CN113502299A - 一种抗金黄色葡萄球菌遗传修饰山羊生产方法 - Google Patents
一种抗金黄色葡萄球菌遗传修饰山羊生产方法 Download PDFInfo
- Publication number
- CN113502299A CN113502299A CN202110819041.3A CN202110819041A CN113502299A CN 113502299 A CN113502299 A CN 113502299A CN 202110819041 A CN202110819041 A CN 202110819041A CN 113502299 A CN113502299 A CN 113502299A
- Authority
- CN
- China
- Prior art keywords
- goat
- tlr2
- protein
- leu
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000283707 Capra Species 0.000 title claims abstract description 123
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 7
- 241000191967 Staphylococcus aureus Species 0.000 title abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 90
- 230000009261 transgenic effect Effects 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 32
- 108091033409 CRISPR Proteins 0.000 claims abstract description 27
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 20
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 20
- 230000003834 intracellular effect Effects 0.000 claims abstract description 16
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 14
- 101100310115 Homo sapiens SETD5 gene Proteins 0.000 claims abstract description 8
- 101150103943 SETD5 gene Proteins 0.000 claims abstract description 8
- 230000000941 anti-staphylcoccal effect Effects 0.000 claims abstract description 3
- 230000001172 regenerating effect Effects 0.000 claims abstract description 3
- 239000013598 vector Substances 0.000 claims description 48
- 102000004169 proteins and genes Human genes 0.000 claims description 44
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 21
- 102100024333 Toll-like receptor 2 Human genes 0.000 claims description 21
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 claims description 20
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 108020004414 DNA Proteins 0.000 claims description 13
- 238000010354 CRISPR gene editing Methods 0.000 claims description 12
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 claims description 11
- 102000002689 Toll-like receptor Human genes 0.000 claims description 11
- 108020000411 Toll-like receptor Proteins 0.000 claims description 11
- 102100039360 Toll-like receptor 4 Human genes 0.000 claims description 11
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 102000005962 receptors Human genes 0.000 claims description 6
- 108020003175 receptors Proteins 0.000 claims description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 238000003975 animal breeding Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 208000004396 mastitis Diseases 0.000 abstract description 14
- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000036039 immunity Effects 0.000 abstract description 4
- 101150076937 TLR2 gene Proteins 0.000 abstract description 3
- 101150082427 Tlr4 gene Proteins 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000010449 nuclear transplantation Methods 0.000 abstract description 3
- 230000015788 innate immune response Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 40
- 210000002540 macrophage Anatomy 0.000 description 35
- 239000013612 plasmid Substances 0.000 description 24
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 11
- 150000001413 amino acids Chemical group 0.000 description 11
- 210000002950 fibroblast Anatomy 0.000 description 11
- 210000000287 oocyte Anatomy 0.000 description 11
- 230000008685 targeting Effects 0.000 description 10
- 108091034057 RNA (poly(A)) Proteins 0.000 description 9
- 238000010367 cloning Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 238000010362 genome editing Methods 0.000 description 7
- 210000005259 peripheral blood Anatomy 0.000 description 7
- 239000011886 peripheral blood Substances 0.000 description 7
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 230000002441 reversible effect Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 230000001605 fetal effect Effects 0.000 description 5
- 238000010363 gene targeting Methods 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 108010034529 leucyl-lysine Proteins 0.000 description 5
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 4
- 108090000364 Ligases Proteins 0.000 description 4
- 206010057249 Phagocytosis Diseases 0.000 description 4
- 101150063416 add gene Proteins 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 210000003101 oviduct Anatomy 0.000 description 4
- 230000000242 pagocytic effect Effects 0.000 description 4
- 230000008782 phagocytosis Effects 0.000 description 4
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 4
- 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 3
- 230000004544 DNA amplification Effects 0.000 description 3
- 229930182566 Gentamicin Natural products 0.000 description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 3
- 101000650669 Homo sapiens Histone-lysine N-methyltransferase SETD5 Proteins 0.000 description 3
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 230000003321 amplification Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 3
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 230000012173 estrus Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 229960002518 gentamicin Drugs 0.000 description 3
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- DAPLJWATMAXPPZ-CIUDSAMLSA-N Asn-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O DAPLJWATMAXPPZ-CIUDSAMLSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 102100027711 Histone-lysine N-methyltransferase SETD5 Human genes 0.000 description 2
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu 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(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 2
- 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 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 206010042573 Superovulation Diseases 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000001728 clone cell Anatomy 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 230000012361 double-strand break repair Effects 0.000 description 2
- 108010045262 enhanced cyan fluorescent protein Proteins 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 210000002503 granulosa cell Anatomy 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108010012058 leucyltyrosine Proteins 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 239000000186 progesterone Substances 0.000 description 2
- 229960003387 progesterone Drugs 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 108010084932 tryptophyl-proline Proteins 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- SVYBEBLNQGDRHF-UHFFFAOYSA-N 4-amino-N-(5-ethyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide Chemical group S1C(CC)=NN=C1NS(=O)(=O)C1=CC=C(N)C=C1 SVYBEBLNQGDRHF-UHFFFAOYSA-N 0.000 description 1
- 206010000050 Abdominal adhesions Diseases 0.000 description 1
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 description 1
- WCBVQNZTOKJWJS-ACZMJKKPSA-N Ala-Cys-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O WCBVQNZTOKJWJS-ACZMJKKPSA-N 0.000 description 1
- CSAHOYQKNHGDHX-ACZMJKKPSA-N Ala-Gln-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CSAHOYQKNHGDHX-ACZMJKKPSA-N 0.000 description 1
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 1
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 description 1
- UWIQWPWWZUHBAO-ZLIFDBKOSA-N Ala-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)CC(C)C)C(O)=O)=CNC2=C1 UWIQWPWWZUHBAO-ZLIFDBKOSA-N 0.000 description 1
- IHMCQESUJVZTKW-UBHSHLNASA-N Ala-Phe-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 IHMCQESUJVZTKW-UBHSHLNASA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-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
- ZTKHZAXGTFXUDD-VEVYYDQMSA-N Arg-Asn-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZTKHZAXGTFXUDD-VEVYYDQMSA-N 0.000 description 1
- RWDVGVPHEWOZMO-GUBZILKMSA-N Arg-Cys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCNC(N)=N)C(O)=O RWDVGVPHEWOZMO-GUBZILKMSA-N 0.000 description 1
- 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 1
- CGWVCWFQGXOUSJ-ULQDDVLXSA-N Arg-Tyr-Leu 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](CC(C)C)C(O)=O CGWVCWFQGXOUSJ-ULQDDVLXSA-N 0.000 description 1
- MEFGKQUUYZOLHM-GMOBBJLQSA-N Asn-Arg-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MEFGKQUUYZOLHM-GMOBBJLQSA-N 0.000 description 1
- MFFOYNGMOYFPBD-DCAQKATOSA-N Asn-Arg-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MFFOYNGMOYFPBD-DCAQKATOSA-N 0.000 description 1
- NVGWESORMHFISY-SRVKXCTJSA-N Asn-Asn-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NVGWESORMHFISY-SRVKXCTJSA-N 0.000 description 1
- YYSYDIYQTUPNQQ-SXTJYALSSA-N Asn-Ile-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YYSYDIYQTUPNQQ-SXTJYALSSA-N 0.000 description 1
- SEKBHZJLARBNPB-GHCJXIJMSA-N Asn-Ile-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O SEKBHZJLARBNPB-GHCJXIJMSA-N 0.000 description 1
- HDHZCEDPLTVHFZ-GUBZILKMSA-N Asn-Leu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O HDHZCEDPLTVHFZ-GUBZILKMSA-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
- DJIMLSXHXKWADV-CIUDSAMLSA-N Asn-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(N)=O DJIMLSXHXKWADV-CIUDSAMLSA-N 0.000 description 1
- ORJQQZIXTOYGGH-SRVKXCTJSA-N Asn-Lys-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ORJQQZIXTOYGGH-SRVKXCTJSA-N 0.000 description 1
- XMHFCUKJRCQXGI-CIUDSAMLSA-N Asn-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O XMHFCUKJRCQXGI-CIUDSAMLSA-N 0.000 description 1
- VLDRQOHCMKCXLY-SRVKXCTJSA-N Asn-Ser-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VLDRQOHCMKCXLY-SRVKXCTJSA-N 0.000 description 1
- PIABYSIYPGLLDQ-XVSYOHENSA-N Asn-Thr-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PIABYSIYPGLLDQ-XVSYOHENSA-N 0.000 description 1
- ZELQAFZSJOBEQS-ACZMJKKPSA-N Asp-Asn-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZELQAFZSJOBEQS-ACZMJKKPSA-N 0.000 description 1
- AKPLMZMNJGNUKT-ZLUOBGJFSA-N Asp-Asp-Cys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(O)=O AKPLMZMNJGNUKT-ZLUOBGJFSA-N 0.000 description 1
- NYQHSUGFEWDWPD-ACZMJKKPSA-N Asp-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N NYQHSUGFEWDWPD-ACZMJKKPSA-N 0.000 description 1
- KLYPOCBLKMPBIQ-GHCJXIJMSA-N Asp-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N KLYPOCBLKMPBIQ-GHCJXIJMSA-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
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-N 0.000 description 1
- PCJOFZYFFMBZKC-PCBIJLKTSA-N Asp-Phe-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PCJOFZYFFMBZKC-PCBIJLKTSA-N 0.000 description 1
- UTLCRGFJFSZWAW-OLHMAJIHSA-N Asp-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UTLCRGFJFSZWAW-OLHMAJIHSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000283708 Capra aegagrus Species 0.000 description 1
- 241000283705 Capra hircus Species 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- AVFGSUXQKHIQJS-QEJZJMRPSA-N Cys-Gln-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CS)N)C(O)=O)=CNC2=C1 AVFGSUXQKHIQJS-QEJZJMRPSA-N 0.000 description 1
- KGIHMGPYGXBYJJ-SRVKXCTJSA-N Cys-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CS KGIHMGPYGXBYJJ-SRVKXCTJSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- LJEPDHWNQXPXMM-NHCYSSNCSA-N Gln-Arg-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O LJEPDHWNQXPXMM-NHCYSSNCSA-N 0.000 description 1
- CYTSBCIIEHUPDU-ACZMJKKPSA-N Gln-Asp-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O CYTSBCIIEHUPDU-ACZMJKKPSA-N 0.000 description 1
- UFNSPPFJOHNXRE-AUTRQRHGSA-N Gln-Gln-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UFNSPPFJOHNXRE-AUTRQRHGSA-N 0.000 description 1
- KCJJFESQRXGTGC-BQBZGAKWSA-N Gln-Glu-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O KCJJFESQRXGTGC-BQBZGAKWSA-N 0.000 description 1
- HHQCBFGKQDMWSP-GUBZILKMSA-N Gln-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)N)N HHQCBFGKQDMWSP-GUBZILKMSA-N 0.000 description 1
- FKXCBKCOSVIGCT-AVGNSLFASA-N Gln-Lys-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O FKXCBKCOSVIGCT-AVGNSLFASA-N 0.000 description 1
- NYCVMJGIJYQWDO-CIUDSAMLSA-N Gln-Ser-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NYCVMJGIJYQWDO-CIUDSAMLSA-N 0.000 description 1
- RSUVOPBMWMTVDI-XEGUGMAKSA-N Glu-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCC(O)=O)C)C(O)=O)=CNC2=C1 RSUVOPBMWMTVDI-XEGUGMAKSA-N 0.000 description 1
- JRCUFCXYZLPSDZ-ACZMJKKPSA-N Glu-Asp-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O JRCUFCXYZLPSDZ-ACZMJKKPSA-N 0.000 description 1
- HILMIYALTUQTRC-XVKPBYJWSA-N Glu-Gly-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HILMIYALTUQTRC-XVKPBYJWSA-N 0.000 description 1
- ZWABFSSWTSAMQN-KBIXCLLPSA-N Glu-Ile-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O ZWABFSSWTSAMQN-KBIXCLLPSA-N 0.000 description 1
- BKRQSECBKKCCKW-HVTMNAMFSA-N Glu-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](CCC(=O)O)N BKRQSECBKKCCKW-HVTMNAMFSA-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
- 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 1
- IOUQWHIEQYQVFD-JYJNAYRXSA-N Glu-Leu-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IOUQWHIEQYQVFD-JYJNAYRXSA-N 0.000 description 1
- CUPSDFQZTVVTSK-GUBZILKMSA-N Glu-Lys-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O CUPSDFQZTVVTSK-GUBZILKMSA-N 0.000 description 1
- RBXSZQRSEGYDFG-GUBZILKMSA-N Glu-Lys-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O RBXSZQRSEGYDFG-GUBZILKMSA-N 0.000 description 1
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- JWNZHMSRZXXGTM-XKBZYTNZSA-N Glu-Ser-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JWNZHMSRZXXGTM-XKBZYTNZSA-N 0.000 description 1
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 1
- MXJYXYDREQWUMS-XKBZYTNZSA-N Glu-Thr-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O MXJYXYDREQWUMS-XKBZYTNZSA-N 0.000 description 1
- UUTGYDAKPISJAO-JYJNAYRXSA-N Glu-Tyr-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 UUTGYDAKPISJAO-JYJNAYRXSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 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
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-N 0.000 description 1
- XLFHCWHXKSFVIB-BQBZGAKWSA-N Gly-Gln-Gln Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLFHCWHXKSFVIB-BQBZGAKWSA-N 0.000 description 1
- 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 1
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 1
- HMHRTKOWRUPPNU-RCOVLWMOSA-N Gly-Ile-Gly Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O HMHRTKOWRUPPNU-RCOVLWMOSA-N 0.000 description 1
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-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
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 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 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
- GMIWMPUGTFQFHK-KCTSRDHCSA-N His-Ala-Trp Chemical compound C[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O GMIWMPUGTFQFHK-KCTSRDHCSA-N 0.000 description 1
- JBJNKUOMNZGQIM-PYJNHQTQSA-N His-Arg-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JBJNKUOMNZGQIM-PYJNHQTQSA-N 0.000 description 1
- BXOLYFJYQQRQDJ-MXAVVETBSA-N His-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CN=CN1)N BXOLYFJYQQRQDJ-MXAVVETBSA-N 0.000 description 1
- SKOKHBGDXGTDDP-MELADBBJSA-N His-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N SKOKHBGDXGTDDP-MELADBBJSA-N 0.000 description 1
- YXXKBPJEIYFGOD-MGHWNKPDSA-N His-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC2=CN=CN2)N YXXKBPJEIYFGOD-MGHWNKPDSA-N 0.000 description 1
- FHKZHRMERJUXRJ-DCAQKATOSA-N His-Ser-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 FHKZHRMERJUXRJ-DCAQKATOSA-N 0.000 description 1
- FRDFAWHTPDKRHG-ULQDDVLXSA-N His-Tyr-Arg Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CN=CN1 FRDFAWHTPDKRHG-ULQDDVLXSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 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
- BEWFWZRGBDVXRP-PEFMBERDSA-N Ile-Glu-Asn Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O BEWFWZRGBDVXRP-PEFMBERDSA-N 0.000 description 1
- KEKTTYCXKGBAAL-VGDYDELISA-N Ile-His-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N KEKTTYCXKGBAAL-VGDYDELISA-N 0.000 description 1
- 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 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
- HXIDVIFHRYRXLZ-NAKRPEOUSA-N Ile-Ser-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)O)N HXIDVIFHRYRXLZ-NAKRPEOUSA-N 0.000 description 1
- DGTOKVBDZXJHNZ-WZLNRYEVSA-N Ile-Thr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N DGTOKVBDZXJHNZ-WZLNRYEVSA-N 0.000 description 1
- ZGKVPOSSTGHJAF-HJPIBITLSA-N Ile-Tyr-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CO)C(=O)O)N ZGKVPOSSTGHJAF-HJPIBITLSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- 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 1
- HBJZFCIVFIBNSV-DCAQKATOSA-N Leu-Arg-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O HBJZFCIVFIBNSV-DCAQKATOSA-N 0.000 description 1
- GRZSCTXVCDUIPO-SRVKXCTJSA-N Leu-Arg-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GRZSCTXVCDUIPO-SRVKXCTJSA-N 0.000 description 1
- STAVRDQLZOTNKJ-RHYQMDGZSA-N Leu-Arg-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STAVRDQLZOTNKJ-RHYQMDGZSA-N 0.000 description 1
- DLCOFDAHNMMQPP-SRVKXCTJSA-N Leu-Asp-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DLCOFDAHNMMQPP-SRVKXCTJSA-N 0.000 description 1
- 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
- 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
- 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 1
- LLBQJYDYOLIQAI-JYJNAYRXSA-N Leu-Glu-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LLBQJYDYOLIQAI-JYJNAYRXSA-N 0.000 description 1
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 1
- XQXGNBFMAXWIGI-MXAVVETBSA-N Leu-His-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 XQXGNBFMAXWIGI-MXAVVETBSA-N 0.000 description 1
- CSFVADKICPDRRF-KKUMJFAQSA-N Leu-His-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=CN=CN1 CSFVADKICPDRRF-KKUMJFAQSA-N 0.000 description 1
- OHZIZVWQXJPBJS-IXOXFDKPSA-N Leu-His-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OHZIZVWQXJPBJS-IXOXFDKPSA-N 0.000 description 1
- KUIDCYNIEJBZBU-AJNGGQMLSA-N Leu-Ile-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O KUIDCYNIEJBZBU-AJNGGQMLSA-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
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-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
- 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 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- HDHQQEDVWQGBEE-DCAQKATOSA-N Leu-Met-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O HDHQQEDVWQGBEE-DCAQKATOSA-N 0.000 description 1
- PTRKPHUGYULXPU-KKUMJFAQSA-N Leu-Phe-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O PTRKPHUGYULXPU-KKUMJFAQSA-N 0.000 description 1
- XWEVVRRSIOBJOO-SRVKXCTJSA-N Leu-Pro-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O XWEVVRRSIOBJOO-SRVKXCTJSA-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
- GOFJOGXGMPHOGL-DCAQKATOSA-N Leu-Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C GOFJOGXGMPHOGL-DCAQKATOSA-N 0.000 description 1
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln 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)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 1
- 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 1
- BTEMNFBEAAOGBR-BZSNNMDCSA-N Leu-Tyr-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BTEMNFBEAAOGBR-BZSNNMDCSA-N 0.000 description 1
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 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
- 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 1
- MQMIRLVJXQNTRJ-SDDRHHMPSA-N Lys-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N)C(=O)O MQMIRLVJXQNTRJ-SDDRHHMPSA-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
- JOSAKOKSPXROGQ-BJDJZHNGSA-N Lys-Ser-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JOSAKOKSPXROGQ-BJDJZHNGSA-N 0.000 description 1
- RPWTZTBIFGENIA-VOAKCMCISA-N Lys-Thr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O RPWTZTBIFGENIA-VOAKCMCISA-N 0.000 description 1
- PHKBGZKVOJCIMZ-SRVKXCTJSA-N Met-Pro-Arg Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PHKBGZKVOJCIMZ-SRVKXCTJSA-N 0.000 description 1
- BVIAOQMSVZHOJM-UHFFFAOYSA-N N(6),N(6)-dimethyladenine Chemical compound CN(C)C1=NC=NC2=C1N=CN2 BVIAOQMSVZHOJM-UHFFFAOYSA-N 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
- 208000002193 Pain Diseases 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- AYPMIIKUMNADSU-IHRRRGAJSA-N Phe-Arg-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O AYPMIIKUMNADSU-IHRRRGAJSA-N 0.000 description 1
- UAMFZRNCIFFMLE-FHWLQOOXSA-N Phe-Glu-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N UAMFZRNCIFFMLE-FHWLQOOXSA-N 0.000 description 1
- YZJKNDCEPDDIDA-BZSNNMDCSA-N Phe-His-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=CN=CN1 YZJKNDCEPDDIDA-BZSNNMDCSA-N 0.000 description 1
- RGZYXNFHYRFNNS-MXAVVETBSA-N Phe-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RGZYXNFHYRFNNS-MXAVVETBSA-N 0.000 description 1
- RORUIHAWOLADSH-HJWJTTGWSA-N Phe-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=CC=C1 RORUIHAWOLADSH-HJWJTTGWSA-N 0.000 description 1
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-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=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 1
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-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
- CZQZSMJXFGGBHM-KKUMJFAQSA-N Phe-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O CZQZSMJXFGGBHM-KKUMJFAQSA-N 0.000 description 1
- GMWNQSGWWGKTSF-LFSVMHDDSA-N Phe-Thr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O GMWNQSGWWGKTSF-LFSVMHDDSA-N 0.000 description 1
- BSTPNLNKHKBONJ-HTUGSXCWSA-N Phe-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O BSTPNLNKHKBONJ-HTUGSXCWSA-N 0.000 description 1
- XNQMZHLAYFWSGJ-HTUGSXCWSA-N Phe-Thr-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XNQMZHLAYFWSGJ-HTUGSXCWSA-N 0.000 description 1
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 1
- FRMKIPSIZSFTTE-HJOGWXRNSA-N Phe-Tyr-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FRMKIPSIZSFTTE-HJOGWXRNSA-N 0.000 description 1
- CYQQWUPHIZVCNY-GUBZILKMSA-N Pro-Arg-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CYQQWUPHIZVCNY-GUBZILKMSA-N 0.000 description 1
- UTAUEDINXUMHLG-FXQIFTODSA-N Pro-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 UTAUEDINXUMHLG-FXQIFTODSA-N 0.000 description 1
- NOXSEHJOXCWRHK-DCAQKATOSA-N Pro-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@@H]1CCCN1 NOXSEHJOXCWRHK-DCAQKATOSA-N 0.000 description 1
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 1
- LQZZPNDMYNZPFT-KKUMJFAQSA-N Pro-Gln-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LQZZPNDMYNZPFT-KKUMJFAQSA-N 0.000 description 1
- NMELOOXSGDRBRU-YUMQZZPRSA-N Pro-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 NMELOOXSGDRBRU-YUMQZZPRSA-N 0.000 description 1
- UEHYFUCOGHWASA-HJGDQZAQSA-N Pro-Glu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 UEHYFUCOGHWASA-HJGDQZAQSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 1
- WHNJMTHJGCEKGA-ULQDDVLXSA-N Pro-Phe-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O WHNJMTHJGCEKGA-ULQDDVLXSA-N 0.000 description 1
- NAIPAPCKKRCMBL-JYJNAYRXSA-N Pro-Pro-Phe Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H]1NCCC1)C1=CC=CC=C1 NAIPAPCKKRCMBL-JYJNAYRXSA-N 0.000 description 1
- RNEFESSBTOQSAC-DCAQKATOSA-N Pro-Ser-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O RNEFESSBTOQSAC-DCAQKATOSA-N 0.000 description 1
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 1
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000005074 Retroviridae Infections Diseases 0.000 description 1
- GXXTUIUYTWGPMV-FXQIFTODSA-N Ser-Arg-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O GXXTUIUYTWGPMV-FXQIFTODSA-N 0.000 description 1
- QFBNNYNWKYKVJO-DCAQKATOSA-N Ser-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CCCN=C(N)N QFBNNYNWKYKVJO-DCAQKATOSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 1
- SQBLRDDJTUJDMV-ACZMJKKPSA-N Ser-Glu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SQBLRDDJTUJDMV-ACZMJKKPSA-N 0.000 description 1
- GYXVUTAOICLGKJ-ACZMJKKPSA-N Ser-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N GYXVUTAOICLGKJ-ACZMJKKPSA-N 0.000 description 1
- 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 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- MOINZPRHJGTCHZ-MMWGEVLESA-N Ser-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N MOINZPRHJGTCHZ-MMWGEVLESA-N 0.000 description 1
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 1
- MQQBBLVOUUJKLH-HJPIBITLSA-N Ser-Ile-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MQQBBLVOUUJKLH-HJPIBITLSA-N 0.000 description 1
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-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
- PMCMLDNPAZUYGI-DCAQKATOSA-N Ser-Lys-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMCMLDNPAZUYGI-DCAQKATOSA-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
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- RXUOAOOZIWABBW-XGEHTFHBSA-N Ser-Thr-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RXUOAOOZIWABBW-XGEHTFHBSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-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
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 1
- 108010053950 Teicoplanin Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- NFMPFBCXABPALN-OWLDWWDNSA-N Thr-Ala-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O NFMPFBCXABPALN-OWLDWWDNSA-N 0.000 description 1
- GARULAKWZGFIKC-RWRJDSDZSA-N Thr-Gln-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GARULAKWZGFIKC-RWRJDSDZSA-N 0.000 description 1
- IGGFFPOIFHZYKC-PBCZWWQYSA-N Thr-His-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O IGGFFPOIFHZYKC-PBCZWWQYSA-N 0.000 description 1
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- VTVVYQOXJCZVEB-WDCWCFNPSA-N Thr-Leu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O VTVVYQOXJCZVEB-WDCWCFNPSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- 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 1
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val 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)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 1
- SOUPNXUJAJENFU-SWRJLBSHSA-N Thr-Trp-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O SOUPNXUJAJENFU-SWRJLBSHSA-N 0.000 description 1
- KPMIQCXJDVKWKO-IFFSRLJSSA-N Thr-Val-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KPMIQCXJDVKWKO-IFFSRLJSSA-N 0.000 description 1
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 1
- OQMQBYOEAHVCGD-GQGQLFGLSA-N Trp-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N OQMQBYOEAHVCGD-GQGQLFGLSA-N 0.000 description 1
- DVIIYMVCSUQOJG-QEJZJMRPSA-N Trp-Glu-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O DVIIYMVCSUQOJG-QEJZJMRPSA-N 0.000 description 1
- CKKFTIQYURNSEI-IHRRRGAJSA-N Tyr-Asn-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CKKFTIQYURNSEI-IHRRRGAJSA-N 0.000 description 1
- PMDWYLVWHRTJIW-STQMWFEESA-N Tyr-Gly-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PMDWYLVWHRTJIW-STQMWFEESA-N 0.000 description 1
- QARCDOCCDOLJSF-HJPIBITLSA-N Tyr-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QARCDOCCDOLJSF-HJPIBITLSA-N 0.000 description 1
- KSCVLGXNQXKUAR-JYJNAYRXSA-N Tyr-Leu-Glu 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](CCC(O)=O)C(O)=O KSCVLGXNQXKUAR-JYJNAYRXSA-N 0.000 description 1
- OBKOPLHSRDATFO-XHSDSOJGSA-N Tyr-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N OBKOPLHSRDATFO-XHSDSOJGSA-N 0.000 description 1
- UUYCNAXCCDNULB-QXEWZRGKSA-N Val-Arg-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O UUYCNAXCCDNULB-QXEWZRGKSA-N 0.000 description 1
- COYSIHFOCOMGCF-WPRPVWTQSA-N Val-Arg-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-WPRPVWTQSA-N 0.000 description 1
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-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
- BWVHQINTNLVWGZ-ZKWXMUAHSA-N Val-Cys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N BWVHQINTNLVWGZ-ZKWXMUAHSA-N 0.000 description 1
- JXGWQYWDUOWQHA-DZKIICNBSA-N Val-Gln-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N JXGWQYWDUOWQHA-DZKIICNBSA-N 0.000 description 1
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 1
- NXRAUQGGHPCJIB-RCOVLWMOSA-N Val-Gly-Asn Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O NXRAUQGGHPCJIB-RCOVLWMOSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 description 1
- OJPRSVJGNCAKQX-SRVKXCTJSA-N Val-Met-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N OJPRSVJGNCAKQX-SRVKXCTJSA-N 0.000 description 1
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 1
- IRAUYEAFPFPVND-UVBJJODRSA-N Val-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 IRAUYEAFPFPVND-UVBJJODRSA-N 0.000 description 1
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- DDTDNCYHLGRFBM-YZEKDTGTSA-N chembl2367892 Chemical compound CC(=O)N[C@H]1[C@@H](O)[C@H](O)[C@H](CO)O[C@H]1O[C@@H]([C@H]1C(N[C@@H](C2=CC(O)=CC(O[C@@H]3[C@H]([C@H](O)[C@H](O)[C@@H](CO)O3)O)=C2C=2C(O)=CC=C(C=2)[C@@H](NC(=O)[C@@H]2NC(=O)[C@@H]3C=4C=C(O)C=C(C=4)OC=4C(O)=CC=C(C=4)[C@@H](N)C(=O)N[C@H](CC=4C=C(Cl)C(O5)=CC=4)C(=O)N3)C(=O)N1)C(O)=O)=O)C(C=C1Cl)=CC=C1OC1=C(O[C@H]3[C@H]([C@@H](O)[C@H](O)[C@H](CO)O3)NC(C)=O)C5=CC2=C1 DDTDNCYHLGRFBM-YZEKDTGTSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000013267 controlled drug release Methods 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 208000013184 decreased milk production Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000007159 enucleation Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010046775 glutamyl-isoleucyl-leucyl-aspartyl-valine Proteins 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 108010013726 glycyl-arginyl-glycyl-glutamyl-serine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 108091005434 innate immune receptors Proteins 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000008316 intracellular mechanism Effects 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 238000002357 laparoscopic surgery Methods 0.000 description 1
- 229960003907 linezolid Drugs 0.000 description 1
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 241000894007 species Species 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
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960001608 teicoplanin Drugs 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- 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
- A01K2227/102—Caprine
-
- 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
- A01K2267/02—Animal zootechnically ameliorated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Veterinary Medicine (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种抗金黄色葡萄球菌遗传修饰山羊生产方法。具体地公开了一种将TLR2‑4嵌合基因(SEQ ID No.1)导入山羊的体细胞中,由转化的体细胞再生出遗传修饰山羊的方法,所述TLR2‑4嵌合基因包括TLR2基因的胞外区、TLR4基因的跨膜区与胞内区。本发明使用CRISPA/Cas9系统将TLR2‑4嵌合基因定点整合到山羊SETD5基因第一内含子上,利用体细胞核移植技术制备表达TLR2‑4嵌合蛋白的转基因山羊,构建的转基因山羊提高了抗金黄色葡萄球菌能力并且增强了免疫力,达到通过山羊自身的先天免疫有效防控金黄色葡萄球菌引起的山羊乳腺炎的目的。为抗金黄色葡萄球菌山羊新种质的制备奠定基础。
Description
技术领域
本发明属于基因工程技术领域,涉及一种抗金黄色葡萄球菌遗传修饰山羊生产方法。具体涉及在山羊细胞中表达嵌合TLR2-4基因抗金黄色葡萄球菌的方法。
背景技术
山羊乳腺炎(goat mastitis)是由病原菌引起的山羊最重要疾病之一。细菌学分析显示,山羊乳房分离的细菌以金黄色葡萄球菌(Staphyloccocus aureus,S.aureus)最多(40.5%)。该病的临床表现为山羊乳房明显异常,疼痛、脓肿、发热,乳汁产量迅速下降,导致山羊泌乳机能受到严重影响,急性的乳腺炎甚至会造成山羊死亡。该病在全世界大范围流行,患病山羊生产的乳品对公共卫生构成严重风险,因为它可能与人类的乳源性疾病有关。除了造成卫生和健康问题外,产奶量下降、奶质差、过早扑杀动物和治疗成本的增加给畜牧业造成巨大的经济损失。山羊乳腺炎是对山羊养殖业危害最严重的疫病之一,因此,针对山羊乳腺炎急需一种良好的解决办法。
抗生素注射是治疗该病的常用手段,常用的乳房炎治疗抗生素有青霉素、四环素和链霉素,但由于S.aureus染色体DNA的固有耐药性以及抗生素的大量使用,S.aureus已经成为耐药菌感染的主要病原菌之一,呈现出对多种抗生素耐药的特点。除了对青霉素类、头孢菌素等常用抗生素耐药外,临床上分离到的S.aureus还对万古霉素、替考拉宁、利奈咗烷和大环内酯类抗生素药物具有不同程度的耐药性。而且抗生素药物残留在山羊体内,还存在严重的食品安全隐患,会给人类的健康带来影响。因此,抗生素治疗山羊乳腺炎的应用频率逐年变低,应用效果也越来越差。
疫苗接种是目前预防山羊乳腺炎的重要措施,既能增强山羊的抵抗能力和免疫能力,还不会有药物残留,能较大程度的保证乳汁安全和卫生,而且操作简便,成本不高。但由于山羊所处的环境、地域、气候各不相同,所以疫苗的免疫效果往往只是短期见效,不具备长期效应,预防效果欠佳。针对这种情况,国内外的相关兽医学专家也正在积极对奶山羊乳腺炎免疫接种进行持续评估,旨在研制效果更为显著的预防山羊乳腺炎的疫苗,但是目前尚未取得突破。因此,如何更早的发现和清除细菌,控制炎症的发生就显得尤为重要,目前针对金黄色葡萄球菌的先天免疫防御研究正受到广泛的关注。
发明内容
本发明所要解决的技术问题是如何从分子水平、早期、有效地防控山羊乳腺炎,增强山羊的免疫力。所要解决的技术问题不限于如所描述的技术主题,本领域技术人员通过以下描述可以清楚地理解本文未提及的其它技术主题。
为解决上述技术问题,本发明首先提供了一种制备遗传修饰山羊的方法,所述方法包括如下步骤:将TLR2-4嵌合基因导入作为转基因受体的山羊体细胞中,得到转化的体细胞,由所述转化的体细胞再生出所述遗传修饰山羊(又称为TLR2-4转基因山羊,表达TLR2-4融合蛋白的山羊),所述TLR2-4嵌合基因编码的嵌合蛋白质包括TLR2的胞外区和TLR4的跨膜区与胞内区。
所述体细胞为离体的细胞。
上述方法中,所述嵌合蛋白质还包括信号肽和/或标签蛋白。
本领域技术人员应当理解,在研究某一目的蛋白功能时,将一个通过各种方法可以检测到的标签蛋白的基因和目的蛋白的基因进行重组融合,产生的融合蛋白除了目的蛋白本身,同时会带上可检测的标签蛋白,这样目的蛋白也一并检测到,其目的在于能够定位目的蛋白或研究蛋白相互作用,因此,适用于本申请的标签蛋白并不局限于特定种类。
所述标签蛋白包括但不限于:GST(谷胱甘肽巯基转移酶)标签蛋白、His6标签蛋白(His-tag)、MBP(麦芽糖结合蛋白)标签蛋白、Flag标签蛋白、SUMO标签蛋白、HA标签蛋白、Myc标签蛋白、eGFP(增强型绿色荧光蛋白)、eCFP(增强型青色荧光蛋白)、eYFP(增强型黄绿色荧光蛋白)、mCherry(单体红色荧光蛋白)或AviTag标签蛋白。
进一步地,所述信号肽可为TLR2的信号肽和/或所述标签蛋白可为Myc。
上述方法中,所述TLR2的胞外区为下述任一种蛋白质:
P1)氨基酸序列是SEQ ID No.2的第31-597位的蛋白质;
P2)将P1)所示的蛋白质经过氨基酸残基的取代和/或缺失和/或添加得到的与P1)所示的蛋白质具有80%以上的同一性且具有相同功能的蛋白质;
所述TLR4的跨膜区与胞内区为下述任一种蛋白质:
Q1)氨基酸序列是SEQ ID No.2的第598-805位的蛋白质;
Q2)将Q1)所示的蛋白质经过氨基酸残基的取代和/或缺失和/或添加得到的与Q1)所示的蛋白质具有80%以上的同一性且具有相同功能的蛋白质。
所述嵌合蛋白质具体可由所述TLR2的胞外区和所述TLR4的跨膜区与胞内区连接而成,也可由所述信号肽、所述TLR2的胞外区和所述TLR4的跨膜区与胞内区连接而成,也可由所述标签蛋白、所述TLR2的胞外区和所述TLR4的跨膜区与胞内区连接而成,也可由所述标签蛋白、所述信号肽、所述TLR2的胞外区和所述TLR4的跨膜区与胞内区连接而成。
上述方法中,所述TLR2-4嵌合基因的核苷酸序列可为SEQ ID No.1。
SEQ ID No.1所示的TLR2-4嵌合基因编码SEQ ID No.2所示的TLR2-4嵌合蛋白质。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化或点突变的方法,对本发明的编码TLR2-4嵌合蛋白质的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明分离得到的TLR2-4嵌合蛋白质的核苷酸序列75%或者更高同一性的核苷酸,只要编码TLR2-4嵌合蛋白质且具有TLR2-4嵌合蛋白质功能,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
上述75%或75%以上同一性,可为80%、85%、90%或95%以上的同一性。
本文中,同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
本文中,所述80%以上的同一性可为至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同一性。
本发明所述TLR2-4嵌合基因能显著提高山羊成纤维细胞对金黄色葡萄球菌的抗性,增加细胞因子和趋化因子的分泌。
上述方法中,所述将TLR2-4嵌合基因导入作为转基因受体的山羊体细胞中为将所述TLR2-4嵌合基因导入到山羊SETD5基因第一内含子中。
所述导入到山羊SETD5基因第一内含子中的具体位点为SETD5-IN;所述SETD5-IN是大小为2kb的双链DNA分子,所述SETD5-IN的核苷酸序列为SEQ ID No.3。
所述导入可为通过显微注射法、逆转录病毒感染法、胚胎干细胞介导法、体细胞核移植法、精子载体介导法、受体介导法等,将所述TLR2-4嵌合基因导入受体动物。
在本发明的一个实施方案中,所述导入可为利用CRISPR/Cas9系统进行。
上述方法中,所述CRISPR/Cas9系统包括如下A1)或A2):
A1)sgRNA,所述sgRNA特异性靶向于SETD5-IN,所述SETD5-IN是大小为2kb的双链DNA分子,所述SETD5-IN的核苷酸序列为SEQ ID No.3;
A2)表达A1)所述sgRNA的CRISPR/Cas9载体。
上述方法中,所述sgRNA识别区的核苷酸序列可为SEQ ID No.4。
所述sgRNA名称为SETD5-IN-sgRNA2,靶点序列(SEQ ID No.5)为:ACTACACATTATAGATGACT(SEQ ID No.3的第868-887),相应sgRNA识别区序列如SEQ ID No.4所示,相应sgRNA识别区的编码DNA序列为:AGTCATCTATAATGTGTAGT。
上述方法中,所述再生出所述遗传修饰山羊为利用体细胞核移植技术进行。
上述方法中,所述方法包括如下步骤:
B1)构建CRISPR/Cas9载体,所述CRISPR/Cas9载体包括SEQ ID No.4所示的sgRNA;
B2)构建供体质粒,所述供体质粒包含SEQ ID No.1所示的TLR2-4嵌合基因;
B3)CRISPR/Cas9载体和供体质粒共转染山羊胎儿成纤维细胞;
B4)筛选、鉴定阳性克隆细胞作为供体细胞;
B5)将所述供体细胞注射到去核的山羊卵母细胞,得到重组胚胎,将所述重组胚胎植入同步发情的受体山羊输卵管中,获得所述遗传修饰山羊。
上述方法中,B2)所述供体质粒为双切型供体质粒,即以pRosa26-promoter为骨架载体,在上下游分别添加BamHI及XbaI酶切位点;双酶切pRosa26-promoter载体,将poly(A)克隆到载体上;用XbaI和ScaI-HF双酶切,通过无缝克隆将右同源臂HA-R插入poly(A)后;用Acc65I和ScalI双酶切载体,在pRosa26-promoter载体上游引入PacI酶切位点并插入左同源臂HA-L,得到重组载体pRosa26-HA,在同源臂外侧各添加一个sgRNA识别位点;用PmlI和EcoRI-HF双酶切pRosa26-HA,在pRosa26-promoter与poly(A)之间插入TLR2-4基因,得到重组载体pRosa26-TLR2-4;用SacI-HF单酶切pRosa26-TLR2-4,在HA-R下游插入CMV-tdtomato,获得完整的pRosa26-TLR2-4供体质粒,简称为pR-T2/4。
上述方法中,所述遗传修饰山羊为抗金黄色葡萄球菌遗传修饰山羊。
所述遗传修饰山羊的巨噬细胞对金黄色葡萄球菌的吞噬能力高于作为转基因受体的山羊的巨噬细胞。
所述遗传修饰山羊的巨噬细胞对金黄色葡萄球菌的清除能力高于作为转基因受体的山羊的巨噬细胞。
本发明还提供了所述方法在动物育种和/或培育抗病山羊中的应用。
本发明提供了表达TLR2-4融合蛋白的山羊制备方法,利用CRISPR/Cas9系统将TLR2-4嵌合基因定点整合到山羊SETD5第一内含子上,筛选中靶细胞,利用体细胞核移植技术制备转基因山羊,从转基因山羊外周血分离TLR2-4巨噬细胞,使宿主机体获得更强的清除S.aureus的能力。
为了实现上述目的,本发明采用如下技术方案:
表达TLR2-4嵌合蛋白的山羊制备方法,具体步骤如下:
(1)构建基因打靶载体pX458-Cas9-sgRNA
a、鉴于基因内含子对突变容忍度较高的特点,选择了其上游基因SETD5的第一内含子上的一段序列进行靶点筛选。该区域内不存在可变剪接结合位点,对其进行基因编辑不会对SETD5基因及邻近基因的表达产生影响。在该区域的DNA双链上寻找PAM区,获得了148个靶点序列,靶点的长度为20nt。与NCBI在线数据库进行比对评估脱靶效应,通过在线预测网站对sgRNA结构和自由能进行评估,筛选出8个CRISPR/Cas9基因编辑靶点,与表达GFP的Cas9载体PX458连接,并电转至山羊成纤维细胞中,通过T7E1酶切鉴定结果评估各靶点的基因编辑效率,综合选出最终靶点;
b、用RT-PCR方法分别扩增出TLR2胞外区和TLR4的跨膜区和胞内区,同时在TLR2外显子的5’端添加信号肽及Myc标签,在TLR2外显子的3’端添加20bp的TLR4胞内同源序列,overlap PCR获得TLR2-4嵌合基因(SEQ ID No.1);为了提高同源重组的效率,构建了双切型供体质粒作为同源重组模板,即在同源臂外侧各添加一个sgRNA识别位点,并且在供体质粒上添加CMV-tdtomato片段,使转染了供体质粒的细胞能够瞬时表达红色荧光;
(2)打靶载体和供体质粒转染山羊胎儿成纤维细胞
将1-3代的山羊胎儿成纤维细胞培养至细胞达到80%汇合时,将打靶载体pX458-Cas9-sgRNA和供体质粒共同电转入山羊胎儿成纤维细胞,筛选出中靶细胞进行接种、扩大培养;
(3)中靶细胞的鉴定
打靶载体pX458-Cas9-sgRNA与供体质粒共转染至山羊成纤维细胞培养3天后,流式分选获得同时表达GFP和tdtomato的细胞。每500个细胞铺至100mm细胞培养皿中,培养约14天,用克隆环挑取细胞单克隆至96孔板中,扩大培养单克隆细胞进行PCR鉴定,细胞单克隆的红色、绿色荧光会在挑取单克隆后的大约7-10天消失;
(4)转TLR2-4基因克隆山羊的制备
将步骤(3)得到的所述中靶细胞核移植到去核后的卵母细胞的透明带下,放入融合液进行融合形成重组胚胎;将重组胚胎植入同步发情的受体羊输卵管中,得到转基因克隆山羊;
(5)转基因克隆山羊的检测
分离出生小羊外周血巨噬细胞,提取基因组DNA和蛋白,进行PCR和WB检测,结果显示TLR2-4基因成功整合并表达;
(6)转TLR2-4基因克隆山羊巨噬细胞清除金黄色葡萄球菌能力的检测
以MOI=10对转TLR2-4基因山羊巨噬细胞和野生型WT山羊巨噬细胞进行攻菌,结果显示转TLR2-4基因山羊巨噬细胞清除S.aureus的能力明显增强。
本发明提供了一种抗金黄色葡萄球菌的遗传修饰山羊的生产方法,通过构建TLR2-4嵌合表达的基因打靶载体,使用CRISPA/Cas9系统将TLR2-4嵌合基因定点整合到山羊SETD5基因座位点(山羊SETD5基因第一内含子上),筛选中靶细胞,利用体细胞核移植技术制备表达TLR2-4嵌合蛋白的转基因山羊,构建的转基因山羊提高了抗金黄色葡萄球菌能力并且增强了免疫力,达到防控金黄色葡萄球菌引起的山羊乳腺炎的目的。经过遗传修饰的TLR2-4转基因山羊巨噬细胞通过TLR2的胞外区域识别金葡菌,再通过TLR4的胞内机制增强对金葡菌的清除能力,达到通过山羊自身的先天免疫有效防控乳腺炎的目的,同时可以避免传统抗生素和疫苗的缺陷。为抗金黄色葡萄球菌山羊新种质的制备奠定基础。
附图说明
图1为本发明提供的TLR2胞外区与TLR4跨膜区和胞内区基因拼接的设计。
图2为本发明提供的打靶载体和供体质粒的结构示意图。
图3为本发明提供的中靶细胞PCR鉴定引物设计方案。
图4为本发明提供的TLR2-4转基因山羊。
图5为本发明提供的转基因山羊外周血巨噬细胞RT-PCR检测TLR2-4的表达结果。
图6为本发明提供的TLR2-4转基因克隆山羊外周血巨噬细胞感染金黄色葡萄球菌后对细菌的吞噬指数。
图7为本发明提供的TLR2-4转基因克隆山羊外周血巨噬细胞感染金黄色葡萄球菌后对细菌的吞噬率。
图8为本发明提供的TLR2-4转基因克隆山羊外周血巨噬细胞感染金黄色葡萄球菌后对细菌的清除率。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的PX458载体、pRosa26-promoter载体购于Addgene公司;pCMV-tdTomato载体购于Clontech公司;限制性核酸内切酶、T7E1酶、T4连接酶及Buffer购于NEB公司;引物合成及序列测定由苏州金唯智公司完成;无缝克隆试剂盒GeneArt GibsonAssembly购于Thermo Fisher公司。金黄色葡萄球菌(S.aureus ATCC29213)购自上海北诺生物科技有限公司
实施例1TLR2-4转基因山羊的制备
1、TLR2-4嵌合基因的制备
参照GenBank中山羊TLR2和TLR4的外显子序列设计并合成2对引物,引物序列如下:
TLR2基因扩增引物:
Forward:5’-GCCTCTGATCAGGCTTCTTC-3’
Reverse:5’-CCTAGGACCTTATTGCAGCT-3’
TLR4基因扩增引物:
Forward:5’-ATCATCAGCGTGTCGGTTGT-3’
Reverse:5’-TCAGGTGGAGGTGGTCGCTT-3’
提取山羊血液RNA,反转录合成cDNA,以此cDNA为模板利用上述引物,用RT-PCR方法扩增出山羊TLR2和TLR4基因片段,并利用下面的引物,在TLR2外显子的5’端添加信号肽及Myc序列,同时在TLR2胞外区外显子的3’端添加20bp的TLR4胞外区同源序列。
Forward:ATGCCACGTGCTTTGTGGACAGCGTGGGTCTGGGCTGTAATCAGCGTGTTCACGGAAGGAGAGCAGAAACTCATCTCTGAAGAGGATCTGGCCTCTGGCCTCTGATCAGGCTTCTTCCCGGTGGCATTCAGAAAGGGA
Reverse:ACAACCGACACGCTGATGATCCGGTGGCATTCAGAAAGGGA
将上述获得的TLR2胞外区外显子序列克隆至平末端载体。利用下面的引物进行Overlap PCR构建TLR2-4嵌合基因(图1)。
Forward:ATGCCACGTGCTTTGTGGAC
Reverse:TCAGGTGGAGGTGGTCGCTT
通过Overlap PCR获得的信号肽-Myc-TLR2-4嵌合基因(以下简称TLR2-4基因)如图1所示,嵌合基因PCR产物为2418bp(SEQ ID No.1)。
TLR2-4嵌合基因的核苷酸序列为SEQ ID No.1,编码的嵌合蛋白TLR2-4的氨基酸序列为SEQ ID No.2。
SEQ ID No.1的第91-2418位是TLR2的胞外区及TLR4的跨膜区与胞内区的核苷酸序列;
SEQ ID No.2的第31-805位是TLR2的胞外区及TLR4的跨膜区与胞内区的氨基酸序列。
SEQ ID No.2的第1-20位是TLR2的信号肽的氨基酸序列;
SEQ ID No.2的第21-30位是标签蛋白Myc的氨基酸序列;
SEQ ID No.2的第31-597位是TLR2的胞外区的氨基酸序列;
SEQ ID No.2的第598-805位是TLR4的跨膜区与胞内区的氨基酸序列;
SEQ ID No.1的第1-60位是TLR2的信号肽的核苷酸序列;
SEQ ID No.1的第61-90位是标签蛋白Myc的核苷酸序列;
SEQ ID No.1的第91-1791位是TLR2的胞外区的核苷酸序列;
SEQ ID No.1的第1792-2418位是TLR4的跨膜区与胞内区的核苷酸序列。
2、基因打靶载体和供体质粒的构建
PX458载体(购于Addgene公司)上已有嵌合gRNA支架结构,只需要将靶点对应的crRNA-oligo连接到酶切位点,其在山羊SETD5基因第一内含子区产生双链断裂,启动DNA双链断裂修复。同时引入连有TLR2-4基因的供体质粒,双链断裂修复过程使TLR2-4基因特异的插入。同时,为了提高转基因细胞的筛选效率,我们在供体质粒上添加CMV-tdtomato片段,使转染了供体质粒的细胞能够瞬时表达tdtomato,构建的基因打靶载体和供体质粒结构如图2所示。
所述sgRNA名称为SETD5-IN-sgRNA2,靶点序列(SEQ ID No.5)为:ACTACACATTATAGATGACT(SEQ ID No.3的第868-887),相应sgRNA识别区序列如SEQ ID No.4所示,相应sgRNA识别区的编码DNA序列为:AGTCATCTATAATGTGTAGT。
crRNA-oligo序列(靶点序列)如下:
ACTACACATTATAGATGACT(SEQ ID No.5)
sgRNA识别区核苷酸序列如下:
ACUACACAUUAUAGAUGACU(SEQ ID No.4)
2-1构建基因打靶载体pX458-Cas9-sgRNA
将SETD5基因的内含子区作为基因编辑靶点筛选区设计基因编辑靶点。在该区域的DNA双链上寻找PAM区(5’-NGG),并将其5’端的20nt作为靶点序列。随后,选取PAM区5’端的13nt作为种子序列,与NCBI在线数据库进行比对以进行脱靶效应的估计,剔除掉除靶点外还有较多脱靶位点的靶点序列。使用sgRNA结构在线预测网站(http://unafold.rna.albany.edu/q=mfold/RNA-Folding-Form/)评估各靶点对应的sgRNA的结构及自由能,筛选CRISPR/Cas9基因编辑靶点并进行细胞水平的验证。
pX458-Cas9-sgRNA打靶载体的具体构建方法如下:将合成的引物SETD5-IN-sgRNA2-F(5’-caccgACTACACATTATAGATGACT-3’)和引物SETD5-IN-sgRNA2-R(5’-aaacAGTCATCTATAATGTGTAGTc-3’)用ddH2O稀释成100μM的溶液,在退火体系中添加SETD5-IN-sgRNA2-F和SETD5-IN-sgRNA2-R引物溶液各1μL,T4连接酶Buffer 1μL,ddH2O 7μL,混匀。将上述退火体系放入PCR仪中:95℃,2min;以-5℃/s的速度降至25℃,随后置于冰上,令sgRNA退火体系中的F和R引物互补配对,形成含有黏性末端的双链寡核苷酸(oligo);并取1μL oligo,用ddH2O稀释100倍,得到oligo稀释液。
将PX458载体(购于Addgene公司)用限制性内切酶BbsI酶切并回收纯化,得到酶切后的线性骨架载体,并在连接体系中添加100ng酶切后的线性骨架载体;在连接体系中加入1μL上述oligo稀释液、1μL T4连接酶Buffer、1μL T4连接酶,并用ddH2O补足至10μL,混匀,置于16℃过夜反应。通过常规转化法进行转化、涂板。待单菌落长成后,挑取数个扩大培养并测序验证,结果显示sgRNA识别区的编码DNA已成功连入骨架载体,得到sgRNA2-Cas9打靶载体pX458-Cas9-sgRNA。
2-2构建供体质粒(pR-T2/4)
为后续的无缝克隆试验,需要在获得的TLR2-4嵌合基因两侧添加同源序列。以山羊基因组DNA为模板,利用表1的引物扩增左右同源臂。
表1 PCR引物
通过表2的引物获得供体质粒pR-T2/4中所需元件CMV-tdtomato和poly(A)。
表2 PCR引物
表中pRosa26-promoter载体购自Addgene公司。pCMV-tdTomato载体购自Clontech公司。
以pRosa26-promoter为骨架载体,在Poly(A)上游引入EcoRI、PmlI酶切位点,上下游分别添加BamHI及XbaI酶切位点;用BamHI及XbaI同时双酶切pRosa26-promoter载体和poly(A),将poly(A)克隆到pRosa26-promoter载体;用XbaI和ScaI-HF双酶切,通过无缝克隆将右同源臂HA-R插入Poly(A)后;用Acc65I和ScalI双酶切载体,在pRosa26-promoter载体上游引入PacI酶切位点并插入左同源臂HA-L,得到重组载体pRosa26-HA。pRosa26-HA是将pRosa26-promoter的限制性核酸内切酶BamHI和Xbal之间的小片段替换为核苷酸序列是SEQ ID No.5所示的DNA片段,保持pRosa26-promoter的其它核苷酸不变得到的重组载体。
用PmlI和EcoRI-HF双酶切pRosa26-HA,在pRosa26-promoter与poly(A)之间插入TLR2-4基因(SEQ ID No.1),得到重组载体pRosa26-TLR2-4;用SacI-HF单酶切pRosa26-TLR2-4,在HA-R下游插入从pCMV-tdTomato载体中获得的CMV-tdtomato,获得完整的pRosa26-TLR2-4供体质粒,简称为pR-T2/4(图2)。
3、TLR2-4定点整合成纤维细胞株的筛选构建的基因编辑载体可表达GFP,供体质粒可表达tdtomato,将打靶载体(pX458-Cas9-sgRNA)与供体质粒(pR-T2/4)共转染至1-3代、80%汇合度的山羊胎儿成纤维细胞,在转染后3天通过流式分选获取同时表达红绿荧光的细胞(即同时表达GFP及tdtomato的细胞),为了获取细胞单克隆,将约500个细胞均匀分散到100mm的细胞培养皿中,并使用含20%FBS的DMEM/F12培养基进行细胞单克隆的培养。培养约10-14天后,可见清晰的细胞单克隆,在倒置显微镜下观察并圈出单克隆的轮廓。用克隆环挑取单克隆并转移至96孔板中,用含20%FBS的DMEM/F12培养基继续培养。待96孔板中的细胞密度达到90%以上,取70%的细胞至新孔中继续培养,剩余细胞在原孔中培养,待新孔中的细胞长满后,消化并裂解细胞,PCR检测同源重组的情况。
4、TLR2-4定点整合成纤维细胞的PCR鉴定
设计鉴定引物如图3所示,利用PCR扩增打靶载体与基因组连接处的序列,确定打靶载体正确整合到打靶位点上。为去除供体质粒残留及NHEJ(非同源性末端接合)介导的随机插入的影响,将用于同源重组鉴定PCR的上游引物Primer U设计在同源臂HA-L上游,下游引物Primer L设计在同源臂外侧基因组上;
上述鉴定引物序列如下:
Primer U:ACAGCCAGTATGAGTGACACC
Primer L:TCCCCCTAAGACTCAGGCATC
Primer L1:AGACACCAGTTGGGTCACAAG
PCR鉴定的阳性克隆细胞(成功定点整合了TLR2-4嵌合基因),即转基因细胞(TLR2-4)作为供体细胞进行下一步的体细胞核移植。
5、体细胞核移植技术制备TLR2-4转基因山羊
实验选用体重40±4.5kg的黑山羊。使用含300mg孕酮的控制药物释放装置(CIDR)给其注射孕酮。供体山羊在去除CIDR前60h开始,每12h注射240IU的卵泡刺激素,共注射6次,进行超数排卵。在停止CIDR时肌肉注射0.1mg前列腺素,并在CIDR停药38h后,肌肉注射100IU促黄体生成素以诱导山羊排卵。在停止供体CIDR的同时停止受体的CIDR,并给受体山羊注射250IU剂量的妊娠母马血清促性腺激素。
为了方便手术和减少术后肠粘连,所有山羊在采集卵母细胞前12h禁食。在CIDR去除后62h进行腹腔镜检查以检测排卵反应,显示有黄体的山羊即对超排处理有反应,用于卵母细胞收集。将套管连接到注射器上插入子宫输卵管,用20ml含0.3%BSA的温PBS溶液冲洗输卵管收集卵母细胞。用体式显微镜放大10到40倍寻找卵母细胞,将细胞转移到M199培养液中并记录每只山羊的卵母细胞总数。将带有颗粒细胞的卵母细胞放入含有0.1%透明质酸酶的M199培养液中消化5分钟,并通过轻轻吹打去除颗粒细胞。将合格的卵母细胞放在含有5g/ml Hoechst 33342和5g/ml细胞松弛素B(CB)的M199中孵育10分钟。在倒置荧光显微镜下,用安装在显微操作器上的去核针吸出第1极体及部分胞质,将TLR2-4成纤维细胞(转基因细胞TLR2-4)作为供体细胞注入到去核卵母细胞的透明带下,然后通过微电极挤压融合,两次直流电脉冲在20V,每次20μs的条件下进行融合。将融合的重构胚胎放入添加7.5μg/ml CB和10%FBS的M199培养液中,在38.5℃、5%CO2、饱合湿度下培养2-3h后观察融合情况。将融合的重构胚胎用含5μmol/L的离子霉素处理4min,然后在含有2mmol/L6-Dimethylaminopurine(6-DMAP)的培养液内培养3h,洗3次后转移到mSOF培养液中进行过夜培养。将这些胚胎移植到受体的输卵管中,胚胎移植后60天通过超声检查评估受体的妊娠状态。
如图4所示,本发明共移植转TLR2-4基因克隆胚胎126枚到12只同期发情的受体山羊体内,一个月后2只建立妊娠,3个月后2只维持妊娠并继续被监测,共生产转基因克隆山羊1只。
实施例2转基因山羊TLR2-4嵌合蛋白的RT-PCR鉴定
提取出生小羊外周血巨噬细胞RNA,进行RT-PCR检测和WB检测,结果显示转基因山羊巨噬细胞cDNA成功扩增出TLR2-4嵌合基因片段,PCR检测结果见图5。引物如下:
Primer P1:(TLR2-4嵌合基因扩增引物)
Forward:TGACTTCCTGTCCTTCACACA
Reverse:CTTTACCAGTTCATTCCGCA
Primer P2:(内参基因GAPDH扩增引物)
Forward:CTGACCTGCCGCCTGGAGAAA
Reverse:GTAGAAGAGTGAGTGTCGCTGTT
结果表明,在转基因山羊中已成功定点整合了TLR2-4嵌合基因并表达出TLR2-4嵌合蛋白。
实施例3转TLR2-4基因山羊巨噬细胞吞噬金黄色葡萄球菌能力的鉴定
FITC标记的金黄色葡萄球菌以MOI=10分别感染TLR2-4巨噬细胞和WT对照组山羊巨噬细胞1小时,PBS洗涤三次后,用含有200μg/ml庆大霉素的DMEM孵育细胞1h以清除黏附在细胞表面的细菌。流式细胞术检测两组细胞的吞噬指数(巨噬细胞中FITC的平均荧光强度)和吞噬率(FITC阳性细胞数/总细胞术×100%)。如图6所示,在感染30min,60min,120min时,TLR2-4巨噬细胞对金黄色葡萄球菌的吞噬指数均显著高于两组野生型山羊巨噬细胞。如图7所示,在感染的所有时间段,TLR2-4巨噬细胞对金黄色葡萄球菌的吞噬率均显著高于两组野生型山羊巨噬细胞。实验结果说明在感染金葡菌后,表达TLR2-4嵌合蛋白能够作为山羊的先天免疫受体识别金葡菌,并且TLR2-4巨噬细胞对金葡菌的吞噬能力显著高于野生型山羊巨噬细胞。采用Origin 8统计软件对数据进行处理,实验结果以平均值±标准偏差表示,采用Student’s t检验,P<0.05(*)表示具有显著性差异,P<0.01(**)表示具有显著性差异,P<0.001(***)表示具有极显著性差异。
实施例4转TLR2-4基因山羊巨噬细胞清除金黄色葡萄球菌能力的鉴定
金黄色葡萄球菌以MOI=10分别感染TLR2-4巨噬细胞和WT对照组山羊巨噬细胞1小时,PBS洗涤三次后,用含有200μg/ml庆大霉素的DMEM培养液孵育细胞1小时以清除黏附在细胞表面的细菌。更换含有200μg/ml庆大霉素的DMEM培养液继续培养4h,8h,12h,24h。裂解细胞,通过cfu菌落计数的方法检测巨噬细胞中的金葡菌数。如图8所示,在感染后的4h,8h,12h,24h,TLR2-4巨噬细胞对金黄色葡萄球菌的清除率均显著高于两组WT山羊巨噬细胞。实验结果表明TLR2-4巨噬细胞比对照组巨噬细胞更有效的清除了金黄色葡萄球菌。采用Origin 8统计软件对数据进行处理,实验结果以平均值±标准偏差表示,采用Student’st检验,P<0.05(*)表示具有显著性差异,P<0.01(**)表示具有显著性差异,P<0.001(***)表示具有极显著性差异。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
SEQUENCE LISTING
<110> 中国农业大学
<120> 一种抗金黄色葡萄球菌遗传修饰山羊生产方法
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 2418
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
atgccacgtg ctttgtggac agcgtgggtc tgggctgtaa tcagcgtgtt cacggaagga 60
gagcagaaac tcatctctga agaggatctg gcctctgatc aggcttcttc tctgtcttgt 120
gacccaactg gtgtctgcga tggccattcc agatctttaa actccatccc ctctggtctc 180
acagcaggtg tgaaaagcct tgacctgtcc gacaacgaga tcacctatgt cggcaacaga 240
gacctgcaga ggtgtgtgaa cctgaagact ctgaggctgg gggccaatga aattcacacg 300
gtggaggaag attctttttt tcacctgagg aatcttgaat atttggactt atcctataat 360
cgcttatcta acttatcatc ttcctggttc aggtcccttt acgtcttgaa attcttaaac 420
ttactgggaa atttatacaa aacacttggg gaaacatctc ttttttctca tctcccaaat 480
ctgcggaccc tgaaagtagg aaatagtaac agcttcactc agattcatga aaaggatttc 540
actggactga cttttcttga ggagcttgag atcagtgctc aaaatctgca gttatatgtg 600
ccaaagagtt taaagtcgat ccagaacatt agccatctga ttcttcatct gaagcagcct 660
gttttactcc tggacattct tatagatatt gtaagttcct tagattattt agaactgaga 720
gatactaatt tgcacacttt ctatttttca gaagcatcca tcagtgaagt taatacatca 780
gttaaaaagc ttatatttag aaatgtgcaa ttcaccgatg aaagttttgt tgaagttgtc 840
aaactgttta actatgtttc tgggatcttg gaagtagagt ttgatgactg tacccatgat 900
ggaattggcg attttacagc actgactttg aacagaatta gatacctagg taacgtggag 960
acgttaacaa tacggaagtt gcatatccca cagtttttct tattttatga tctgagtagt 1020
atatatccac tcacaggcaa agttaaaaga gtcacaatag aaaacagtaa ggttttcctg 1080
gttccttgtt tactttcaca acatttaaaa tcactagaat atttggatct cagtgaaaac 1140
ttaatgtctg aagaaacctt gagaaactca gcctgtgagc atgcctggcc cttccttcaa 1200
accctggttt taaggcagaa tcgtttgaaa tcactagaaa aaactggaga acttttgctt 1260
actctgaaaa atctgaataa ccttgatatc agtaagaata attttctttc aatgcctgaa 1320
acttgtcagt ggccaggaaa aatgaaacag ttgaacttat ccagcacgag gatacacagt 1380
ttaacccagt gccttcccca gaccctggaa attttagatg ttagcaataa caatctcgat 1440
tcattttctt tgattttgcc gcaactcaaa gaactttata tttccagaaa taagttgaag 1500
actctaccag atgcctcctt cttacccgtg ttatcagtta tgagaattag cggaaatata 1560
ataaatactt tctcgaagga acaacttgat tcttttccac aactgaaggc tttggaggcc 1620
ggtggcaaca acttcatttg ctcctgtgac ttcctgtcct tcacacaggg acagcaggca 1680
ctggcccgtg tcctggtcga ctggccagat ggctaccgct gtgacgctcc ctcgcacgtg 1740
cggggccagc gggtgcagga cgcccggctc tccctttctg aatgccaccg gatcatcagc 1800
gtgtcggttg tcactgtact cctggtatct gtggtaggag tcctagtcta taagttctat 1860
ttccacctga tgcttcttgc tggctgcaaa aagtatggca gaggtgaaag cacctatgat 1920
gcctttgtaa tctactcgag ccaggatgaa gcctgggtgc ggaatgaact ggtaaagaac 1980
ttggaggagg gcgtgccccc ctttcagctc tgccttcact acagggactt tattcctggg 2040
gtggccatcg ccgccaacat catccaggaa ggtttccaca agagccgtaa ggtgattgtc 2100
gtggtgtccc agcacttcat ccagagccga tggtgtatct tcgagtatga gattgcccag 2160
acctggcagt ttctgagcag ccgtgctggc atcatcttca tcgtcctgca gaagctggag 2220
aagtctctcc tgcggcagca ggtggaactc tatcgccttc tgaacaggaa cacctacctg 2280
gagtgggagg acagtgtcct ggggcggcat gtcttctgga gaagactcag aaaagccttg 2340
ctggctggta aaccccggag tccagaagga acagcagatg cagagaccaa cccgcaggaa 2400
gcgaccacct ccacctga 2418
<210> 2
<211> 805
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 2
Met Pro Arg Ala Leu Trp Thr Ala Trp Val Trp Ala Val Ile Ser Val
1 5 10 15
Phe Thr Glu Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ala Ser
20 25 30
Asp Gln Ala Ser Ser Leu Ser Cys Asp Pro Thr Gly Val Cys Asp Gly
35 40 45
His Ser Arg Ser Leu Asn Ser Ile Pro Ser Gly Leu Thr Ala Gly Val
50 55 60
Lys Ser Leu Asp Leu Ser Asp Asn Glu Ile Thr Tyr Val Gly Asn Arg
65 70 75 80
Asp Leu Gln Arg Cys Val Asn Leu Lys Thr Leu Arg Leu Gly Ala Asn
85 90 95
Glu Ile His Thr Val Glu Glu Asp Ser Phe Phe His Leu Arg Asn Leu
100 105 110
Glu Tyr Leu Asp Leu Ser Tyr Asn Arg Leu Ser Asn Leu Ser Ser Ser
115 120 125
Trp Phe Arg Ser Leu Tyr Val Leu Lys Phe Leu Asn Leu Leu Gly Asn
130 135 140
Leu Tyr Lys Thr Leu Gly Glu Thr Ser Leu Phe Ser His Leu Pro Asn
145 150 155 160
Leu Arg Thr Leu Lys Val Gly Asn Ser Asn Ser Phe Thr Gln Ile His
165 170 175
Glu Lys Asp Phe Thr Gly Leu Thr Phe Leu Glu Glu Leu Glu Ile Ser
180 185 190
Ala Gln Asn Leu Gln Leu Tyr Val Pro Lys Ser Leu Lys Ser Ile Gln
195 200 205
Asn Ile Ser His Leu Ile Leu His Leu Lys Gln Pro Val Leu Leu Leu
210 215 220
Asp Ile Leu Ile Asp Ile Val Ser Ser Leu Asp Tyr Leu Glu Leu Arg
225 230 235 240
Asp Thr Asn Leu His Thr Phe Tyr Phe Ser Glu Ala Ser Ile Ser Glu
245 250 255
Val Asn Thr Ser Val Lys Lys Leu Ile Phe Arg Asn Val Gln Phe Thr
260 265 270
Asp Glu Ser Phe Val Glu Val Val Lys Leu Phe Asn Tyr Val Ser Gly
275 280 285
Ile Leu Glu Val Glu Phe Asp Asp Cys Thr His Asp Gly Ile Gly Asp
290 295 300
Phe Thr Ala Leu Thr Leu Asn Arg Ile Arg Tyr Leu Gly Asn Val Glu
305 310 315 320
Thr Leu Thr Ile Arg Lys Leu His Ile Pro Gln Phe Phe Leu Phe Tyr
325 330 335
Asp Leu Ser Ser Ile Tyr Pro Leu Thr Gly Lys Val Lys Arg Val Thr
340 345 350
Ile Glu Asn Ser Lys Val Phe Leu Val Pro Cys Leu Leu Ser Gln His
355 360 365
Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser Glu Asn Leu Met Ser Glu
370 375 380
Glu Thr Leu Arg Asn Ser Ala Cys Glu His Ala Trp Pro Phe Leu Gln
385 390 395 400
Thr Leu Val Leu Arg Gln Asn Arg Leu Lys Ser Leu Glu Lys Thr Gly
405 410 415
Glu Leu Leu Leu Thr Leu Lys Asn Leu Asn Asn Leu Asp Ile Ser Lys
420 425 430
Asn Asn Phe Leu Ser Met Pro Glu Thr Cys Gln Trp Pro Gly Lys Met
435 440 445
Lys Gln Leu Asn Leu Ser Ser Thr Arg Ile His Ser Leu Thr Gln Cys
450 455 460
Leu Pro Gln Thr Leu Glu Ile Leu Asp Val Ser Asn Asn Asn Leu Asp
465 470 475 480
Ser Phe Ser Leu Ile Leu Pro Gln Leu Lys Glu Leu Tyr Ile Ser Arg
485 490 495
Asn Lys Leu Lys Thr Leu Pro Asp Ala Ser Phe Leu Pro Val Leu Ser
500 505 510
Val Met Arg Ile Ser Gly Asn Ile Ile Asn Thr Phe Ser Lys Glu Gln
515 520 525
Leu Asp Ser Phe Pro Gln Leu Lys Ala Leu Glu Ala Gly Gly Asn Asn
530 535 540
Phe Ile Cys Ser Cys Asp Phe Leu Ser Phe Thr Gln Gly Gln Gln Ala
545 550 555 560
Leu Ala Arg Val Leu Val Asp Trp Pro Asp Gly Tyr Arg Cys Asp Ala
565 570 575
Pro Ser His Val Arg Gly Gln Arg Val Gln Asp Ala Arg Leu Ser Leu
580 585 590
Ser Glu Cys His Arg Ile Ile Ser Val Ser Val Val Thr Val Leu Leu
595 600 605
Val Ser Val Val Gly Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met
610 615 620
Leu Leu Ala Gly Cys Lys Lys Tyr Gly Arg Gly Glu Ser Thr Tyr Asp
625 630 635 640
Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Ala Trp Val Arg Asn Glu
645 650 655
Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu
660 665 670
His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile
675 680 685
Gln Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln
690 695 700
His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln
705 710 715 720
Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu
725 730 735
Gln Lys Leu Glu Lys Ser Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg
740 745 750
Leu Leu Asn Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly
755 760 765
Arg His Val Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Ala Gly Lys
770 775 780
Pro Arg Ser Pro Glu Gly Thr Ala Asp Ala Glu Thr Asn Pro Gln Glu
785 790 795 800
Ala Thr Thr Ser Thr
805
<210> 3
<211> 2000
<212> DNA
<213> 山羊(Capra hircus)
<400> 3
ttaaaaatgc catttcatca catactttgg aatttttggc agcaaggaat ggtgtcaatc 60
attatttatc ataggtacat tacactttga ggaatgactg aatttttttc tttttacaaa 120
taaccgccag ttgtattact aaatatatac tatctcataa ttttgcaggg ttgaagtttc 180
atttgtccta aataacaaaa aataagaaaa ctttatttta ttcataatcc actttttcac 240
tggtaagatg gatccaacaa tcactgattg tctttagctt ctgcccaata tttcataaat 300
taattgaata tgaagaggat aacgtaaaag tggttgtctg cacatatgca ttttgaacaa 360
tattattttc ctgtccaaag ggaaaaaatg aagtggccat cctcctccag aaaggccaaa 420
ataagtcccc aaagtatgta catttctaga cacaaaacac tatcagaaga cagtaaaaat 480
ttcaagcatg ctggtaaaat aaattctatg aaaaggcctt ctatgctctt aaggaaatga 540
tcttacgtta agatctcaaa ggagttccaa acaggaaata caagttaaaa acaattattt 600
tataaggtag ctttcatatt atataaaaga caacttacag acatttgtat ctatgttaaa 660
cagccagtat gagtgacacc atacttatct gatatggcat cttttatatg tgatagtaat 720
aagaattaga accacctaaa atgttatcca ttaaaggttc tgtttcatta ttccccttgc 780
aacactgaaa gacaatgcaa gtaacatttg tgaatgacaa aactcatttc cattctttct 840
ggttcccgcc acaaacgcca gagtcatact acacattata gatgactggg ttcatttaca 900
caaaaccgaa catactaaaa ttctgaagga aaaaaaaaat atatatatat ataggatgcc 960
tgagtcttag ggggagacaa taacaggcat ctatctgtta agtatcagat gtaactgatg 1020
gtatattggg ctggtgattc tccttcaagt acttccatat ttaacaaact gttctgctta 1080
atcagagatc ttcctaaact gtgggaaccg tggaagaaga cagactacat aaggggccta 1140
gatgctatca tctcaagtat cctgtatact agatttattc aatttctgag tattccttct 1200
agtctgtcat atttcaaatg gatcactgtc aaacatatag atccttgtga tcttagacaa 1260
ctatactcaa cacatattat aaatcaatat cacaggtcaa attctaaaga aataatctaa 1320
tttggagaaa tctattacta acacagaaaa taagaaaata cttgatattt atacatagaa 1380
atcgttctgc aaaggacagt tgtttctaga gtttaagacc ctaaatatag taaaatgttt 1440
agcaaaacat ctgttcaata aggtaactat ggtctaacaa ctggaaggaa gaagacgtac 1500
aagtattaaa attgatttaa tgattaaaaa gatggaggaa aacatgccag ctgcagaaaa 1560
atcaactgat attttctcca ttctggttct catgaagatc cttaaattct tattaccttg 1620
gttttaaaag gattgaaaaa ttaaaactca aatggaactt ctggtcctat ccaagcaaaa 1680
tccccagatt aaaaccaaaa atctgccaca aaaaccaaac agaaaagtct ttaactacgg 1740
ttcagtttcc aaatgctgat atccataaat gggtagaagg ttatgaattg ctaattctca 1800
tttccccacc aataaaatgc aaaccagcac agcagtgaaa attcccagga aagcaaggtt 1860
tccgtcttag ccctgaagca gccattgcct aagtgcagct ccctgcagcc aacagcatta 1920
atggacgctg cactgctgtc cttccctgga gacagcagcc agcactactc aagctcctca 1980
cgtagcaacc agagctccag 2000
<210> 4
<211> 20
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 4
acuacacauu auagaugacu 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
actacacatt atagatgact 20
<210> 6
<211> 5599
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 6
actacacatt atagatgact gggcccaagc tatggggtat ctactcaatt cagaatgaat 60
atacagcaga acatttttca taagactaac tgattaaaca aaatatgtac tgagaaatta 120
aagaaatgat aatctatcaa cctctcatga actaccctga atcttcacca caagagacat 180
cactgcgtca acagaaaggt taaaaatgcc atttcatcac atactttgga atttttggca 240
gcaaggaatg gtgtcaatca ttatttatca taggtacatt acactttgag gaatgactga 300
atttttttct ttttacaaat aaccgccagt tgtattacta aatatatact atctcataat 360
tttgcagggt tgaagtttca tttgtcctaa ataacaaaaa ataagaaaac tttattttat 420
tcataatcca ctttttcact ggtaagatgg atccaacaat cactgattgt ctttagcttc 480
tgcccaatat ttcataaatt aattgaatat gaagaggata acgtaaaagt ggttgtctgc 540
acatatgcat tttgaacaat attattttcc tgtccaaagg gaaaaaatga agtggccatc 600
ctcctccaga aaggccaaaa taagtcccca aagtatgtac atttctagac acaaaacact 660
atcagaagac agtaaaaatt tcaagcatgc tggtaaaata aattctatga aaaggccttc 720
tatgctctta aggaaatgat cttacgttaa gatctcaaag gagttccaaa caggaaatac 780
aagttaaaaa caattatttt ataaggtagc tttcatatta tataaaagac aacttacaga 840
catttgtatc tatgttaaac agccagtatg agtgacacca tacttatctg atatggcatc 900
ttttatatgt gatagtaata agaattagaa ccacctaaaa tgttatccat taaaggttct 960
gtttcattat tccccttgca acactgaaag acaatgcaag taacatttgt gaatgacaaa 1020
actcatttcc attctttctg gttcccgcca caaacgccag agtcatacta cacattatag 1080
atttaattaa gtcgactaga tgaaggagag cctttctctc tgggcaagag cggtgcaatg 1140
gtgtgtaaag gtagctgaga agacgaaaag ggcaagcatc ttcctgctac caggctgggg 1200
aggcccaggc ccacgacccc gaggagaggg aacgcaggga gactgaggtg acccttcttt 1260
cccccggggc ccggtcgtgt ggttcggtgt ctcttttctg ttggaccctt accttgaccc 1320
aggcgctgcc ggggcctggg cccgggctgc ggcgcacggc actcccggga ggcagcgaga 1380
ctcgagttag gcccaacgcg gcgccacggc gtttcctggc cgggaatggc ccgtacccgt 1440
gaggtggggg tggggggcag aaaaggcgga gcgagcccga ggcggggagg gggagggcca 1500
ggggcggagg gggccggcac tactgtgttg gcggactggc gggactaggg ctgcgtgagt 1560
ctctgagcgc aggcgggcgg cggccgcccc tcccccggcg gcggcagcgg cggcagcggc 1620
ggcagctcac tcagcccgct gcccgagcgg aaacgccact gaccgcacgg ggattcccag 1680
tgccggcgcc aggggcacgc gggacacgcc ccctcccgcc gcgccattgg cctctccgcc 1740
caccgcccca cacttattgg ccggtgcgcc gccaatcagc ggaggctgcc ggggccgcct 1800
aaagaagagg ctgtgctttg gggctccggc tcctcagaga gcctcggcta ggtaggggat 1860
cgggactctg gcgggagggc ggcttggtgc gtttgcgggg atccgaattc atgccacgtg 1920
ctttgtggac agcgtgggtc tgggctgtaa tcagcgtgtt cacggaagga gagcagaaac 1980
tcatctctga agaggatctg gcctctgatc aggcttcttc tctgtcttgt gacccaactg 2040
gtgtctgcga tggccattcc agatctttaa actccatccc ctctggtctc acagcaggtg 2100
tgaaaagcct tgacctgtcc gacaacgaga tcacctatgt cggcaacaga gacctgcaga 2160
ggtgtgtgaa cctgaagact ctgaggctgg gggccaatga aattcacacg gtggaggaag 2220
attctttttt tcacctgagg aatcttgaat atttggactt atcctataat cgcttatcta 2280
acttatcatc ttcctggttc aggtcccttt acgtcttgaa attcttaaac ttactgggaa 2340
atttatacaa aacacttggg gaaacatctc ttttttctca tctcccaaat ctgcggaccc 2400
tgaaagtagg aaatagtaac agcttcactc agattcatga aaaggatttc actggactga 2460
cttttcttga ggagcttgag atcagtgctc aaaatctgca gttatatgtg ccaaagagtt 2520
taaagtcgat ccagaacatt agccatctga ttcttcatct gaagcagcct gttttactcc 2580
tggacattct tatagatatt gtaagttcct tagattattt agaactgaga gatactaatt 2640
tgcacacttt ctatttttca gaagcatcca tcagtgaagt taatacatca gttaaaaagc 2700
ttatatttag aaatgtgcaa ttcaccgatg aaagttttgt tgaagttgtc aaactgttta 2760
actatgtttc tgggatcttg gaagtagagt ttgatgactg tacccatgat ggaattggcg 2820
attttacagc actgactttg aacagaatta gatacctagg taacgtggag acgttaacaa 2880
tacggaagtt gcatatccca cagtttttct tattttatga tctgagtagt atatatccac 2940
tcacaggcaa agttaaaaga gtcacaatag aaaacagtaa ggttttcctg gttccttgtt 3000
tactttcaca acatttaaaa tcactagaat atttggatct cagtgaaaac ttaatgtctg 3060
aagaaacctt gagaaactca gcctgtgagc atgcctggcc cttccttcaa accctggttt 3120
taaggcagaa tcgtttgaaa tcactagaaa aaactggaga acttttgctt actctgaaaa 3180
atctgaataa ccttgatatc agtaagaata attttctttc aatgcctgaa acttgtcagt 3240
ggccaggaaa aatgaaacag ttgaacttat ccagcacgag gatacacagt ttaacccagt 3300
gccttcccca gaccctggaa attttagatg ttagcaataa caatctcgat tcattttctt 3360
tgattttgcc gcaactcaaa gaactttata tttccagaaa taagttgaag actctaccag 3420
atgcctcctt cttacccgtg ttatcagtta tgagaattag cggaaatata ataaatactt 3480
tctcgaagga acaacttgat tcttttccac aactgaaggc tttggaggcc ggtggcaaca 3540
acttcatttg ctcctgtgac ttcctgtcct tcacacaggg acagcaggca ctggcccgtg 3600
tcctggtcga ctggccagat ggctaccgct gtgacgctcc ctcgcacgtg cggggccagc 3660
gggtgcagga cgcccggctc tccctttctg aatgccaccg gatcatcagc gtgtcggttg 3720
tcactgtact cctggtatct gtggtaggag tcctagtcta taagttctat ttccacctga 3780
tgcttcttgc tggctgcaaa aagtatggca gaggtgaaag cacctatgat gcctttgtaa 3840
tctactcgag ccaggatgaa gcctgggtgc ggaatgaact ggtaaagaac ttggaggagg 3900
gcgtgccccc ctttcagctc tgccttcact acagggactt tattcctggg gtggccatcg 3960
ccgccaacat catccaggaa ggtttccaca agagccgtaa ggtgattgtc gtggtgtccc 4020
agcacttcat ccagagccga tggtgtatct tcgagtatga gattgcccag acctggcagt 4080
ttctgagcag ccgtgctggc atcatcttca tcgtcctgca gaagctggag aagtctctcc 4140
tgcggcagca ggtggaactc tatcgccttc tgaacaggaa cacctacctg gagtgggagg 4200
acagtgtcct ggggcggcat gtcttctgga gaagactcag aaaagccttg ctggctggta 4260
aaccccggag tccagaagga acagcagatg cagagaccaa cccgcaggaa gcgaccacct 4320
ccacctgaca cgtgctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 4380
gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 4440
tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 4500
caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggt 4560
ctagagggtt catttacaca aaaccgaaca tactaaaatt ctgaaggaaa aaaaaaatat 4620
atatatatat aggatgcctg agtcttaggg ggagacaata acaggcatct atctgttaag 4680
tatcagatgt aactgatggt atattgggct ggtgattctc cttcaagtac ttccatattt 4740
aacaaactgt tctgcttaat cagagatctt cctaaactgt gggaaccgtg gaagaagaca 4800
gactacataa ggggcctaga tgctatcatc tcaagtatcc tgtatactag atttattcaa 4860
tttctgagta ttccttctag tctgtcatat ttcaaatgga tcactgtcaa acatatagat 4920
ccttgtgatc ttagacaact atactcaaca catattataa atcaatatca caggtcaaat 4980
tctaaagaaa taatctaatt tggagaaatc tattactaac acagaaaata agaaaatact 5040
tgatatttat acatagaaat cgttctgcaa aggacagttg tttctagagt ttaagaccct 5100
aaatatagta aaatgtttag caaaacatct gttcaataag gtaactatgg tctaacaact 5160
ggaaggaaga agacgtacaa gtattaaaat tgatttaatg attaaaaaga tggaggaaaa 5220
catgccagct gcagaaaaat caactgatat tttctccatt ctggttctca tgaagatcct 5280
taaattctta ttaccttggt tttaaaagga ttgaaaaatt aaaactcaaa tggaacttct 5340
ggtcctatcc aagcaaaatc cccagattaa aaccaaaaat ctgccacaaa aaccaaacag 5400
aaaagtcttt aactacggtt cagtttccaa atgctgatat ccataaatgg gtagaaggtt 5460
atgaattgct aattctcatt tccccaccaa taaaatgcaa accagcacag cagtgaaaat 5520
tcccaggaaa gcaaggtttc cgtcttagcc ctgaagcagc cattgcctaa gtgcagctcc 5580
ctgcagccaa cagcattaa 5599
Claims (10)
1.一种制备遗传修饰山羊的方法,其特征在于,所述方法包括如下步骤:将TLR2-4嵌合基因导入作为转基因受体的山羊体细胞中,得到转化的体细胞,由所述转化的体细胞再生出所述遗传修饰山羊,所述TLR2-4嵌合基因编码的嵌合蛋白质包括TLR2的胞外区和TLR4的跨膜区与胞内区。
2.根据权利要求1所述的方法,其特征在于,所述嵌合蛋白质还包括信号肽和/或标签蛋白。
3.根据权利要求1或2所述的方法,其特征在于,所述TLR2的胞外区为下述任一种蛋白质:
P1)氨基酸序列是SEQ ID No.2的第31-597位的蛋白质;
P2)将P1)所示的蛋白质经过氨基酸残基的取代和/或缺失和/或添加得到的与P1)所示的蛋白质具有80%以上的同一性且具有相同功能的蛋白质;
所述TLR4的跨膜区与胞内区为下述任一种蛋白质:
Q1)氨基酸序列是SEQ ID No.2的第598-805位的蛋白质;
Q2)将Q1)所示的蛋白质经过氨基酸残基的取代和/或缺失和/或添加得到的与Q1)所示的蛋白质具有80%以上的同一性且具有相同功能的蛋白质。
4.根据权利要求1所述的方法,其特征在于,所述TLR2-4嵌合基因的核苷酸序列为SEQID No.1。
5.根据权利要求1-4中任一所述的方法,其特征在于,所述将TLR2-4嵌合基因导入作为转基因受体的山羊体细胞中为将所述TLR2-4嵌合基因导入到山羊SETD5基因第一内含子中。
6.根据权利要求1-5中任一所述的方法,其特征在于,所述导入为利用CRISPR/Cas9系统进行。
7.根据权利要求6所述的方法,其特征在于,所述CRISPR/Cas9系统包括如下A1)或A2):
A1)sgRNA,所述sgRNA特异性靶向于SETD5-IN,所述SETD5-IN是大小为2kb的双链DNA分子,所述SETD5-IN的核苷酸序列为SEQ ID No.3;
A2)表达A1)所述sgRNA的CRISPR/Cas9载体。
8.根据权利要求7所述的方法,其特征在于,所述sgRNA识别区的核苷酸序列为SEQ IDNo.4。
9.根据权利要求1-8任一所述的方法,其特征在于,所述遗传修饰山羊为抗金黄色葡萄球菌遗传修饰山羊。
10.权利要求1-9任一所述的方法在动物育种和/或培育抗病山羊中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110819041.3A CN113502299B (zh) | 2021-07-20 | 2021-07-20 | 一种抗金黄色葡萄球菌遗传修饰山羊生产方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110819041.3A CN113502299B (zh) | 2021-07-20 | 2021-07-20 | 一种抗金黄色葡萄球菌遗传修饰山羊生产方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113502299A true CN113502299A (zh) | 2021-10-15 |
CN113502299B CN113502299B (zh) | 2023-03-14 |
Family
ID=78013965
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110819041.3A Active CN113502299B (zh) | 2021-07-20 | 2021-07-20 | 一种抗金黄色葡萄球菌遗传修饰山羊生产方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113502299B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1263555A (zh) * | 1997-05-07 | 2000-08-16 | 先灵公司 | 人toll样受体蛋白、相关试剂和方法 |
-
2021
- 2021-07-20 CN CN202110819041.3A patent/CN113502299B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1263555A (zh) * | 1997-05-07 | 2000-08-16 | 先灵公司 | 人toll样受体蛋白、相关试剂和方法 |
Non-Patent Citations (6)
Title |
---|
MUHAMMAD AKHTAR: "常春藤苷C通过抑制TLR2&TLR4及其下游NF-κB和MAPKS的信号级联效应发挥抗炎作用", 《CNKI博士学位论文全文库》 * |
TAKEUCHI ET AL: "Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.", 《IMMUNITY》 * |
WIELAND等: "Host defence during Klebsiella pneumonia relies on haematopoietic-expressed Toll-like receptors 4 and 2", 《EUR RESPIR J.》 * |
沈媛: "大肠杆菌感染过程中TLR2/4和NLRP3介导小鼠炎症反应发生的机制研究", 《CNKI博士学位论文全文库》 * |
袁建龙: "利用体细胞核移植技术生产转基因绒山羊的研究", 《CNKI博士学位论文全文库》 * |
郝斐: "利用CRISPR-Cas9系统与体细胞核移植技术制备EDAR基因打靶绒山羊的研究", 《CNKI博士学位论文全文库》 * |
Also Published As
Publication number | Publication date |
---|---|
CN113502299B (zh) | 2023-03-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2022201329B2 (en) | Genetically modified cells, tissues, and organs for treating disease | |
KR20210091740A (ko) | 세포-매개된 종양용해 바이러스 요법용 향상된 시스템 | |
JP2022518329A (ja) | CRISPR-Cas12j酵素およびシステム | |
CN110337493A (zh) | 用于治疗肌强直性营养不良的组合物和方法 | |
CN113748124A (zh) | 工程化以定植肿瘤、肿瘤驻留免疫细胞和肿瘤微环境的免疫刺激性细菌 | |
CN113957069B (zh) | 用于pAPN基因第736位和第738位氨基酸同时修饰的组合物及其应用 | |
AU2015404563A1 (en) | Pathogen-resistant animals having modified CD163 genes | |
KR101760618B1 (ko) | Sox 유전자가 이입된 비바이러스성 미니써클 벡터 및 이의 제조방법 | |
CN116445454B (zh) | 一种用于培育抗tgev感染的猪品种的成套系统及其应用 | |
CN113502299B (zh) | 一种抗金黄色葡萄球菌遗传修饰山羊生产方法 | |
CN107075501B (zh) | 炎症报道系统 | |
CN110904046B (zh) | Islr基因在制备治疗肥胖及改善胰岛素抵抗的药物中的应用 | |
CN113073114A (zh) | 一种抗非洲猪瘟克隆猪的制备方法 | |
CN116769016B (zh) | pAPN突变体和用于pAPN基因定点修饰的组合物及应用 | |
CN113501885B (zh) | 嵌合抗病基因及其相关生物材料与应用 | |
CN115322993B (zh) | 一种用于猪基因组定点整合外源基因的安全位点及用其构建猪育种群方法 | |
CN114717200B (zh) | 一株马立克病病毒特超强毒株及其基因缺失毒株的构建和应用 | |
CN109207521B (zh) | 人GDNF基因在牛β-casein基因座定位整合的载体及其应用 | |
LU503556B1 (en) | Monoclonal antibody or derivative generated based on transgenic goat and application thereof | |
JP2019092417A (ja) | 遺伝子改変非ヒト動物及びその使用 | |
CN109266678B (zh) | 一种人Epo基因在牛β-casein基因座上定位整合的基因打靶方法 | |
CN111621500B (zh) | 一种基于myl4基因编辑的心房颤动/心房心肌病大鼠模型及其构建方法 | |
CN114908097A (zh) | 一种Dox调控的记录猪组织分化与器官发生的谱系示踪技术 | |
WO2015163711A1 (ko) | 마이오스타틴 유전자를 표적으로 하는 talen 및 이를 이용한 마이오스타틴 유전자가 녹아웃된 동물을 제조하는 방법 | |
KR100460852B1 (ko) | 미꾸라지 렉틴 유전자의 조절부위를 포함하는 발현벡터 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |