CN115232811A - Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application - Google Patents
Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application Download PDFInfo
- Publication number
- CN115232811A CN115232811A CN202110718541.8A CN202110718541A CN115232811A CN 115232811 A CN115232811 A CN 115232811A CN 202110718541 A CN202110718541 A CN 202110718541A CN 115232811 A CN115232811 A CN 115232811A
- Authority
- CN
- China
- Prior art keywords
- hbb
- protein
- lys
- leu
- sickle cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000007056 sickle cell anemia Diseases 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 21
- 101150013707 HBB gene Proteins 0.000 title abstract description 21
- 238000010449 nuclear transplantation Methods 0.000 title abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 175
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 154
- 239000002773 nucleotide Substances 0.000 claims abstract description 68
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 68
- 108091033409 CRISPR Proteins 0.000 claims abstract description 42
- 241000282887 Suidae Species 0.000 claims abstract description 23
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 19
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 19
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 210000000056 organ Anatomy 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 12
- 238000013334 tissue model Methods 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 8
- 230000008506 pathogenesis Effects 0.000 claims abstract description 7
- 239000013612 plasmid Substances 0.000 claims description 78
- 108020004414 DNA Proteins 0.000 claims description 64
- 102000053602 DNA Human genes 0.000 claims description 36
- 108010077850 Nuclear Localization Signals Proteins 0.000 claims description 30
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 21
- 108010013369 Enteropeptidase Proteins 0.000 claims description 19
- 102100029727 Enteropeptidase Human genes 0.000 claims description 15
- 241001052560 Thallis Species 0.000 claims description 11
- 238000000746 purification Methods 0.000 claims description 11
- 108091005804 Peptidases Proteins 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 10
- 238000001042 affinity chromatography Methods 0.000 claims description 9
- 238000005520 cutting process Methods 0.000 claims description 9
- 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 9
- 108010006519 Molecular Chaperones Proteins 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 239000012460 protein solution Substances 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- 102000005431 Molecular Chaperones Human genes 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 230000007017 scission Effects 0.000 claims description 7
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 5
- 101150038500 cas9 gene Proteins 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 235000019750 Crude protein Nutrition 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 3
- 238000002659 cell therapy Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 230000000857 drug effect Effects 0.000 claims description 3
- 238000001415 gene therapy Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 4
- 206010064571 Gene mutation Diseases 0.000 abstract description 6
- 235000018102 proteins Nutrition 0.000 description 96
- 210000004027 cell Anatomy 0.000 description 83
- 241000282898 Sus scrofa Species 0.000 description 50
- 210000002950 fibroblast Anatomy 0.000 description 48
- 230000035772 mutation Effects 0.000 description 41
- 238000013518 transcription Methods 0.000 description 32
- 230000035897 transcription Effects 0.000 description 32
- 238000012163 sequencing technique Methods 0.000 description 29
- 239000000243 solution Substances 0.000 description 27
- 230000014509 gene expression Effects 0.000 description 18
- 238000010362 genome editing Methods 0.000 description 18
- 108020005004 Guide RNA Proteins 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- 108091026890 Coding region Proteins 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- 238000001962 electrophoresis Methods 0.000 description 13
- 230000002441 reversible effect Effects 0.000 description 13
- 108010092854 aspartyllysine Proteins 0.000 description 12
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 229910052754 neon Inorganic materials 0.000 description 10
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 108020004705 Codon Proteins 0.000 description 9
- 238000004520 electroporation Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 8
- 210000000349 chromosome Anatomy 0.000 description 8
- 238000012217 deletion Methods 0.000 description 8
- 230000037430 deletion Effects 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 108010054155 lysyllysine Proteins 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 108010054147 Hemoglobins Proteins 0.000 description 7
- 102000001554 Hemoglobins Human genes 0.000 description 7
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 108010034529 leucyl-lysine Proteins 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 6
- 241000282412 Homo Species 0.000 description 6
- 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 6
- 238000012408 PCR amplification Methods 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 101100394333 Sus scrofa HBB gene Proteins 0.000 description 6
- 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 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 108010038633 aspartylglutamate Proteins 0.000 description 6
- 238000012761 co-transfection Methods 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 6
- 108010050848 glycylleucine Proteins 0.000 description 6
- 238000002744 homologous recombination Methods 0.000 description 6
- 230000006801 homologous recombination Effects 0.000 description 6
- 108010017391 lysylvaline Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 235000019419 proteases Nutrition 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241000288906 Primates Species 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 108010005233 alanylglutamic acid Proteins 0.000 description 5
- 108010013835 arginine glutamate Proteins 0.000 description 5
- 108010008355 arginyl-glutamine Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 108010057821 leucylproline Proteins 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000008055 phosphate buffer solution Substances 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
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 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 4
- 101001012262 Bos taurus Enteropeptidase Proteins 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 4
- 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 4
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 4
- CMNMPCTVCWWYHY-MXAVVETBSA-N Ile-His-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(C)C)C(=O)O)N CMNMPCTVCWWYHY-MXAVVETBSA-N 0.000 description 4
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 4
- IZPVWNSAVUQBGP-CIUDSAMLSA-N Leu-Ser-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IZPVWNSAVUQBGP-CIUDSAMLSA-N 0.000 description 4
- WVJNGSFKBKOKRV-AJNGGQMLSA-N Lys-Leu-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVJNGSFKBKOKRV-AJNGGQMLSA-N 0.000 description 4
- 108010047562 NGR peptide Proteins 0.000 description 4
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 108010062796 arginyllysine Proteins 0.000 description 4
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000011536 extraction buffer Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000012224 gene deletion Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 210000001322 periplasm Anatomy 0.000 description 4
- 101150009573 phoA gene Proteins 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 108010073969 valyllysine Proteins 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 3
- BTJVOUQWFXABOI-IHRRRGAJSA-N Arg-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCNC(N)=N BTJVOUQWFXABOI-IHRRRGAJSA-N 0.000 description 3
- IZSMEUDYADKZTJ-KJEVXHAQSA-N Arg-Tyr-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IZSMEUDYADKZTJ-KJEVXHAQSA-N 0.000 description 3
- OPEPUCYIGFEGSW-WDSKDSINSA-N Asn-Gly-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OPEPUCYIGFEGSW-WDSKDSINSA-N 0.000 description 3
- NVFSJIXJZCDICF-SRVKXCTJSA-N Asp-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N NVFSJIXJZCDICF-SRVKXCTJSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PJBVXVBTTFZPHJ-GUBZILKMSA-N Glu-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N PJBVXVBTTFZPHJ-GUBZILKMSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 3
- YRRCOJOXAJNSAX-IHRRRGAJSA-N Leu-Pro-Lys Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N YRRCOJOXAJNSAX-IHRRRGAJSA-N 0.000 description 3
- VJGQRELPQWNURN-JYJNAYRXSA-N Leu-Tyr-Glu 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](CCC(O)=O)C(O)=O VJGQRELPQWNURN-JYJNAYRXSA-N 0.000 description 3
- 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 3
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- KYYMILWEGJYPQZ-IHRRRGAJSA-N Phe-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KYYMILWEGJYPQZ-IHRRRGAJSA-N 0.000 description 3
- WPTYDQPGBMDUBI-QWRGUYRKSA-N Phe-Gly-Asn Chemical compound N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O WPTYDQPGBMDUBI-QWRGUYRKSA-N 0.000 description 3
- LRBSWBVUCLLRLU-BZSNNMDCSA-N Phe-Leu-Lys Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(O)=O LRBSWBVUCLLRLU-BZSNNMDCSA-N 0.000 description 3
- DSGYZICNAMEJOC-AVGNSLFASA-N Ser-Glu-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DSGYZICNAMEJOC-AVGNSLFASA-N 0.000 description 3
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 3
- 102000002933 Thioredoxin Human genes 0.000 description 3
- VRUFCJZQDACGLH-UVOCVTCTSA-N Thr-Leu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VRUFCJZQDACGLH-UVOCVTCTSA-N 0.000 description 3
- VLOYGOZDPGYWFO-LAEOZQHASA-N Val-Asp-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VLOYGOZDPGYWFO-LAEOZQHASA-N 0.000 description 3
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 3
- JVGHIFMSFBZDHH-WPRPVWTQSA-N Val-Met-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)O)N JVGHIFMSFBZDHH-WPRPVWTQSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 3
- 108010044940 alanylglutamine Proteins 0.000 description 3
- 108010070944 alanylhistidine Proteins 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 108010003700 lysyl aspartic acid Proteins 0.000 description 3
- 108010064235 lysylglycine Proteins 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108010012581 phenylalanylglutamate Proteins 0.000 description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 description 3
- 108010071207 serylmethionine Proteins 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108060008226 thioredoxin Proteins 0.000 description 3
- 229940094937 thioredoxin Drugs 0.000 description 3
- 108010051110 tyrosyl-lysine Proteins 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- MVBWLRJESQOQTM-ACZMJKKPSA-N Ala-Gln-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O MVBWLRJESQOQTM-ACZMJKKPSA-N 0.000 description 2
- TZDNWXDLYFIFPT-BJDJZHNGSA-N Ala-Ile-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O TZDNWXDLYFIFPT-BJDJZHNGSA-N 0.000 description 2
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 2
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 2
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 2
- WEZNQZHACPSMEF-QEJZJMRPSA-N Ala-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 WEZNQZHACPSMEF-QEJZJMRPSA-N 0.000 description 2
- VYMJAWXRWHJIMS-LKTVYLICSA-N Ala-Tyr-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N VYMJAWXRWHJIMS-LKTVYLICSA-N 0.000 description 2
- MUXONAMCEUBVGA-DCAQKATOSA-N Arg-Arg-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(O)=O MUXONAMCEUBVGA-DCAQKATOSA-N 0.000 description 2
- VNFWDYWTSHFRRG-SRVKXCTJSA-N Arg-Gln-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O VNFWDYWTSHFRRG-SRVKXCTJSA-N 0.000 description 2
- UZGFHWIJWPUPOH-IHRRRGAJSA-N Arg-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UZGFHWIJWPUPOH-IHRRRGAJSA-N 0.000 description 2
- FSNVAJOPUDVQAR-AVGNSLFASA-N Arg-Lys-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FSNVAJOPUDVQAR-AVGNSLFASA-N 0.000 description 2
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 2
- KSUALAGYYLQSHJ-RCWTZXSCSA-N Arg-Met-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSUALAGYYLQSHJ-RCWTZXSCSA-N 0.000 description 2
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 2
- BDMIFVIWCNLDCT-CIUDSAMLSA-N Asn-Arg-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O BDMIFVIWCNLDCT-CIUDSAMLSA-N 0.000 description 2
- OLGCWMNDJTWQAG-GUBZILKMSA-N Asn-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(N)=O OLGCWMNDJTWQAG-GUBZILKMSA-N 0.000 description 2
- DDPXDCKYWDGZAL-BQBZGAKWSA-N Asn-Gly-Arg Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N DDPXDCKYWDGZAL-BQBZGAKWSA-N 0.000 description 2
- BZWRLDPIWKOVKB-ZPFDUUQYSA-N Asn-Leu-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BZWRLDPIWKOVKB-ZPFDUUQYSA-N 0.000 description 2
- OOXUBGLNDRGOKT-FXQIFTODSA-N Asn-Ser-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OOXUBGLNDRGOKT-FXQIFTODSA-N 0.000 description 2
- VWADICJNCPFKJS-ZLUOBGJFSA-N Asn-Ser-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O VWADICJNCPFKJS-ZLUOBGJFSA-N 0.000 description 2
- 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 2
- UGKZHCBLMLSANF-CIUDSAMLSA-N Asp-Asn-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UGKZHCBLMLSANF-CIUDSAMLSA-N 0.000 description 2
- KHGPWGKPYHPOIK-QWRGUYRKSA-N Asp-Gly-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KHGPWGKPYHPOIK-QWRGUYRKSA-N 0.000 description 2
- QSFHZPQUAAQHAQ-CIUDSAMLSA-N Asp-Ser-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O QSFHZPQUAAQHAQ-CIUDSAMLSA-N 0.000 description 2
- WAEDSQFVZJUHLI-BYULHYEWSA-N Asp-Val-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WAEDSQFVZJUHLI-BYULHYEWSA-N 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- LHJDLVVQRJIURS-SRVKXCTJSA-N Cys-Phe-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CS)N LHJDLVVQRJIURS-SRVKXCTJSA-N 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- JFOKLAPFYCTNHW-SRVKXCTJSA-N Gln-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N JFOKLAPFYCTNHW-SRVKXCTJSA-N 0.000 description 2
- KVXVVDFOZNYYKZ-DCAQKATOSA-N Gln-Gln-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O KVXVVDFOZNYYKZ-DCAQKATOSA-N 0.000 description 2
- FTIJVMLAGRAYMJ-MNXVOIDGSA-N Gln-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(N)=O FTIJVMLAGRAYMJ-MNXVOIDGSA-N 0.000 description 2
- ZNTDJIMJKNNSLR-RWRJDSDZSA-N Gln-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZNTDJIMJKNNSLR-RWRJDSDZSA-N 0.000 description 2
- SHAUZYVSXAMYAZ-JYJNAYRXSA-N Gln-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N SHAUZYVSXAMYAZ-JYJNAYRXSA-N 0.000 description 2
- IOFDDSNZJDIGPB-GVXVVHGQSA-N Gln-Leu-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IOFDDSNZJDIGPB-GVXVVHGQSA-N 0.000 description 2
- XKPACHRGOWQHFH-IRIUXVKKSA-N Gln-Thr-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XKPACHRGOWQHFH-IRIUXVKKSA-N 0.000 description 2
- AFODTOLGSZQDSL-PEFMBERDSA-N Glu-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N AFODTOLGSZQDSL-PEFMBERDSA-N 0.000 description 2
- 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 2
- PAQUJCSYVIBPLC-AVGNSLFASA-N Glu-Asp-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PAQUJCSYVIBPLC-AVGNSLFASA-N 0.000 description 2
- QQLBPVKLJBAXBS-FXQIFTODSA-N Glu-Glu-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O QQLBPVKLJBAXBS-FXQIFTODSA-N 0.000 description 2
- VGOFRWOTSXVPAU-SDDRHHMPSA-N Glu-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CCC(=O)O)N)C(=O)O VGOFRWOTSXVPAU-SDDRHHMPSA-N 0.000 description 2
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 2
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 2
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 2
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 2
- HFXJIZNEXNIZIJ-BQBZGAKWSA-N Gly-Glu-Gln Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFXJIZNEXNIZIJ-BQBZGAKWSA-N 0.000 description 2
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 2
- YYPFZVIXAVDHIK-IUCAKERBSA-N Gly-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN YYPFZVIXAVDHIK-IUCAKERBSA-N 0.000 description 2
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 2
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 2
- DEOQGJUXUQGUJN-KKUMJFAQSA-N His-Lys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N DEOQGJUXUQGUJN-KKUMJFAQSA-N 0.000 description 2
- RWIKBYVJQAJYDP-BJDJZHNGSA-N Ile-Ala-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RWIKBYVJQAJYDP-BJDJZHNGSA-N 0.000 description 2
- SACHLUOUHCVIKI-GMOBBJLQSA-N Ile-Arg-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SACHLUOUHCVIKI-GMOBBJLQSA-N 0.000 description 2
- YKRIXHPEIZUDDY-GMOBBJLQSA-N Ile-Asn-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKRIXHPEIZUDDY-GMOBBJLQSA-N 0.000 description 2
- UAVQIQOOBXFKRC-BYULHYEWSA-N Ile-Asn-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O UAVQIQOOBXFKRC-BYULHYEWSA-N 0.000 description 2
- CSQNHSGHAPRGPQ-YTFOTSKYSA-N Ile-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)O)N CSQNHSGHAPRGPQ-YTFOTSKYSA-N 0.000 description 2
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 2
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 2
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 2
- CIDLJWVDMNDKPT-FIRPJDEBSA-N Ile-Phe-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N CIDLJWVDMNDKPT-FIRPJDEBSA-N 0.000 description 2
- HJDZMPFEXINXLO-QPHKQPEJSA-N Ile-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N HJDZMPFEXINXLO-QPHKQPEJSA-N 0.000 description 2
- BCISUQVFDGYZBO-QSFUFRPTSA-N Ile-Val-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O BCISUQVFDGYZBO-QSFUFRPTSA-N 0.000 description 2
- 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 2
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 2
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 2
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 2
- WGNOPSQMIQERPK-GARJFASQSA-N Leu-Asn-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N WGNOPSQMIQERPK-GARJFASQSA-N 0.000 description 2
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 2
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 2
- DBSLVQBXKVKDKJ-BJDJZHNGSA-N Leu-Ile-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DBSLVQBXKVKDKJ-BJDJZHNGSA-N 0.000 description 2
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 2
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 2
- ZAVCJRJOQKIOJW-KKUMJFAQSA-N Leu-Phe-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=CC=C1 ZAVCJRJOQKIOJW-KKUMJFAQSA-N 0.000 description 2
- PJWOOBTYQNNRBF-BZSNNMDCSA-N Leu-Phe-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)O)N PJWOOBTYQNNRBF-BZSNNMDCSA-N 0.000 description 2
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 2
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 2
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 2
- JGKHAFUAPZCCDU-BZSNNMDCSA-N Leu-Tyr-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=C(O)C=C1 JGKHAFUAPZCCDU-BZSNNMDCSA-N 0.000 description 2
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 2
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 2
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 2
- VHNOAIFVYUQOOY-XUXIUFHCSA-N Lys-Arg-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VHNOAIFVYUQOOY-XUXIUFHCSA-N 0.000 description 2
- DNEJSAIMVANNPA-DCAQKATOSA-N Lys-Asn-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DNEJSAIMVANNPA-DCAQKATOSA-N 0.000 description 2
- HQVDJTYKCMIWJP-YUMQZZPRSA-N Lys-Asn-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O HQVDJTYKCMIWJP-YUMQZZPRSA-N 0.000 description 2
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 2
- LLSUNJYOSCOOEB-GUBZILKMSA-N Lys-Glu-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O LLSUNJYOSCOOEB-GUBZILKMSA-N 0.000 description 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 2
- PGLGNCVOWIORQE-SRVKXCTJSA-N Lys-His-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O PGLGNCVOWIORQE-SRVKXCTJSA-N 0.000 description 2
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 2
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 2
- YUAXTFMFMOIMAM-QWRGUYRKSA-N Lys-Lys-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O YUAXTFMFMOIMAM-QWRGUYRKSA-N 0.000 description 2
- ATNKHRAIZCMCCN-BZSNNMDCSA-N Lys-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N ATNKHRAIZCMCCN-BZSNNMDCSA-N 0.000 description 2
- GZGWILAQHOVXTD-DCAQKATOSA-N Lys-Met-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O GZGWILAQHOVXTD-DCAQKATOSA-N 0.000 description 2
- MTBLFIQZECOEBY-IHRRRGAJSA-N Lys-Met-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O MTBLFIQZECOEBY-IHRRRGAJSA-N 0.000 description 2
- RMKJOQSYLQQRFN-KKUMJFAQSA-N Lys-Tyr-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O RMKJOQSYLQQRFN-KKUMJFAQSA-N 0.000 description 2
- IEIHKHYMBIYQTH-YESZJQIVSA-N Lys-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCCN)N)C(=O)O IEIHKHYMBIYQTH-YESZJQIVSA-N 0.000 description 2
- OHXUUQDOBQKSNB-AVGNSLFASA-N Lys-Val-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OHXUUQDOBQKSNB-AVGNSLFASA-N 0.000 description 2
- NYTDJEZBAAFLLG-IHRRRGAJSA-N Lys-Val-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O NYTDJEZBAAFLLG-IHRRRGAJSA-N 0.000 description 2
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 2
- RRIHXWPHQSXHAQ-XUXIUFHCSA-N Met-Ile-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(O)=O RRIHXWPHQSXHAQ-XUXIUFHCSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- NEHSHYOUIWBYSA-DCPHZVHLSA-N Phe-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC3=CC=CC=C3)N NEHSHYOUIWBYSA-DCPHZVHLSA-N 0.000 description 2
- LGBVMDMZZFYSFW-HJWJTTGWSA-N Phe-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CC=CC=C1)N LGBVMDMZZFYSFW-HJWJTTGWSA-N 0.000 description 2
- WMGVYPPIMZPWPN-SRVKXCTJSA-N Phe-Asp-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N WMGVYPPIMZPWPN-SRVKXCTJSA-N 0.000 description 2
- KRYSMKKRRRWOCZ-QEWYBTABSA-N Phe-Ile-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O KRYSMKKRRRWOCZ-QEWYBTABSA-N 0.000 description 2
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 2
- YKUGPVXSDOOANW-KKUMJFAQSA-N Phe-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YKUGPVXSDOOANW-KKUMJFAQSA-N 0.000 description 2
- APMXLWHMIVWLLR-BZSNNMDCSA-N Phe-Tyr-Ser Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 APMXLWHMIVWLLR-BZSNNMDCSA-N 0.000 description 2
- VOZIBWWZSBIXQN-SRVKXCTJSA-N Pro-Glu-Lys Chemical compound NCCCC[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O VOZIBWWZSBIXQN-SRVKXCTJSA-N 0.000 description 2
- CHYAYDLYYIJCKY-OSUNSFLBSA-N Pro-Thr-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CHYAYDLYYIJCKY-OSUNSFLBSA-N 0.000 description 2
- 108010003201 RGH 0205 Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- KAAPNMOKUUPKOE-SRVKXCTJSA-N Ser-Asn-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KAAPNMOKUUPKOE-SRVKXCTJSA-N 0.000 description 2
- UOLGINIHBRIECN-FXQIFTODSA-N Ser-Glu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UOLGINIHBRIECN-FXQIFTODSA-N 0.000 description 2
- QJKPECIAWNNKIT-KKUMJFAQSA-N Ser-Lys-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QJKPECIAWNNKIT-KKUMJFAQSA-N 0.000 description 2
- 108010076818 TEV protease Proteins 0.000 description 2
- MQBTXMPQNCGSSZ-OSUNSFLBSA-N Thr-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)CCCN=C(N)N MQBTXMPQNCGSSZ-OSUNSFLBSA-N 0.000 description 2
- IRKWVRSEQFTGGV-VEVYYDQMSA-N Thr-Asn-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IRKWVRSEQFTGGV-VEVYYDQMSA-N 0.000 description 2
- JMGJDTNUMAZNLX-RWRJDSDZSA-N Thr-Glu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JMGJDTNUMAZNLX-RWRJDSDZSA-N 0.000 description 2
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 2
- KRDSCBLRHORMRK-JXUBOQSCSA-N Thr-Lys-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O KRDSCBLRHORMRK-JXUBOQSCSA-N 0.000 description 2
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 description 2
- LXXCHJKHJYRMIY-FQPOAREZSA-N Thr-Tyr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O LXXCHJKHJYRMIY-FQPOAREZSA-N 0.000 description 2
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 2
- 101150110111 Ttn gene Proteins 0.000 description 2
- UABYBEBXFFNCIR-YDHLFZDLSA-N Tyr-Asp-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UABYBEBXFFNCIR-YDHLFZDLSA-N 0.000 description 2
- FBHBVXUBTYVCRU-BZSNNMDCSA-N Tyr-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CN=CN1 FBHBVXUBTYVCRU-BZSNNMDCSA-N 0.000 description 2
- JLKVWTICWVWGSK-JYJNAYRXSA-N Tyr-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JLKVWTICWVWGSK-JYJNAYRXSA-N 0.000 description 2
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 2
- VBFVQTPETKJCQW-RPTUDFQQSA-N Tyr-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VBFVQTPETKJCQW-RPTUDFQQSA-N 0.000 description 2
- RCMWNNJFKNDKQR-UFYCRDLUSA-N Tyr-Pro-Phe Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 RCMWNNJFKNDKQR-UFYCRDLUSA-N 0.000 description 2
- HZDQUVQEVVYDDA-ACRUOGEOSA-N Tyr-Tyr-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HZDQUVQEVVYDDA-ACRUOGEOSA-N 0.000 description 2
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 description 2
- ISERLACIZUGCDX-ZKWXMUAHSA-N Val-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N ISERLACIZUGCDX-ZKWXMUAHSA-N 0.000 description 2
- APQIVBCUIUDSMB-OSUNSFLBSA-N Val-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N APQIVBCUIUDSMB-OSUNSFLBSA-N 0.000 description 2
- 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 2
- XBJKAZATRJBDCU-GUBZILKMSA-N Val-Pro-Ala Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XBJKAZATRJBDCU-GUBZILKMSA-N 0.000 description 2
- MIAZWUMFUURQNP-YDHLFZDLSA-N Val-Tyr-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N MIAZWUMFUURQNP-YDHLFZDLSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 2
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 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 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 2
- 108010059459 arginyl-threonyl-phenylalanine Proteins 0.000 description 2
- 108010084758 arginyl-tyrosyl-aspartic acid Proteins 0.000 description 2
- 108010060035 arginylproline Proteins 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 238000010370 cell cloning Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 102000018146 globin Human genes 0.000 description 2
- 108060003196 globin Proteins 0.000 description 2
- 108010079547 glutamylmethionine Proteins 0.000 description 2
- HPAIKDPJURGQLN-UHFFFAOYSA-N glycyl-L-histidyl-L-phenylalanine Natural products C=1C=CC=CC=1CC(C(O)=O)NC(=O)C(NC(=O)CN)CC1=CN=CN1 HPAIKDPJURGQLN-UHFFFAOYSA-N 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 2
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 2
- 108010015792 glycyllysine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 108010078274 isoleucylvaline Proteins 0.000 description 2
- 210000001503 joint Anatomy 0.000 description 2
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 2
- 108010030617 leucyl-phenylalanyl-valine Proteins 0.000 description 2
- 108010012058 leucyltyrosine Proteins 0.000 description 2
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 2
- 108010009298 lysylglutamic acid Proteins 0.000 description 2
- 108010063431 methionyl-aspartyl-glycine Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000030648 nucleus localization Effects 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 108010025488 pinealon Proteins 0.000 description 2
- 230000002028 premature Effects 0.000 description 2
- 108010031719 prolyl-serine Proteins 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 108700029760 synthetic LTSP Proteins 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108010087967 type I signal peptidase Proteins 0.000 description 2
- 108010020532 tyrosyl-proline Proteins 0.000 description 2
- IGXNPQWXIRIGBF-KEOOTSPTSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IGXNPQWXIRIGBF-KEOOTSPTSA-N 0.000 description 1
- NBQSSMDPPLHNDC-GSOGOTFGSA-N (2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s,3r)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-(1h-indol-3-yl)pr Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 NBQSSMDPPLHNDC-GSOGOTFGSA-N 0.000 description 1
- GYNQVPIDAQTZOY-ROUUACIJSA-N (2s)-2-[[2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]acetyl]amino]-3-phenylpropanoic acid Chemical compound C([C@H](N)C(=O)NCC(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 GYNQVPIDAQTZOY-ROUUACIJSA-N 0.000 description 1
- RRBGTUQJDFBWNN-MUGJNUQGSA-N (2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2,6-diaminohexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O RRBGTUQJDFBWNN-MUGJNUQGSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- LWUWMHIOBPTZBA-DCAQKATOSA-N Ala-Arg-Lys Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O LWUWMHIOBPTZBA-DCAQKATOSA-N 0.000 description 1
- XEXJJJRVTFGWIC-FXQIFTODSA-N Ala-Asn-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XEXJJJRVTFGWIC-FXQIFTODSA-N 0.000 description 1
- NXSFUECZFORGOG-CIUDSAMLSA-N Ala-Asn-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXSFUECZFORGOG-CIUDSAMLSA-N 0.000 description 1
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-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
- LSLIRHLIUDVNBN-CIUDSAMLSA-N Ala-Asp-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LSLIRHLIUDVNBN-CIUDSAMLSA-N 0.000 description 1
- FVSOUJZKYWEFOB-KBIXCLLPSA-N Ala-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)N FVSOUJZKYWEFOB-KBIXCLLPSA-N 0.000 description 1
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 1
- NWVVKQZOVSTDBQ-CIUDSAMLSA-N Ala-Glu-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NWVVKQZOVSTDBQ-CIUDSAMLSA-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
- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 1
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 1
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 1
- FDAZDMAFZYTHGS-XVYDVKMFSA-N Ala-His-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O FDAZDMAFZYTHGS-XVYDVKMFSA-N 0.000 description 1
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 1
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- VCSABYLVNWQYQE-SRVKXCTJSA-N Ala-Lys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O VCSABYLVNWQYQE-SRVKXCTJSA-N 0.000 description 1
- KQESEZXHYOUIIM-CQDKDKBSSA-N Ala-Lys-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KQESEZXHYOUIIM-CQDKDKBSSA-N 0.000 description 1
- MDNAVFBZPROEHO-DCAQKATOSA-N Ala-Lys-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O MDNAVFBZPROEHO-DCAQKATOSA-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
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 description 1
- WQLDNOCHHRISMS-NAKRPEOUSA-N Ala-Pro-Ile Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WQLDNOCHHRISMS-NAKRPEOUSA-N 0.000 description 1
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 1
- NHWYNIZWLJYZAG-XVYDVKMFSA-N Ala-Ser-His Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N NHWYNIZWLJYZAG-XVYDVKMFSA-N 0.000 description 1
- OMCKWYSDUQBYCN-FXQIFTODSA-N Ala-Ser-Met Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O OMCKWYSDUQBYCN-FXQIFTODSA-N 0.000 description 1
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 1
- ZJLORAAXDAJLDC-CQDKDKBSSA-N Ala-Tyr-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O ZJLORAAXDAJLDC-CQDKDKBSSA-N 0.000 description 1
- IIABBYGHLYWVOS-FXQIFTODSA-N Arg-Asn-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O IIABBYGHLYWVOS-FXQIFTODSA-N 0.000 description 1
- SQKPKIJVWHAWNF-DCAQKATOSA-N Arg-Asp-Lys Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(O)=O SQKPKIJVWHAWNF-DCAQKATOSA-N 0.000 description 1
- RRGPUNYIPJXJBU-GUBZILKMSA-N Arg-Asp-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O RRGPUNYIPJXJBU-GUBZILKMSA-N 0.000 description 1
- QAODJPUKWNNNRP-DCAQKATOSA-N Arg-Glu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QAODJPUKWNNNRP-DCAQKATOSA-N 0.000 description 1
- MZRBYBIQTIKERR-GUBZILKMSA-N Arg-Glu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MZRBYBIQTIKERR-GUBZILKMSA-N 0.000 description 1
- OGUPCHKBOKJFMA-SRVKXCTJSA-N Arg-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N OGUPCHKBOKJFMA-SRVKXCTJSA-N 0.000 description 1
- DJAIOAKQIOGULM-DCAQKATOSA-N Arg-Glu-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O DJAIOAKQIOGULM-DCAQKATOSA-N 0.000 description 1
- GOWZVQXTHUCNSQ-NHCYSSNCSA-N Arg-Glu-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GOWZVQXTHUCNSQ-NHCYSSNCSA-N 0.000 description 1
- QKSAZKCRVQYYGS-UWVGGRQHSA-N Arg-Gly-His Chemical compound N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O QKSAZKCRVQYYGS-UWVGGRQHSA-N 0.000 description 1
- RKQRHMKFNBYOTN-IHRRRGAJSA-N Arg-His-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N RKQRHMKFNBYOTN-IHRRRGAJSA-N 0.000 description 1
- CRCCTGPNZUCAHE-DCAQKATOSA-N Arg-His-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 CRCCTGPNZUCAHE-DCAQKATOSA-N 0.000 description 1
- HCIUUZGFTDTEGM-NAKRPEOUSA-N Arg-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N HCIUUZGFTDTEGM-NAKRPEOUSA-N 0.000 description 1
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 1
- YBZMTKUDWXZLIX-UWVGGRQHSA-N Arg-Leu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YBZMTKUDWXZLIX-UWVGGRQHSA-N 0.000 description 1
- MJINRRBEMOLJAK-DCAQKATOSA-N Arg-Lys-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N MJINRRBEMOLJAK-DCAQKATOSA-N 0.000 description 1
- MTYLORHAQXVQOW-AVGNSLFASA-N Arg-Lys-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(O)=O MTYLORHAQXVQOW-AVGNSLFASA-N 0.000 description 1
- GRRXPUAICOGISM-RWMBFGLXSA-N Arg-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GRRXPUAICOGISM-RWMBFGLXSA-N 0.000 description 1
- PYZPXCZNQSEHDT-GUBZILKMSA-N Arg-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N PYZPXCZNQSEHDT-GUBZILKMSA-N 0.000 description 1
- YTMKMRSYXHBGER-IHRRRGAJSA-N Arg-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YTMKMRSYXHBGER-IHRRRGAJSA-N 0.000 description 1
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 1
- NGYHSXDNNOFHNE-AVGNSLFASA-N Arg-Pro-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O NGYHSXDNNOFHNE-AVGNSLFASA-N 0.000 description 1
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 1
- AOJYORNRFWWEIV-IHRRRGAJSA-N Arg-Tyr-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 AOJYORNRFWWEIV-IHRRRGAJSA-N 0.000 description 1
- PSUXEQYPYZLNER-QXEWZRGKSA-N Arg-Val-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PSUXEQYPYZLNER-QXEWZRGKSA-N 0.000 description 1
- SLKLLQWZQHXYSV-CIUDSAMLSA-N Asn-Ala-Lys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O SLKLLQWZQHXYSV-CIUDSAMLSA-N 0.000 description 1
- XWGJDUSDTRPQRK-ZLUOBGJFSA-N Asn-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O XWGJDUSDTRPQRK-ZLUOBGJFSA-N 0.000 description 1
- XHFXZQHTLJVZBN-FXQIFTODSA-N Asn-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N XHFXZQHTLJVZBN-FXQIFTODSA-N 0.000 description 1
- QISZHYWZHJRDAO-CIUDSAMLSA-N Asn-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N QISZHYWZHJRDAO-CIUDSAMLSA-N 0.000 description 1
- WPOLSNAQGVHROR-GUBZILKMSA-N Asn-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N WPOLSNAQGVHROR-GUBZILKMSA-N 0.000 description 1
- OKZOABJQOMAYEC-NUMRIWBASA-N Asn-Gln-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OKZOABJQOMAYEC-NUMRIWBASA-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
- BZMWJLLUAKSIMH-FXQIFTODSA-N Asn-Glu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BZMWJLLUAKSIMH-FXQIFTODSA-N 0.000 description 1
- JREOBWLIZLXRIS-GUBZILKMSA-N Asn-Glu-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JREOBWLIZLXRIS-GUBZILKMSA-N 0.000 description 1
- ASCGFDYEKSRNPL-CIUDSAMLSA-N Asn-Glu-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O ASCGFDYEKSRNPL-CIUDSAMLSA-N 0.000 description 1
- HYQYLOSCICEYTR-YUMQZZPRSA-N Asn-Gly-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O HYQYLOSCICEYTR-YUMQZZPRSA-N 0.000 description 1
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 1
- UDSVWSUXKYXSTR-QWRGUYRKSA-N Asn-Gly-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UDSVWSUXKYXSTR-QWRGUYRKSA-N 0.000 description 1
- ANPFQTJEPONRPL-UGYAYLCHSA-N Asn-Ile-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O ANPFQTJEPONRPL-UGYAYLCHSA-N 0.000 description 1
- LVHMEJJWEXBMKK-GMOBBJLQSA-N Asn-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)N)N LVHMEJJWEXBMKK-GMOBBJLQSA-N 0.000 description 1
- SPCONPVIDFMDJI-QSFUFRPTSA-N Asn-Ile-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O SPCONPVIDFMDJI-QSFUFRPTSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 1
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 1
- OROMFUQQTSWUTI-IHRRRGAJSA-N Asn-Phe-Arg Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N OROMFUQQTSWUTI-IHRRRGAJSA-N 0.000 description 1
- LSJQOMAZIKQMTJ-SRVKXCTJSA-N Asn-Phe-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O LSJQOMAZIKQMTJ-SRVKXCTJSA-N 0.000 description 1
- HZZIFFOVHLWGCS-KKUMJFAQSA-N Asn-Phe-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O HZZIFFOVHLWGCS-KKUMJFAQSA-N 0.000 description 1
- RVHGJNGNKGDCPX-KKUMJFAQSA-N Asn-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N RVHGJNGNKGDCPX-KKUMJFAQSA-N 0.000 description 1
- YUUIAUXBNOHFRJ-IHRRRGAJSA-N Asn-Phe-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O YUUIAUXBNOHFRJ-IHRRRGAJSA-N 0.000 description 1
- FTNRWCPWDWRPAV-BZSNNMDCSA-N Asn-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CC(N)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FTNRWCPWDWRPAV-BZSNNMDCSA-N 0.000 description 1
- JTXVXGXTRXMOFJ-FXQIFTODSA-N Asn-Pro-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O JTXVXGXTRXMOFJ-FXQIFTODSA-N 0.000 description 1
- NJSNXIOKBHPFMB-GMOBBJLQSA-N Asn-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N)N NJSNXIOKBHPFMB-GMOBBJLQSA-N 0.000 description 1
- AWXDRZJQCVHCIT-DCAQKATOSA-N Asn-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(N)=O AWXDRZJQCVHCIT-DCAQKATOSA-N 0.000 description 1
- HPASIOLTWSNMFB-OLHMAJIHSA-N Asn-Thr-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O HPASIOLTWSNMFB-OLHMAJIHSA-N 0.000 description 1
- KTDWFWNZLLFEFU-KKUMJFAQSA-N Asn-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O KTDWFWNZLLFEFU-KKUMJFAQSA-N 0.000 description 1
- CGYKCTPUGXFPMG-IHPCNDPISA-N Asn-Tyr-Trp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O CGYKCTPUGXFPMG-IHPCNDPISA-N 0.000 description 1
- JZLFYAAGGYMRIK-BYULHYEWSA-N Asn-Val-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O JZLFYAAGGYMRIK-BYULHYEWSA-N 0.000 description 1
- JNCRAQVYJZGIOW-QSFUFRPTSA-N Asn-Val-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNCRAQVYJZGIOW-QSFUFRPTSA-N 0.000 description 1
- GHWWTICYPDKPTE-NGZCFLSTSA-N Asn-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N GHWWTICYPDKPTE-NGZCFLSTSA-N 0.000 description 1
- VPPXTHJNTYDNFJ-CIUDSAMLSA-N Asp-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N VPPXTHJNTYDNFJ-CIUDSAMLSA-N 0.000 description 1
- BLQBMRNMBAYREH-UWJYBYFXSA-N Asp-Ala-Tyr Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O BLQBMRNMBAYREH-UWJYBYFXSA-N 0.000 description 1
- WSOKZUVWBXVJHX-CIUDSAMLSA-N Asp-Arg-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O WSOKZUVWBXVJHX-CIUDSAMLSA-N 0.000 description 1
- MFMJRYHVLLEMQM-DCAQKATOSA-N Asp-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N MFMJRYHVLLEMQM-DCAQKATOSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- JDHOJQJMWBKHDB-CIUDSAMLSA-N Asp-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N JDHOJQJMWBKHDB-CIUDSAMLSA-N 0.000 description 1
- KNMRXHIAVXHCLW-ZLUOBGJFSA-N Asp-Asn-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N)C(=O)O KNMRXHIAVXHCLW-ZLUOBGJFSA-N 0.000 description 1
- RDRMWJBLOSRRAW-BYULHYEWSA-N Asp-Asn-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O RDRMWJBLOSRRAW-BYULHYEWSA-N 0.000 description 1
- VPSHHQXIWLGVDD-ZLUOBGJFSA-N Asp-Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O VPSHHQXIWLGVDD-ZLUOBGJFSA-N 0.000 description 1
- SBHUBSDEZQFJHJ-CIUDSAMLSA-N Asp-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O SBHUBSDEZQFJHJ-CIUDSAMLSA-N 0.000 description 1
- CELPEWWLSXMVPH-CIUDSAMLSA-N Asp-Asp-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O CELPEWWLSXMVPH-CIUDSAMLSA-N 0.000 description 1
- DXQOQMCLWWADMU-ACZMJKKPSA-N Asp-Gln-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DXQOQMCLWWADMU-ACZMJKKPSA-N 0.000 description 1
- VILLWIDTHYPSLC-PEFMBERDSA-N Asp-Glu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VILLWIDTHYPSLC-PEFMBERDSA-N 0.000 description 1
- RRKCPMGSRIDLNC-AVGNSLFASA-N Asp-Glu-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RRKCPMGSRIDLNC-AVGNSLFASA-N 0.000 description 1
- DTNUIAJCPRMNBT-WHFBIAKZSA-N Asp-Gly-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O DTNUIAJCPRMNBT-WHFBIAKZSA-N 0.000 description 1
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 1
- TVIZQBFURPLQDV-DJFWLOJKSA-N Asp-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)O)N TVIZQBFURPLQDV-DJFWLOJKSA-N 0.000 description 1
- SEMWSADZTMJELF-BYULHYEWSA-N Asp-Ile-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O SEMWSADZTMJELF-BYULHYEWSA-N 0.000 description 1
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 1
- JNNVNVRBYUJYGS-CIUDSAMLSA-N Asp-Leu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O JNNVNVRBYUJYGS-CIUDSAMLSA-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
- UMHUHHJMEXNSIV-CIUDSAMLSA-N Asp-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UMHUHHJMEXNSIV-CIUDSAMLSA-N 0.000 description 1
- ORRJQLIATJDMQM-HJGDQZAQSA-N Asp-Leu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O ORRJQLIATJDMQM-HJGDQZAQSA-N 0.000 description 1
- UZFHNLYQWMGUHU-DCAQKATOSA-N Asp-Lys-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UZFHNLYQWMGUHU-DCAQKATOSA-N 0.000 description 1
- CTWCFPWFIGRAEP-CIUDSAMLSA-N Asp-Lys-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O CTWCFPWFIGRAEP-CIUDSAMLSA-N 0.000 description 1
- MYLZFUMPZCPJCJ-NHCYSSNCSA-N Asp-Lys-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O MYLZFUMPZCPJCJ-NHCYSSNCSA-N 0.000 description 1
- WQSXAPPYLGNMQL-IHRRRGAJSA-N Asp-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](CC(=O)O)N WQSXAPPYLGNMQL-IHRRRGAJSA-N 0.000 description 1
- GYWQGGUCMDCUJE-DLOVCJGASA-N Asp-Phe-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O GYWQGGUCMDCUJE-DLOVCJGASA-N 0.000 description 1
- LIJXJYGRSRWLCJ-IHRRRGAJSA-N Asp-Phe-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LIJXJYGRSRWLCJ-IHRRRGAJSA-N 0.000 description 1
- AHWRSSLYSGLBGD-CIUDSAMLSA-N Asp-Pro-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AHWRSSLYSGLBGD-CIUDSAMLSA-N 0.000 description 1
- UAXIKORUDGGIGA-DCAQKATOSA-N Asp-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O UAXIKORUDGGIGA-DCAQKATOSA-N 0.000 description 1
- ZBYLEBZCVKLPCY-FXQIFTODSA-N Asp-Ser-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZBYLEBZCVKLPCY-FXQIFTODSA-N 0.000 description 1
- KGHLGJAXYSVNJP-WHFBIAKZSA-N Asp-Ser-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O KGHLGJAXYSVNJP-WHFBIAKZSA-N 0.000 description 1
- DRCOAZZDQRCGGP-GHCJXIJMSA-N Asp-Ser-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DRCOAZZDQRCGGP-GHCJXIJMSA-N 0.000 description 1
- HRVQDZOWMLFAOD-BIIVOSGPSA-N Asp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N)C(=O)O HRVQDZOWMLFAOD-BIIVOSGPSA-N 0.000 description 1
- DKQCWCQRAMAFLN-UBHSHLNASA-N Asp-Trp-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O DKQCWCQRAMAFLN-UBHSHLNASA-N 0.000 description 1
- BJDHEININLSZOT-KKUMJFAQSA-N Asp-Tyr-Lys Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(O)=O BJDHEININLSZOT-KKUMJFAQSA-N 0.000 description 1
- SQIARYGNVQWOSB-BZSNNMDCSA-N Asp-Tyr-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SQIARYGNVQWOSB-BZSNNMDCSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 101100118093 Drosophila melanogaster eEF1alpha2 gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000701832 Enterobacteria phage T3 Species 0.000 description 1
- 101900345593 Escherichia coli Alkaline phosphatase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108010044495 Fetal Hemoglobin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- DTMLKCYOQKZXKZ-HJGDQZAQSA-N Gln-Arg-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DTMLKCYOQKZXKZ-HJGDQZAQSA-N 0.000 description 1
- PHZYLYASFWHLHJ-FXQIFTODSA-N Gln-Asn-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PHZYLYASFWHLHJ-FXQIFTODSA-N 0.000 description 1
- ZPDVKYLJTOFQJV-WDSKDSINSA-N Gln-Asn-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O ZPDVKYLJTOFQJV-WDSKDSINSA-N 0.000 description 1
- CKNUKHBRCSMKMO-XHNCKOQMSA-N Gln-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O CKNUKHBRCSMKMO-XHNCKOQMSA-N 0.000 description 1
- XEYMBRRKIFYQMF-GUBZILKMSA-N Gln-Asp-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O XEYMBRRKIFYQMF-GUBZILKMSA-N 0.000 description 1
- SNLOOPZHAQDMJG-CIUDSAMLSA-N Gln-Glu-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SNLOOPZHAQDMJG-CIUDSAMLSA-N 0.000 description 1
- PNENQZWRFMUZOM-DCAQKATOSA-N Gln-Glu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O PNENQZWRFMUZOM-DCAQKATOSA-N 0.000 description 1
- ITZWDGBYBPUZRG-KBIXCLLPSA-N Gln-Ile-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O ITZWDGBYBPUZRG-KBIXCLLPSA-N 0.000 description 1
- LGIKBBLQVSWUGK-DCAQKATOSA-N Gln-Leu-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LGIKBBLQVSWUGK-DCAQKATOSA-N 0.000 description 1
- CAXXTYYGFYTBPV-IUCAKERBSA-N Gln-Leu-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CAXXTYYGFYTBPV-IUCAKERBSA-N 0.000 description 1
- PSERKXGRRADTKA-MNXVOIDGSA-N Gln-Leu-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PSERKXGRRADTKA-MNXVOIDGSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- IULKWYSYZSURJK-AVGNSLFASA-N Gln-Leu-Lys Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O IULKWYSYZSURJK-AVGNSLFASA-N 0.000 description 1
- ZBKUIQNCRIYVGH-SDDRHHMPSA-N Gln-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZBKUIQNCRIYVGH-SDDRHHMPSA-N 0.000 description 1
- IHSGESFHTMFHRB-GUBZILKMSA-N Gln-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(N)=O IHSGESFHTMFHRB-GUBZILKMSA-N 0.000 description 1
- SXGMGNZEHFORAV-IUCAKERBSA-N Gln-Lys-Gly Chemical compound C(CCN)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N SXGMGNZEHFORAV-IUCAKERBSA-N 0.000 description 1
- ZVQZXPADLZIQFF-FHWLQOOXSA-N Gln-Phe-Tyr Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 ZVQZXPADLZIQFF-FHWLQOOXSA-N 0.000 description 1
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 1
- KPNWAJMEMRCLAL-GUBZILKMSA-N Gln-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N KPNWAJMEMRCLAL-GUBZILKMSA-N 0.000 description 1
- FITIQFSXXBKFFM-NRPADANISA-N Gln-Val-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FITIQFSXXBKFFM-NRPADANISA-N 0.000 description 1
- FYBSCGZLICNOBA-XQXXSGGOSA-N Glu-Ala-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FYBSCGZLICNOBA-XQXXSGGOSA-N 0.000 description 1
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 1
- NLKVNZUFDPWPNL-YUMQZZPRSA-N Glu-Arg-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O NLKVNZUFDPWPNL-YUMQZZPRSA-N 0.000 description 1
- PBEQPAZRHDVJQI-SRVKXCTJSA-N Glu-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N PBEQPAZRHDVJQI-SRVKXCTJSA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- KEBACWCLVOXFNC-DCAQKATOSA-N Glu-Arg-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O KEBACWCLVOXFNC-DCAQKATOSA-N 0.000 description 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 description 1
- YKLNMGJYMNPBCP-ACZMJKKPSA-N Glu-Asn-Asp Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YKLNMGJYMNPBCP-ACZMJKKPSA-N 0.000 description 1
- MLCPTRRNICEKIS-FXQIFTODSA-N Glu-Asn-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MLCPTRRNICEKIS-FXQIFTODSA-N 0.000 description 1
- CKRUHITYRFNUKW-WDSKDSINSA-N Glu-Asn-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CKRUHITYRFNUKW-WDSKDSINSA-N 0.000 description 1
- VAZZOGXDUQSVQF-NUMRIWBASA-N Glu-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)O VAZZOGXDUQSVQF-NUMRIWBASA-N 0.000 description 1
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 1
- QPRZKNOOOBWXSU-CIUDSAMLSA-N Glu-Asp-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N QPRZKNOOOBWXSU-CIUDSAMLSA-N 0.000 description 1
- NTBDVNJIWCKURJ-ACZMJKKPSA-N Glu-Asp-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NTBDVNJIWCKURJ-ACZMJKKPSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- OXEMJGCAJFFREE-FXQIFTODSA-N Glu-Gln-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O OXEMJGCAJFFREE-FXQIFTODSA-N 0.000 description 1
- XHUCVVHRLNPZSZ-CIUDSAMLSA-N Glu-Gln-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XHUCVVHRLNPZSZ-CIUDSAMLSA-N 0.000 description 1
- UMIRPYLZFKOEOH-YVNDNENWSA-N Glu-Gln-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UMIRPYLZFKOEOH-YVNDNENWSA-N 0.000 description 1
- LVCHEMOPBORRLB-DCAQKATOSA-N Glu-Gln-Lys Chemical compound NCCCC[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O LVCHEMOPBORRLB-DCAQKATOSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- LGYZYFFDELZWRS-DCAQKATOSA-N Glu-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O LGYZYFFDELZWRS-DCAQKATOSA-N 0.000 description 1
- YLJHCWNDBKKOEB-IHRRRGAJSA-N Glu-Glu-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YLJHCWNDBKKOEB-IHRRRGAJSA-N 0.000 description 1
- MTAOBYXRYJZRGQ-WDSKDSINSA-N Glu-Gly-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MTAOBYXRYJZRGQ-WDSKDSINSA-N 0.000 description 1
- CUXJIASLBRJOFV-LAEOZQHASA-N Glu-Gly-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CUXJIASLBRJOFV-LAEOZQHASA-N 0.000 description 1
- VOORMNJKNBGYGK-YUMQZZPRSA-N Glu-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N VOORMNJKNBGYGK-YUMQZZPRSA-N 0.000 description 1
- XOFYVODYSNKPDK-AVGNSLFASA-N Glu-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCC(=O)O)N XOFYVODYSNKPDK-AVGNSLFASA-N 0.000 description 1
- NJPQBTJSYCKCNS-HVTMNAMFSA-N Glu-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)O)N NJPQBTJSYCKCNS-HVTMNAMFSA-N 0.000 description 1
- CXRWMMRLEMVSEH-PEFMBERDSA-N Glu-Ile-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O CXRWMMRLEMVSEH-PEFMBERDSA-N 0.000 description 1
- ITBHUUMCJJQUSC-LAEOZQHASA-N Glu-Ile-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O ITBHUUMCJJQUSC-LAEOZQHASA-N 0.000 description 1
- ZCOJVESMNGBGLF-GRLWGSQLSA-N Glu-Ile-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZCOJVESMNGBGLF-GRLWGSQLSA-N 0.000 description 1
- WTMZXOPHTIVFCP-QEWYBTABSA-N Glu-Ile-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 WTMZXOPHTIVFCP-QEWYBTABSA-N 0.000 description 1
- ZSWGJYOZWBHROQ-RWRJDSDZSA-N Glu-Ile-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSWGJYOZWBHROQ-RWRJDSDZSA-N 0.000 description 1
- IRXNJYPKBVERCW-DCAQKATOSA-N Glu-Leu-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IRXNJYPKBVERCW-DCAQKATOSA-N 0.000 description 1
- NWOUBJNMZDDGDT-AVGNSLFASA-N Glu-Leu-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 NWOUBJNMZDDGDT-AVGNSLFASA-N 0.000 description 1
- SWRVAQHFBRZVNX-GUBZILKMSA-N Glu-Lys-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O SWRVAQHFBRZVNX-GUBZILKMSA-N 0.000 description 1
- OCJRHJZKGGSPRW-IUCAKERBSA-N Glu-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O OCJRHJZKGGSPRW-IUCAKERBSA-N 0.000 description 1
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 1
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 description 1
- ZQYZDDXTNQXUJH-CIUDSAMLSA-N Glu-Met-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(=O)O)N ZQYZDDXTNQXUJH-CIUDSAMLSA-N 0.000 description 1
- FGSGPLRPQCZBSQ-AVGNSLFASA-N Glu-Phe-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O FGSGPLRPQCZBSQ-AVGNSLFASA-N 0.000 description 1
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 1
- BXSZPACYCMNKLS-AVGNSLFASA-N Glu-Ser-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BXSZPACYCMNKLS-AVGNSLFASA-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
- TWYSSILQABLLME-HJGDQZAQSA-N Glu-Thr-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWYSSILQABLLME-HJGDQZAQSA-N 0.000 description 1
- JVYNYWXHZWVJEF-NUMRIWBASA-N Glu-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O JVYNYWXHZWVJEF-NUMRIWBASA-N 0.000 description 1
- RGJKYNUINKGPJN-RWRJDSDZSA-N Glu-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(=O)O)N RGJKYNUINKGPJN-RWRJDSDZSA-N 0.000 description 1
- GWKBAXRZPLSWJS-QEJZJMRPSA-N Glu-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N GWKBAXRZPLSWJS-QEJZJMRPSA-N 0.000 description 1
- MFYLRRCYBBJYPI-JYJNAYRXSA-N Glu-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O MFYLRRCYBBJYPI-JYJNAYRXSA-N 0.000 description 1
- YQPFCZVKMUVZIN-AUTRQRHGSA-N Glu-Val-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQPFCZVKMUVZIN-AUTRQRHGSA-N 0.000 description 1
- HQTDNEZTGZUWSY-XVKPBYJWSA-N Glu-Val-Gly Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)NCC(O)=O HQTDNEZTGZUWSY-XVKPBYJWSA-N 0.000 description 1
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 1
- NZAFOTBEULLEQB-WDSKDSINSA-N Gly-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CN NZAFOTBEULLEQB-WDSKDSINSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- XCLCVBYNGXEVDU-WHFBIAKZSA-N Gly-Asn-Ser Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O XCLCVBYNGXEVDU-WHFBIAKZSA-N 0.000 description 1
- FMNHBTKMRFVGRO-FOHZUACHSA-N Gly-Asn-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CN FMNHBTKMRFVGRO-FOHZUACHSA-N 0.000 description 1
- MHHUEAIBJZWDBH-YUMQZZPRSA-N Gly-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN MHHUEAIBJZWDBH-YUMQZZPRSA-N 0.000 description 1
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-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
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 1
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 1
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 1
- UUWOBINZFGTFMS-UWVGGRQHSA-N Gly-His-Met Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCSC)C(O)=O UUWOBINZFGTFMS-UWVGGRQHSA-N 0.000 description 1
- SXJHOPPTOJACOA-QXEWZRGKSA-N Gly-Ile-Arg Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SXJHOPPTOJACOA-QXEWZRGKSA-N 0.000 description 1
- DENRBIYENOKSEX-PEXQALLHSA-N Gly-Ile-His Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 DENRBIYENOKSEX-PEXQALLHSA-N 0.000 description 1
- YTSVAIMKVLZUDU-YUMQZZPRSA-N Gly-Leu-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YTSVAIMKVLZUDU-YUMQZZPRSA-N 0.000 description 1
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 1
- NTBOEZICHOSJEE-YUMQZZPRSA-N Gly-Lys-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O NTBOEZICHOSJEE-YUMQZZPRSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 description 1
- BBTCXWTXOXUNFX-IUCAKERBSA-N Gly-Met-Arg Chemical compound CSCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O BBTCXWTXOXUNFX-IUCAKERBSA-N 0.000 description 1
- QSQXZZCGPXQBPP-BQBZGAKWSA-N Gly-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)CN)C(=O)N[C@@H](CS)C(=O)O QSQXZZCGPXQBPP-BQBZGAKWSA-N 0.000 description 1
- HFPVRZWORNJRRC-UWVGGRQHSA-N Gly-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN HFPVRZWORNJRRC-UWVGGRQHSA-N 0.000 description 1
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 1
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 1
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 1
- LLWQVJNHMYBLLK-CDMKHQONSA-N Gly-Thr-Phe Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LLWQVJNHMYBLLK-CDMKHQONSA-N 0.000 description 1
- RCHFYMASWAZQQZ-ZANVPECISA-N Gly-Trp-Ala Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CN)=CNC2=C1 RCHFYMASWAZQQZ-ZANVPECISA-N 0.000 description 1
- PNUFMLXHOLFRLD-KBPBESRZSA-N Gly-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 PNUFMLXHOLFRLD-KBPBESRZSA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 108010027616 Hemoglobin A2 Proteins 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 108091005886 Hemoglobin subunit gamma Proteins 0.000 description 1
- 102100038617 Hemoglobin subunit gamma-2 Human genes 0.000 description 1
- IPIVXQQRZXEUGW-UWJYBYFXSA-N His-Ala-His Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IPIVXQQRZXEUGW-UWJYBYFXSA-N 0.000 description 1
- XINDHUAGVGCNSF-QSFUFRPTSA-N His-Ala-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XINDHUAGVGCNSF-QSFUFRPTSA-N 0.000 description 1
- SVHKVHBPTOMLTO-DCAQKATOSA-N His-Arg-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SVHKVHBPTOMLTO-DCAQKATOSA-N 0.000 description 1
- MVADCDSCFTXCBT-CIUDSAMLSA-N His-Asp-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MVADCDSCFTXCBT-CIUDSAMLSA-N 0.000 description 1
- WGVPDSNCHDEDBP-KKUMJFAQSA-N His-Asp-Phe Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WGVPDSNCHDEDBP-KKUMJFAQSA-N 0.000 description 1
- LDTJBEOANMQRJE-CIUDSAMLSA-N His-Cys-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N LDTJBEOANMQRJE-CIUDSAMLSA-N 0.000 description 1
- DVHGLDYMGWTYKW-GUBZILKMSA-N His-Gln-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DVHGLDYMGWTYKW-GUBZILKMSA-N 0.000 description 1
- IMCHNUANCIGUKS-SRVKXCTJSA-N His-Glu-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IMCHNUANCIGUKS-SRVKXCTJSA-N 0.000 description 1
- XMENRVZYPBKBIL-AVGNSLFASA-N His-Glu-His Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O XMENRVZYPBKBIL-AVGNSLFASA-N 0.000 description 1
- IDQNVIWPPWAFSY-AVGNSLFASA-N His-His-Gln Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O IDQNVIWPPWAFSY-AVGNSLFASA-N 0.000 description 1
- IWXMHXYOACDSIA-PYJNHQTQSA-N His-Ile-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O IWXMHXYOACDSIA-PYJNHQTQSA-N 0.000 description 1
- ZSKJIISDJXJQPV-BZSNNMDCSA-N His-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 ZSKJIISDJXJQPV-BZSNNMDCSA-N 0.000 description 1
- TTYKEFZRLKQTHH-MELADBBJSA-N His-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O TTYKEFZRLKQTHH-MELADBBJSA-N 0.000 description 1
- SVVULKPWDBIPCO-BZSNNMDCSA-N His-Phe-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SVVULKPWDBIPCO-BZSNNMDCSA-N 0.000 description 1
- WCHONUZTYDQMBY-PYJNHQTQSA-N His-Pro-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WCHONUZTYDQMBY-PYJNHQTQSA-N 0.000 description 1
- KFQDSSNYWKZFOO-LSJOCFKGSA-N His-Val-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KFQDSSNYWKZFOO-LSJOCFKGSA-N 0.000 description 1
- 101100230565 Homo sapiens HBB gene Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- LQSBBHNVAVNZSX-GHCJXIJMSA-N Ile-Ala-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N LQSBBHNVAVNZSX-GHCJXIJMSA-N 0.000 description 1
- YKRYHWJRQUSTKG-KBIXCLLPSA-N Ile-Ala-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKRYHWJRQUSTKG-KBIXCLLPSA-N 0.000 description 1
- VAXBXNPRXPHGHG-BJDJZHNGSA-N Ile-Ala-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)O)N VAXBXNPRXPHGHG-BJDJZHNGSA-N 0.000 description 1
- YOTNPRLPIPHQSB-XUXIUFHCSA-N Ile-Arg-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOTNPRLPIPHQSB-XUXIUFHCSA-N 0.000 description 1
- QYZYJFXHXYUZMZ-UGYAYLCHSA-N Ile-Asn-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N QYZYJFXHXYUZMZ-UGYAYLCHSA-N 0.000 description 1
- NBJAAWYRLGCJOF-UGYAYLCHSA-N Ile-Asp-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N NBJAAWYRLGCJOF-UGYAYLCHSA-N 0.000 description 1
- NKRJALPCDNXULF-BYULHYEWSA-N Ile-Asp-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O NKRJALPCDNXULF-BYULHYEWSA-N 0.000 description 1
- HGNUKGZQASSBKQ-PCBIJLKTSA-N Ile-Asp-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HGNUKGZQASSBKQ-PCBIJLKTSA-N 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- LKACSKJPTFSBHR-MNXVOIDGSA-N Ile-Gln-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N LKACSKJPTFSBHR-MNXVOIDGSA-N 0.000 description 1
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 1
- MTFVYKQRLXYAQN-LAEOZQHASA-N Ile-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O MTFVYKQRLXYAQN-LAEOZQHASA-N 0.000 description 1
- DFJJAVZIHDFOGQ-MNXVOIDGSA-N Ile-Glu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N DFJJAVZIHDFOGQ-MNXVOIDGSA-N 0.000 description 1
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 1
- KFVUBLZRFSVDGO-BYULHYEWSA-N Ile-Gly-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O KFVUBLZRFSVDGO-BYULHYEWSA-N 0.000 description 1
- NYEYYMLUABXDMC-NHCYSSNCSA-N Ile-Gly-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)O)N NYEYYMLUABXDMC-NHCYSSNCSA-N 0.000 description 1
- PDTMWFVVNZYWTR-NHCYSSNCSA-N Ile-Gly-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O PDTMWFVVNZYWTR-NHCYSSNCSA-N 0.000 description 1
- DFFTXLCCDFYRKD-MBLNEYKQSA-N Ile-Gly-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N DFFTXLCCDFYRKD-MBLNEYKQSA-N 0.000 description 1
- UASTVUQJMLZWGG-PEXQALLHSA-N Ile-His-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N UASTVUQJMLZWGG-PEXQALLHSA-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
- HUWYGQOISIJNMK-SIGLWIIPSA-N Ile-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HUWYGQOISIJNMK-SIGLWIIPSA-N 0.000 description 1
- PKGGWLOLRLOPGK-XUXIUFHCSA-N Ile-Leu-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PKGGWLOLRLOPGK-XUXIUFHCSA-N 0.000 description 1
- OUUCIIJSBIBCHB-ZPFDUUQYSA-N Ile-Leu-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O OUUCIIJSBIBCHB-ZPFDUUQYSA-N 0.000 description 1
- YGDWPQCLFJNMOL-MNXVOIDGSA-N Ile-Leu-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YGDWPQCLFJNMOL-MNXVOIDGSA-N 0.000 description 1
- TVYWVSJGSHQWMT-AJNGGQMLSA-N Ile-Leu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N TVYWVSJGSHQWMT-AJNGGQMLSA-N 0.000 description 1
- PHRWFSFCNJPWRO-PPCPHDFISA-N Ile-Leu-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N PHRWFSFCNJPWRO-PPCPHDFISA-N 0.000 description 1
- DSDPLOODKXISDT-XUXIUFHCSA-N Ile-Leu-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O DSDPLOODKXISDT-XUXIUFHCSA-N 0.000 description 1
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 1
- GVNNAHIRSDRIII-AJNGGQMLSA-N Ile-Lys-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N GVNNAHIRSDRIII-AJNGGQMLSA-N 0.000 description 1
- IDMNOFVUXYYZPF-DKIMLUQUSA-N Ile-Lys-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IDMNOFVUXYYZPF-DKIMLUQUSA-N 0.000 description 1
- GLYJPWIRLBAIJH-FQUUOJAGSA-N Ile-Lys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N GLYJPWIRLBAIJH-FQUUOJAGSA-N 0.000 description 1
- RVNOXPZHMUWCLW-GMOBBJLQSA-N Ile-Met-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)N)C(=O)O)N RVNOXPZHMUWCLW-GMOBBJLQSA-N 0.000 description 1
- FGBRXCZYVRFNKQ-MXAVVETBSA-N Ile-Phe-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N FGBRXCZYVRFNKQ-MXAVVETBSA-N 0.000 description 1
- BJECXJHLUJXPJQ-PYJNHQTQSA-N Ile-Pro-His Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N BJECXJHLUJXPJQ-PYJNHQTQSA-N 0.000 description 1
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 1
- YCKPUHHMCFSUMD-IUKAMOBKSA-N Ile-Thr-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCKPUHHMCFSUMD-IUKAMOBKSA-N 0.000 description 1
- YBKKLDBBPFIXBQ-MBLNEYKQSA-N Ile-Thr-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)O)N YBKKLDBBPFIXBQ-MBLNEYKQSA-N 0.000 description 1
- KBDIBHQICWDGDL-PPCPHDFISA-N Ile-Thr-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N KBDIBHQICWDGDL-PPCPHDFISA-N 0.000 description 1
- DLEBSGAVWRPTIX-PEDHHIEDSA-N Ile-Val-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)[C@@H](C)CC DLEBSGAVWRPTIX-PEDHHIEDSA-N 0.000 description 1
- UYODHPPSCXBNCS-XUXIUFHCSA-N Ile-Val-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C UYODHPPSCXBNCS-XUXIUFHCSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010065390 Inflammatory pain Diseases 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 1
- 101800005093 LVV-hemorphin-7 Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- PBCHMHROGNUXMK-DLOVCJGASA-N Leu-Ala-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 PBCHMHROGNUXMK-DLOVCJGASA-N 0.000 description 1
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 1
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 1
- IGUOAYLTQJLPPD-DCAQKATOSA-N Leu-Asn-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IGUOAYLTQJLPPD-DCAQKATOSA-N 0.000 description 1
- BPANDPNDMJHFEV-CIUDSAMLSA-N Leu-Asp-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O BPANDPNDMJHFEV-CIUDSAMLSA-N 0.000 description 1
- ILJREDZFPHTUIE-GUBZILKMSA-N Leu-Asp-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ILJREDZFPHTUIE-GUBZILKMSA-N 0.000 description 1
- JQSXWJXBASFONF-KKUMJFAQSA-N Leu-Asp-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JQSXWJXBASFONF-KKUMJFAQSA-N 0.000 description 1
- PVMPDMIKUVNOBD-CIUDSAMLSA-N Leu-Asp-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O PVMPDMIKUVNOBD-CIUDSAMLSA-N 0.000 description 1
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 1
- LOLUPZNNADDTAA-AVGNSLFASA-N Leu-Gln-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LOLUPZNNADDTAA-AVGNSLFASA-N 0.000 description 1
- FQZPTCNSNPWHLJ-AVGNSLFASA-N Leu-Gln-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O FQZPTCNSNPWHLJ-AVGNSLFASA-N 0.000 description 1
- CIVKXGPFXDIQBV-WDCWCFNPSA-N Leu-Gln-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CIVKXGPFXDIQBV-WDCWCFNPSA-N 0.000 description 1
- YVKSMSDXKMSIRX-GUBZILKMSA-N Leu-Glu-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O YVKSMSDXKMSIRX-GUBZILKMSA-N 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 1
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 1
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- SGIIOQQGLUUMDQ-IHRRRGAJSA-N Leu-His-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N SGIIOQQGLUUMDQ-IHRRRGAJSA-N 0.000 description 1
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 1
- JFSGIJSCJFQGSZ-MXAVVETBSA-N Leu-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(C)C)N JFSGIJSCJFQGSZ-MXAVVETBSA-N 0.000 description 1
- SEMUSFOBZGKBGW-YTFOTSKYSA-N Leu-Ile-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SEMUSFOBZGKBGW-YTFOTSKYSA-N 0.000 description 1
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- UBZGNBKMIJHOHL-BZSNNMDCSA-N Leu-Leu-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 UBZGNBKMIJHOHL-BZSNNMDCSA-N 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- JLWZLIQRYCTYBD-IHRRRGAJSA-N Leu-Lys-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JLWZLIQRYCTYBD-IHRRRGAJSA-N 0.000 description 1
- RZXLZBIUTDQHJQ-SRVKXCTJSA-N Leu-Lys-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O RZXLZBIUTDQHJQ-SRVKXCTJSA-N 0.000 description 1
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 1
- AIRUUHAOKGVJAD-JYJNAYRXSA-N Leu-Phe-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIRUUHAOKGVJAD-JYJNAYRXSA-N 0.000 description 1
- SYRTUBLKWNDSDK-DKIMLUQUSA-N Leu-Phe-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYRTUBLKWNDSDK-DKIMLUQUSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- KZZCOWMDDXDKSS-CIUDSAMLSA-N Leu-Ser-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KZZCOWMDDXDKSS-CIUDSAMLSA-N 0.000 description 1
- AKVBOOKXVAMKSS-GUBZILKMSA-N Leu-Ser-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O AKVBOOKXVAMKSS-GUBZILKMSA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 1
- DAYQSYGBCUKVKT-VOAKCMCISA-N Leu-Thr-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O DAYQSYGBCUKVKT-VOAKCMCISA-N 0.000 description 1
- KLSUAWUZBMAZCL-RHYQMDGZSA-N Leu-Thr-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(O)=O KLSUAWUZBMAZCL-RHYQMDGZSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 1
- XFIHDSBIPWEYJJ-YUMQZZPRSA-N Lys-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN XFIHDSBIPWEYJJ-YUMQZZPRSA-N 0.000 description 1
- WQWZXKWOEVSGQM-DCAQKATOSA-N Lys-Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN WQWZXKWOEVSGQM-DCAQKATOSA-N 0.000 description 1
- IXHKPDJKKCUKHS-GARJFASQSA-N Lys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IXHKPDJKKCUKHS-GARJFASQSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- CKSXSQUVEYCDIW-AVGNSLFASA-N Lys-Arg-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N CKSXSQUVEYCDIW-AVGNSLFASA-N 0.000 description 1
- NTSPQIONFJUMJV-AVGNSLFASA-N Lys-Arg-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O NTSPQIONFJUMJV-AVGNSLFASA-N 0.000 description 1
- FACUGMGEFUEBTI-SRVKXCTJSA-N Lys-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCCCN FACUGMGEFUEBTI-SRVKXCTJSA-N 0.000 description 1
- NCTDKZKNBDZDOL-GARJFASQSA-N Lys-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N)C(=O)O NCTDKZKNBDZDOL-GARJFASQSA-N 0.000 description 1
- DGWXCIORNLWGGG-CIUDSAMLSA-N Lys-Asn-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O DGWXCIORNLWGGG-CIUDSAMLSA-N 0.000 description 1
- KPJJOZUXFOLGMQ-CIUDSAMLSA-N Lys-Asp-Asn Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N KPJJOZUXFOLGMQ-CIUDSAMLSA-N 0.000 description 1
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 1
- LMVOVCYVZBBWQB-SRVKXCTJSA-N Lys-Asp-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LMVOVCYVZBBWQB-SRVKXCTJSA-N 0.000 description 1
- QIJVAFLRMVBHMU-KKUMJFAQSA-N Lys-Asp-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QIJVAFLRMVBHMU-KKUMJFAQSA-N 0.000 description 1
- PHHYNOUOUWYQRO-XIRDDKMYSA-N Lys-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)N PHHYNOUOUWYQRO-XIRDDKMYSA-N 0.000 description 1
- WTZUSCUIVPVCRH-SRVKXCTJSA-N Lys-Gln-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WTZUSCUIVPVCRH-SRVKXCTJSA-N 0.000 description 1
- NNCDAORZCMPZPX-GUBZILKMSA-N Lys-Gln-Ser Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N NNCDAORZCMPZPX-GUBZILKMSA-N 0.000 description 1
- DCRWPTBMWMGADO-AVGNSLFASA-N Lys-Glu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DCRWPTBMWMGADO-AVGNSLFASA-N 0.000 description 1
- DUTMKEAPLLUGNO-JYJNAYRXSA-N Lys-Glu-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DUTMKEAPLLUGNO-JYJNAYRXSA-N 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 1
- XNKDCYABMBBEKN-IUCAKERBSA-N Lys-Gly-Gln Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O XNKDCYABMBBEKN-IUCAKERBSA-N 0.000 description 1
- NNKLKUUGESXCBS-KBPBESRZSA-N Lys-Gly-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NNKLKUUGESXCBS-KBPBESRZSA-N 0.000 description 1
- OWRUUFUVXFREBD-KKUMJFAQSA-N Lys-His-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O OWRUUFUVXFREBD-KKUMJFAQSA-N 0.000 description 1
- SPCHLZUWJTYZFC-IHRRRGAJSA-N Lys-His-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O SPCHLZUWJTYZFC-IHRRRGAJSA-N 0.000 description 1
- JYXBNQOKPRQNQS-YTFOTSKYSA-N Lys-Ile-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JYXBNQOKPRQNQS-YTFOTSKYSA-N 0.000 description 1
- ZXFRGTAIIZHNHG-AJNGGQMLSA-N Lys-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](CCCCN)N ZXFRGTAIIZHNHG-AJNGGQMLSA-N 0.000 description 1
- ONPDTSFZAIWMDI-AVGNSLFASA-N Lys-Leu-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ONPDTSFZAIWMDI-AVGNSLFASA-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
- XIZQPFCRXLUNMK-BZSNNMDCSA-N Lys-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCCCN)N XIZQPFCRXLUNMK-BZSNNMDCSA-N 0.000 description 1
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 1
- VUTWYNQUSJWBHO-BZSNNMDCSA-N Lys-Leu-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VUTWYNQUSJWBHO-BZSNNMDCSA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- XOQMURBBIXRRCR-SRVKXCTJSA-N Lys-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN XOQMURBBIXRRCR-SRVKXCTJSA-N 0.000 description 1
- ALGGDNMLQNFVIZ-SRVKXCTJSA-N Lys-Lys-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N ALGGDNMLQNFVIZ-SRVKXCTJSA-N 0.000 description 1
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 1
- PLDJDCJLRCYPJB-VOAKCMCISA-N Lys-Lys-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PLDJDCJLRCYPJB-VOAKCMCISA-N 0.000 description 1
- WWEWGPOLIJXGNX-XUXIUFHCSA-N Lys-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)N WWEWGPOLIJXGNX-XUXIUFHCSA-N 0.000 description 1
- KVNLHIXLLZBAFQ-RWMBFGLXSA-N Lys-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N KVNLHIXLLZBAFQ-RWMBFGLXSA-N 0.000 description 1
- BOJYMMBYBNOOGG-DCAQKATOSA-N Lys-Pro-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BOJYMMBYBNOOGG-DCAQKATOSA-N 0.000 description 1
- QBHGXFQJFPWJIH-XUXIUFHCSA-N Lys-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN QBHGXFQJFPWJIH-XUXIUFHCSA-N 0.000 description 1
- WQDKIVRHTQYJSN-DCAQKATOSA-N Lys-Ser-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WQDKIVRHTQYJSN-DCAQKATOSA-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
- JMNRXRPBHFGXQX-GUBZILKMSA-N Lys-Ser-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JMNRXRPBHFGXQX-GUBZILKMSA-N 0.000 description 1
- ZUGVARDEGWMMLK-SRVKXCTJSA-N Lys-Ser-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN ZUGVARDEGWMMLK-SRVKXCTJSA-N 0.000 description 1
- QVTDVTONTRSQMF-WDCWCFNPSA-N Lys-Thr-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CCCCN QVTDVTONTRSQMF-WDCWCFNPSA-N 0.000 description 1
- DLCAXBGXGOVUCD-PPCPHDFISA-N Lys-Thr-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DLCAXBGXGOVUCD-PPCPHDFISA-N 0.000 description 1
- RQILLQOQXLZTCK-KBPBESRZSA-N Lys-Tyr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O RQILLQOQXLZTCK-KBPBESRZSA-N 0.000 description 1
- PSVAVKGDUAKZKU-BZSNNMDCSA-N Lys-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCCN)N)O PSVAVKGDUAKZKU-BZSNNMDCSA-N 0.000 description 1
- LMMBAXJRYSXCOQ-ACRUOGEOSA-N Lys-Tyr-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O LMMBAXJRYSXCOQ-ACRUOGEOSA-N 0.000 description 1
- SQRLLZAQNOQCEG-KKUMJFAQSA-N Lys-Tyr-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 SQRLLZAQNOQCEG-KKUMJFAQSA-N 0.000 description 1
- VKCPHIOZDWUFSW-ONGXEEELSA-N Lys-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN VKCPHIOZDWUFSW-ONGXEEELSA-N 0.000 description 1
- RPWQJSBMXJSCPD-XUXIUFHCSA-N Lys-Val-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(O)=O RPWQJSBMXJSCPD-XUXIUFHCSA-N 0.000 description 1
- OZVXDDFYCQOPFD-XQQFMLRXSA-N Lys-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N OZVXDDFYCQOPFD-XQQFMLRXSA-N 0.000 description 1
- QAHFGYLFLVGBNW-DCAQKATOSA-N Met-Ala-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN QAHFGYLFLVGBNW-DCAQKATOSA-N 0.000 description 1
- HDNOQCZWJGGHSS-VEVYYDQMSA-N Met-Asn-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HDNOQCZWJGGHSS-VEVYYDQMSA-N 0.000 description 1
- KQBJYJXPZBNEIK-DCAQKATOSA-N Met-Glu-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQBJYJXPZBNEIK-DCAQKATOSA-N 0.000 description 1
- QGRJTULYDZUBAY-ZPFDUUQYSA-N Met-Ile-Glu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O QGRJTULYDZUBAY-ZPFDUUQYSA-N 0.000 description 1
- MSSJHBAKDDIRMJ-SRVKXCTJSA-N Met-Lys-Gln Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O MSSJHBAKDDIRMJ-SRVKXCTJSA-N 0.000 description 1
- QEDGNYFHLXXIDC-DCAQKATOSA-N Met-Pro-Gln Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O QEDGNYFHLXXIDC-DCAQKATOSA-N 0.000 description 1
- WXXNVZMWHOLNRJ-AVGNSLFASA-N Met-Pro-Lys Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O WXXNVZMWHOLNRJ-AVGNSLFASA-N 0.000 description 1
- KSIPKXNIQOWMIC-RCWTZXSCSA-N Met-Thr-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KSIPKXNIQOWMIC-RCWTZXSCSA-N 0.000 description 1
- VEKRTVRZDMUOQN-AVGNSLFASA-N Met-Val-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 VEKRTVRZDMUOQN-AVGNSLFASA-N 0.000 description 1
- 108010061951 Methemoglobin Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 108010090127 Periplasmic Proteins Proteins 0.000 description 1
- BKWJQWJPZMUWEG-LFSVMHDDSA-N Phe-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BKWJQWJPZMUWEG-LFSVMHDDSA-N 0.000 description 1
- CGOMLCQJEMWMCE-STQMWFEESA-N Phe-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CGOMLCQJEMWMCE-STQMWFEESA-N 0.000 description 1
- QCHNRQQVLJYDSI-DLOVCJGASA-N Phe-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 QCHNRQQVLJYDSI-DLOVCJGASA-N 0.000 description 1
- WIVCOAKLPICYGY-KKUMJFAQSA-N Phe-Asp-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N WIVCOAKLPICYGY-KKUMJFAQSA-N 0.000 description 1
- GDBOREPXIRKSEQ-FHWLQOOXSA-N Phe-Gln-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GDBOREPXIRKSEQ-FHWLQOOXSA-N 0.000 description 1
- MPFGIYLYWUCSJG-AVGNSLFASA-N Phe-Glu-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MPFGIYLYWUCSJG-AVGNSLFASA-N 0.000 description 1
- KJJROSNFBRWPHS-JYJNAYRXSA-N Phe-Glu-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O KJJROSNFBRWPHS-JYJNAYRXSA-N 0.000 description 1
- JWQWPTLEOFNCGX-AVGNSLFASA-N Phe-Glu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 JWQWPTLEOFNCGX-AVGNSLFASA-N 0.000 description 1
- JEBWZLWTRPZQRX-QWRGUYRKSA-N Phe-Gly-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O JEBWZLWTRPZQRX-QWRGUYRKSA-N 0.000 description 1
- SWCOXQLDICUYOL-ULQDDVLXSA-N Phe-His-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SWCOXQLDICUYOL-ULQDDVLXSA-N 0.000 description 1
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 1
- KZRQONDKKJCAOL-DKIMLUQUSA-N Phe-Leu-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KZRQONDKKJCAOL-DKIMLUQUSA-N 0.000 description 1
- YCCUXNNKXDGMAM-KKUMJFAQSA-N Phe-Leu-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YCCUXNNKXDGMAM-KKUMJFAQSA-N 0.000 description 1
- PTLMYJOMJLTMCB-KKUMJFAQSA-N Phe-Met-Gln Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N PTLMYJOMJLTMCB-KKUMJFAQSA-N 0.000 description 1
- TXJJXEXCZBHDNA-ACRUOGEOSA-N Phe-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N TXJJXEXCZBHDNA-ACRUOGEOSA-N 0.000 description 1
- RBRNEFJTEHPDSL-ACRUOGEOSA-N Phe-Phe-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 RBRNEFJTEHPDSL-ACRUOGEOSA-N 0.000 description 1
- IPFXYNKCXYGSSV-KKUMJFAQSA-N Phe-Ser-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N IPFXYNKCXYGSSV-KKUMJFAQSA-N 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- BPIFSOUEUYDJRM-DCPHZVHLSA-N Phe-Trp-Ala Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(O)=O)C1=CC=CC=C1 BPIFSOUEUYDJRM-DCPHZVHLSA-N 0.000 description 1
- DBNGDEAQXGFGRA-ACRUOGEOSA-N Phe-Tyr-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCCN)C(=O)O)N DBNGDEAQXGFGRA-ACRUOGEOSA-N 0.000 description 1
- YUPRIZTWANWWHK-DZKIICNBSA-N Phe-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N YUPRIZTWANWWHK-DZKIICNBSA-N 0.000 description 1
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 1
- FZHBZMDRDASUHN-NAKRPEOUSA-N Pro-Ala-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1)C(O)=O FZHBZMDRDASUHN-NAKRPEOUSA-N 0.000 description 1
- OBVCYFIHIIYIQF-CIUDSAMLSA-N Pro-Asn-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O OBVCYFIHIIYIQF-CIUDSAMLSA-N 0.000 description 1
- VOHFZDSRPZLXLH-IHRRRGAJSA-N Pro-Asn-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VOHFZDSRPZLXLH-IHRRRGAJSA-N 0.000 description 1
- DIFXZGPHVCIVSQ-CIUDSAMLSA-N Pro-Gln-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DIFXZGPHVCIVSQ-CIUDSAMLSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- KIPIKSXPPLABPN-CIUDSAMLSA-N Pro-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 KIPIKSXPPLABPN-CIUDSAMLSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- SSWJYJHXQOYTSP-SRVKXCTJSA-N Pro-His-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O SSWJYJHXQOYTSP-SRVKXCTJSA-N 0.000 description 1
- SOACYAXADBWDDT-CYDGBPFRSA-N Pro-Ile-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SOACYAXADBWDDT-CYDGBPFRSA-N 0.000 description 1
- BBFRBZYKHIKFBX-GMOBBJLQSA-N Pro-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@@H]1CCCN1 BBFRBZYKHIKFBX-GMOBBJLQSA-N 0.000 description 1
- AQGUSRZKDZYGGV-GMOBBJLQSA-N Pro-Ile-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O AQGUSRZKDZYGGV-GMOBBJLQSA-N 0.000 description 1
- VZKBJNBZMZHKRC-XUXIUFHCSA-N Pro-Ile-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O VZKBJNBZMZHKRC-XUXIUFHCSA-N 0.000 description 1
- BCNRNJWSRFDPTQ-HJWJTTGWSA-N Pro-Ile-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BCNRNJWSRFDPTQ-HJWJTTGWSA-N 0.000 description 1
- BRJGUPWVFXKBQI-XUXIUFHCSA-N Pro-Leu-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BRJGUPWVFXKBQI-XUXIUFHCSA-N 0.000 description 1
- FKYKZHOKDOPHSA-DCAQKATOSA-N Pro-Leu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FKYKZHOKDOPHSA-DCAQKATOSA-N 0.000 description 1
- JUJCUYWRJMFJJF-AVGNSLFASA-N Pro-Lys-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 JUJCUYWRJMFJJF-AVGNSLFASA-N 0.000 description 1
- DWGFLKQSGRUQTI-IHRRRGAJSA-N Pro-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 DWGFLKQSGRUQTI-IHRRRGAJSA-N 0.000 description 1
- WCNVGGZRTNHOOS-ULQDDVLXSA-N Pro-Lys-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O WCNVGGZRTNHOOS-ULQDDVLXSA-N 0.000 description 1
- FNGOXVQBBCMFKV-CIUDSAMLSA-N Pro-Ser-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O FNGOXVQBBCMFKV-CIUDSAMLSA-N 0.000 description 1
- RSTWKJFWBKFOFC-JYJNAYRXSA-N Pro-Trp-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O RSTWKJFWBKFOFC-JYJNAYRXSA-N 0.000 description 1
- QDDJNKWPTJHROJ-UFYCRDLUSA-N Pro-Tyr-Tyr Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 QDDJNKWPTJHROJ-UFYCRDLUSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 1
- ZXLUWXWISXIFIX-ACZMJKKPSA-N Ser-Asn-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZXLUWXWISXIFIX-ACZMJKKPSA-N 0.000 description 1
- BYIROAKULFFTEK-CIUDSAMLSA-N Ser-Asp-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO BYIROAKULFFTEK-CIUDSAMLSA-N 0.000 description 1
- OLIJLNWFEQEFDM-SRVKXCTJSA-N Ser-Asp-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OLIJLNWFEQEFDM-SRVKXCTJSA-N 0.000 description 1
- HEQPKICPPDOSIN-SRVKXCTJSA-N Ser-Asp-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HEQPKICPPDOSIN-SRVKXCTJSA-N 0.000 description 1
- YQQKYAZABFEYAF-FXQIFTODSA-N Ser-Glu-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQQKYAZABFEYAF-FXQIFTODSA-N 0.000 description 1
- SNVIOQXAHVORQM-WDSKDSINSA-N Ser-Gly-Gln Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O SNVIOQXAHVORQM-WDSKDSINSA-N 0.000 description 1
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 1
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 1
- JIPVNVNKXJLFJF-BJDJZHNGSA-N Ser-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N JIPVNVNKXJLFJF-BJDJZHNGSA-N 0.000 description 1
- VZQRNAYURWAEFE-KKUMJFAQSA-N Ser-Leu-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VZQRNAYURWAEFE-KKUMJFAQSA-N 0.000 description 1
- PPNPDKGQRFSCAC-CIUDSAMLSA-N Ser-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPNPDKGQRFSCAC-CIUDSAMLSA-N 0.000 description 1
- GVMUJUPXFQFBBZ-GUBZILKMSA-N Ser-Lys-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GVMUJUPXFQFBBZ-GUBZILKMSA-N 0.000 description 1
- CRJZZXMAADSBBQ-SRVKXCTJSA-N Ser-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO CRJZZXMAADSBBQ-SRVKXCTJSA-N 0.000 description 1
- UGTZYIPOBYXWRW-SRVKXCTJSA-N Ser-Phe-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O UGTZYIPOBYXWRW-SRVKXCTJSA-N 0.000 description 1
- BUYHXYIUQUBEQP-AVGNSLFASA-N Ser-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N BUYHXYIUQUBEQP-AVGNSLFASA-N 0.000 description 1
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- PCJLFYBAQZQOFE-KATARQTJSA-N Ser-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N)O PCJLFYBAQZQOFE-KATARQTJSA-N 0.000 description 1
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- 108010016797 Sickle Hemoglobin Proteins 0.000 description 1
- 101000582398 Staphylococcus aureus Replication initiation protein Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- FQPQPTHMHZKGFM-XQXXSGGOSA-N Thr-Ala-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O FQPQPTHMHZKGFM-XQXXSGGOSA-N 0.000 description 1
- NAXBBCLCEOTAIG-RHYQMDGZSA-N Thr-Arg-Lys Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O NAXBBCLCEOTAIG-RHYQMDGZSA-N 0.000 description 1
- CEXFELBFVHLYDZ-XGEHTFHBSA-N Thr-Arg-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CEXFELBFVHLYDZ-XGEHTFHBSA-N 0.000 description 1
- SKHPKKYKDYULDH-HJGDQZAQSA-N Thr-Asn-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SKHPKKYKDYULDH-HJGDQZAQSA-N 0.000 description 1
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 description 1
- RKDFEMGVMMYYNG-WDCWCFNPSA-N Thr-Gln-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O RKDFEMGVMMYYNG-WDCWCFNPSA-N 0.000 description 1
- LAFLAXHTDVNVEL-WDCWCFNPSA-N Thr-Gln-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O LAFLAXHTDVNVEL-WDCWCFNPSA-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
- BNGDYRRHRGOPHX-IFFSRLJSSA-N Thr-Glu-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O BNGDYRRHRGOPHX-IFFSRLJSSA-N 0.000 description 1
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 1
- BVOVIGCHYNFJBZ-JXUBOQSCSA-N Thr-Leu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O BVOVIGCHYNFJBZ-JXUBOQSCSA-N 0.000 description 1
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 1
- WYLAVUAWOUVUCA-XVSYOHENSA-N Thr-Phe-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O WYLAVUAWOUVUCA-XVSYOHENSA-N 0.000 description 1
- IWAVRIPRTCJAQO-HSHDSVGOSA-N Thr-Pro-Trp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O IWAVRIPRTCJAQO-HSHDSVGOSA-N 0.000 description 1
- MFMGPEKYBXFIRF-SUSMZKCASA-N Thr-Thr-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MFMGPEKYBXFIRF-SUSMZKCASA-N 0.000 description 1
- NHQVWACSJZJCGJ-FLBSBUHZSA-N Thr-Thr-Ile 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@@H](C)CC)C(O)=O NHQVWACSJZJCGJ-FLBSBUHZSA-N 0.000 description 1
- ABCLYRRGTZNIFU-BWAGICSOSA-N Thr-Tyr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O ABCLYRRGTZNIFU-BWAGICSOSA-N 0.000 description 1
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 1
- PWONLXBUSVIZPH-RHYQMDGZSA-N Thr-Val-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O PWONLXBUSVIZPH-RHYQMDGZSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- BIJDDZBDSJLWJY-PJODQICGSA-N Trp-Ala-Val Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O BIJDDZBDSJLWJY-PJODQICGSA-N 0.000 description 1
- HJTYJQVRIQXMHM-XIRDDKMYSA-N Trp-Asp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N HJTYJQVRIQXMHM-XIRDDKMYSA-N 0.000 description 1
- DLZKEQQWXODGGZ-KWQFWETISA-N Tyr-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KWQFWETISA-N 0.000 description 1
- AYPAIRCDLARHLM-KKUMJFAQSA-N Tyr-Asn-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O AYPAIRCDLARHLM-KKUMJFAQSA-N 0.000 description 1
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 1
- RYSNTWVRSLCAJZ-RYUDHWBXSA-N Tyr-Gln-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RYSNTWVRSLCAJZ-RYUDHWBXSA-N 0.000 description 1
- SLCSPPCQWUHPPO-JYJNAYRXSA-N Tyr-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SLCSPPCQWUHPPO-JYJNAYRXSA-N 0.000 description 1
- AKLNEFNQWLHIGY-QWRGUYRKSA-N Tyr-Gly-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N)O AKLNEFNQWLHIGY-QWRGUYRKSA-N 0.000 description 1
- FIRUOPRJKCBLST-KKUMJFAQSA-N Tyr-His-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O FIRUOPRJKCBLST-KKUMJFAQSA-N 0.000 description 1
- LFCQXIXJQXWZJI-BZSNNMDCSA-N Tyr-His-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N)O LFCQXIXJQXWZJI-BZSNNMDCSA-N 0.000 description 1
- KIJLSRYAUGGZIN-CFMVVWHZSA-N Tyr-Ile-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O KIJLSRYAUGGZIN-CFMVVWHZSA-N 0.000 description 1
- GULIUBBXCYPDJU-CQDKDKBSSA-N Tyr-Leu-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 GULIUBBXCYPDJU-CQDKDKBSSA-N 0.000 description 1
- BSCBBPKDVOZICB-KKUMJFAQSA-N Tyr-Leu-Asp 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(O)=O)C(O)=O BSCBBPKDVOZICB-KKUMJFAQSA-N 0.000 description 1
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 1
- CWVHKVVKAQIJKY-ACRUOGEOSA-N Tyr-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CC=C(C=C2)O)N CWVHKVVKAQIJKY-ACRUOGEOSA-N 0.000 description 1
- PMHLLBKTDHQMCY-ULQDDVLXSA-N Tyr-Lys-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMHLLBKTDHQMCY-ULQDDVLXSA-N 0.000 description 1
- PSALWJCUIAQKFW-ACRUOGEOSA-N Tyr-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N PSALWJCUIAQKFW-ACRUOGEOSA-N 0.000 description 1
- QPOUERMDWKKZEG-HJPIBITLSA-N Tyr-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 QPOUERMDWKKZEG-HJPIBITLSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- AKRHKDCELJLTMD-BVSLBCMMSA-N Tyr-Trp-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N AKRHKDCELJLTMD-BVSLBCMMSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- SQUMHUZLJDUROQ-YDHLFZDLSA-N Tyr-Val-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O SQUMHUZLJDUROQ-YDHLFZDLSA-N 0.000 description 1
- ABSXSJZNRAQDDI-KJEVXHAQSA-N Tyr-Val-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ABSXSJZNRAQDDI-KJEVXHAQSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- LIQJSDDOULTANC-QSFUFRPTSA-N Val-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N LIQJSDDOULTANC-QSFUFRPTSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- PVPAOIGJYHVWBT-KKHAAJSZSA-N Val-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N)O PVPAOIGJYHVWBT-KKHAAJSZSA-N 0.000 description 1
- VUTHNLMCXKLLFI-LAEOZQHASA-N Val-Asp-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N VUTHNLMCXKLLFI-LAEOZQHASA-N 0.000 description 1
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 1
- YODDULVCGFQRFZ-ZKWXMUAHSA-N Val-Asp-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YODDULVCGFQRFZ-ZKWXMUAHSA-N 0.000 description 1
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 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
- VLDMQVZZWDOKQF-AUTRQRHGSA-N Val-Glu-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N VLDMQVZZWDOKQF-AUTRQRHGSA-N 0.000 description 1
- SZTTYWIUCGSURQ-AUTRQRHGSA-N Val-Glu-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SZTTYWIUCGSURQ-AUTRQRHGSA-N 0.000 description 1
- VCAWFLIWYNMHQP-UKJIMTQDSA-N Val-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N VCAWFLIWYNMHQP-UKJIMTQDSA-N 0.000 description 1
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 1
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-N 0.000 description 1
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 1
- KZKMBGXCNLPYKD-YEPSODPASA-N Val-Gly-Thr Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O KZKMBGXCNLPYKD-YEPSODPASA-N 0.000 description 1
- VXDSPJJQUQDCKH-UKJIMTQDSA-N Val-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N VXDSPJJQUQDCKH-UKJIMTQDSA-N 0.000 description 1
- HGJRMXOWUWVUOA-GVXVVHGQSA-N Val-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N HGJRMXOWUWVUOA-GVXVVHGQSA-N 0.000 description 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 1
- ZHQWPWQNVRCXAX-XQQFMLRXSA-N Val-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZHQWPWQNVRCXAX-XQQFMLRXSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 1
- WBAJDGWKRIHOAC-GVXVVHGQSA-N Val-Lys-Gln Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O WBAJDGWKRIHOAC-GVXVVHGQSA-N 0.000 description 1
- IJGPOONOTBNTFS-GVXVVHGQSA-N Val-Lys-Glu Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O IJGPOONOTBNTFS-GVXVVHGQSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- YMTOEGGOCHVGEH-IHRRRGAJSA-N Val-Lys-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O YMTOEGGOCHVGEH-IHRRRGAJSA-N 0.000 description 1
- VPGCVZRRBYOGCD-AVGNSLFASA-N Val-Lys-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O VPGCVZRRBYOGCD-AVGNSLFASA-N 0.000 description 1
- UEPLNXPLHJUYPT-AVGNSLFASA-N Val-Met-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O UEPLNXPLHJUYPT-AVGNSLFASA-N 0.000 description 1
- GQMNEJMFMCJJTD-NHCYSSNCSA-N Val-Pro-Gln Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O GQMNEJMFMCJJTD-NHCYSSNCSA-N 0.000 description 1
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 1
- UVHFONIHVHLDDQ-IFFSRLJSSA-N Val-Thr-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O UVHFONIHVHLDDQ-IFFSRLJSSA-N 0.000 description 1
- SUGRIIAOLCDLBD-ZOBUZTSGSA-N Val-Trp-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SUGRIIAOLCDLBD-ZOBUZTSGSA-N 0.000 description 1
- VTIAEOKFUJJBTC-YDHLFZDLSA-N Val-Tyr-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VTIAEOKFUJJBTC-YDHLFZDLSA-N 0.000 description 1
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 1
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 108010080488 arginyl-arginyl-leucine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010059898 glycyl-tyrosyl-lysine Proteins 0.000 description 1
- 108010020688 glycylhistidine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 208000034737 hemoglobinopathy Diseases 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 108010087810 leucyl-seryl-glutamyl-leucine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108010074082 phenylalanyl-alanyl-lysine Proteins 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010060175 trypsinogen activation peptide Proteins 0.000 description 1
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 1
- 108010012567 tyrosyl-glycyl-glycyl-phenylalanyl Proteins 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0381—Animal model for diseases of the hematopoietic system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Diabetes (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Endocrinology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for constructing sickle cell anemia model pig nuclear transplantation donor cells with HBB gene mutation and application thereof. The invention provides application of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein in preparation of a kit. The application of the kit is as follows: preparing a recombinant cell; preparing sickle cell anemia model pigs; preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model. HBB-gU1 is sgRNA, and the binding region of the target sequence is shown as SEQ ID NO:18 from nucleotide 3 to nucleotide 22. HBB-gD3 is sgRNA, and the binding region of the target sequence is shown in SEQ ID NO:19 at nucleotides 3 to 22. HBB-mutant-ss174 is SEQ ID NO:20 in the sequence listing. The NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein. The invention has great application value for researching and developing sickle cell anemia drugs and disclosing the pathogenesis of the sickle cell anemia drugs.
Description
Technical Field
The invention belongs to the technical field of biology, particularly belongs to the technical field of gene editing, and more particularly relates to a method for constructing HBB gene mutated sickle cell anemia model pig nuclear transplantation donor cells by applying a CRISPR/Cas9 system and an ssoDN homologous recombination technology and application thereof.
Background
Sickle cell anemia is a global high-incidence hemoglobinopathy, and when the red blood cells of a patient lack oxygen, the red blood cells become sickle-shaped (normally in a disc shape) and lose the function of oxygen delivery. The disease is clinically manifested by chronic hemolytic anemia, susceptibility to infection and recurrent pain, so that chronic ischemia causes organ and tissue damage and even death. Sickle cell anemia is common in africa and american blacks, also in mediterranean coastal countries such as middle east, greek, italy, and the like, and india, indian, and the like, and cases are also found in southern China, and the disease incidence range is widely distributed.
Hemoglobin consists of a ferrihemoglobin ring and four globin chains: two alpha chains and two non-alpha chains. The composition of the four globin chains determines the type of haemoglobin: fetal hemoglobin (HbF) consists of two alpha chains and two gamma chains (alpha 2-gamma 2), adult hemoglobin a (HbA) consists of two alpha chains and two beta chains (alpha 2 beta 2), and hemoglobin A2 (HbA 2) consists of two alpha chains and two delta chains (alpha 2 delta 2). At birth, hbF accounts for approximately 80% of hemoglobin and HbA accounts for approximately 20%. The transition from gamma-globin synthesis (HbF) to beta-globin synthesis (HbA) starts before birth. At about 6 months of age, hemoglobin in healthy infants will shift to contain primarily HbA, a small amount of HbA2 and negligible HbF.
Sickle cell anemia is an autosomal recessive genetic disease, the 6 th amino acid of a hemoglobin beta-peptide chain (coded by HBB gene) of a patient is mutated from glutamic acid to valine, the formed sickle hemoglobin (HbS) replaces normal HbA, hbS intermolecular interaction is changed into a spiral polymer with low solubility when the oxygen partial pressure is reduced, and red blood cells are distorted into sickle cells, namely sickle cells. Most often, both parents have an abnormal gene called homozygous SS, type SS has no normal HbA at all, more than 80% of the normal HbA is replaced by HbS, and type SS has a severe prognosis, with only 14% of the patients living into adults and most of the patients dying before the age of 30 years. The heterozygote type, normal HbA mixed with abnormal HbS, is also called AS. In Africa, 35% of patients have AS type genes, which vary widely, and in the mild case, the adult can live, and in the severe case, crisis attacks can occur repeatedly.
The initial stage of erythroid sickling is reversible, giving oxygen to reverse the sickling process. However, when the sickle has severely damaged the red blood cell membrane, the sickle becomes irreversible and the red blood cells remain sickle even when the cells are put under aerobic conditions. Sickle red blood cells are rigid, poorly deformable, and can be destroyed by vascular mechanisms and phagocytosed by mononuclear macrophages to cause hemolysis. The sickle red blood cells also increase blood viscosity, slow blood flow, poor deformability, easy blockage of capillaries, local hypoxia and inflammatory reaction, and pain in corresponding parts, which are commonly seen in muscles, bones, joints of four limbs, chest and abdomen, especially joints and chest and abdomen.
At present, no definite therapy or medicine can be used for treating sickle cell anemia. The research on the molecular mechanism of sickle cell anemia caused by HBB mutation and the research and development of corresponding medicaments are both required to be carried out on the basis of animal models. The current common animal model is a mouse model, however, the mouse is different from the human body in the aspects of body type, organ size, physiology, pathology and the like, and the normal physiological and pathological states of the human body cannot be simulated really. The pig as a large animal is a main meat food supply animal for human for a long time, the size and physiological function of the pig are similar to those of the human, the pig is easy to breed and feed in a large scale, and the pig is low in requirements on ethics, animal protection and the like and is an ideal human disease model animal.
Gene editing is a biotechnology that has been under significant development in recent years, and includes editing technologies from homologous recombination-based gene editing to nuclease-based ZFNs, TALENs, CRISPR/Cas9, and the like, where CRISPR/Cas9 technology is one of the most advanced gene editing technologies at present. Currently, gene editing techniques are increasingly applied to the production of animal models.
Homologous recombination (HDR) is the exchange of DNA sequence information by sequence homology: that is, the repair template contains the desired insert, and the recombination arms with sequence homology near the insertion site are at both ends of the repair template. In the past, double-stranded DNA (dsDNA) was commonly used as a repair template, but recent studies have revealed the superiority of single-stranded oligodeoxynucleotides (ssodns) as HDR donor templates. First, ssODN is more specific as a donor template than the insertion site of a dsDNA template, which is susceptible to random insertions. Second, ssODN requires shorter lengths for homologous recombination arms than dsDNA templates, and 30-60 base-on-a-side recombination arm designs can yield highly efficient and stable HDR, which provides higher insertion efficiency than similar dsDNA templates. Third, dsDNA is easily incorporated by the NHEJ repair pathway, resulting in duplication of the homology arms or partial integration of the dsDNA template, which is not easily produced by ssODN. In addition, dsDNAs are detrimental to the cultured cells, have low transfection efficiencies for linear or plasmid dsDNAs, and cause adverse reactions in the cells, and the ssODN templates are more advantageous in these respects.
Disclosure of Invention
The invention aims to provide a method for constructing a sickle cell anemia model pig nuclear transplantation donor cell with HBB gene mutation and application thereof.
The invention provides application of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein in preparation of a kit.
The invention also provides application of the HBB-gU1, HBB-gD3, HBB-mutant-ss174 and PRONCN proteins in preparation of the kit.
The invention also provides application of the HBB-gU1, the HBB-gD3, the HBB-mutant-ss174 and the idiosyncratic particles in preparation of the kit.
The invention also provides a kit which comprises HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein.
The invention also provides a kit which comprises HBB-gU1, HBB-gD3, HBB-mutant-ss174 and PRONCN protein.
The invention also provides a kit which comprises HBB-gU1, HBB-gD3, HBB-mutant-ss174 and specific plasmids.
Any of the kits above further comprising porcine cells.
The use of any one of the above kits is (a) or (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
The invention also provides a method for preparing recombinant cells, which comprises the following steps: using the nucleotide sequence of SEQ ID NO:20 to substitute the DNA molecule shown in SEQ ID NO:21 to obtain a recombinant cell.
Using the nucleotide sequence of SEQ ID NO:20 to substitute the DNA molecule shown in SEQ ID NO:21 is realized by the following steps: HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein were co-transfected into porcine cells.
The co-transfection is specifically a shock transfection mode.
The parameters for electroporation transfection may specifically be: 1450V, 10ms, 3pulse.
The co-transfection may be specifically carried out using a mammalian nuclear transfection kit (Neon kit, thermofoisher) and a Neon TM transfection system electrotransfer apparatus.
The proportions of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein are as follows in sequence: 0.8-1.2. Mu.g HBB-gU1: 0.8-1.2. Mu.g HBB-gD3: 1.8-2.2. Mu.g HBB-mutant-ss174: 3-5. Mu.g NCN protein.
The proportions of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein are as follows in sequence: 1. Mu.g HBB-gU1:1 μ g HBB-gD3:2 μ g HBB-mutant-ss174: mu.g NCN protein.
The proportions of the pig cells, the HBB-gU1, the HBB-gD3, the HBB-mutant-ss174 and the NCN protein are as follows in sequence: 10 ten thousand porcine cells: 0.8-1.2. Mu.g HBB-gU1: 0.8-1.2. Mu.g HBB-gD3: 1.8-2.2. Mu.g HBB-mutant-ss174: 3-5. Mu.g NCN protein.
The proportions of the pig cells, the HBB-gU1, the HBB-gD3, the HBB-mutant-ss174 and the NCN protein are as follows in sequence: 10 ten thousand porcine cells: 1. Mu.g HBB-gU1:1 μ g HBB-gD3:2 μ g HBB-mutant-ss174: mu.g NCN protein.
Any one of the HBB-gU1 is sgRNA, and a target sequence binding region of the sgRNA is shown in SEQ ID NO:18 from nucleotide 3 to nucleotide 22.
Any one of the HBB-gD3 is sgRNA, and the target sequence binding region is shown as SEQ ID NO:19 at nucleotides 3 to 22.
Any one of the HBB-mutant-ss174 is SEQ ID NO:20, or a single-stranded DNA molecule as set forth in fig. 20.
The NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein.
Specifically, the NCN protein is shown as SEQ ID NO:3, respectively.
Specifically, the HBB-gU1 is shown as SEQ ID NO:18, respectively.
Specifically, the HBB-gD3 is shown as SEQ ID NO:19, respectively.
Specifically, the HBB-gU1 is shown as SEQ ID NO: shown at 10.
Specifically, the HBB-gD3 is shown as SEQ ID NO: shown at 15.
Any one of the above porcine cells is a porcine fibroblast.
Any of the above porcine cells are porcine primary fibroblasts.
The preparation method of the NCN protein comprises the following steps:
(1) Introducing the plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain recombinant bacteria;
(2) Culturing the recombinant bacteria by adopting a liquid culture medium at 30 ℃, then adding IPTG (isopropyl-beta-D-thiogalactoside) and carrying out induced culture at 25 ℃, and then collecting bacteria;
(3) Crushing the collected thalli, and collecting a crude protein solution;
(4) Purification of the crude protein solution with His by affinity chromatography 6 A fusion protein of the tag;
(5) By using a compound having His 6 Tagged enterokinase cleavage with His 6 The tagged fusion protein was then removed with His using Ni-NTA resin 6 A tagged protein, resulting in a purified NCN protein;
plasmid pKG-GE4 has the sequence shown in SEQ ID NO:1, 5209 to 9852 th nucleotide.
The preparation method of the NCN protein specifically comprises the following steps:
(1) The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.
(2) Inoculating the recombinant bacteria obtained in the step (1) to a liquid LB culture medium containing ampicillin, and carrying out shake culture;
(3) Inoculating the bacterial liquid obtained in the step (2) to a liquid LB culture medium, and carrying out shaking culture at 30 ℃ and 230rpm until the bacterial liquid is OD 600nm The value =1.0, then IPTG was added so that the concentration thereof in the system became 0.5mM, followed by shaking culture at 230rpm at 25 ℃ for 12 hours, and then the cells were collected by centrifugation;
(4) Taking the thalli obtained in the step (3), and washing the thalli with a PBS (phosphate buffer solution);
(5) Adding the crude extraction buffer solution into the thalli obtained in the step (4), suspending the thalli, then crushing the thalli, then centrifugally collecting supernate, filtering by adopting a filter membrane with the aperture of 0.22 mu m, and collecting filtrate;
(6) Purifying the filtrate obtained in step (5) by affinity chromatography to obtain a purified product having His 6 A fusion protein of the tag (a fusion protein shown in SEQ ID NO: 2);
(7) Taking the post-column solution collected in the step (6), concentrating by using an ultrafiltration tube, and then diluting with 25mM Tris-HCl (pH8.0);
(8) Will have His 6 Adding the labeled recombinant bovine enterokinase into the solution obtained in the step (7), and performing enzyme digestion;
(9) Mixing the solution obtained in the step (8) with Ni-NTA resin, incubating, and centrifuging to collect supernatant;
(10) And (5) taking the supernatant obtained in the step (9), concentrating by using an ultrafiltration tube, and then adding the supernatant into an enzyme stock solution to obtain the NCN protein solution.
Purifying the filtrate obtained in step (5) by affinity chromatography to obtain a purified product having His 6 The specific method of the tagged fusion protein is as follows:
firstly, balancing a Ni-NTA agarose column by using a balance solution with 5 column volumes (the flow rate is 1 ml/min); then, 50ml of the filtrate obtained in the step (5) is loaded (the flow rate is 0.5-1 ml/min); the column was then washed with 5 column volumes of equilibration solution (flow rate 1 ml/min); the column was then washed with 5 column volumes of buffer (flow rate 1 ml/min) to remove contaminating proteins; then eluting with 10 column volumes of eluent at a flow rate of 0.5-1ml/min, and collecting the solution (90-100 ml) after passing through the column.
Any one of the PRONCN proteins sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, cas9 protein, nuclear localization signal.
The signal peptide has the function of promoting protein secretion expression. The signal peptide may be selected from the group consisting of the escherichia coli alkaline phosphatase (phoA) signal peptide, the staphylococcus aureus protein a signal peptide, the escherichia coli outer membrane protein (ompa) signal peptide or the signal peptide of any other prokaryotic gene, preferably the alkaline phosphatase signal peptide (phoA signal peptide). The signal peptide of alkaline phosphatase is used to guide the secretory expression of the target protein into the bacterial periplasm cavity so as to be separated from the protein in the bacterial cell, and the target protein secreted into the bacterial periplasm cavity is soluble expression and can be cleaved by the signal peptidase in the bacterial periplasm cavity.
The chaperone protein functions to increase the solubility of the protein. The chaperone may be any protein that helps to form disulfide bonds, preferably a thioredoxin (TrxA protein). The thioredoxin can be used as a molecular chaperone to help a co-expressed target protein (such as a Cas9 protein) to form a disulfide bond, so that the stability and the folding correctness of the protein are improved, and the solubility and the activity of the target protein are increased.
The protein tag functions for protein purification. The Tag can be His Tag (His-Tag, his) 6 Protein tag), GST tag, flag tag, HA tag, c-Myc tag, or any other protein tag, more preferably His tag. The His tag can be combined with a Ni column, and the target protein can be purified by one-step Ni column affinity chromatography, so that the purification process of the target protein can be greatly simplified.
The protease cleavage site functions to cleave non-functional segments after purification to release the native form of the Cas9 protein. The protease may be selected from Enterokinase (Enterokinase), factor Xa (Factor Xa), thrombin (thrombobin), TEV protease (TEV protease), HRV 3C protease (HRV 3C protease), WELQut protease or any other endoprotease, further preferably Enterokinase. EK is an enterokinase enzyme cutting site, so that fused TrxA-His segment can be conveniently cut by enterokinase to obtain the Cas9 protein in a natural form. After the fusion protein is digested by using the commercial enterokinase with the His label, the TrxA-His section and the enterokinase with the His label can be removed through one-time affinity chromatography to obtain the Cas9 protein in a natural form, so that the damage and loss of target protein caused by multiple times of purification and dialysis are avoided.
The nuclear localization signal may be any nuclear localization signal, preferably an SV40 nuclear localization signal and/or a nucleocapsin nuclear localization signal. NLS is a nuclear localization signal, and NLS sites are respectively designed at the N end and the C end of Cas9, so that Cas9 can more effectively enter a cell nucleus for gene editing.
The Cas9 protein may be saCas9 or spCas9, preferably is a spCas9 protein.
The PRONCN protein is specifically shown as SEQ ID NO:2, respectively.
Any one of the above specific plasmids sequentially comprises the following elements from upstream to downstream: promoter, operator, ribosome binding site, PRONCN protein coding gene and terminator.
The promoter may specifically be a T7 promoter. The T7 promoter is a prokaryotic expression strong promoter and can efficiently drive the expression of exogenous genes.
The operon may specifically be a Lac operon. The Lac operon is a regulatory element for lactose-induced expression, and IPTG can be used for inducing the expression of the target protein at low temperature after bacteria grow to a certain amount, so that the influence of the premature expression of the target protein on the growth of host bacteria can be avoided, and the solubility of the expressed target protein can be obviously improved by inducing expression at low temperature.
The ribosome binding site is a ribosome binding site for translation of a protein, and is essential for translation of a protein.
The terminator may specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the end of the target gene, and prevent other downstream sequences except the target gene from being transcribed and translated.
For the codon of the spCas9 protein, the codon is optimized, so that the codon preference of the escherichia coli high-efficiency expression strain E.coli BL21 (DE 3) selected by the application is completely adapted, and the expression level of the Cas9 protein is improved.
The T7 promoter is shown as SEQ ID NO:1 from nucleotide 5121 to nucleotide 5139.
The Lac operon is shown as SEQ ID NO:1 from nucleotide 5140 to nucleotide 5164.
The ribosome binding site is shown as SEQ ID NO:1, nucleotides 5178 to 5201.
The coding sequence of the alkaline phosphatase signal peptide is shown as SEQ ID NO:1, nucleotides 5209-5271.
The coding sequence of the TrxA protein is shown as SEQ ID NO:1, nucleotides 5272-5598.
The coding sequence of His-Tag is shown in SEQ ID NO:1 from nucleotide 5620 to 5637.
The coding sequence of the enterokinase enzyme cutting site is shown as SEQ ID NO:1 from nucleotide 5638 to nucleotide 5652.
The coding sequence of the nuclear localization signal is shown as SEQ ID NO:1, nucleotides 5656-5670.
The coding sequence of the spCas9 protein is shown in SEQ ID NO:1, nucleotides 5701-9801.
The coding sequence of the nuclear localization signal is shown as SEQ ID NO:1, nucleotides 9802 to 9849.
The T7 terminator is shown as SEQ ID NO: nucleotides 9902-9949 of 1.
Specifically, the specific plasmid is plasmid pKG-GE4.
Plasmid pKG-GE4 has the sequence shown in SEQ ID NO:1, nucleotides 5121-9949.
Specifically, any one of the plasmids pKG-GE4 is shown as SEQ ID NO:1 is shown.
The invention also protects the recombinant cell prepared by any one of the methods.
The invention also protects the application of the recombinant cell in preparing sickle cell anemia model pigs.
The recombinant cell is used as a nuclear transfer donor cell to clone somatic cells, so that a cloned pig, namely a sickle cell anemia model pig can be obtained.
The invention also protects pig tissues of the model pig prepared by the recombinant cells, namely a sickle cell anemia tissue model.
The invention also protects a pig organ of a model pig prepared by the recombinant cell, namely a sickle cell anemia organ model.
The invention also protects pig cells of a model pig prepared by the recombinant cells, namely a sickle cell anemia cell model.
The invention also protects the application of the recombinant cell, the sickle-cell anemia tissue model, the sickle-cell anemia organ model, the sickle-cell anemia cell model or the sickle-cell anemia model pig, which is (d 1) or (d 2) or (d 3) or (d 4) as follows:
(d1) Screening a medicament for treating sickle cell anemia;
(d2) Evaluating the drug effect of the sickle cell anemia drug;
(d3) Evaluating the curative effect of gene therapy and/or cell therapy of sickle cell anemia;
(d4) The pathogenesis of sickle cell anemia was studied.
Any one of the above pigs may be a fragrant pig from Yangjiang.
Any one of the sickle cell anemia can be sickle cell anemia caused by HBB gene mutation.
Any one of the sickle-cell anemia can be sickle-cell anemia caused by E6V mutation (the codon for coding the 6 th amino acid residue is mutated from glutamic acid codon to valine codon) of HBB gene.
Pig HBB gene information: encodes a hemoglobin β subunit; is located on chromosome 9; geneID was 407066, sus scrofa. The amino acid sequence of the protein coded by the pig HBB gene is shown in SEQ ID NO. 8. The pig HBB gene has the nucleotide sequence shown in SEQ ID NO:9, or a fragment thereof.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The subject of the invention (pig) has better applicability than other animals (rats, mice, primates).
Rodents such as rats and mice have great differences from humans in body types, organ sizes, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that over 95% of drugs validated to be effective in large mice are not effective in human clinical trials. In large animals, primates are animals that have a close relationship with humans, but are small in size, late in sexual maturity (mating starts at age 6-7), and are single-birth animals, and the population propagation speed is extremely slow, and the raising cost is high. In addition, primate cloning efficiency is low, difficulty is high, and cost is high.
However, pigs, which are animals related to humans other than primates, do not have the above-mentioned disadvantages, and have body types, body weights, organ sizes, and the like similar to those of humans, and are very similar to those of humans in terms of anatomy, physiology, immunology, nutritional metabolism, disease pathogenesis, and the like. Meanwhile, the pigs have early sexual maturity (4-6 months), high reproductive capacity and multiple piglets in one birth, and can form a large group within 2-3 years. In addition, the cloning technology of the pig is very mature, and the cloning and breeding cost is much lower than that of the primate. Pigs are therefore very suitable animals as models for human disease.
(2) The vector constructed by the invention uses a strong promoter T7-lac which can efficiently express the target protein to express the target protein, and uses a signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) to guide the secretion and expression of the target protein to a bacterial periplasm cavity, so that the target protein is separated from the bacterial intracellular protein and is expressed in a soluble way. Meanwhile, the thioredoxin TrxA and the Cas9 protein are fused and expressed, the TrxA can help the coexpressed target protein to form a disulfide bond, the stability and the folding correctness of the protein are improved, and the solubility and the activity of the target protein are increased. In order to facilitate the purification of the target protein, the His label is designed, and the target protein can be purified through one-step Ni column affinity chromatography, so that the purification process of the target protein is greatly simplified. Meanwhile, an enterokinase enzyme cutting site is designed behind the His tag, so that the fused TrxA-His polypeptide fragment can be conveniently cut off, and the Cas9 protein in a natural form can be obtained. After the fusion protein is digested by using the enterokinase with the His tag, the TrxA-His polypeptide fragment and the enterokinase with the His tag can be removed through one-time affinity chromatography to obtain the Cas9 protein in a natural form, so that the damage and loss of the target protein caused by multiple times of purification dialysis are avoided. Meanwhile, the invention also designs an NLS site at the N end and the C end of the Cas9 respectively, so that the Cas9 can enter the cell nucleus more effectively for gene editing. In addition, the E.coli BL21 (DE 3) strain is selected as a target protein expression strain, and the strain can efficiently express and clone the exogenous gene of an expression vector (such as pET-32 a) containing a bacteriophage T7 promoter. Meanwhile, as for the codon of the Cas9 protein, the codon optimization is carried out, so that the codon preference of the expression strain is completely adapted, and the expression level of the target protein is improved. In addition, after the bacteria grow to a certain amount, IPTG is used for inducing the expression of the target protein at low temperature, so that the influence of the premature expression of the target protein on the growth of host bacteria can be avoided, and the solubility of the expressed target protein is also obviously improved by inducing the expression at low temperature. Through the optimization design and experimental implementation of the above items, the activity of the obtained Cas9 protein is significantly improved compared with that of a commercial Cas9 protein.
(3) The constructed and expressed Cas9 efficient protein is combined with the gRNA transcribed in vitro to carry out gene editing, the optimal dosage ratio of Cas9 and gRNA is optimized, and the synthesized ssODN is matched to be used as Donor DNA, so that the single cell cloning rate of the target site point mutation is up to 17.5 percent and the point mutation efficiency is far higher than the conventional point mutation efficiency (less than 5 percent).
(4) The cloned pig containing target gene point mutation can be directly obtained by cloning somatic cell nuclear transfer animals by using the obtained target gene point mutation unicellular cloned strain, and the mutation can be stably inherited.
The method for embryo transplantation after injecting gene editing materials into fertilized eggs in the mouse model making is not suitable for making large animal (such as pig) models with longer gestation period because the probability of directly obtaining point mutation offspring is very low (less than 1 percent) and the offspring hybridization breeding is needed. Therefore, the method adopts the primary cell in-vitro editing with great technical difficulty and high challenge, the ssODN homologous recombination and the screening positive editing single cell clone, and directly obtains the corresponding disease model pig through the somatic cell nuclear transfer animal cloning technology in the later stage, thereby greatly shortening the model pig manufacturing period and saving manpower, material resources and financial resources.
The invention adopts CRISPR/Cas9 technology combined with ssODN homologous recombination technology to carry out site-directed mutagenesis of HBB gene, obtains single cell clone of HBB gene site-directed mutagenesis, and lays a foundation for breeding sickle cell anemia disease model pigs by somatic cell nuclear transfer animal cloning technology in later period. The model pig provides a powerful experimental tool for researching pathogenesis of sickle cell anemia and drug research and development.
The invention lays a solid foundation for obtaining sickle cell anemia model pigs with HBB gene mutation by means of gene editing, is helpful for researching and disclosing the pathogenesis of sickle cell anemia caused by HBB gene mutation, can also be used for researching drug screening, drug effect detection, gene therapy, cell therapy and the like, can provide effective experimental data for further clinical application, and further provides a powerful experimental means for successfully treating human sickle cell anemia. The invention has great application value for researching and developing sickle cell anemia drugs and disclosing the pathogenesis of the sickle cell anemia drugs.
Drawings
FIG. 1 is a schematic diagram of the structure of plasmid pET-32 a.
FIG. 2 is a schematic diagram of the structure of plasmid pKG-GE4.
FIG. 3 is an electrophoretogram of optimized dosage ratio of gRNA and NCN protein in example 3.
Fig. 4 is an electrophoretogram comparing gene editing efficiency of NCN protein and a commercial Cas9 protein in example 3.
FIG. 5 is an electrophoretogram of PCR amplification using different primer pairs using the extracted genome of ear tissue of pig designated as 1 as a template in example 4.
FIG. 6 is an electrophoretogram of PCR amplification using primer pairs consisting of HBB-E1-F287 and HBB-E1-R789 in example 4 using genomic DNA of 18 pigs, respectively, as a template.
FIG. 7 is an electropherogram comparing the editing efficiency of different targets in example 4.
FIG. 8 is an electrophoretogram in example 5.
FIG. 9 is a forward and reverse sequencing of single cell clone numbered 1 simultaneously aligned with the target site wild type sequence.
FIG. 10 is a forward and reverse sequencing of single cell clone numbered 4 simultaneously aligned with the target site wild type sequence.
FIG. 11 is a forward and reverse sequencing of single cell clone numbered 5 aligned simultaneously with the target site wild type sequence.
FIG. 12 is a forward and reverse sequencing of single cell clone numbered 7 aligned simultaneously with the target site wild type sequence.
FIG. 13 is a forward and reverse sequencing of single cell clone numbered 9 aligned simultaneously with the target site wild type sequence.
FIG. 14 is a forward and reverse sequencing of single cell clone numbered 2 aligned simultaneously with the target site wild type sequence.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The recombinant plasmids constructed in the examples were all sequence verified. The commercial Cas9-A protein is commercially available the Cas9 protein with good effect. The commercial Cas9-B protein is a commercial Cas9 protein with good effect. Complete culture broth (% by volume): 15% fetal bovine serum (Gibco) +83% DMEM medium (Gibco) +1% Penicilin-Streptomyces (Gibco) +1% HEPES (Solarbio). Cell culture conditions: 37 ℃ C., 5% CO 2 、5%O 2 The constant temperature incubator.
The porcine primary fibroblasts used in the examples were all prepared from porcine ear tissue, which was freshly obtained from Jiangxiang pigs. The method for preparing the primary pig fibroblasts comprises the following steps: (1) taking 0.5g of pig ear tissue, removing hair and bone tissue, soaking in 75% alcohol for 30-40s, washing with PBS buffer containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) for 5 times, and washing with PBS buffer for one time; (2) shearing the tissue with scissors, digesting with 5mL of 0.1% collagenase solution (Sigma) at 37 ℃ for 1h, centrifuging at 500g for 5min, and discarding the supernatant; (3) resuspending the precipitate with 1mL of complete culture solution, spreading into a 10cm diameter cell culture dish containing 10mL of complete culture solution and sealed with 0.2% gelatin (VWR), and culturing until the bottom of the dish is 60% full of cells; (4) after completion of step (3), the cells were digested with trypsin and collected, and then resuspended in complete medium. Used for carrying out subsequent electrotransfer experiments.
Example 1 construction of plasmids
1. Construction of prokaryotic Cas9 high-efficiency expression vector
The structure of plasmid pET-32a is schematically shown in FIG. 1.
The plasmid pKG-GE4 is obtained by modifying a plasmid pET-32a serving as a starting plasmid. Plasmid pET32a-T7lac-phoA SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (plasmid pKG-GE4 for short) as shown in SEQ ID NO:1, which is circular plasmid, and the structural schematic diagram is shown in figure 2.
The amino acid sequence of SEQ ID NO:1, the 5121-5139 th nucleotide constitutes T7 promoter, the 5140-5164 th nucleotide encodes Lac operator (Lac operator), the 5178-5201 th nucleotide constitutes Ribosome Binding Site (RBS), the 5209-5271 th nucleotide encodes alkaline phosphatase signal peptide (phoA signal peptide), the 5272-5598 th nucleotide encodes TrxA protein, the 5620-567 th nucleotide encodes His-Tag, the 5638-5652 th nucleotide encodes enterokinase cleavage site (EK cleavage site), the 5656-5670 th nucleotide encodes nuclear localization signal, the 5701-9801 th nucleotide encodes SPCas9 protein, the 9802-9849 th nucleotide encodes nuclear localization signal, and the 9902-9949 th nucleotide constitutes T7 terminator. The nucleotides encoding the spCas9 protein have been codon optimized for the e.coli BL21 (DE 3) strain.
The main modifications of plasmid pKG-GE4 are as follows: (1) the encoding region of the TrxA protein is reserved, and the TrxA protein can help the expressed target protein to form a disulfide bond and increase the solubility and the activity of the target protein; adding a coding sequence of an alkaline phosphatase signal peptide before a coding region of the TrxA protein, wherein the alkaline phosphatase signal peptide can guide the expressed target protein to be secreted into the periplasmic cavity of the bacteria and can be cut by prokaryotic periplasmic signal peptidase; (2) adding a coding sequence of His-Tag after the coding sequence of the TrxA protein, wherein the His-Tag can be used for enriching the expressed target protein; (3) adding the coding sequence of an enterokinase enzyme cutting site DDDDK (Asp-Asp-Asp-Asp-Lys) at the downstream of the coding sequence of the His-Tag, and removing the His-Tag and the upstream fused TrxA protein by the purified protein under the action of enterokinase; (4) the Cas9 gene which is suitable for being expressed by an escherichia coli BL21 (DE 3) strain after codon optimization is inserted, and meanwhile, the nuclear localization signal coding sequence is added at the upstream and the downstream of the gene, so that the nuclear localization capability of the Cas9 protein purified at the later stage is improved.
The fusion gene in plasmid pKG-GE4 is shown in SEQ ID NO:1, nucleotide 5209-9852 of SEQ ID NO:2 (fusion protein TrxA-His-EK-NLS-spCas9-NLS, abbreviated as PRONCN protein). Due to the presence of the alkaline phosphatase signal peptide and the enterokinase cleavage site, the fusion protein is cleaved by enterokinase to form SEQ ID NO:3, and SEQ ID NO:3 is designated as NCN protein.
2. Plasmid pKG-GE3
Plasmid pKG-GE3, a circular plasmid, as described in patent application 202010084343.6, SEQ ID NO:2, respectively. SEQ ID NO:2, the nucleotide at positions 395 to 680 constitutes CMV enhancer, the nucleotide at positions 682 to 890 constitutes EF1a promoter, the nucleotide at positions 986 to 1006 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 1016 to 1036 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 1037 to 5161 encodes Cas9 protein, the nucleotide at positions 5162 to 5209 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 5219 to 5266 encodes a Nuclear Localization Signal (NLS), the nucleotide at positions 5276 to 5332 encodes self-cleaving polypeptide P2A (the amino acid sequence of self-cleaving polypeptide P2A is "ATNFSLSLLKKQAKGDAKGDVEENPGP", the position of self-cleaving is between the first and second amino acid residues from the C-terminus of the sequence), the nucleotide at positions 5333 to 6046 encodes EGFP protein, the nucleotide at positions 6056 to 539 encodes self-cleaving polypeptide T2A (the amino acid sequence of self-cleaving polypeptide T2A is "EGSLRGSLRGPLGVEGDVEGFENP", the nucleotide at positions 73610739 and the nucleotide at positions 7373769 to 677647), the nucleotide at positions WPBYb 6747 encodes the nucleotide sequence (the nucleotide sequence of the nucleotide at positions WPSbSLRGBW 679) and the nucleotide sequence of the nucleotide at positions 677610 to 677647), and the nucleotide sequence of WPSbRGSLRG 677610 to 677647, the sequence (WPSbRG 679). SEQ ID NO:2, the 911-6706 th nucleotides form fusion gene to express fusion protein. Due to the presence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously forms the following three proteins: proteins with Cas9 protein, proteins with EGFP protein and proteins with Puro protein.
3. Plasmid pKG-U6gRNA
The pKG-U6gRNA vector, plasmid pKG-U6gRNA, is a circular plasmid, as described in patent application 202010084343.6 in SEQ ID NO:3, respectively. SEQ ID NO:3, the 2280 th to 2539 th nucleotides form the hU6 promoter, and the 2558 th to 2637 th nucleotides are used for transcription to form a gRNA framework. When the recombinant plasmid is used, a DNA molecule (a target sequence binding region for forming gRNA through transcription) of about 20bp is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid, and the recombinant plasmid is transcribed in a cell to obtain the gRNA.
Example 2 preparation and purification of NCN protein
1. Inducible expression
1. The plasmid pKG-GE4 was introduced into E.coli BL21 (DE 3) to obtain a recombinant strain.
2. The recombinant strain obtained in step 1 was inoculated into a liquid LB medium containing 100. Mu.g/ml ampicillin and cultured overnight at 37 ℃ with shaking at 200 rpm.
3. Inoculating the bacterial liquid obtained in the step 2 to a liquid LB culture medium, and carrying out shaking culture at 30 ℃ and 230rpm until the bacterial liquid is OD 600nm The value =1.0, isopropyl thiogalactoside (IPTG) was added to make the concentration in the system 0.5mM, and the mixture was subjected to shaking culture at 230rpm at 25 ℃ for 12 hours, then centrifuged at 10000 ℃ for 15 minutes at 4 ℃ to collect the cells.
4. The cells obtained in step 3 were washed with PBS buffer.
2. Purification of fusion protein TrxA-His-EK-NLS-spCas9-NLS
1. And (2) adding the crude extraction buffer solution into the thalli obtained in the step one, suspending the thalli, then crushing the thalli by a homogenizer (three times of 1000par circulation), then centrifuging for 30min at 4 ℃ at 15000g, collecting supernate, filtering the supernate by a filter membrane with the aperture of 0.22 mu m, and collecting filtrate. In this step, 10ml of crude extraction buffer solution is prepared for each g of wet-weight thallus.
Crude extraction buffer: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, 1mM PMSF, and the balance ddH 2 O。
2. The fusion protein was purified by affinity chromatography.
Firstly, balancing a Ni-NTA agarose column by using a balancing solution with 5 column volumes (the flow rate is 1 ml/min); then 50ml of the filtrate obtained in step 1 was loaded (flow rate 0.5-1 ml/min); the column was then washed with 5 column volumes of equilibration solution (flow rate 1 ml/min); the column was then washed with 5 column volumes of buffer (flow rate 1 ml/min) to remove contaminating proteins; then eluting with 10 column volumes of eluent at a flow rate of 0.5-1ml/min, and collecting the solution (90-100 ml) after passing through the column.
Ni-NTA agarose column: ausrey, L00250/L00250-C, 10ml of filler.
Balance liquid: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 5mM Imidazole, and the balance ddH 2 O。
Buffer solution: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 50mM Imidazole, and the balance ddH 2 O。
Eluent: containing 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 500mM Imidazole, and the balance ddH 2 O。
3. Enzyme digestion of fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein
1. 15ml of the post-column solution collected in step two was concentrated to 200. Mu.l using Amicon ultrafiltration tube (Sigma, UFC9100, capacity 15 ml) and then diluted to 1ml with 25mM Tris-HCl (pH 8.0). 6 ultrafiltration tubes were used to give a total of 6ml.
2. The product is obtained from commercial sources and has His 6 Tagged recombinant bovine enterokinase (biol., C620031, recombinant bovine enterokinase light chain, his-bearing 6 The tag, recombinan Bovine Enterokinase Light Chain, his), was added to the solution (about 6 ml) obtained in step 1, and cleaved at 25 ℃ for 16 hours. 2 units of enterokinase are added in the amount of each 50 mug protein.
3. The solution (about 6 ml) that completed step 2 was taken and mixed with 480. Mu.l of Ni-NTA resin (Kinseri, L00250/L00250-C), mixed by rotation at room temperature for 15min, and then 7000g was centrifuged for 3min, and the supernatant (4-5.5 ml) was collected.
4. And (3) taking the supernatant obtained in the step (3), concentrating the supernatant to 200 mu l by using an Amicon ultrafiltration tube (Sigma, UFC9100, the volume of which is 15 ml), adding the concentrated solution into an enzyme stock solution, and adjusting the protein concentration to be 5mg/ml to obtain the NCN protein solution.
And (3) sequencing the protein in the NCN protein solution, wherein the 15N-terminal amino acid residues are shown as SEQ ID NO:3, positions 1 to 15, i.e., the NCN protein.
The NCN proteins used in the subsequent examples were all provided by NCN protein solutions.
Enzyme stock solution (ph 7.4): containing 10mM Tris,300mM NaCl,0.1mM EDTA,1mM DTT,50% (by volume) glycerol, and the balance ddH 2 O。
Example 3 Performance of NCN proteins
The 2 gRNA targets targeting the TTN gene were selected as follows:
TTN-gRNA1:AGAGCACAGTCAGCCTGGCG;
TTN-gRNA2:CTTCCAGAATTGGATCTCCG。
primers used to identify target fragments comprising grnas in the TTN gene were as follows:
TTN-F55:TACGGAATTGGGGAGCCAGCGGA;
TTN-R560:CAAAGTTAACTCTCTGTGTCT。
1. preparation of gRNA
1. Preparation of TTN-T7-gRNA1 transcription template and TTN-T7-gRNA2 transcription template
The TTN-T7-gRNA1 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO:4, respectively.
The TTN-T7-gRNA2 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO:5, respectively.
2. In vitro transcription to obtain gRNA
Taking TTN-T7-gRNA1 Transcription template, adopting a Transcription Aid T7 High Yield Transcription Kit (Fermentas, K0441) to carry out in vitro Transcription, and then using MEGA clear TM The Transcription Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain TTN-gRNA1.TTN-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO: and 6, respectively.
Taking TTN-T7-gRNA2 Transcription template, adopting a Transcription Aid T7 High Yield Transcription Kit (Fermentas, K0441) to carry out in vitro Transcription, and then using MEGA clear TM The Transcription Clean-Up Kit (Thermo, AM 1908) was recovered and purified to obtain TTN-gRNA2.TTN-gRNA2 is a single-stranded RNA, as shown in SEQ ID NO: shown at 7.
2. gRNA and NCN protein dosage proportion optimization
1. Co-transfected porcine primary fibroblasts
A first group: co-transfecting primary pig fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN protein. Proportioning: about 10 ten thousand porcine primary fibroblasts: 0.5 μ g TTN-gRNA1:0.5 μ g TTN-gRNA2: mu.g NCN protein.
Second group: co-transfecting primary pig fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN protein. Proportioning: about 10 million porcine primary fibroblasts: 0.75 μ g TTN-gRNA1:0.75 μ g TTN-gRNA2: mu.g NCN protein.
Third group: co-transfecting primary pig fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN protein. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2: mu.g NCN protein.
And a fourth group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. Proportioning: about 10 ten thousand porcine primary fibroblasts: 1.25 μ g TTN-gRNA1:1.25 μ g TTN-gRNA2: mu.g NCN protein.
And a fifth group: co-transfecting TTN-gRNA1 and TTN-gRNA2 to a porcine primary fibroblast. Proportioning: about 10 ten thousand porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were digested with trypsin and collected, genomic DNA was extracted, PCR amplified using a primer pair consisting of TTN-F55 and TTN-R560, and then subjected to 1% agarose gel electrophoresis.
The electrophoretogram is shown in FIG. 3. The 505bp band is wild type band (WT), and the about 254bp band (251 bp band is theoretically deleted from 505bp band of wild type) is deletion mutant band (MT).
Gene deletion mutation efficiency = (MT grayscale/MT band bp number)/(WT grayscale/WT band bp number + MT grayscale/MT band bp number) × 100%. The deletion mutation efficiency of the first group of genes is 19.9 percent, the deletion mutation efficiency of the second group of genes is 39.9 percent, the deletion mutation efficiency of the third group of genes is 79.9 percent, and the deletion mutation efficiency of the fourth group of genes is 44.3 percent. The fifth group was not mutated.
The result shows that when the mass ratio of the two gRNAs to the NCN protein is 1:1:4, actual dosage is 1 mu g:1 μ g: the gene editing efficiency is highest at 4 mug. Thus, the optimal amount of two grnas and NCN proteins was determined to be 1 μ g:1 μ g:4 μ g.
3. Comparison of Gene editing efficiency of NCN protein with that of the commercial Cas9 protein
1. Co-transfected porcine primary fibroblasts
Cas9-a group: co-transfecting the TTN-gRNA1, the TTN-gRNA2 and a commercial Cas9-A protein into a pig primary fibroblast. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2:4 μ g Cas9-A protein.
pKG-GE4 group: co-transfecting the porcine primary fibroblasts with TTN-gRNA1, TTN-gRNA2 and NCN proteins. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2: mu.g NCN protein.
Cas9-B set: co-transfecting the TTN-gRNA1, the TTN-gRNA2 and a commercial Cas9-B protein into a pig primary fibroblast. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2:4 μ g Cas9-B protein.
Control group: co-transfecting the TTN-gRNA1 and the TTN-gRNA2 to the pig primary fibroblasts. Proportioning: about 10 million porcine primary fibroblasts: 1 μ g TTN-gRNA1:1 μ g TTN-gRNA2.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were digested and collected with trypsin, genomic DNA was extracted, PCR amplified using a primer pair consisting of TTN-F55 and TTN-R560, and then subjected to 1% agarose gel electrophoresis.
The electrophoretogram is shown in FIG. 4. The gene deletion mutation efficiency with the commercial Cas9-a protein was 28.5%, the gene deletion mutation efficiency with the NCN protein was 85.6%, and the gene deletion mutation efficiency with the commercial Cas9-B protein was 16.6%.
The result shows that compared with the Cas9 protein which adopts a commodity, the NCN protein prepared by the invention can obviously improve the gene editing efficiency.
Example 4 screening of HBB Gene efficient gRNA target
Pig HBB gene information: encodes a hemoglobin β subunit; is located on chromosome 9; geneID was 407066, sus scrofa. The amino acid sequence of the protein coded by the pig HBB gene is shown in SEQ ID NO. 8. In the genomic DNA, the porcine HBB gene has 3 exons. In the pig genome DNA, the partial sequence (containing the 1 st exon, the 1 st intron, the 2 nd exon and partial 2 nd intron) of the HBB gene is shown as SEQ ID NO: shown at 9. Human sickle cell anemia associated HBB is mutated into E6V (amino acid residue 6 is mutated from glutamic acid into valine), and the mutation corresponds to exon 1 of pig HBB gene.
1. Conservative analysis of HBB gene pre-set point mutation site and adjacent genome sequence
18 newborn Jiangxiang pigs, wherein 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) and 8 males (named A, B, C, D, E, F, G and H respectively) are provided.
HBB-E1-F287:GGAACGCCGAAGACAGGTATACT;
HBB-E1-R690:CTGAGGGGTGAAAAGAGCGCAA;
HBB-E1-F310:GTCAACATTCAAGCCTCACCCTG;
HBB-E1-R789:TGGGCCTTCACCTTGGGATTG。
The porcine ear tissue designated 1 was used to extract the genome as a template, PCR amplified with different primer pairs, and then subjected to 1% agarose gel electrophoresis. The electrophoretogram is shown in FIG. 5. In FIG. 5: group 1: HBB-E1-F287/HBB-E1-R690; group 2: HBB-E1-F287/HBB-E1-R789; group 3: HBB-E1-F310/HBB-E1-R690; group 4: HBB-E1-F310/HBB-E1-R789. As a result, it is preferable to amplify the target fragment using a primer pair consisting of HBB-E1-F287 and HBB-E1-R789.
The genomic DNA of 18 pigs was used as a template, and PCR amplification was carried out using a primer pair consisting of HBB-E1-F287 and HBB-E1-R789, followed by 1% agarose gel electrophoresis. The electrophoretogram is shown in FIG. 6. And recovering PCR amplification products, sequencing, and comparing and analyzing a sequencing result with an HBB gene sequence in a public database. A common conserved region in 18 pigs is selected for designing a gRNA target.
2. Screening target spots
And (3) primarily screening a plurality of targets by screening NGG (avoiding possible mutation sites), and further screening 6 targets from the NGG through a preliminary experiment.
The 6 targets were as follows:
HBB-E1-gU1:AACTGCACAAACAGACAACA;
HBB-E1-gU2:CATGGTGCATCTGTCTGCTG;
HBB-E1-gU3:TGTGCAGTTGCTAGTGAACA;
HBB-E1-gD1:TCACTTTGCCCCACAGGCCG;
HBB-E1-gD2:GAGGCCGTCCTCGGCCTGTG;
HBB-E1-gD3:CCTGTGGGGCAAAGTGAATG。
3. preparation of recombinant plasmid
The plasmid pKG-U6gRNA was digested with the restriction enzyme BbsI, and the vector backbone (approximately 3kb linear large fragment) was recovered.
HBB-E1-gU1-S and HBB-E1-gU1-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. Connecting double-stranded DNA molecules with cohesive ends with a carrier skeleton to obtainTo plasmid pKG-U6gRNA (HBB-E1-gU 1). Plasmid pKG-U6gRNA (HBB-E1-gU 1) expresses the nucleic acid sequence of SEQ ID NO:10 sgRNA HBB-E1-gU1 。sgRNA HBB-E1-gU1 (SEQ ID NO:10):
AACUGCACAAACAGACAACAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gU2-S and HBB-E1-gU2-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having a cohesive end was ligated to a vector backbone to obtain a plasmid pKG-U6gRNA (HBB-E1-gU 2). Plasmid pKG-U6gRNA (HBB-E1-gU 2) expresses the nucleic acid sequence of SEQ ID NO:11 sgRNA HBB-E1-gU2 。sgRNA HBB-E1-gU2 (SEQ ID NO:11):
CAUGGUGCAUCUGUCUGCUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gU3-S and HBB-E1-gU3-A are synthesized separately, and then mixed and annealed to obtain a double-stranded DNA molecule having a cohesive end. The double-stranded DNA molecule having a cohesive end was ligated to a vector backbone to obtain a plasmid pKG-U6gRNA (HBB-E1-gU 3). Plasmid pKG-U6gRNA (HBB-E1-gU 3) expresses the nucleic acid sequence of SEQ ID NO:12 sgRNA HBB-E1-gU3 。sgRNA HBB-E1-gU3 (SEQ ID NO:12):
UGUGCAGUUGCUAGUGAACAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gD1-S and HBB-E1-gD1-A are synthesized separately, and then mixed and annealed to obtain a double-stranded DNA molecule with a cohesive end. The double-stranded DNA molecule having a cohesive end was ligated to a vector backbone to obtain a plasmid pKG-U6gRNA (HBB-E1-gD 1). Plasmid pKG-U6gRNA (HBB-E1-gD 1) expresses the nucleic acid sequence of SEQ ID NO:13 sgRNA HBB-E1-gD1 。sgRNA HBB-E1-gD1 (SEQ ID NO:13):
UCACUUUGCCCCACAGGCCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gD2-S and HBB-E1-gD2-A are synthesized respectively, and then mixed and annealed to obtain a double-stranded DNA molecule with a sticky end. Will have sticky endsThe double-stranded DNA molecule is connected with a vector framework to obtain a plasmid pKG-U6gRNA (HBB-E1-gD 2). Plasmid pKG-U6gRNA (HBB-E1-gD 2) expresses the nucleic acid sequence of SEQ ID NO:14 sgRNA HBB-E1-gD2 。sgRNA HBB-E1-gD2 (SEQ ID NO:14):
GAGGCCGUCCUCGGCCUGUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gD3-S and HBB-E1-gD3-A are synthesized respectively, and then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule having the cohesive ends was ligated to a vector backbone to obtain plasmid pKG-U6gRNA (HBB-E1-gD 3). Plasmid pKG-U6gRNA (HBB-E1-gD 3) expresses the nucleic acid sequence of SEQ ID NO:15 sgRNA HBB-E1-gD3 。sgRNA HBB-E1-gD3 (SEQ ID NO:15):
CCUGUGGGGCAAAGUGAAUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu。
HBB-E1-gU1-S:caccgAACTGCACAAACAGACAACA;
HBB-E1-gU1-A:aaacTGTTGTCTGTTTGTGCAGTTc;
HBB-E1-gU2-S:caccgCATGGTGCATCTGTCTGCTG;
HBB-E1-gU2-A:aaacCAGCAGACAGATGCACCATGc;
HBB-E1-gU3-S:caccgTGTGCAGTTGCTAGTGAACA;
HBB-E1-gU3-A:aaacTGTTCACTAGCAACTGCACAc;
HBB-E1-gD1-S:caccgTCACTTTGCCCCACAGGCCG;
HBB-E1-gD1-A:aaacCGGCCTGTGGGGCAAAGTGAc;
HBB-E1-gD2-S:caccGAGGCCGTCCTCGGCCTGTG;
HBB-E1-gD2-A:aaacCACAGGCCGAGGACGGCCTC;
HBB-E1-gD3-S:caccgCCTGTGGGGCAAAGTGAATG;
HBB-E1-gD3-A:aaacCATTCACTTTGCCCCACAGGc。
HBB-E1-gU1-S, HBB-E1-gU1-A, HBB-E1-gU2-S, HBB-E1-gU2-A, HBB-E1-gU3-S, HBB-E1-gU3-A, HBB-E1-gD1-S, HBB-E1-gD1-A, HBB-E1-gD2-S, HBB-E1-gD2-A, HBB-E1-gD3-S, HBB-E1-gD3-A are single-stranded DNA molecules.
4. Comparison of editing efficiency of different targets
1. Cotransfection
A first group: the plasmid pKG-U6gRNA (HBB-E1-gU 1) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gU 1): 1.08. Mu.g of plasmid pKG-GE3.
Second group: the plasmid pKG-U6gRNA (HBB-E1-gU 2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gU 2): 1.08. Mu.g of plasmid pKG-GE3.
Third group: the plasmid pKG-U6gRNA (HBB-E1-gU 3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 ten thousand porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gU 3): 1.08. Mu.g of plasmid pKG-GE3.
And a fourth group: the plasmid pKG-U6gRNA (HBB-E1-gD 1) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gD 1): 1.08. Mu.g of plasmid pKG-GE3.
A fifth group: the plasmid pKG-U6gRNA (HBB-E1-gD 2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gD 2): 1.08. Mu.g of plasmid pKG-GE3.
A sixth group: the plasmid pKG-U6gRNA (HBB-E1-gD 3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Proportioning: about 20 million porcine primary fibroblasts: 0.92. Mu.g of plasmid pKG-U6gRNA (HBB-E1-gD 3): 1.08. Mu.g of plasmid pKG-GE3.
A seventh group: carrying out electrotransformation operation on primary pig fibroblasts with the same electrotransformation parameters and without plasmids.
Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, thermofeisher) and a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 12 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After step 2 was completed, cells were digested and collected with trypsin, lysed, genomic DNA was extracted, PCR amplified using a primer pair consisting of HBB-E1-F287 and HBB-E1-R789, and then subjected to 1% agarose gel electrophoresis. The mutation of the target gene of the cell is detected, and the electrophoretogram is shown in FIG. 7.
And cutting and recovering the target product, sending the target product to a sequencing company for sequencing, and analyzing a sequencing peak map by using a webpage version Synthego ICE tool to obtain the gene editing efficiency of different targets. The gene editing efficiencies of the first to sixth groups were 81%, 36%, 53%, 29%, 43%, and 76% in this order, and no gene editing occurred in the seventh group. The result shows that the editing efficiency of HBB-E1-gU1 and HBB-E1-gD3 is higher.
Example 5 preparation of HBB Gene site-directed mutagenesis Single cell clone by somatic cloning method
Two high-efficiency gRNA targets (HBB-E1-gU 1 and HBB-E1-gD 3) screened in example 4 were selected.
1. Preparation of gRNA
1. Preparation of HBB-E1-gU1 transcription template and HBB-E1-gD3 transcription template
The HBB-E1-gU1 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO: shown at 16.
The HBB-E1-gD3 transcription template is a double-stranded DNA molecule, and is shown as SEQ ID NO: shown at 17.
2. In vitro transcription to obtain gRNA
Taking HBB-E1-gU1 Transcription template, adopting a Transcription Aid T7 High Yield Transcription Kit (Fermentas, K0441) to perform in vitro Transcription, and then using MEGA clear TM HBB-gU1 was obtained by recovering and purifying the Transcription Clean-Up Kit (Thermo, AM 1908). HBB-gU1 is single-stranded RNA, and is shown in SEQ ID NO:18, respectively.
Taking HBB-E1-gD3 Transcription template, adopting Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441) to carry out in vitro Transcription, and then using MEGA clear TM HBB-gD3 was obtained by recovering and purifying the Transcription clear-Up Kit (Thermo, AM 1908). HBB-gD3 is a single-stranded RNA, as shown in SEQ ID NO:19, respectively.
2. Synthesis of Single-stranded Donor DNA containing HBB mutation site
Synthesizing single-stranded DNA corresponding to the human HBB E6V mutation as Donor DNA, wherein the single-stranded DNA contains the synonymous mutation of HBB-E1-gU1 and HBB-E1-gD3 target PAM or the 3' end sequence adjacent to PAM besides the target site mutation. The single-stranded Donor DNA was designated as HBB-mutant-ss174.
HBB-mutant-ss174 is shown as SEQ ID NO: shown at 20.
3. Transfection of porcine primary fibroblasts
1.HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein were co-transfected into porcine primary fibroblasts. Proportioning: about 10 million porcine primary fibroblasts: 1. Mu.g HBB-gU1: 1. Mu.g HBB-gD3:2 μ g HBB-mutant-ss174: mu.g NCN protein. Co-transfection was performed by electroporation using a mammalian Nuclear transfection kit (Neon kit, thermofisiher) with a Neon TM transfection system electrotransfer instrument (parameters set at 1450V, 10ms, 3 pulses).
2. After step 1, the culture is carried out for 16 to 18 hours by using the complete culture solution, and then the culture is carried out by replacing the complete culture solution with a new one. The total time of incubation after electroporation was 48 hours.
3. After completion of step 2, cells were trypsinized and collected, then washed with complete medium, then resuspended with complete medium, and then each individual monoclonal was picked up and transferred to a 96-well plate (1 cell per well with 100. Mu.l of complete medium per well) for 2 weeks (replacement of new complete medium every 2-3 days).
4. After completion of step 3, cells were trypsinized and collected (cells obtained per well, approximately 2/3 of which were seeded into 6-well plates containing complete medium, the remaining 1/3 of which were collected in 1.5mL centrifuge tubes).
5. The 6-well plate of step 4 was taken, cultured until the cells grew to 80% confluency, trypsinized and collected, and the cells were cryopreserved using cell cryopreserving (90% complete medium +10% dmso by volume).
6. And (4) taking the centrifuge tube in the step (4), taking cells, carrying out cell lysis and extracting genome DNA, carrying out PCR amplification by adopting a primer pair consisting of HBB-E1-F287 and HBB-E1-R789, and then carrying out electrophoresis. Porcine primary fibroblasts were used as wild type control (WT). The electrophoretogram is shown in FIG. 8. Lane numbers in fig. 8 are consistent with cell numbers in table 1.
7. After completion of step 6, the PCR amplification product was recovered and sequenced.
The sequencing result of the primary pig fibroblast is only one, and the genotype of the primary pig fibroblast is homozygous wild type. If the sequencing result of a single-cell clone has two types, one type is consistent with the sequencing result of the pig primary fibroblast, and the other type has mutation (mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the pig primary fibroblast, the genotype of the single-cell clone is heterozygote; if the sequencing result of one single-cell clone is two types, the two types of single-cell clones are mutated (the mutation comprises deletion, insertion or substitution of one or more nucleotides) compared with the sequencing result of the primary porcine fibroblast, and the genotype of the single-cell clone is a biallelic different mutant type; if the sequencing result of a single-cell clone is one and mutation (mutation comprises deletion, insertion or substitution of one or more nucleotides) is generated compared with the sequencing result of the pig primary fibroblast, the genotype of the single-cell clone is a biallelic identical mutant; if the sequencing result of a single cell clone is one and is consistent with the sequencing result of the primary pig fibroblast, the genotype of the single cell clone is homozygous wild type.
The results are shown in Table 1. The genotypes of the single cell clones numbered 1, 6, 11, 17, 25, 30, 37 were homozygous wild-types. The genotypes of the single cell clones numbered 4, 8, 9, 12, 13, 15, 19, 22, 23, 24, 27, 33, 34, 35, 36 are heterozygous. The genotypes of single cell clones numbered 3, 5, 10, 14, 16, 20, 26, 32, 39 were biallelic different mutants. The genotypes of the single cell clones numbered 2, 7, 18, 21, 28, 29, 31, 38, 40 are biallelic and identical mutants. Single cell clones numbered 3, 16, 32, 39 were biallelic different mutants with the target site mutation (one of the two homologous chromosomes completed the single-stranded Donor DNA replacement). Single cell clone No. 9 was heterozygous for the mutation at the target site (i.e. one of the two homologous chromosomes completed the single-stranded Donor DNA replacement). Single cell clones numbered 2, 29 were biallelic mutants of the target site mutation (i.e., both homologous chromosomes completed the single-stranded Donor DNA replacement). The rate of obtaining single-cell clones with HBB gene editing was 82.5%, and the rate of obtaining single-cell clones with mutant target sites (i.e., single-cell clones numbered 2, 3, 9, 16, 29, 32, and 39) was 17.5%.
Exemplary sequencing alignments are shown in figures 9 to 14. FIG. 9 is the results of a simultaneous forward and reverse sequencing of single cell clone numbered 1 aligned with the target site wild type sequence, being homozygous wild type. FIG. 10 is a forward and reverse sequencing of single cell clone numbered 4 aligned simultaneously with the target site wild type sequence, as a heterozygous. FIG. 11 is a forward and reverse sequencing alignment of single cell clone numbered 5 with the target site wild type sequence, as biallelic different mutants. FIG. 12 is a forward and reverse sequencing alignment of single cell clone numbered 7 with the target site wild type sequence, as a biallelic identity mutant. FIG. 13 is the result of a simultaneous forward and reverse sequencing of single cell clone numbered 9 and alignment with the target site wild type sequence, heterozygous for the target site mutation. FIG. 14 is a alignment of forward sequencing of single cell clone number 2 and the target site wild type sequence, a biallelic mutant to the target site mutation.
TABLE 1 genotype determination results of HBB gene point mutation single cell clones
Note: the target site mutation means that the replacement of the single-stranded Donor DNA is completed; substitution of single-stranded Donor DNA was with SEQ ID NO:20 replaces the DNA molecule shown in SEQ ID NO: 21.
Single cell clones numbered 3, 16, 32, 39 were biallelic different mutants with a mutation at the target site (one of the two homologous chromosomes completed the replacement of the single stranded Donor DNA). The single cell clone numbered 9 was heterozygous for the mutation at the target site (i.e. one of the two homologous chromosomes has completed the single-stranded Donor DNA replacement). Single cell clones numbered 2, 29 were biallelic mutants of the target site mutation (i.e., both homologous chromosomes completed the single-stranded Donor DNA replacement).
Recombinant cells with target site mutations, whether heterozygous or homozygous, can be used for subsequent cloned pig production. The cells are taken as nuclear transplantation donor cells to carry out somatic cell cloning, and cloned pigs, namely sickle cell anemia model pigs, can be obtained.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Nanjing King Gene engineering Co., ltd
<120> method for constructing sickle cell anemia model pig nuclear transplantation donor cells with HBB gene mutation and application
<130> GNCYX211984
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9974
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160
ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gaaacaaagc 5220
actattgcac tggcactctt accgttactg tttacccctg tgacaaaagc catgagcgat 5280
aaaattattc acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg 5340
atcctcgtcg atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg 5400
gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa 5460
aaccctggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 5520
aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 5580
ctcgacgcta acctggccgg ttctggttct ggccatatgc accatcatca tcatcatgac 5640
gatgacgata agatgcccaa aaagaaacga aaggtgggta tccacggagt cccagcagcc 5700
gacaaaaaat atagcatcgg cctggacatc ggtaccaaca gcgttggctg ggcagtgatc 5760
actgatgaat acaaagttcc atccaaaaaa tttaaagtac tgggcaacac cgaccgtcac 5820
tctatcaaaa aaaacctgat tggtgctctg ctgtttgaca gcggcgaaac tgctgaggct 5880
acccgtctga aacgtacggc tcgccgtcgc tacactcgtc gtaaaaaccg catctgttat 5940
ctgcaggaaa ttttctctaa cgaaatggca aaagttgatg atagcttctt tcatcgtctg 6000
gaagagagct tcctggtgga agaagataaa aaacacgaac gtcacccgat tttcggtaac 6060
attgtggatg aggttgccta ccacgagaaa tatccgacca tctaccatct gcgtaaaaaa 6120
ctggttgata gcactgacaa agcggatctg cgtctgatct acctggctct ggcacacatg 6180
atcaaattcc gtggtcactt cctgatcgaa ggtgatctga accctgataa ctccgacgtg 6240
gacaaactgt tcattcagct ggttcagacc tataaccagc tgttcgaaga aaacccgatc 6300
aacgcgtccg gtgtagacgc taaggcaatt ctgtctgcgc gtctgtctaa gtctcgtcgt 6360
ctggaaaacc tgattgcgca actgccaggt gaaaagaaaa acggcctgtt cggcaatctg 6420
atcgccctgt ccctgggtct gactccgaac tttaaatcca actttgacct ggcggaagat 6480
gccaagctgc agctgagcaa agatacctat gacgatgacc tggataacct gctggcacag 6540
atcggtgatc agtatgccga tctgttcctg gccgcgaaaa acctgtctga tgcgattctg 6600
ctgtctgata tcctgcgcgt taacactgaa attactaaag cgccgctgag cgcatccatg 6660
attaaacgtt acgatgaaca ccaccaggat ctgaccctgc tgaaagcgct ggtgcgtcag 6720
cagctgccgg aaaaatacaa ggagatcttc ttcgaccaga gcaaaaacgg ttacgcgggc 6780
tacattgatg gtggtgcatc tcaggaggaa ttctacaaat tcattaaacc gatcctggaa 6840
aaaatggatg gtactgaaga gctgctggtt aaactgaatc gtgaagatct gctgcgcaaa 6900
cagcgtacct tcgataacgg ttccatcccg catcagattc atctgggcga actgcacgct 6960
atcctgcgcc gtcaggaaga cttttatccg ttcctgaaag acaaccgtga gaaaattgaa 7020
aaaatcctga ccttccgtat tccgtactat gtaggtccgc tggcgcgtgg taactcccgt 7080
ttcgcttgga tgacccgcaa aagcgaagaa accatcaccc cgtggaattt cgaagaagtc 7140
gttgacaaag gcgcgtccgc gcagtctttc atcgaacgca tgacgaactt cgacaaaaac 7200
ctgccgaacg agaaagtgct gccgaaacac tctctgctgt acgagtactt cactgtgtac 7260
aacgaactga ccaaagtgaa atacgtcacc gaaggtatgc gtaaaccggc attcctgtcc 7320
ggtgagcaaa aaaaagcaat cgtggatctg ctgttcaaaa ccaaccgtaa agtaaccgtg 7380
aaacagctga aggaagacta tttcaagaaa atcgaatgtt ttgattctgt tgaaatctcc 7440
ggcgtggaag atcgcttcaa tgcgtccctg ggtacgtatc acgacctgct gaaaattatc 7500
aaagacaaag attttctgga caacgaggaa aacgaagaca tcctggagga tattgtactg 7560
accctgaccc tgttcgaaga ccgtgagatg atcgaagaac gcctgaaaac ctacgcccac 7620
ctgttcgatg acaaggtaat gaagcagctg aaacgtcgtc gttataccgg ctggggtcgt 7680
ctgtcccgta aactgatcaa tggcatccgt gataaacagt ctggcaaaac catcctggac 7740
ttcctgaaat ccgacggttt cgcgaatcgt aacttcatgc aactgattca tgacgattct 7800
ctgactttca aagaagacat ccagaaagca caggtttccg gccagggtga ctctctgcac 7860
gagcacattg ccaatctggc tggttctccg gctattaaaa agggtattct gcagactgtg 7920
aaagtagttg atgagctggt caaagtaatg ggccgtcaca agccggaaaa cattgtgatc 7980
gaaatggcac gtgaaaacca gacgacccag aaaggtcaga aaaactctcg tgaacgcatg 8040
aaacgtatcg aagaaggcat caaagaactg ggctctcaga tcctgaagga acaccctgta 8100
gaaaataccc agctgcagaa cgaaaagctg tatctgtatt acctgcagaa cggccgcgat 8160
atgtatgtgg accaggaact ggatatcaac cgcctgtccg attacgatgt agatcacatc 8220
gtgccgcaaa gcttcctgaa agacgacagc attgacaaca aagtactgac ccgttctgat 8280
aagaaccgtg gcaaatccga taacgtcccg tctgaagaag ttgttaaaaa aatgaaaaac 8340
tattggcgtc agctgctgaa cgcgaaactg atcacccagc gtaagttcga caatctgact 8400
aaagctgagc gcggtggtct gtccgaactg gataaagcgg gttttatcaa acgccagctg 8460
gttgaaaccc gtcagatcac gaagcacgtt gcgcagattc tggactctcg tatgaacacc 8520
aaatacgacg aaaacgacaa actgatccgc gaggttaagg ttatcaccct gaaaagcaaa 8580
ctggtatccg attttcgtaa agactttcag ttctacaaag tgcgcgaaat taacaactat 8640
caccacgctc acgatgcata tctgaatgca gttgttggca cggcgctgat caaaaagtat 8700
ccgaaactgg aatctgaatt cgtatacggc gattacaaag tgtatgacgt tcgtaagatg 8760
atcgcaaaat ccgagcagga aattggtaag gcgacggcga aatacttctt ttattccaat 8820
attatgaact ttttcaaaac cgaaatcacc ctggcgaatg gtgaaattcg taaacgcccg 8880
ctgatcgaaa ccaacggtga aactggtgaa atcgtttggg acaaaggccg cgacttcgcg 8940
accgtgcgta aagttctgtc tatgccgcaa gtgaacatcg tcaagaagac cgaagtacaa 9000
accggcggtt ttagcaaaga gagcattctg ccaaaacgta actccgacaa actgatcgcg 9060
cgcaagaaag actgggatcc gaaaaaatac ggtggtttcg attctccaac cgttgcttat 9120
tccgttctgg tggtagccaa agttgagaaa ggtaaaagca aaaaactgaa atccgtaaag 9180
gaactgctgg gtattactat catggagcgt agctccttcg aaaaaaaccc gatcgatttt 9240
ctggaagcga aaggctataa agaagtcaaa aaggacctga tcatcaaact gccaaaatac 9300
agcctgttcg agctggaaaa cggccgtaaa cgtatgctgg catctgcggg cgaactgcag 9360
aaaggcaacg agctggctct gccgtccaaa tacgtgaact ttctgtacct ggcctctcac 9420
tacgaaaaac tgaaaggttc cccggaagac aacgaacaga aacagctgtt cgtagagcag 9480
cacaaacact acctggacga gatcatcgaa cagatttctg aattttctaa acgtgtgatt 9540
ctggctgatg cgaatctgga taaagttctg tctgcctata acaagcatcg tgacaaaccg 9600
atccgcgaac aggctgagaa catcatccac ctgttcactc tgactaacct gggcgcgcca 9660
gcggctttca agtactttga taccaccatt gaccgcaagc gttacacctc cactaaagaa 9720
gtgctggacg cgactctgat ccaccagtcc atcaccggtc tgtacgagac ccgtatcgat 9780
ctgagccagc tgggcggtga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 9840
aagaaaaagt gacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9900
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9960
aactatatcc ggat 9974
<210> 2
<211> 1547
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr
1 5 10 15
Pro Val Thr Lys Ala Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp
20 25 30
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp
35 40 45
Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu
50 55 60
Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu
65 70 75 80
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly
85 90 95
Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
100 105 110
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn
115 120 125
Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His Asp
130 135 140
Asp Asp Asp Lys Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly
145 150 155 160
Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr
165 170 175
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
180 185 190
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
195 200 205
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
210 215 220
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
225 230 235 240
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
245 250 255
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
260 265 270
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
275 280 285
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
290 295 300
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
305 310 315 320
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
325 330 335
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
340 345 350
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
355 360 365
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
370 375 380
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
385 390 395 400
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
405 410 415
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
420 425 430
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
435 440 445
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
450 455 460
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
465 470 475 480
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
485 490 495
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
500 505 510
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
515 520 525
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
530 535 540
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
545 550 555 560
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
565 570 575
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
580 585 590
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
595 600 605
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
610 615 620
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
625 630 635 640
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
645 650 655
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
660 665 670
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
675 680 685
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser
690 695 700
Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg
705 710 715 720
Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu
725 730 735
Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala
740 745 750
Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp
755 760 765
Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu
770 775 780
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
785 790 795 800
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg
805 810 815
Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly
820 825 830
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
835 840 845
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser
850 855 860
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
865 870 875 880
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
885 890 895
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
900 905 910
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
915 920 925
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
930 935 940
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys
945 950 955 960
Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
965 970 975
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
980 985 990
Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
995 1000 1005
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp
1010 1015 1020
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1025 1030 1035 1040
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr
1045 1050 1055
Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser
1060 1065 1070
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1075 1080 1085
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
1090 1095 1100
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1105 1110 1115 1120
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
1125 1130 1135
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1140 1145 1150
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1155 1160 1165
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1170 1175 1180
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
1185 1190 1195 1200
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1205 1210 1215
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1220 1225 1230
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
1235 1240 1245
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
1250 1255 1260
Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1265 1270 1275 1280
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
1285 1290 1295
Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
1300 1305 1310
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1315 1320 1325
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
1330 1335 1340
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1345 1350 1355 1360
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
1365 1370 1375
Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1380 1385 1390
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1395 1400 1405
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1410 1415 1420
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
1425 1430 1435 1440
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1445 1450 1455
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1460 1465 1470
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys
1475 1480 1485
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu
1490 1495 1500
Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1505 1510 1515 1520
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala
1525 1530 1535
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1540 1545
<210> 3
<211> 1399
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala
1 5 10 15
Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
20 25 30
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
35 40 45
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
50 55 60
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
65 70 75 80
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
85 90 95
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
100 105 110
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
115 120 125
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
130 135 140
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
145 150 155 160
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
165 170 175
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
180 185 190
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
195 200 205
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
210 215 220
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
225 230 235 240
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
245 250 255
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
260 265 270
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
275 280 285
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
290 295 300
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
305 310 315 320
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
325 330 335
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
340 345 350
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
355 360 365
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
370 375 380
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
385 390 395 400
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
405 410 415
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
420 425 430
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
435 440 445
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
450 455 460
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
465 470 475 480
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
485 490 495
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
500 505 510
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
515 520 525
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
530 535 540
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
545 550 555 560
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
565 570 575
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
580 585 590
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
595 600 605
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
610 615 620
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
625 630 635 640
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
645 650 655
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
660 665 670
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
675 680 685
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
690 695 700
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
705 710 715 720
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
725 730 735
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
740 745 750
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
755 760 765
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
770 775 780
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
785 790 795 800
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
805 810 815
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
820 825 830
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
835 840 845
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
850 855 860
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
865 870 875 880
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
885 890 895
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
900 905 910
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
915 920 925
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
930 935 940
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
945 950 955 960
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
965 970 975
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
980 985 990
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
995 1000 1005
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
1010 1015 1020
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1025 1030 1035 1040
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1045 1050 1055
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1060 1065 1070
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val
1075 1080 1085
Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
1090 1095 1100
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1105 1110 1115 1120
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1125 1130 1135
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1140 1145 1150
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1155 1160 1165
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1170 1175 1180
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1185 1190 1195 1200
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1205 1210 1215
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1220 1225 1230
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1235 1240 1245
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1250 1255 1260
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1265 1270 1275 1280
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
1285 1290 1295
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1300 1305 1310
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1315 1320 1325
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
1330 1335 1340
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1345 1350 1355 1360
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
1365 1370 1375
Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala
1380 1385 1390
Gly Gln Ala Lys Lys Lys Lys
1395
<210> 4
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggagagc acagtcagcc tggcggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 5
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggcttcc agaattggat ctccggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 6
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggagagcaca gucagccugg cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 7
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggcuuccaga auuggaucuc cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 8
<211> 147
<212> PRT
<213> Sus scrofa
<400> 8
Met Val His Leu Ser Ala Glu Glu Lys Glu Ala Val Leu Gly Leu Trp
1 5 10 15
Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu
20 25 30
Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp
35 40 45
Leu Ser Asn Ala Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His
50 55 60
Gly Lys Lys Val Leu Gln Ser Phe Ser Asp Gly Leu Lys His Leu Asp
65 70 75 80
Asn Leu Lys Gly Thr Phe Ala Lys Leu Ser Glu Leu His Cys Asp Gln
85 90 95
Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Ile Val
100 105 110
Val Val Leu Ala Arg Arg Leu Gly His Asp Phe Asn Pro Asn Val Gln
115 120 125
Ala Ala Phe Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His
130 135 140
Lys Tyr His
145
<210> 9
<211> 892
<212> DNA
<213> Sus scrofa
<400> 9
caaatccctc cactttctgc tccttggatt cttcgtttgt gtactaagaa aatggggagg 60
cagtctctaa gagattgcta cagtgggact caactctaaa agttgtacag acttgctaag 120
gaggatgaaa ttagtagcac tttgcactgt gaggatggga cctagagctc cccagagaag 180
ggctgaaggt ctgaagttgg tgccaggaac gccgaagaca ggtatactgt caacattcaa 240
gcctcaccct gtggaaccac gccctggcct gggccaatct gctcccagaa gcagggaggg 300
caggaggctg ggggggcata aaaggaagag cagagccagc agccacctac atttgcttct 360
gacacaaccg tgttcactag caactgcaca aacagacaac atggtgcatc tgtctgctga 420
ggagaaggag gccgtcctcg gcctgtgggg caaagtgaat gtggacgaag ttggtggtga 480
ggccctgggc aggttggtat ccagggcttc aggagaggga gcgggaggtg ggcaggtggg 540
gacagagcca cccctgcctt tctgacaggt gctgactccc tcgggccttg cgctcttttc 600
acccctcagg ctgctggttg tctacccctg gactcagagg ttcttcgagt cctttgggga 660
cctgtccaat gccgatgccg tcatgggcaa tcccaaggtg aaggcccacg gcaagaaggt 720
gctccagtcc ttcagtgacg gcctgaaaca tctcgacaac ctcaagggca cctttgctaa 780
gctgagcgag ctgcactgtg accagctgca cgtggatcct gagaacttca gggtgagtct 840
gggggaccct cacgttctcc ttctgctcct tggtcatggc tgagctcgtg tc 892
<210> 10
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aacugcacaa acagacaaca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 11
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cauggugcau cugucugcug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 12
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ugugcaguug cuagugaaca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 13
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ucacuuugcc ccacaggccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 14
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaggccgucc ucggccugug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 15
<211> 100
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ccuguggggc aaagugaaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 16
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggaactg cacaaacaga caacagtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 17
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60
taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120
ataggcctgt ggggcaaagt gaatggtttt agagctagaa atagcaagtt aaaataaggc 180
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225
<210> 18
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggaacugcac aaacagacaa caguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 19
<211> 102
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggccuguggg gcaaagugaa ugguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60
uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102
<210> 20
<211> 174
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acctacattt gcttctgaca caaccgtgtt cactagcaac tgcacaaaca gacaatatgg 60
tgcatctgtc tgctgtggag aaggaggccg tcctcggcct gtggggcaaa gtgaatgtag 120
acgaagttgg tggtgaggcc ctgggcaggt tggtatccag ggcttcagga gagg 174
<210> 21
<211> 174
<212> DNA
<213> Sus scrofa
<400> 21
acctacattt gcttctgaca caaccgtgtt cactagcaac tgcacaaaca gacaacatgg 60
tgcatctgtc tgctgaggag aaggaggccg tcctcggcct gtggggcaaa gtgaatgtgg 120
acgaagttgg tggtgaggcc ctgggcaggt tggtatccag ggcttcagga gagg 174
Claims (15)
- The application of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein in the preparation of a kit;the HBB-gU1 is sgRNA, and a target sequence binding region is shown as SEQ ID NO:18, nucleotides 3 to 22; the HBB-gD3 is sgRNA, and a target sequence binding region of the HBB-gD3 is shown in SEQ ID NO:19 at nucleotides 3-22; the HBB-mutant-ss174 is SEQ ID NO:20, a single-stranded DNA molecule; the NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 2, application of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and PRONCN protein in preparation of a kit;HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is the HBB-gD3 of claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 according to claim 1;the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 3, application of HBB-gU1, HBB-gD3, HBB-mutant-ss174 and the very-heterogeneous particles in preparation of the kit;HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is HBB-gD3 according to claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 according to claim 1;the specific plasmid comprises the following elements from upstream to downstream in sequence: a promoter, an operator, a ribosome binding site, a PRONCN protein coding gene and a terminator; the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 4. A kit comprising HBB-gU1, HBB-gD3, HBB-mutant-ss174, and NCN protein;HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is HBB-gD3 according to claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 of claim 1; the NCN protein is the NCN protein described in claim 1;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 5. A kit comprises HBB-gU1, HBB-gD3, HBB-mutant-ss174, and PRONCN protein;HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is HBB-gD3 according to claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 according to claim 1;the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 6. A kit comprises HBB-gU1, HBB-gD3, HBB-mutant-ss174 and specific plasmids;HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is HBB-gD3 according to claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 of claim 1;the specific plasmid sequentially comprises the following elements from upstream to downstream: a promoter, an operator, a ribosome binding site, a PRONCN protein encoding gene, and a terminator; the PRONCN protein sequentially comprises the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease enzyme cutting site, nuclear localization signal, cas9 protein and nuclear localization signal;the application of the kit is as follows (a), (b) or (c): (a) preparing a recombinant cell; (b) preparing sickle cell anemia model pigs; (c) Preparing sickle cell anemia cell model or sickle cell anemia tissue model or sickle cell anemia organ model.
- 7. A method of making a recombinant cell comprising the steps of: using the nucleotide sequence of SEQ ID NO:20 to substitute the DNA molecule shown in SEQ ID NO:21 to obtain a recombinant cell.
- 8. The method of claim 7, wherein: using the nucleotide sequence of SEQ ID NO:20 to substitute the DNA molecule shown in SEQ ID NO:21 is as follows: co-transfecting the HBB-gU1, HBB-gD3, HBB-mutant-ss174 and NCN protein into pig cells; HBB-gU1 is HBB-gU1 as defined in claim 1; HBB-gD3 is HBB-gD3 according to claim 1; HBB-mutant-ss174 is HBB-mutant-ss174 of claim 1; the NCN protein is the NCN protein according to claim 1.
- 9. The method of claim 8, wherein: the proportions of the pig cells, the HBB-gU1, the HBB-gD3, the HBB-mutant-ss174 and the NCN protein are as follows in sequence: 10 ten thousand porcine cells: 0.8-1.2. Mu.g HBB-gU1: 0.8-1.2. Mu.g HBB-gD3: 1.8-2.2. Mu.g HBB-mutant-ss174: 3-5. Mu.g NCN protein.
- 10. The use according to claim 1 or the kit according to claim 4 or the method according to claim 8 or 9, characterized in that: the NCN protein is shown as SEQ ID NO:3, respectively.
- 11. The use or kit or method of claim 10, wherein:the preparation method of the NCN protein comprises the following steps:(1) Introducing the plasmid pKG-GE4 into escherichia coli BL21 (DE 3) to obtain a recombinant strain;(2) Culturing the recombinant strain by adopting a liquid culture medium at 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) and carrying out induced culture at 25 ℃, and then collecting thalli;(3) Crushing the collected thalli, and collecting a crude protein solution;(4) Purification of the crude protein solution with His by affinity chromatography 6 A tagged fusion protein;(5) By means of a compound having His 6 Tagged enterokinase cleavage with His 6 Tag fusion protein, then removing His in the protein with Ni-NTA resin 6 A tagged protein, resulting in a purified NCN protein;plasmid pKG-GE4 has the sequence SEQ ID NO:1, 5209 to 9852 th nucleotide.
- 12. Recombinant cell produced by the method according to any one of claims 7 to 11.
- 13. Use of the recombinant cell of claim 12 for the preparation of sickle cell anemia model pigs.
- 14. Porcine tissue, porcine organ or porcine cells of a sickle cell anemia model pig prepared using the recombinant cells of claim 12.
- 15. Use of the recombinant cell of claim 12, the porcine tissue of claim 14, the porcine organ of claim 14, the porcine cell of claim 14, or the sickle cell anemia model porcine produced using the recombinant cell of claim 12, wherein the recombinant cell is (d 1) or (d 2) or (d 3) or (d 4):(d1) Screening a medicament for treating sickle cell anemia;(d2) Evaluating the drug effect of the sickle cell anemia drug;(d3) Evaluating the curative effect of gene therapy and/or cell therapy of sickle cell anemia;(d4) The pathogenesis of sickle cell anemia was studied.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110718541.8A CN115232811A (en) | 2021-06-28 | 2021-06-28 | Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110718541.8A CN115232811A (en) | 2021-06-28 | 2021-06-28 | Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115232811A true CN115232811A (en) | 2022-10-25 |
Family
ID=83666951
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110718541.8A Pending CN115232811A (en) | 2021-06-28 | 2021-06-28 | Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115232811A (en) |
-
2021
- 2021-06-28 CN CN202110718541.8A patent/CN115232811A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101307880B1 (en) | Recombinant expression of multiprotein complexes using polygenes | |
DK3087178T3 (en) | PROCEDURE FOR PREPARING OXALATE OXIDASES WITH ACTIVITY OPTIMUM NEAR PHYSIOLOGICAL PH AND APPLICATION OF SUCH RECOMBINANT OXALATE OXIDASES IN THE TREATMENT OF OXALATE-RELATED DISEASES | |
CN110923183A (en) | Construction method of lanosterol-producing escherichia coli strain | |
CN113278061B (en) | Method for preparing cable Ma Lutai by biochemical method | |
CN112553176B (en) | Glutamine transaminase with improved thermal stability | |
CN115247173A (en) | Gene editing system for constructing TMPRSS6 gene mutant iron deficiency anemia pig nuclear transplantation donor cells and application thereof | |
CN115161335B (en) | Gene editing system for constructing ALS model pig nuclear transfer donor cells with TARDBP gene mutation and application of gene editing system | |
CN115232811A (en) | Method for constructing HBB gene mutant sickle cell anemia model pig nuclear transplantation donor cell and application | |
CN115247153A (en) | Gene editing system for constructing diabetes model pig nuclear transplantation donor cells with HNF1A gene mutation and application thereof | |
CN115232834A (en) | Gene editing system for constructing nuclear transplantation donor cells of OCA-1A albinism model pigs and application thereof | |
CN115232794A (en) | Gene editing system and application thereof in construction of OCA-1B albinism model pig nuclear transplantation donor cells | |
CN115247174A (en) | Gene editing system for constructing congenital gamma globulin deficiency model pig nuclear transplantation donor cell and application thereof | |
CN115232818A (en) | Gene editing system for constructing congenital myasthenia model pig nuclear transplantation donor cells with DOK7 gene mutation and application thereof | |
CN115232812A (en) | Method for constructing nuclear transplantation donor cells of MRAP2 gene mutation severe early obesity model pigs | |
CN115232814A (en) | Method and system for constructing congenital cataract disease model pig nuclear transplantation donor cells with CRYBB2 gene mutation | |
CN115232796A (en) | Gene editing system for constructing central precocity model pig nuclear transplantation donor cells with MKRN3 gene mutation and application thereof | |
CN115232813A (en) | Gene editing system for constructing von willebrand model pig nuclear transplantation donor cells with vWF gene mutation and application of gene editing system | |
CN115247164A (en) | Gene editing system for constructing adenomatous polyposis model pig and colorectal cancer model pig and application thereof | |
CN115232816A (en) | CRISPR system for constructing cataract model pig nuclear transplantation donor cells with YAP1 gene mutation and application thereof | |
CN115232793A (en) | Gene editing system for constructing ALS model pig nuclear transplantation donor cells with SOD1 gene mutation and application thereof | |
CN115247175A (en) | Gene editing system for constructing epigenetic dysregulation model pig nuclear transplantation donor cell of SETDB1 gene mutation and application thereof | |
CN115232836A (en) | Gene editing system for constructing congenital cataract model pig nuclear transplantation donor cell with CRYGC gene mutation and application thereof | |
CN115232817A (en) | Gene editing system for constructing three-gene combined mutant miniature pig nuclear transplantation donor cells and application thereof | |
CN115232815A (en) | Gene editing system for constructing MIP gene mutation cataract disease model pig nuclear transplantation donor cell and application thereof | |
CN115247191A (en) | Gene editing system and application thereof in construction of double-gene-mutation nevus basal cell carcinoma syndrome pig nuclear transplantation donor cell |
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 |