WT的表达盒的核苷酸序列。
CYP4V2表达盒可以包装在rAAV中具有来自任何AAV血清型或其杂交体或变体的衣壳的载体。
病毒载体通过质粒共转染方法获得。将含有AAV2外壳蛋白基因和能帮助AAV复制的基因的辅助质粒和含CYP4V2目的基因表达盒的穿梭质粒pAAV2-CYP4V2 WT或质粒pAAV2-CYP4V2 opt共转染HEK 293T细胞,初步形成重组腺相关病毒载体。
1.2腺病毒载体的分离和纯化
AAV载体的生产采用三质粒系统,即用含CYP4V2目的基因的穿梭质粒、带AAV载体repcap基因的pRepCap质粒和辅助质粒pHelper,以PEI为转染试剂,共同转染HEK293细胞,重组包装出AAV病毒载体。转染后48-72小时进行收获,收获液经亲和层析纯化后,通过阴离子层析进一步纯化,超滤浓缩,置换缓冲液。纯化后的重组AAV病毒载体测定基因组滴度,除菌过滤分装后备用。
实施例2:密码子优化的CYP4V2 opt的体外表达检测
本实施例在HEK293和ARPE-19两种细胞系中评估了密码子优化的CYP4V2 opt的体外表达水平。其中,HEK293细胞系采用质粒转染的方法,ARPE-19细胞系中采用AAV病毒侵染的方法,将目的基因导入细胞。
在HEK293中,瞬时转染了表达CYP4V2 WT的质粒AAV2-CYP4V2 WT或表达CYP4V2opt的质粒AAV2-CYP4V2 opt,并通过Western印迹分析评估了细胞裂解物中的蛋白质表达(图2)。密码子优化后CYP4V2的基因opt6、opt7、opt8、opt18(SEQ ID NO 8、6、7、5)在HEK293细胞中的表达水平都明显高于野生型人源CYP4V2基因(WT)。
在ARPE-19中,通过质粒AAV2-CYP4V2 WT或质粒AAV2-CYP4V2 opt侵染后,Western印迹分析评估了细胞裂解物中的蛋白质表达(图3)。
由图2和图3可见,和野生型人源CYP4V2 WT基因相比,密码子优化后CYP4V2 opt的基因,尤其是opt18明显表现出更高的表达水平。
实施例3:BCD细胞模型的构建和鉴定
为了评定AAV2-CYP4V2 WT和opt基因在BCD基因疗法中的功效,本实施例用CRISPR-Cas9方法在HEK293和ARPE-19两种细胞系中构建了带有CYP4V2突变的细胞模型(图4和图5)。
细胞模型的构建策略如图4A所示。在外显子5附近设计了Exon5-sgRNA1(其核苷酸序列如表1所示),在外显子7附近设计了Exon7-sgRNA1、Exon7-sgRNA2和Exon7-sgRNA3(其核苷酸序列分别如表1所示),四种sgRNA均构建至Cas9-sgRNA载体(Addgene,#58766),获得Cas9-sgRNA质粒;将所述Cas9-sgRNA质粒分别瞬时转染至HEK293和ARPE-19两种细胞,转染48h后,加入嘌呤霉素去除转染不成功细胞,然后通过有限稀释法将细胞接种96孔培养板中培养,2~3周后PCR鉴定96孔板中单克隆细胞基因型。所用引物序列详见表1。
表1用CRIPSR/Cas9构建CYP4V2突变细胞所用的sgRNA和引物序列
单克隆细胞筛选扩增后,通过基因测序验证带有正确突变的细胞克隆,结果如图4B和5A所示。对细胞克隆的基因型,蛋白产物的描述见表格2。野生型CYP4V2蛋白全长序列如SEQ ID NO 27所示,其核苷酸序列如SEQ ID NO 28所示。CYP4V2外显子5的突变细胞在全长序列的223-224aa发生移码突变,外显子7的突变细胞在268-271aa发生移码突变,表达比野生型截短的蛋白。
表2.用CRIPSR/Cas9构建CYP4V2突变细胞
用通过检测细胞增殖和自噬体功能对HEK293带有CYP4V2突变的细胞进行表型鉴定。文献报导,在由BCD病人的iPSC细胞分化的RPE细胞中存在细胞增殖率降低和自噬体的功能缺失(Hata,M.,et al.(2018)."Reduction of lipid accumulation rescuesBietti's crystalline dystrophy phenotypes."Proc Natl Acad Sci U S A 115(15):3936-3941)。本发明人发现,与HEK293 WT细胞相比,HEK293突变细胞的胞增殖率显著降低。本发明人评估了在HEK293 WT和突变细胞中的自噬体标志物微管相关蛋白1轻链3(LC3)的蛋白水平。LC3蛋白的C端处理产生LC3-I。在自噬体的形成过程中引发修饰,使LC3-I变为LC3-II。结果显示LC3-II蛋白的表达水平在HEK293突变细胞中比在HEK293 WT细胞中高。Bafilomycin-A1,一种液泡H+-ATPase抑制剂,能增加WT细胞中的LC3-II蛋白水平,但不能增加突变细胞中的LC3-II蛋白水平。表明突变细胞中自噬体累积但自噬通量受损。这些观察结果显示CYP4V2突变引起细胞正常功能缺失,在HEK293细胞中造成细胞增殖降低和自噬体功能缺陷(图4C-E)。图4以5-C14细胞为代表展示鉴定结果。其他的细胞克隆也有相似的表型结果。
在特征更接近RPE细胞的ARPE-19细胞系中,CYP4V2突变也引起了细胞表型的变化。ARPE-19细胞是源自人视网膜色素上皮细胞,细胞表面表达有RPE细胞的表面标记CRALBP和RPE-65的表达。采用CRISPR-Cas9技术获得CYP4V2突变的ARPE-19细胞(图5A),与ARPE-19WT细胞相比,ARPE-19突变细胞的细胞增殖率显著降低(图5B),CYP4V2基因在眼部RPE细胞层编码参与脂肪酸代谢的羟基化酶,维持视网膜多聚不饱和脂肪酸的稳态平衡。在CYP4V2突变的ARPE-19细胞系中,细胞用160μM花生四烯酸(AA)连续处理4天,然后用5μMBODIPY 493/503脂质染色后可见脂肪颗粒沉积显著高于野生型细胞(图5C-5D)。透射电镜观察结果显示,突变细胞中出现了更多数目的自噬泡(图5E)。这些观察结果显示CYP4V2突变引起细胞正常功能缺失,在ARPE-19细胞中造成细胞增殖降低,脂肪代谢异常和自噬体功能缺陷。图5以5-C13细胞为代表展示鉴定结果。
实施例4:密码子优化的AAV2-CYP4V2 opt的在BCD细胞模型中的体外药效评价
在带有CYP4V2突变的HEK293和ARPE-19两种BCD细胞模型中用AAV病毒侵染评估了密码子优化的CYP4V2 opt的体外药效。
AAV2介导的CYP4V2 WT和opt在HEK293 CYP4V2突变的BCD细胞模型中的表达如图6A所示。在相同病毒感染量的条件下,侵染的CYP4V2 opt的表达量高于WT。
CYP4V2 WT和opt的表达在HEK293 BCD细胞模型中修复了突变细胞的细胞增殖率缺陷,并且存在剂量依赖性(图6B-C)。CYP4V2 opt对突变细胞的功能修复后细胞增殖水平仍低于野生型HEK293细胞,表明CYP4V2 opt的表达没有使细胞过度活化(图6D)。
和部分修复细胞增殖降低的结果一致,CYP4V2 WT和opt的表达也部分恢复了突变细胞的自噬体功能缺陷。突变细胞中表达AAV2-CYP4V2 WT或opt和EGFP对照相比,没有显著修复自噬体累积(未加入Baf处理的情况:NT条件下)。但经Bafilomycin-A1(Baf)处理的条件下,有AAV2-CYP4V2 WT或opt表达的突变细胞中自噬通量显著上升,表明自噬功能被部分恢复(图6E-F)。
同样,AAV2介导的CYP4V2 WT和opt在ARPE-19CYP4V2突变的BCD细胞模型也表现出治疗效果。AAV2-WT与AAV2-opt18病毒侵染ARPE-19突变细胞5-C13,如图5C-5D显示,AAV2-WT和AAV2-opt18病毒侵染的ARPE-19突变细胞5-C13内,和未经侵染的突变细胞比较,脂质沉积颗粒明显减少,达到野生型ARPE-19细胞水平。优化基因opt18降低脂质沉积的效果与WT相比更好,具有明显统计学差异。(图5C-5D)。
图5-6以CYP4V2 opt18为代表展示结果。其他的opt基因的表达也有相似的表型结果。
这些结果表明,在HEK293和ARPE-19BCD细胞模型中观察到的表型缺陷是由CYP4V2基因的功能缺失引起。补充CYP4V2基因的表达能实现恢复这些细胞功能缺陷,具有治疗BCD的效果。
实施例5:密码子优化的CYP4V2 opt的体内表达检测
采用视网膜下腔注射,对4周龄野生型C57BL/6小鼠的眼球注射1μl 1.5×1012vg/ml的AAV2-CYP4V2 WT和opt重组病毒颗粒,注射后每隔4周进行眼部组织取样,小鼠眼部组织匀浆后加入RIPA裂解液裂解组织,匀浆裂解。通过SDS-PAGE分离等量的总蛋白,然后进行免疫印迹,蛋白定量后Western检测CYP4V2表达水平。基因的表达水平通过Western印迹检测进行分析。密码子优化后CYP4V2 opt基因的蛋白表达水平和WT进行比较。注射后4周的结果表明,AAV2-CYP4V2 opt基因在小鼠眼部以比WT更高的水平表达(图7)。
实施例6:高脂喂养的CYP4V3 KO BCD小鼠模型的表型鉴定
人源CYP4V2基因在小鼠的同源基因CYP4V3的敲除(knockout,KO)模型与BCD患者相比,具有较少的生理和功能变化,而且表型出现远晚于病人。6个月小鼠(相当于人类20-30岁)有零星眼部脂质沉积,12个月(相当于人类50岁,已经出现眼盲)有明显脂质沉积但没有眼睛ERG功能和视力缺失(Lockhart,C.M.,et al.(2014)."Generation andcharacterization of a murine model of Bietti crystalline dystrophy."InvestOphthalmol Vis Sci 55(9):5572-5581)。给予高脂饮食(high fat diet,HFD)后,视网膜病变的发生在CYP4V3 KO小鼠模型中加速和聚集(Qu,B.,et al.(2020)."Treating Bietticrystalline dystrophy in a high-fat diet-exacerbated murine model using genetherapy."Gene Ther 27(7-8):370-382)。本实施例采取了在小鼠出生离乳后进行HFD喂养,4周周龄后每隔4周进行眼底成像观察,OCT和ERG功能检测。
实施例7:密码子优化的AAV2-CYP4V2 opt的在BCD小鼠模型中的体内药效评价
采用视网膜下腔注射,对高脂喂养出现眼底病理变化的CYP4V3 KO BCD小鼠的眼球注射1μl 5e12 vg/ml的AAV2-GFP或CYP4V2 opt病毒颗粒。注射后每隔4周进行OCT和ERG功能检测,比较分析AAV2-GFP和CYP4V2opt病毒颗粒的治疗效果。
序列表
<110> 苏州诺洁贝生物技术有限公司
<120> 优化的CPY4V2基因及其用途
<130> MTI21296
<160> 28
<210> 1
<211> 145
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 1
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcct 145
<210> 2
<211> 380
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 2
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg 380
<210> 3
<211> 278
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 3
tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60
ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120
ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 180
cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240
ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcg 278
<210> 4
<211> 1017
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 4
ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg 60
ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct 120
ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa 180
agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc 240
gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc 300
tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg 360
ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg 420
tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac 480
ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt 540
ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg 600
gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc 660
ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg 720
cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc 780
ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag 840
ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg 900
cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg 960
accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacag 1017
<210> 5
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 5
gctggcctgt ggctgggcct ggtgtggcag aagctgttgc tgtggggggc tgcctctgcc 60
ctgagcctgg ctggggctag cctggtgttg agcctgctgc agagagtggc tagctatgct 120
agaaagtggc agcagatgag acccatcccc acagtggcaa gagcctaccc tctggtgggc 180
catgccctgc tgatgaagcc tgatggcaga gagttctttc agcagatcat agagtacaca 240
gaggagtaca gacacatgcc cctgctgaaa ctgtgggtgg gccctgtgcc catggtggct 300
ctgtacaatg ctgagaatgt ggaggtgatt ctgacaagca gcaagcagat tgacaagagc 360
tccatgtaca agttcctgga gccctggctg ggcctgggac tgctgacaag cactggcaac 420
aagtggagaa gcagaagaaa gatgctgacc cccaccttcc acttcaccat cctggaggac 480
ttcctggaca ttatgaatga gcaagccaac atcctggtga aaaagctgga aaagcacatc 540
aaccaagagg ccttcaactg cttcttctac atcaccctgt gtgccctgga catcatctgt 600
gagacagcca tgggcaagaa catcggggcc cagagcaatg atgactctga gtatgtgaga 660
gctgtgtaca gaatgtctga gatgatcttc agaagaatca agatgccctg gctgtggctg 720
gacctgtggt acctgatgtt caaggagggc tgggagcaca aaaagagcct gcagatcctg 780
cacaccttca ccaactctgt gatcgccgag agagccaatg agatgaatgc caatgaggac 840
tgcagagggg atggcagagg ctctgcccct agcaagaaca agagaagagc cttcctggac 900
ctgttgctgt ctgtgaccga cgatgagggc aacagactga gccatgagga catcagagaa 960
gaggtggaca catttatgtt tgagggccat gacaccacag ctgctgccat aaactggagc 1020
ctgtacctgc tgggcagcaa ccctgaggtg cagaagaagg tggaccatga gctggatgat 1080
gtgtttggca agtctgacag acctgccaca gtggaggacc tgaagaagct gagatacctg 1140
gagtgtgtga tcaaggagac cctgagactg ttcccctctg tgcccctgtt tgctagatct 1200
gtgtctgagg actgtgaggt ggctggctat agagtgctga agggcacaga ggctgtgatc 1260
atcccctatg ccctgcacag agaccctaga tacttcccca accctgagga gtttcagcct 1320
gagagattct tccctgagaa tgcccaaggc agacacccct atgcctatgt gccattctct 1380
gctggcccaa gaaactgcat tgggcagaag tttgctgtga tggaggagaa gaccatcctg 1440
agctgcatct tgagacactt ctggattgag agcaatcaga agagagagga gctgggcctg 1500
gaggggcagc tgatactgag accaagcaat ggcatctgga tcaagctgaa gagaagaaat 1560
gctgatgaga ga 1572
<210> 6
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 6
gctggcctgt ggctgggcct ggtgtggcag aagctgctgc tgtggggagc tgcctctgcc 60
ctgtctctgg ctggagccag cctggtgctg agcctgctgc agagagtggc cagctatgcc 120
aggaaatggc aacagatgag acctatcccc acagtggcca gagcctaccc cctggttggc 180
catgccctgc tgatgaagcc tgatggcaga gagttcttcc agcagatcat tgagtacaca 240
gaagagtaca gacacatgcc tctgctgaag ctgtgggtgg gccctgtgcc catggtggcc 300
ctgtacaatg cagagaatgt ggaagtgatc ctgaccagca gcaagcagat agacaagtcc 360
agcatgtaca agttcctgga accttggctg ggcctgggcc tgctcacctc cacaggcaac 420
aagtggagaa gcagaaggaa gatgctgacc ccaaccttcc acttcaccat cctggaggac 480
tttctggaca tcatgaatga gcaggccaac atcctggtca aaaaactgga aaagcacatc 540
aaccaggaag ccttcaactg cttcttctac atcaccctgt gtgccctgga catcatctgc 600
gagacagcca tgggcaagaa catcggagcc cagagcaatg atgactctga atatgtcagg 660
gcagtgtaca gaatgtctga gatgatcttc cggcggatca agatgccttg gctgtggctg 720
gacctgtggt acctgatgtt caaagagggc tgggagcaca agaagagcct gcagatcctg 780
cacaccttca ccaacagcgt gattgctgag cgggccaatg aaatgaacgc caatgaggac 840
tgcagaggag atggcagagg ctctgccccc agcaagaaca agagaagagc cttcctggac 900
ctgctgctgt ccgtgacaga tgatgagggc aacagactga gccacgagga catcagagag 960
gaagtggaca cctttatgtt tgaaggccat gacaccacag ctgctgccat caactggagc 1020
ctgtacctcc tgggcagcaa ccctgaggtg cagaagaagg tggaccatga gctggatgat 1080
gtgtttggca agtctgacag acctgccaca gtggaagacc tgaagaaact caggtacctg 1140
gaatgtgtga tcaaagagac cctgagactg ttcccaagtg tgcctctgtt tgccagatct 1200
gtctctgagg actgtgaagt ggctggctac agagtgctga agggcacaga ggcagttatc 1260
atcccctatg ccctgcacag agaccccaga tacttcccca accctgaaga gttccagcct 1320
gagagattct tcccagagaa tgcccagggc agacaccctt atgcctatgt gcccttctca 1380
gctggaccta gaaactgcat aggccaaaag tttgcagtga tggaagagaa gaccatcctg 1440
agctgcatcc tgaggcactt ctggattgag agcaaccaga agagagagga actgggcctg 1500
gaaggacagc tgatcctgag gcctagcaat ggcatctgga tcaagctgaa aagaagaaat 1560
gctgatgaga gg 1572
<210> 7
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 7
gctggcctgt ggctgggcct ggtgtggcag aaactgctgc tgtggggagc tgcctctgcc 60
ctgtctctgg ctggagcctc ccttgtcctg tccctcctgc aaagagttgc cagctatgcc 120
agaaagtggc agcagatgag acccatcccc acagtggcca gggcctaccc actggtgggc 180
catgccctgc tgatgaagcc tgatggcaga gaattctttc agcagatcat agagtacaca 240
gaggagtaca gacacatgcc tctgctgaag ctgtgggtgg gccctgtgcc tatggtggcc 300
ctgtacaatg ctgagaatgt ggaagtgatc ctgaccagca gcaagcagat tgacaagagc 360
agcatgtaca agttcctgga gccttggctg ggcctgggcc tgctgaccag cacaggcaac 420
aagtggagaa gcaggagaaa gatgctgacc cccaccttcc acttcaccat cctggaagac 480
ttcctggaca tcatgaatga gcaggccaac atcctggtga aaaagctgga aaaacacatc 540
aaccaggaag ccttcaactg cttcttctac attaccctgt gtgccctgga catcatctgc 600
gagacagcca tgggaaaaaa catcggagct cagagcaatg atgacagcga gtatgtgaga 660
gcagtgtacc ggatgagcga aatgatcttc agacggatca agatgccctg gctgtggctg 720
gacctgtggt acctcatgtt taaggagggc tgggaacaca agaagagcct gcagatcctg 780
cacaccttca caaacagtgt gatcgctgaa agggccaacg agatgaatgc caatgaggat 840
tgcagaggcg atggccgcgg ctccgcccct agcaagaaca agagaagagc cttcctggac 900
ctgctgctgt ctgtcaccga tgacgagggc aaccggctgt ctcatgagga catcagagaa 960
gaggtggaca ccttcatgtt tgagggccac gacaccacag ccgccgccat caactggagc 1020
ctgtacctgc ttggcagcaa ccctgaggtg caaaagaagg tggaccatga gctggatgat 1080
gtttttggca aatctgacag acctgccaca gtggaggacc tgaagaaact gagatacctg 1140
gagtgtgtga tcaaggaaac cctgagactc ttccctagtg tgcctctgtt tgccagatct 1200
gtctcagagg actgtgaggt ggctggctac agagtgctga agggcacaga agcagtgatc 1260
atcccctatg ccctgcacag agaccccaga tacttcccca accctgagga attccagcca 1320
gagaggttct tccctgagaa tgcccaggga agacacccat atgcctatgt gcctttctct 1380
gctggcccca gaaactgcat tggacagaag tttgctgtga tggaggaaaa gaccatcctg 1440
agctgcatcc tgagacactt ctggatagaa agcaaccaga agagggaaga gctgggcctg 1500
gaaggccagc tgatcctgag gcctagcaat ggcatctgga tcaaactgaa gagaagaaat 1560
gcagatgaga ga 1572
<210> 8
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 8
gctggcctgt ggctgggcct ggtgtggcag aagctgctgc tgtggggagc tgcctctgcc 60
ctgtccctgg ctggagcctc tctggtgctg agcctgctgc agagagtggc cagctatgcc 120
aggaaatggc aacagatgag acccatccca acagtggcca gagcctaccc cctggttggc 180
catgccctgc tgatgaagcc tgatggcaga gagttcttcc agcagatcat tgagtacaca 240
gaggaataca gacacatgcc tctgctgaag ctgtgggtgg gccctgtgcc catggtggcc 300
ctgtacaatg cagagaatgt ggaagtgatc ctgacctcca gcaagcagat agacaagagc 360
agcatgtaca agttcctgga accttggctg ggccttggcc tgctcaccag cacaggcaac 420
aagtggagaa gcagaaggaa gatgctgacc cctaccttcc acttcaccat cctggaggac 480
tttctggaca tcatgaatga gcaggccaac atcctggtca aaaagctgga aaagcacatc 540
aaccaggagg ccttcaactg cttcttctac atcaccctgt gtgccctgga catcatctgc 600
gagacagcca tgggcaaaaa catcggagcc cagagcaatg atgactctga gtatgtgcgg 660
gctgtgtacc ggatgagcga aatgatcttc agacggatca agatgccctg gctgtggctg 720
gacctctggt acctgatgtt caaagaaggc tgggagcaca agaagagcct gcagatcctg 780
cacaccttca ccaacagcgt gattgcagaa agagccaatg agatgaatgc caacgaggac 840
tgcagaggag atggcagagg ctctgcccct agcaagaaca agagaagggc ctttctggac 900
ctgctgctgt ctgtcacaga tgatgagggc aacagactga gccacgagga catcagagag 960
gaagtggaca ccttcatgtt tgaaggccat gacaccacag ctgctgccat caactggtcc 1020
ctgtacctgc tgggcagcaa ccctgaggtg cagaagaagg tggaccatga gctggatgat 1080
gtgtttggca agtctgacag acctgccaca gtggaagacc tgaaaaagct gagatacctg 1140
gaatgtgtga tcaaagagac cctgagactg ttcccttctg tgcctctgtt tgccagaagt 1200
gtttctgagg actgtgaagt ggctggctac agagtgctga agggcacaga ggcagtcatc 1260
atcccctatg ccctgcacag agaccccagg tacttcccca accctgagga gttccagcct 1320
gaaagattct tcccagagaa tgcccagggc agacacccat atgcctatgt gcccttctca 1380
gctggaccta gaaactgcat aggacaaaag tttgcagtga tggaagagaa gaccatcctg 1440
agctgcatcc tgaggcactt ctggattgag agcaaccaga aaagagagga actgggcctg 1500
gaaggccagc tgatcctcag gcctagcaat ggcatctgga tcaagctgaa gagaaggaat 1560
gctgatgaga ga 1572
<210> 9
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 9
gcggggctct ggctggggct cgtgtggcag aagctgctgc tgtggggcgc ggcgagtgcc 60
ctttccctgg ccggcgccag tctggtcctg agcctgctgc agagggtggc gagctacgcg 120
cggaaatggc agcagatgcg gcccatcccc acggtggccc gcgcctaccc actggtgggc 180
cacgcgctgc tgatgaagcc ggacgggcga gaattttttc agcagatcat tgagtacaca 240
gaggaatacc gccacatgcc gctgctgaag ctctgggtcg ggccagtgcc catggtggcc 300
ctttataatg cagaaaatgt ggaggtaatt ttaactagtt caaagcaaat tgacaaatcc 360
tctatgtaca agtttttaga accatggctt ggcctaggac ttcttacaag tactggaaac 420
aaatggcgct ccaggagaaa gatgttaaca cccactttcc attttaccat tctggaagat 480
ttcttagata tcatgaatga acaagcaaat atattggtta agaaacttga aaaacacatt 540
aaccaagaag catttaactg ctttttttac atcactcttt gtgccttaga tatcatctgt 600
gaaacagcta tggggaagaa tattggtgct caaagtaatg atgattccga gtatgtccgt 660
gcagtttata gaatgagtga gatgatattt cgaagaataa agatgccctg gctttggctt 720
gatctctggt accttatgtt taaagaagga tgggaacaca aaaagagcct tcagatccta 780
catactttta ccaacagtgt catcgctgaa cgggccaatg aaatgaacgc caatgaagac 840
tgtagaggtg atggcagggg ctctgccccc tccaaaaata aacgcagggc ctttcttgac 900
ttgcttttaa gtgtgactga tgacgaaggg aacaggctaa gtcatgaaga tattcgagaa 960
gaagttgaca ccttcatgtt tgaggggcac gatacaactg cagctgcaat aaactggtcc 1020
ttatacctgt tgggttctaa cccagaagtc cagaaaaaag tggatcatga attggatgac 1080
gtgtttggga agtctgaccg tcccgctaca gtagaagacc tgaagaaact tcggtatctg 1140
gaatgtgtta ttaaggagac ccttcgcctt tttccttctg ttcctttatt tgcccgtagt 1200
gttagtgaag attgtgaagt ggcaggttac agagttctaa aaggcactga agccgtcatc 1260
attccctatg cattgcacag agatccgaga tacttcccca accccgagga gttccagcct 1320
gagcggttct tccccgagaa tgcacaaggg cgccatccat atgcctacgt gcccttctct 1380
gctggcccca ggaactgtat aggtcaaaag tttgctgtga tggaagaaaa gaccattctt 1440
tcgtgcatcc tgaggcactt ttggatagaa tccaaccaga aaagagaaga gcttggtcta 1500
gaaggacagt tgattcttcg tccaagtaat ggcatctgga tcaagttgaa gaggagaaat 1560
gcagatgaac gc 1572
<210> 10
<211> 225
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 10
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225
<210> 11
<211> 145
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 11
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120
gagcgcgcag agagggagtg gccaa 145
<210> 12
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 12
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctctcga ggagcttggc ccattgcata cgttgtatcc 180
atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttactagtg 240
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 300
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 360
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 420
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 480
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 540
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 600
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 660
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 720
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 780
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 840
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 900
gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 960
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1020
tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 1080
aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 1140
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 1200
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1260
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1320
gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1380
cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1440
tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1500
ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1560
cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1620
gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1680
cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1740
gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1800
gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1860
gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1920
cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcgcgcc 1980
gccaccatgg ctggcctgtg gctgggcctg gtgtggcaga agctgctgct gtggggagct 2040
gcctctgccc tgtccctggc tggagcctct ctggtgctga gcctgctgca gagagtggcc 2100
agctatgcca ggaaatggca acagatgaga cccatcccaa cagtggccag agcctacccc 2160
ctggttggcc atgccctgct gatgaagcct gatggcagag agttcttcca gcagatcatt 2220
gagtacacag aggaatacag acacatgcct ctgctgaagc tgtgggtggg ccctgtgccc 2280
atggtggccc tgtacaatgc agagaatgtg gaagtgatcc tgacctccag caagcagata 2340
gacaagagca gcatgtacaa gttcctggaa ccttggctgg gccttggcct gctcaccagc 2400
acaggcaaca agtggagaag cagaaggaag atgctgaccc ctaccttcca cttcaccatc 2460
ctggaggact ttctggacat catgaatgag caggccaaca tcctggtcaa aaagctggaa 2520
aagcacatca accaggaggc cttcaactgc ttcttctaca tcaccctgtg tgccctggac 2580
atcatctgcg agacagccat gggcaaaaac atcggagccc agagcaatga tgactctgag 2640
tatgtgcggg ctgtgtaccg gatgagcgaa atgatcttca gacggatcaa gatgccctgg 2700
ctgtggctgg acctctggta cctgatgttc aaagaaggct gggagcacaa gaagagcctg 2760
cagatcctgc acaccttcac caacagcgtg attgcagaaa gagccaatga gatgaatgcc 2820
aacgaggact gcagaggaga tggcagaggc tctgccccta gcaagaacaa gagaagggcc 2880
tttctggacc tgctgctgtc tgtcacagat gatgagggca acagactgag ccacgaggac 2940
atcagagagg aagtggacac cttcatgttt gaaggccatg acaccacagc tgctgccatc 3000
aactggtccc tgtacctgct gggcagcaac cctgaggtgc agaagaaggt ggaccatgag 3060
ctggatgatg tgtttggcaa gtctgacaga cctgccacag tggaagacct gaaaaagctg 3120
agatacctgg aatgtgtgat caaagagacc ctgagactgt tcccttctgt gcctctgttt 3180
gccagaagtg tttctgagga ctgtgaagtg gctggctaca gagtgctgaa gggcacagag 3240
gcagtcatca tcccctatgc cctgcacaga gaccccaggt acttccccaa ccctgaggag 3300
ttccagcctg aaagattctt cccagagaat gcccagggca gacacccata tgcctatgtg 3360
cccttctcag ctggacctag aaactgcata ggacaaaagt ttgcagtgat ggaagagaag 3420
accatcctga gctgcatcct gaggcacttc tggattgaga gcaaccagaa aagagaggaa 3480
ctgggcctgg aaggccagct gatcctcagg cctagcaatg gcatctggat caagctgaag 3540
agaaggaatg ctgatgagag ataaagatct gcctcgactg tgccttctag ttgccagcca 3600
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3660
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3720
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3780
ggggatgcgg tgggctctat ggatccccta actacaagga acccctagtg atggagttgg 3840
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 3900
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3960
a 3961
<210> 13
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 13
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctctcga ggagcttggc ccattgcata cgttgtatcc 180
atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttactagtg 240
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 300
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 360
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 420
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 480
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 540
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 600
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 660
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 720
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 780
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 840
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 900
gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 960
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1020
tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 1080
aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 1140
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 1200
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1260
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1320
gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1380
cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1440
tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1500
ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1560
cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1620
gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1680
cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1740
gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1800
gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1860
gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1920
cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcgcgcc 1980
gccaccatgg ctggcctgtg gctgggcctg gtgtggcaga agctgctgct gtggggagct 2040
gcctctgccc tgtctctggc tggagccagc ctggtgctga gcctgctgca gagagtggcc 2100
agctatgcca ggaaatggca acagatgaga cctatcccca cagtggccag agcctacccc 2160
ctggttggcc atgccctgct gatgaagcct gatggcagag agttcttcca gcagatcatt 2220
gagtacacag aagagtacag acacatgcct ctgctgaagc tgtgggtggg ccctgtgccc 2280
atggtggccc tgtacaatgc agagaatgtg gaagtgatcc tgaccagcag caagcagata 2340
gacaagtcca gcatgtacaa gttcctggaa ccttggctgg gcctgggcct gctcacctcc 2400
acaggcaaca agtggagaag cagaaggaag atgctgaccc caaccttcca cttcaccatc 2460
ctggaggact ttctggacat catgaatgag caggccaaca tcctggtcaa aaaactggaa 2520
aagcacatca accaggaagc cttcaactgc ttcttctaca tcaccctgtg tgccctggac 2580
atcatctgcg agacagccat gggcaagaac atcggagccc agagcaatga tgactctgaa 2640
tatgtcaggg cagtgtacag aatgtctgag atgatcttcc ggcggatcaa gatgccttgg 2700
ctgtggctgg acctgtggta cctgatgttc aaagagggct gggagcacaa gaagagcctg 2760
cagatcctgc acaccttcac caacagcgtg attgctgagc gggccaatga aatgaacgcc 2820
aatgaggact gcagaggaga tggcagaggc tctgccccca gcaagaacaa gagaagagcc 2880
ttcctggacc tgctgctgtc cgtgacagat gatgagggca acagactgag ccacgaggac 2940
atcagagagg aagtggacac ctttatgttt gaaggccatg acaccacagc tgctgccatc 3000
aactggagcc tgtacctcct gggcagcaac cctgaggtgc agaagaaggt ggaccatgag 3060
ctggatgatg tgtttggcaa gtctgacaga cctgccacag tggaagacct gaagaaactc 3120
aggtacctgg aatgtgtgat caaagagacc ctgagactgt tcccaagtgt gcctctgttt 3180
gccagatctg tctctgagga ctgtgaagtg gctggctaca gagtgctgaa gggcacagag 3240
gcagttatca tcccctatgc cctgcacaga gaccccagat acttccccaa ccctgaagag 3300
ttccagcctg agagattctt cccagagaat gcccagggca gacaccctta tgcctatgtg 3360
cccttctcag ctggacctag aaactgcata ggccaaaagt ttgcagtgat ggaagagaag 3420
accatcctga gctgcatcct gaggcacttc tggattgaga gcaaccagaa gagagaggaa 3480
ctgggcctgg aaggacagct gatcctgagg cctagcaatg gcatctggat caagctgaaa 3540
agaagaaatg ctgatgagag gtaaagatct gcctcgactg tgccttctag ttgccagcca 3600
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3660
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3720
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3780
ggggatgcgg tgggctctat ggatccccta actacaagga acccctagtg atggagttgg 3840
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 3900
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3960
a 3961
<210> 14
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 14
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctctcga ggagcttggc ccattgcata cgttgtatcc 180
atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttactagtg 240
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 300
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 360
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 420
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 480
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 540
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 600
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 660
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 720
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 780
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 840
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 900
gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 960
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1020
tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 1080
aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 1140
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 1200
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1260
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1320
gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1380
cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1440
tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1500
ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1560
cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1620
gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1680
cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1740
gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1800
gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1860
gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1920
cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcgcgcc 1980
gccaccatgg ctggcctgtg gctgggcctg gtgtggcaga aactgctgct gtggggagct 2040
gcctctgccc tgtctctggc tggagcctcc cttgtcctgt ccctcctgca aagagttgcc 2100
agctatgcca gaaagtggca gcagatgaga cccatcccca cagtggccag ggcctaccca 2160
ctggtgggcc atgccctgct gatgaagcct gatggcagag aattctttca gcagatcata 2220
gagtacacag aggagtacag acacatgcct ctgctgaagc tgtgggtggg ccctgtgcct 2280
atggtggccc tgtacaatgc tgagaatgtg gaagtgatcc tgaccagcag caagcagatt 2340
gacaagagca gcatgtacaa gttcctggag ccttggctgg gcctgggcct gctgaccagc 2400
acaggcaaca agtggagaag caggagaaag atgctgaccc ccaccttcca cttcaccatc 2460
ctggaagact tcctggacat catgaatgag caggccaaca tcctggtgaa aaagctggaa 2520
aaacacatca accaggaagc cttcaactgc ttcttctaca ttaccctgtg tgccctggac 2580
atcatctgcg agacagccat gggaaaaaac atcggagctc agagcaatga tgacagcgag 2640
tatgtgagag cagtgtaccg gatgagcgaa atgatcttca gacggatcaa gatgccctgg 2700
ctgtggctgg acctgtggta cctcatgttt aaggagggct gggaacacaa gaagagcctg 2760
cagatcctgc acaccttcac aaacagtgtg atcgctgaaa gggccaacga gatgaatgcc 2820
aatgaggatt gcagaggcga tggccgcggc tccgccccta gcaagaacaa gagaagagcc 2880
ttcctggacc tgctgctgtc tgtcaccgat gacgagggca accggctgtc tcatgaggac 2940
atcagagaag aggtggacac cttcatgttt gagggccacg acaccacagc cgccgccatc 3000
aactggagcc tgtacctgct tggcagcaac cctgaggtgc aaaagaaggt ggaccatgag 3060
ctggatgatg tttttggcaa atctgacaga cctgccacag tggaggacct gaagaaactg 3120
agatacctgg agtgtgtgat caaggaaacc ctgagactct tccctagtgt gcctctgttt 3180
gccagatctg tctcagagga ctgtgaggtg gctggctaca gagtgctgaa gggcacagaa 3240
gcagtgatca tcccctatgc cctgcacaga gaccccagat acttccccaa ccctgaggaa 3300
ttccagccag agaggttctt ccctgagaat gcccagggaa gacacccata tgcctatgtg 3360
cctttctctg ctggccccag aaactgcatt ggacagaagt ttgctgtgat ggaggaaaag 3420
accatcctga gctgcatcct gagacacttc tggatagaaa gcaaccagaa gagggaagag 3480
ctgggcctgg aaggccagct gatcctgagg cctagcaatg gcatctggat caaactgaag 3540
agaagaaatg cagatgagag ataaagatct gcctcgactg tgccttctag ttgccagcca 3600
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3660
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3720
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3780
ggggatgcgg tgggctctat ggatccccta actacaagga acccctagtg atggagttgg 3840
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 3900
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3960
a 3961
<210> 15
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 15
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctctcga ggagcttggc ccattgcata cgttgtatcc 180
atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttactagtg 240
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 300
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 360
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 420
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 480
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 540
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 600
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 660
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 720
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 780
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 840
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 900
gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 960
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1020
tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 1080
aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 1140
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 1200
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1260
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1320
gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1380
cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1440
tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1500
ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1560
cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1620
gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1680
cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1740
gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1800
gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1860
gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1920
cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcgcgcc 1980
gccaccatgg ctggcctgtg gctgggcctg gtgtggcaga agctgttgct gtggggggct 2040
gcctctgccc tgagcctggc tggggctagc ctggtgttga gcctgctgca gagagtggct 2100
agctatgcta gaaagtggca gcagatgaga cccatcccca cagtggcaag agcctaccct 2160
ctggtgggcc atgccctgct gatgaagcct gatggcagag agttctttca gcagatcata 2220
gagtacacag aggagtacag acacatgccc ctgctgaaac tgtgggtggg ccctgtgccc 2280
atggtggctc tgtacaatgc tgagaatgtg gaggtgattc tgacaagcag caagcagatt 2340
gacaagagct ccatgtacaa gttcctggag ccctggctgg gcctgggact gctgacaagc 2400
actggcaaca agtggagaag cagaagaaag atgctgaccc ccaccttcca cttcaccatc 2460
ctggaggact tcctggacat tatgaatgag caagccaaca tcctggtgaa aaagctggaa 2520
aagcacatca accaagaggc cttcaactgc ttcttctaca tcaccctgtg tgccctggac 2580
atcatctgtg agacagccat gggcaagaac atcggggccc agagcaatga tgactctgag 2640
tatgtgagag ctgtgtacag aatgtctgag atgatcttca gaagaatcaa gatgccctgg 2700
ctgtggctgg acctgtggta cctgatgttc aaggagggct gggagcacaa aaagagcctg 2760
cagatcctgc acaccttcac caactctgtg atcgccgaga gagccaatga gatgaatgcc 2820
aatgaggact gcagagggga tggcagaggc tctgccccta gcaagaacaa gagaagagcc 2880
ttcctggacc tgttgctgtc tgtgaccgac gatgagggca acagactgag ccatgaggac 2940
atcagagaag aggtggacac atttatgttt gagggccatg acaccacagc tgctgccata 3000
aactggagcc tgtacctgct gggcagcaac cctgaggtgc agaagaaggt ggaccatgag 3060
ctggatgatg tgtttggcaa gtctgacaga cctgccacag tggaggacct gaagaagctg 3120
agatacctgg agtgtgtgat caaggagacc ctgagactgt tcccctctgt gcccctgttt 3180
gctagatctg tgtctgagga ctgtgaggtg gctggctata gagtgctgaa gggcacagag 3240
gctgtgatca tcccctatgc cctgcacaga gaccctagat acttccccaa ccctgaggag 3300
tttcagcctg agagattctt ccctgagaat gcccaaggca gacaccccta tgcctatgtg 3360
ccattctctg ctggcccaag aaactgcatt gggcagaagt ttgctgtgat ggaggagaag 3420
accatcctga gctgcatctt gagacacttc tggattgaga gcaatcagaa gagagaggag 3480
ctgggcctgg aggggcagct gatactgaga ccaagcaatg gcatctggat caagctgaag 3540
agaagaaatg ctgatgagag ataaagatct gcctcgactg tgccttctag ttgccagcca 3600
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3660
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3720
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3780
ggggatgcgg tgggctctat ggatccccta actacaagga acccctagtg atggagttgg 3840
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 3900
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3960
a 3961
<210> 16
<211> 3961
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 16
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctctcga ggagcttggc ccattgcata cgttgtatcc 180
atatcataat atgtacattt atattggctc atgtccaaca ttaccgccat gttactagtg 240
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 300
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 360
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 420
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 480
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 540
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 600
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 660
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 720
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 780
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 840
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 900
gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 960
gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 1020
tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 1080
aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 1140
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 1200
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 1260
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1320
gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1380
cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1440
tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1500
ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1560
cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1620
gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1680
cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1740
gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1800
gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1860
gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1920
cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcggcgcgcc 1980
gccaccatgg cggggctctg gctggggctc gtgtggcaga agctgctgct gtggggcgcg 2040
gcgagtgccc tttccctggc cggcgccagt ctggtcctga gcctgctgca gagggtggcg 2100
agctacgcgc ggaaatggca gcagatgcgg cccatcccca cggtggcccg cgcctaccca 2160
ctggtgggcc acgcgctgct gatgaagccg gacgggcgag aattttttca gcagatcatt 2220
gagtacacag aggaataccg ccacatgccg ctgctgaagc tctgggtcgg gccagtgccc 2280
atggtggccc tttataatgc agaaaatgtg gaggtaattt taactagttc aaagcaaatt 2340
gacaaatcct ctatgtacaa gtttttagaa ccatggcttg gcctaggact tcttacaagt 2400
actggaaaca aatggcgctc caggagaaag atgttaacac ccactttcca ttttaccatt 2460
ctggaagatt tcttagatat catgaatgaa caagcaaata tattggttaa gaaacttgaa 2520
aaacacatta accaagaagc atttaactgc tttttttaca tcactctttg tgccttagat 2580
atcatctgtg aaacagctat ggggaagaat attggtgctc aaagtaatga tgattccgag 2640
tatgtccgtg cagtttatag aatgagtgag atgatatttc gaagaataaa gatgccctgg 2700
ctttggcttg atctctggta ccttatgttt aaagaaggat gggaacacaa aaagagcctt 2760
cagatcctac atacttttac caacagtgtc atcgctgaac gggccaatga aatgaacgcc 2820
aatgaagact gtagaggtga tggcaggggc tctgccccct ccaaaaataa acgcagggcc 2880
tttcttgact tgcttttaag tgtgactgat gacgaaggga acaggctaag tcatgaagat 2940
attcgagaag aagttgacac cttcatgttt gaggggcacg atacaactgc agctgcaata 3000
aactggtcct tatacctgtt gggttctaac ccagaagtcc agaaaaaagt ggatcatgaa 3060
ttggatgacg tgtttgggaa gtctgaccgt cccgctacag tagaagacct gaagaaactt 3120
cggtatctgg aatgtgttat taaggagacc cttcgccttt ttccttctgt tcctttattt 3180
gcccgtagtg ttagtgaaga ttgtgaagtg gcaggttaca gagttctaaa aggcactgaa 3240
gccgtcatca ttccctatgc attgcacaga gatccgagat acttccccaa ccccgaggag 3300
ttccagcctg agcggttctt ccccgagaat gcacaagggc gccatccata tgcctacgtg 3360
cccttctctg ctggccccag gaactgtata ggtcaaaagt ttgctgtgat ggaagaaaag 3420
accattcttt cgtgcatcct gaggcacttt tggatagaat ccaaccagaa aagagaagag 3480
cttggtctag aaggacagtt gattcttcgt ccaagtaatg gcatctggat caagttgaag 3540
aggagaaatg cagatgaacg ctaaagatct gcctcgactg tgccttctag ttgccagcca 3600
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3660
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3720
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3780
ggggatgcgg tgggctctat ggatccccta actacaagga acccctagtg atggagttgg 3840
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 3900
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3960
a 3961
<210> 17
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 17
gccacc 6
<210> 18
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 18
agtatgtccg tgcagtttat agg 23
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 19
catacaggtc atcgctgaac ggg 23
<210> 20
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 20
tcatacaggt catcgctgaa cgg 23
<210> 21
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 21
gattatcatt caaatcatac agg 23
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 22
gaaatcacac tccaccggga 20
<210> 23
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 23
acctttactg cttaaacaca tgct 24
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 24
caggcagcag aaatcgcaag 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 25
agcctgttcc cttcgtcatc 20
<210> 26
<211> 102
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 26
taactagggt gcatccaagt ccaaacagaa gcatgtgatt atcattcaaa gcgaacgggc 60
caatgaaatg aacgccaatg aagactgtag aggtgatggc ag 102
<210> 27
<211> 525
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成蛋白
<400> 27
Met Ala Gly Leu Trp Leu Gly Leu Val Trp Gln Lys Leu Leu Leu Trp
1 5 10 15
Gly Ala Ala Ser Ala Leu Ser Leu Ala Gly Ala Ser Leu Val Leu Ser
20 25 30
Leu Leu Gln Arg Val Ala Ser Tyr Ala Arg Lys Trp Gln Gln Met Arg
35 40 45
Pro Ile Pro Thr Val Ala Arg Ala Tyr Pro Leu Val Gly His Ala Leu
50 55 60
Leu Met Lys Pro Asp Gly Arg Glu Phe Phe Gln Gln Ile Ile Glu Tyr
65 70 75 80
Thr Glu Glu Tyr Arg His Met Pro Leu Leu Lys Leu Trp Val Gly Pro
85 90 95
Val Pro Met Val Ala Leu Tyr Asn Ala Glu Asn Val Glu Val Ile Leu
100 105 110
Thr Ser Ser Lys Gln Ile Asp Lys Ser Ser Met Tyr Lys Phe Leu Glu
115 120 125
Pro Trp Leu Gly Leu Gly Leu Leu Thr Ser Thr Gly Asn Lys Trp Arg
130 135 140
Ser Arg Arg Lys Met Leu Thr Pro Thr Phe His Phe Thr Ile Leu Glu
145 150 155 160
Asp Phe Leu Asp Ile Met Asn Glu Gln Ala Asn Ile Leu Val Lys Lys
165 170 175
Leu Glu Lys His Ile Asn Gln Glu Ala Phe Asn Cys Phe Phe Tyr Ile
180 185 190
Thr Leu Cys Ala Leu Asp Ile Ile Cys Glu Thr Ala Met Gly Lys Asn
195 200 205
Ile Gly Ala Gln Ser Asn Asp Asp Ser Glu Tyr Val Arg Ala Val Tyr
210 215 220
Arg Met Ser Glu Met Ile Phe Arg Arg Ile Lys Met Pro Trp Leu Trp
225 230 235 240
Leu Asp Leu Trp Tyr Leu Met Phe Lys Glu Gly Trp Glu His Lys Lys
245 250 255
Ser Leu Gln Ile Leu His Thr Phe Thr Asn Ser Val Ile Ala Glu Arg
260 265 270
Ala Asn Glu Met Asn Ala Asn Glu Asp Cys Arg Gly Asp Gly Arg Gly
275 280 285
Ser Ala Pro Ser Lys Asn Lys Arg Arg Ala Phe Leu Asp Leu Leu Leu
290 295 300
Ser Val Thr Asp Asp Glu Gly Asn Arg Leu Ser His Glu Asp Ile Arg
305 310 315 320
Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp Thr Thr Ala Ala
325 330 335
Ala Ile Asn Trp Ser Leu Tyr Leu Leu Gly Ser Asn Pro Glu Val Gln
340 345 350
Lys Lys Val Asp His Glu Leu Asp Asp Val Phe Gly Lys Ser Asp Arg
355 360 365
Pro Ala Thr Val Glu Asp Leu Lys Lys Leu Arg Tyr Leu Glu Cys Val
370 375 380
Ile Lys Glu Thr Leu Arg Leu Phe Pro Ser Val Pro Leu Phe Ala Arg
385 390 395 400
Ser Val Ser Glu Asp Cys Glu Val Ala Gly Tyr Arg Val Leu Lys Gly
405 410 415
Thr Glu Ala Val Ile Ile Pro Tyr Ala Leu His Arg Asp Pro Arg Tyr
420 425 430
Phe Pro Asn Pro Glu Glu Phe Gln Pro Glu Arg Phe Phe Pro Glu Asn
435 440 445
Ala Gln Gly Arg His Pro Tyr Ala Tyr Val Pro Phe Ser Ala Gly Pro
450 455 460
Arg Asn Cys Ile Gly Gln Lys Phe Ala Val Met Glu Glu Lys Thr Ile
465 470 475 480
Leu Ser Cys Ile Leu Arg His Phe Trp Ile Glu Ser Asn Gln Lys Arg
485 490 495
Glu Glu Leu Gly Leu Glu Gly Gln Leu Ile Leu Arg Pro Ser Asn Gly
500 505 510
Ile Trp Ile Lys Leu Lys Arg Arg Asn Ala Asp Glu Arg
515 520 525
<210> 28
<211> 1575
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 合成核酸
<400> 28
atggcggggc tctggctggg gctcgtgtgg cagaagctgc tgctgtgggg cgcggcgagt 60
gccctttccc tggccggcgc cagtctggtc ctgagcctgc tgcagagggt ggcgagctac 120
gcgcggaaat ggcagcagat gcggcccatc cccacggtgg cccgcgccta cccactggtg 180
ggccacgcgc tgctgatgaa gccggacggg cgagaatttt ttcagcagat cattgagtac 240
acagaggaat accgccacat gccgctgctg aagctctggg tcgggccagt gcccatggtg 300
gccctttata atgcagaaaa tgtggaggta attttaacta gttcaaagca aattgacaaa 360
tcctctatgt acaagttttt agaaccatgg cttggcctag gacttcttac aagtactgga 420
aacaaatggc gctccaggag aaagatgtta acacccactt tccattttac cattctggaa 480
gatttcttag atatcatgaa tgaacaagca aatatattgg ttaagaaact tgaaaaacac 540
attaaccaag aagcatttaa ctgctttttt tacatcactc tttgtgcctt agatatcatc 600
tgtgaaacag ctatggggaa gaatattggt gctcaaagta atgatgattc cgagtatgtc 660
cgtgcagttt atagaatgag tgagatgata tttcgaagaa taaagatgcc ctggctttgg 720
cttgatctct ggtaccttat gtttaaagaa ggatgggaac acaaaaagag ccttcagatc 780
ctacatactt ttaccaacag tgtcatcgct gaacgggcca atgaaatgaa cgccaatgaa 840
gactgtagag gtgatggcag gggctctgcc ccctccaaaa ataaacgcag ggcctttctt 900
gacttgcttt taagtgtgac tgatgacgaa gggaacaggc taagtcatga agatattcga 960
gaagaagttg acaccttcat gtttgagggg cacgatacaa ctgcagctgc aataaactgg 1020
tccttatacc tgttgggttc taacccagaa gtccagaaaa aagtggatca tgaattggat 1080
gacgtgtttg ggaagtctga ccgtcccgct acagtagaag acctgaagaa acttcggtat 1140
ctggaatgtg ttattaagga gacccttcgc ctttttcctt ctgttccttt atttgcccgt 1200
agtgttagtg aagattgtga agtggcaggt tacagagttc taaaaggcac tgaagccgtc 1260
atcattccct atgcattgca cagagatccg agatacttcc ccaaccccga ggagttccag 1320
cctgagcggt tcttccccga gaatgcacaa gggcgccatc catatgccta cgtgcccttc 1380
tctgctggcc ccaggaactg tataggtcaa aagtttgctg tgatggaaga aaagaccatt 1440
ctttcgtgca tcctgaggca cttttggata gaatccaacc agaaaagaga agagcttggt 1500
ctagaaggac agttgattct tcgtccaagt aatggcatct ggatcaagtt gaagaggaga 1560
aatgcagatg aacgc 1575
Claims (10)
1.一种编码CYP4V2蛋白的多核苷酸,其包含与SEQ ID NO 5、SEQ ID NO 6、SEQ ID NO7、SEQ ID NO 8或SEQ ID NO 9所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;
更优选地,所述多核苷酸如SEQ ID NO 5、SEQ ID NO 6、SEQ ID NO 7、SEQ ID NO 8或SEQ ID NO 9所示;
更优选地,所述多核苷酸如SEQ ID NO 5所示。
2.一种表达盒,其包含权利要求1所述的多核苷酸以及与所述多核苷酸可操作连接的启动子。
3.一种表达载体,其包含权利要求1所述的多核苷酸或权利要求2所述的表达盒。
4.权利要求3所述的表达载体,其还包含表达控制元件,所述编码CYP4V2蛋白的多核苷酸与所述表达控制元件可操作地连接;
优选地,所述表达控制元件选自转录/翻译控制信号、复制起点、启动子、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和/或蛋白质的稳定性的元件中的一种或多种;
优选地,所述表达载体包含复制起点;优选地,所述复制起点序列选自f1噬菌体ori、RK2oriV、pUC ori以及pSC101ori;
优选地,所述表达载体还包含5’ITR;优选地,所述5’ITR包含与SEQ ID NO 1所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 1所示;
优选地,所述表达载体还包含3’ITR;优选地,所述3’ITR包含与SEQ ID NO 11所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 11所示;
优选地,所述表达载体还包含增强子;优选地,所述增强子为CMV增强子;更优选地,所述增强子包含与SEQ ID NO 2所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 2所示;
优选地,所述表达载体还包含启动子;优选地,所述启动子为特异性或非特异性启动子;优选地,所述启动子选自CBA启动子、CMV启动子、SV40启动子、hPGK启动子和TRE3GS启动子;更优选地,所述启动子为CBA启动子,优选地,所述CBA启动子包含与SEQ ID NO 3所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 3所示;优选地,所述启动子是可诱导启动子优选地,所述可诱导系统包含四环素调控启动子、醇调控启动子、类固醇调控启动子、金属调控启动子、致病调控启动子、温度/热诱导型启动子和光调控启动子、IPTG诱导型系统中的一种或多种;优选地,所述四环素调控启动子选自Tet on启动子、Tet off的启动子和TetActivator启动子;优选地,所述醇调控启动子选自醇脱氢酶I(alcA)基因启动子、响应于醇反式激活蛋白(AlcR)的启动子;优选地,所述类固醇调控启动子选自大鼠糖皮质激素受体启动子、人雌激素受体启动子、蛾蜕皮激素受体启动子,类视黄醇启动子和甲状腺受体超家族启动子;优选地,所述金属调控启动子选自酵母、小鼠和人的金属硫蛋白启动子;优选地,所述致病调控启动子选自由水杨酸调控启动子、乙烯调控启动子和苯并噻二唑调控(BTH)启动子;优选地,所述温度/热诱导型启动子选自HSP-70启动、HSP-90启动和大豆热激启动子;优选地,所述光调控启动子是植物细胞的光应答型启动子;
优选地,所述表达载体还包含外显子和内含子;优选地,所述外显子和内含子为鸡β-actin基因的第一个外显子和第一个内含子;优选地,所述外显子和内含子包含与SEQ IDNO 4所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 4所示;
优选地,所述表达载体还包含polyA信号;优选地,所述polyA信号为牛生长激素poly A(BGH poly A)、短poly A、SV40 polyA、和/或人β珠蛋白poly A;优选地,所述polyA信号包含与SEQ ID NO 10所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 10所示;
优选地,所述表达载体包含与SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO15或SEQ ID NO 16所示核苷酸序列具有90%或以上同一性的核苷酸序列,优选具有91%、92%、93%、94%、95%、96%、97%、98%、99%以上同一性的核苷酸列,更优选具有98%或99%以上同一性的核苷酸序列;更优选地,所述多核苷酸如SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15或SEQ ID NO 16所示;
优选地,所述载体包含转录后调节元件;优选地,所述转录后调节元件为土拨鼠肝炎病毒转录后调节元件(WPRE);
优选地,所述载体还包含编码标志物基因,优选地,所述标志物选自抗生素抗性蛋白质、毒素抗性蛋白质、有色的或荧光的或发光的蛋白质和介导增强的细胞生长和/或基因扩增的蛋白质中的一种或多种;
优选地,所述抗生素选自氨苄青霉素、新霉素、G418、嘌呤霉素和杀稻瘟素;
优选地,所述毒素选自炭疽毒素和白喉毒素;
优选地,所述有色的或荧光的或发光的蛋白质选自绿色荧光蛋白、增强型绿色荧光蛋白、红色荧光蛋白和萤光素酶;优选地,所述介导增强的细胞生长和/或基因扩增的蛋白质为二氢叶酸还原酶(DHFR)。
5.权利要求4所述的表达载体,所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子;
优选地,所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603和pCMV;
优选地,所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如,腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如,流感病毒)、弹状病毒(例如,狂犬病和水疱性口炎病毒)、副粘病毒(例如,麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒,和双链DNA病毒,所述双链DNA病毒包括腺病毒、疱疹病毒(例如,单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如,牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒;
优选地,所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒和泡沫病毒;
优选地,所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV和绵羊脱髓鞘性脑白质炎慢病毒。
6.权利要求3-5任一项所述的表达载体,其为腺相关病毒;
优选地,所述腺相关病毒选自AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV 9型、AAV 10型、禽AAV、牛AAV、犬AAV、马AAV和绵羊AAV等;
优选地,所述表达载体还包含缩短的嵌合内含子和Kozak起始序列;
优选地,所述缩短的嵌合内含子如SEQ ID NO 4所示;
优选地,所述Kozak起始序列如SEQ ID NO 17所示。
7.一种病毒颗粒,其包含权利要求1所述的多核苷酸、权利要求2所述的表达盒和/或权利要求3-6任一项所述的表达载体。
8.一种治疗BCD的药物组合物,其包含权利要求1所述的多核苷酸、权利要求2所述的表达盒、权利要求3-6任一所述的表达载体和/或权利要求7所述的病毒颗粒,以及药学上可接受的载体,其中,所述的药物组合物表达野生型或密码子优化的CYP4V2蛋白。
9.权利要求1所述的多核苷酸、权利要求2所述的表达盒、权利要求3-6任一所述的表达载体、权利要求7所述的病毒颗粒和/或权利要求8所述的药物组合物在制备治疗Bietti结晶性营养不良(BCD)中的用途。
10.构建带有CYP4V2突变的BCD细胞模型的方法,其包括以下步骤:
(1)设计针对CYP4V2基因的sgRNA;
(2)将步骤(1)获得的所述sgRNA构建至Cas9-sgRNA载体,获得Cas9-sgRNA质粒;
(3)将步骤(2)获得的所述Cas9-sgRNA质粒导入细胞,获得具有CYP4V2基因突变的细胞;
优选地,所述方法还包括步骤(4)筛选具有CYP4V2基因突变的细胞;
优选地,所述方法还包括步骤(5)对所述CYP4V2基因突变细胞进行鉴定;
优选地,所述sgRNA选自SEQ ID NO 18-21中的一种或多种;
优选地,所述鉴定选自细胞增殖鉴定、自噬变化鉴定及脂肪颗粒沉积鉴定。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111580873.0A CN114381465B (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
CN202311813311.5A CN118028318A (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
KR1020247024292A KR20240116854A (ko) | 2021-12-22 | 2022-12-21 | 최적화된 cyp4v2 유전자 및 이의 응용 |
PCT/CN2022/140586 WO2023116745A1 (zh) | 2021-12-22 | 2022-12-21 | 优化的cyp4v2基因及其用途 |
TW111149339A TW202328454A (zh) | 2021-12-22 | 2022-12-22 | 優化的cyp4v2基因及其用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111580873.0A CN114381465B (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311813311.5A Division CN118028318A (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114381465A true CN114381465A (zh) | 2022-04-22 |
CN114381465B CN114381465B (zh) | 2024-01-16 |
Family
ID=81198874
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111580873.0A Active CN114381465B (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
CN202311813311.5A Pending CN118028318A (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311813311.5A Pending CN118028318A (zh) | 2021-12-22 | 2021-12-22 | 优化的cyp4v2基因及其用途 |
Country Status (4)
Country | Link |
---|---|
KR (1) | KR20240116854A (zh) |
CN (2) | CN114381465B (zh) |
TW (1) | TW202328454A (zh) |
WO (1) | WO2023116745A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023116745A1 (zh) * | 2021-12-22 | 2023-06-29 | 苏州诺洁贝生物技术有限公司 | 优化的cyp4v2基因及其用途 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2024524707A (ja) * | 2021-07-15 | 2024-07-05 | シャンハイ ヴァイタルゲン バイオファーマ カンパニー リミテッド | ビエッティ結晶性ジストロフィー治療のための遺伝子組換アデノ随伴ウイルスベクター |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020117898A1 (en) * | 2018-12-05 | 2020-06-11 | Abeona Therapeutics Inc. | Recombinant adeno-associated viral vector for gene delivery |
US20200255859A1 (en) * | 2017-07-31 | 2020-08-13 | Reflection Biotechnologies Limited | Cellular models of and therapies for ocular diseases |
WO2020174368A1 (en) * | 2019-02-25 | 2020-09-03 | Novartis Ag | Compositions and methods to treat bietti crystalline dystrophy |
WO2020174369A2 (en) * | 2019-02-25 | 2020-09-03 | Novartis Ag | Compositions and methods to treat bietti crystalline dystrophy |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114381465B (zh) * | 2021-12-22 | 2024-01-16 | 苏州诺洁贝生物技术有限公司 | 优化的cyp4v2基因及其用途 |
-
2021
- 2021-12-22 CN CN202111580873.0A patent/CN114381465B/zh active Active
- 2021-12-22 CN CN202311813311.5A patent/CN118028318A/zh active Pending
-
2022
- 2022-12-21 KR KR1020247024292A patent/KR20240116854A/ko unknown
- 2022-12-21 WO PCT/CN2022/140586 patent/WO2023116745A1/zh unknown
- 2022-12-22 TW TW111149339A patent/TW202328454A/zh unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20200255859A1 (en) * | 2017-07-31 | 2020-08-13 | Reflection Biotechnologies Limited | Cellular models of and therapies for ocular diseases |
CN111630170A (zh) * | 2017-07-31 | 2020-09-04 | 映像生物有限公司 | 眼部疾病的细胞模型及用于眼部疾病的疗法 |
WO2020117898A1 (en) * | 2018-12-05 | 2020-06-11 | Abeona Therapeutics Inc. | Recombinant adeno-associated viral vector for gene delivery |
WO2020174368A1 (en) * | 2019-02-25 | 2020-09-03 | Novartis Ag | Compositions and methods to treat bietti crystalline dystrophy |
WO2020174369A2 (en) * | 2019-02-25 | 2020-09-03 | Novartis Ag | Compositions and methods to treat bietti crystalline dystrophy |
CN113474461A (zh) * | 2019-02-25 | 2021-10-01 | 诺华股份有限公司 | 治疗bietti晶体营养不良的组合物和方法 |
CN113677801A (zh) * | 2019-02-25 | 2021-11-19 | 诺华股份有限公司 | 治疗bietti晶体营养不良的组合物和方法 |
Non-Patent Citations (5)
Title |
---|
HATA M 等: "Reduction of lipid accumulation rescues Bietti\'s crystalline dystrophy phenotypes", vol. 115, no. 15, pages 4 - 5 * |
QU B 等: "Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy", vol. 27, no. 7, pages 370 - 382, XP037223533, DOI: 10.1038/s41434-020-0159-3 * |
WANG J H 等: "Validation of in vitro gene therapy for Bietti crystalline dystrophy", vol. 60, no. 9, pages 3420 - 3420 * |
许可 等: "携带CYP4V2基因突变的结晶样视网膜色素变性患者的表型特征", vol. 29, no. 2, pages 93 - 97 * |
贾睿璇 等: "Cyp4v3 基因敲除小鼠模型的表型分析", vol. 53, no. 6, pages 1101 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023116745A1 (zh) * | 2021-12-22 | 2023-06-29 | 苏州诺洁贝生物技术有限公司 | 优化的cyp4v2基因及其用途 |
Also Published As
Publication number | Publication date |
---|---|
TW202328454A (zh) | 2023-07-16 |
KR20240116854A (ko) | 2024-07-30 |
CN114381465B (zh) | 2024-01-16 |
WO2023116745A1 (zh) | 2023-06-29 |
CN118028318A (zh) | 2024-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108350467B (zh) | 基因构建体 | |
TWI702955B (zh) | 使用腺相關病毒(aav)sflt-1治療老年性黃斑部退化(amd) | |
JP6290185B2 (ja) | 網膜形成不全を治療するためのウイルスベクター | |
US20220040333A1 (en) | Use of neuroglobin agonist for preventing or treating mitochondrial RCCI and/or RCCIII deficiency disease | |
AU2016370487C1 (en) | Gene therapy for ocular disorders | |
WO2023116745A1 (zh) | 优化的cyp4v2基因及其用途 | |
CA2543189A1 (en) | In vivo gene therapy of parkinson's disease | |
JP2023116709A (ja) | 眼疾患のための遺伝子療法 | |
KR20230044522A (ko) | 척수성 근위축을 치료하기 위한 핵산 구축물 및 이의 용도 | |
EP4165193A2 (en) | Aav-mediated gene transfer for retinopathy | |
US20210277417A1 (en) | Methods of treating clrn1-associated hearing loss and/or vision loss | |
US20230398235A1 (en) | Variant txnip compositions and methods of use thereof for the treatment of degenerative ocular diseases | |
Bellingrath | Optimising Gene Therapy for X-linked Retinitis Pigmentosa | |
CN117337331A (zh) | 用于增强视觉功能的组合物和方法 | |
JP2024522072A (ja) | 視覚機能を増強するための組成物及び方法 | |
KR20220152550A (ko) | Nmnat1-연관 망막 변성의 유전자 치료법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |