CN112979781B - Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof - Google Patents
Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof Download PDFInfo
- Publication number
- CN112979781B CN112979781B CN202110292721.4A CN202110292721A CN112979781B CN 112979781 B CN112979781 B CN 112979781B CN 202110292721 A CN202110292721 A CN 202110292721A CN 112979781 B CN112979781 B CN 112979781B
- Authority
- CN
- China
- Prior art keywords
- recombinant protein
- beta
- protein
- tag
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000193 Interleukin-1 beta Proteins 0.000 title claims abstract description 97
- 102000003777 Interleukin-1 beta Human genes 0.000 title claims abstract description 93
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 66
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 66
- 241001582957 Sebastes schlegelii Species 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 claims abstract description 11
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 claims abstract description 10
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 150000001413 amino acids Chemical group 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 241000588724 Escherichia coli Species 0.000 claims description 15
- 239000013604 expression vector Substances 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 9
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 claims description 8
- 238000001976 enzyme digestion Methods 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 108010076818 TEV protease Proteins 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 3
- 108010093488 His-His-His-His-His-His Proteins 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 230000002101 lytic effect Effects 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000001042 affinity chromatography Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 14
- 239000013598 vector Substances 0.000 abstract description 11
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 230000006698 induction Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 2
- 238000000605 extraction Methods 0.000 abstract 1
- 230000009465 prokaryotic expression Effects 0.000 description 10
- 241000251468 Actinopterygii Species 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 108700026244 Open Reading Frames Proteins 0.000 description 7
- 238000004153 renaturation Methods 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 210000003000 inclusion body Anatomy 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- ACRYGQFHAQHDSF-ZLUOBGJFSA-N Asn-Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ACRYGQFHAQHDSF-ZLUOBGJFSA-N 0.000 description 5
- TZFQICWZWFNIKU-KKUMJFAQSA-N Asn-Leu-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 TZFQICWZWFNIKU-KKUMJFAQSA-N 0.000 description 5
- 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 5
- 210000004027 cell Anatomy 0.000 description 5
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 4
- 108010065920 Insulin Lispro Proteins 0.000 description 4
- 238000001261 affinity purification Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 3
- NKVZTQVGUNLLQW-JBDRJPRFSA-N Ile-Ala-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)O)N NKVZTQVGUNLLQW-JBDRJPRFSA-N 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 108010038633 aspartylglutamate Proteins 0.000 description 3
- 108010092854 aspartyllysine Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 230000032696 parturition Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 108010061238 threonyl-glycine Proteins 0.000 description 3
- ZQJHYRVSKHGGJY-YPKJBDGSSA-N (2s,3r)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoic acid Chemical compound C([C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 ZQJHYRVSKHGGJY-YPKJBDGSSA-N 0.000 description 2
- XCVRVWZTXPCYJT-BIIVOSGPSA-N Ala-Asn-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N XCVRVWZTXPCYJT-BIIVOSGPSA-N 0.000 description 2
- MVBWLRJESQOQTM-ACZMJKKPSA-N Ala-Gln-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O MVBWLRJESQOQTM-ACZMJKKPSA-N 0.000 description 2
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 2
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 2
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 2
- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-N 0.000 description 2
- CJQAEJMHBAOQHA-DLOVCJGASA-N Ala-Phe-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CJQAEJMHBAOQHA-DLOVCJGASA-N 0.000 description 2
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 2
- TVUFMYKTYXTRPY-HERUPUMHSA-N Ala-Trp-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O TVUFMYKTYXTRPY-HERUPUMHSA-N 0.000 description 2
- XKHLBBQNPSOGPI-GUBZILKMSA-N Ala-Val-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N XKHLBBQNPSOGPI-GUBZILKMSA-N 0.000 description 2
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 2
- KMFPQTITXUKJOV-DCAQKATOSA-N Arg-Ser-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O KMFPQTITXUKJOV-DCAQKATOSA-N 0.000 description 2
- XRNXPIGJPQHCPC-RCWTZXSCSA-N Arg-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)O)C(O)=O XRNXPIGJPQHCPC-RCWTZXSCSA-N 0.000 description 2
- RZVVKNIACROXRM-ZLUOBGJFSA-N Asn-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N RZVVKNIACROXRM-ZLUOBGJFSA-N 0.000 description 2
- ANPFQTJEPONRPL-UGYAYLCHSA-N Asn-Ile-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O ANPFQTJEPONRPL-UGYAYLCHSA-N 0.000 description 2
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 2
- DATSKXOXPUAOLK-KKUMJFAQSA-N Asn-Tyr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DATSKXOXPUAOLK-KKUMJFAQSA-N 0.000 description 2
- JDHOJQJMWBKHDB-CIUDSAMLSA-N Asp-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N JDHOJQJMWBKHDB-CIUDSAMLSA-N 0.000 description 2
- POTCZYQVVNXUIG-BQBZGAKWSA-N Asp-Gly-Pro Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O POTCZYQVVNXUIG-BQBZGAKWSA-N 0.000 description 2
- NHSDEZURHWEZPN-SXTJYALSSA-N Asp-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CC(=O)O)N NHSDEZURHWEZPN-SXTJYALSSA-N 0.000 description 2
- LBOVBQONZJRWPV-YUMQZZPRSA-N Asp-Lys-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LBOVBQONZJRWPV-YUMQZZPRSA-N 0.000 description 2
- GKWFMNNNYZHJHV-SRVKXCTJSA-N Asp-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(O)=O GKWFMNNNYZHJHV-SRVKXCTJSA-N 0.000 description 2
- ZKAOJVJQGVUIIU-GUBZILKMSA-N Asp-Pro-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZKAOJVJQGVUIIU-GUBZILKMSA-N 0.000 description 2
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 2
- 101150071146 COX2 gene Proteins 0.000 description 2
- RMOCFPBLHAOTDU-ACZMJKKPSA-N Gln-Asn-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RMOCFPBLHAOTDU-ACZMJKKPSA-N 0.000 description 2
- VNTGPISAOMAXRK-CIUDSAMLSA-N Gln-Pro-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O VNTGPISAOMAXRK-CIUDSAMLSA-N 0.000 description 2
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 2
- IVGJYOOGJLFKQE-AVGNSLFASA-N Glu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N IVGJYOOGJLFKQE-AVGNSLFASA-N 0.000 description 2
- MFNUFCFRAZPJFW-JYJNAYRXSA-N Glu-Lys-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFNUFCFRAZPJFW-JYJNAYRXSA-N 0.000 description 2
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 description 2
- JBRBACJPBZNFMF-YUMQZZPRSA-N Gly-Ala-Lys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN JBRBACJPBZNFMF-YUMQZZPRSA-N 0.000 description 2
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 2
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 2
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 2
- UHPAZODVFFYEEL-QWRGUYRKSA-N Gly-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN UHPAZODVFFYEEL-QWRGUYRKSA-N 0.000 description 2
- ISSDODCYBOWWIP-GJZGRUSLSA-N Gly-Pro-Trp Chemical compound [H]NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ISSDODCYBOWWIP-GJZGRUSLSA-N 0.000 description 2
- SBVMXEZQJVUARN-XPUUQOCRSA-N Gly-Val-Ser Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O SBVMXEZQJVUARN-XPUUQOCRSA-N 0.000 description 2
- JENKOCSDMSVWPY-SRVKXCTJSA-N His-Leu-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O JENKOCSDMSVWPY-SRVKXCTJSA-N 0.000 description 2
- BZAQOPHNBFOOJS-DCAQKATOSA-N His-Pro-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O BZAQOPHNBFOOJS-DCAQKATOSA-N 0.000 description 2
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 2
- QLRMMMQNCWBNPQ-QXEWZRGKSA-N Ile-Arg-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)O)N QLRMMMQNCWBNPQ-QXEWZRGKSA-N 0.000 description 2
- QADCTXFNLZBZAB-GHCJXIJMSA-N Ile-Asn-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C)C(=O)O)N QADCTXFNLZBZAB-GHCJXIJMSA-N 0.000 description 2
- UAVQIQOOBXFKRC-BYULHYEWSA-N Ile-Asn-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O UAVQIQOOBXFKRC-BYULHYEWSA-N 0.000 description 2
- OVDKXUDMKXAZIV-ZPFDUUQYSA-N Ile-Lys-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OVDKXUDMKXAZIV-ZPFDUUQYSA-N 0.000 description 2
- BATWGBRIZANGPN-ZPFDUUQYSA-N Ile-Pro-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BATWGBRIZANGPN-ZPFDUUQYSA-N 0.000 description 2
- JNLSTRPWUXOORL-MMWGEVLESA-N Ile-Ser-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N JNLSTRPWUXOORL-MMWGEVLESA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 2
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 2
- GZAUZBUKDXYPEH-CIUDSAMLSA-N Leu-Cys-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N GZAUZBUKDXYPEH-CIUDSAMLSA-N 0.000 description 2
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 2
- DPWGZWUMUUJQDT-IUCAKERBSA-N Leu-Gln-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O DPWGZWUMUUJQDT-IUCAKERBSA-N 0.000 description 2
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 2
- DBSLVQBXKVKDKJ-BJDJZHNGSA-N Leu-Ile-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DBSLVQBXKVKDKJ-BJDJZHNGSA-N 0.000 description 2
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 2
- 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 2
- MVVSHHJKJRZVNY-ACRUOGEOSA-N Leu-Phe-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MVVSHHJKJRZVNY-ACRUOGEOSA-N 0.000 description 2
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 2
- PWPBLZXWFXJFHE-RHYQMDGZSA-N Leu-Pro-Thr Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O PWPBLZXWFXJFHE-RHYQMDGZSA-N 0.000 description 2
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 2
- 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 2
- HQVDJTYKCMIWJP-YUMQZZPRSA-N Lys-Asn-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O HQVDJTYKCMIWJP-YUMQZZPRSA-N 0.000 description 2
- HKCCVDWHHTVVPN-CIUDSAMLSA-N Lys-Asp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O HKCCVDWHHTVVPN-CIUDSAMLSA-N 0.000 description 2
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 2
- LMVOVCYVZBBWQB-SRVKXCTJSA-N Lys-Asp-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LMVOVCYVZBBWQB-SRVKXCTJSA-N 0.000 description 2
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 2
- LCMWVZLBCUVDAZ-IUCAKERBSA-N Lys-Gly-Glu Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CCC([O-])=O LCMWVZLBCUVDAZ-IUCAKERBSA-N 0.000 description 2
- YXTKSLRSRXKXNV-IHRRRGAJSA-N Lys-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCCN)N YXTKSLRSRXKXNV-IHRRRGAJSA-N 0.000 description 2
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 2
- ZJSZPXISKMDJKQ-JYJNAYRXSA-N Lys-Phe-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=CC=C1 ZJSZPXISKMDJKQ-JYJNAYRXSA-N 0.000 description 2
- XYLSGAWRCZECIQ-JYJNAYRXSA-N Lys-Tyr-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 XYLSGAWRCZECIQ-JYJNAYRXSA-N 0.000 description 2
- CKRRYCJKCRZOCU-GUBZILKMSA-N Met-Cys-Met Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCSC CKRRYCJKCRZOCU-GUBZILKMSA-N 0.000 description 2
- SJDQOYTYNGZZJX-SRVKXCTJSA-N Met-Glu-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SJDQOYTYNGZZJX-SRVKXCTJSA-N 0.000 description 2
- MPCKIRSXNKACRF-GUBZILKMSA-N Met-Pro-Asn Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O MPCKIRSXNKACRF-GUBZILKMSA-N 0.000 description 2
- DBMLDOWSVHMQQN-XGEHTFHBSA-N Met-Ser-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DBMLDOWSVHMQQN-XGEHTFHBSA-N 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- 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 2
- KDYPMIZMXDECSU-JYJNAYRXSA-N Phe-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 KDYPMIZMXDECSU-JYJNAYRXSA-N 0.000 description 2
- AUJWXNGCAQWLEI-KBPBESRZSA-N Phe-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O AUJWXNGCAQWLEI-KBPBESRZSA-N 0.000 description 2
- BPIFSOUEUYDJRM-DCPHZVHLSA-N Phe-Trp-Ala Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(O)=O)C1=CC=CC=C1 BPIFSOUEUYDJRM-DCPHZVHLSA-N 0.000 description 2
- ZYBUKTMPPFQSHL-JYJNAYRXSA-N Pro-Asp-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ZYBUKTMPPFQSHL-JYJNAYRXSA-N 0.000 description 2
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 2
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 description 2
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 2
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 2
- DYJTXTCEXMCPBF-UFYCRDLUSA-N Pro-Tyr-Phe Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O DYJTXTCEXMCPBF-UFYCRDLUSA-N 0.000 description 2
- 241001481818 Sebastes Species 0.000 description 2
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 2
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 2
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- FQPQPTHMHZKGFM-XQXXSGGOSA-N Thr-Ala-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O FQPQPTHMHZKGFM-XQXXSGGOSA-N 0.000 description 2
- GFDUZZACIWNMPE-KZVJFYERSA-N Thr-Ala-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O GFDUZZACIWNMPE-KZVJFYERSA-N 0.000 description 2
- LXWZOMSOUAMOIA-JIOCBJNQSA-N Thr-Asn-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O LXWZOMSOUAMOIA-JIOCBJNQSA-N 0.000 description 2
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 2
- PAXANSWUSVPFNK-IUKAMOBKSA-N Thr-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N PAXANSWUSVPFNK-IUKAMOBKSA-N 0.000 description 2
- XIULAFZYEKSGAJ-IXOXFDKPSA-N Thr-Leu-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XIULAFZYEKSGAJ-IXOXFDKPSA-N 0.000 description 2
- DXPURPNJDFCKKO-RHYQMDGZSA-N Thr-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DXPURPNJDFCKKO-RHYQMDGZSA-N 0.000 description 2
- KZURUCDWKDEAFZ-XVSYOHENSA-N Thr-Phe-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O KZURUCDWKDEAFZ-XVSYOHENSA-N 0.000 description 2
- WNQJTLATMXYSEL-OEAJRASXSA-N Thr-Phe-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O WNQJTLATMXYSEL-OEAJRASXSA-N 0.000 description 2
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 2
- VGNKUXWYFFDWDH-BEMMVCDISA-N Thr-Trp-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N)O VGNKUXWYFFDWDH-BEMMVCDISA-N 0.000 description 2
- PXQPYPMSLBQHJJ-WFBYXXMGSA-N Trp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N PXQPYPMSLBQHJJ-WFBYXXMGSA-N 0.000 description 2
- XGEUYEOEZYFHRL-KKXDTOCCSA-N Tyr-Ala-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 XGEUYEOEZYFHRL-KKXDTOCCSA-N 0.000 description 2
- GFHYISDTIWZUSU-QWRGUYRKSA-N Tyr-Asn-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GFHYISDTIWZUSU-QWRGUYRKSA-N 0.000 description 2
- YKCXQOBTISTQJD-BZSNNMDCSA-N Tyr-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N YKCXQOBTISTQJD-BZSNNMDCSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- REJBPZVUHYNMEN-LSJOCFKGSA-N Val-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N REJBPZVUHYNMEN-LSJOCFKGSA-N 0.000 description 2
- IVXJODPZRWHCCR-JYJNAYRXSA-N Val-Arg-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IVXJODPZRWHCCR-JYJNAYRXSA-N 0.000 description 2
- CGGVNFJRZJUVAE-BYULHYEWSA-N Val-Asp-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CGGVNFJRZJUVAE-BYULHYEWSA-N 0.000 description 2
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 2
- UZDHNIJRRTUKKC-DLOVCJGASA-N Val-Gln-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N UZDHNIJRRTUKKC-DLOVCJGASA-N 0.000 description 2
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 0.000 description 2
- SYOMXKPPFZRELL-ONGXEEELSA-N Val-Gly-Lys Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N SYOMXKPPFZRELL-ONGXEEELSA-N 0.000 description 2
- MYLNLEIZWHVENT-VKOGCVSHSA-N Val-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](C(C)C)N MYLNLEIZWHVENT-VKOGCVSHSA-N 0.000 description 2
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 2
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 108010081404 acein-2 Proteins 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 108010070944 alanylhistidine Proteins 0.000 description 2
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 2
- 108010066988 asparaginyl-alanyl-glycyl-alanine Proteins 0.000 description 2
- 108010077245 asparaginyl-proline Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 108010081447 cytochrophin-4 Proteins 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 108010079547 glutamylmethionine Proteins 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010037850 glycylvaline Proteins 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 108010085325 histidylproline Proteins 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108010053037 kyotorphin Proteins 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 108010056582 methionylglutamic acid Proteins 0.000 description 2
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108010004914 prolylarginine Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 108010071207 serylmethionine Proteins 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 2
- IGXNPQWXIRIGBF-KEOOTSPTSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IGXNPQWXIRIGBF-KEOOTSPTSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 1
- AWAXZRDKUHOPBO-GUBZILKMSA-N Ala-Gln-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O AWAXZRDKUHOPBO-GUBZILKMSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- MFMDKJIPHSWSBM-GUBZILKMSA-N Ala-Lys-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFMDKJIPHSWSBM-GUBZILKMSA-N 0.000 description 1
- KYDYGANDJHFBCW-DRZSPHRISA-N Ala-Phe-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N KYDYGANDJHFBCW-DRZSPHRISA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- XSLGWYYNOSUMRM-ZKWXMUAHSA-N Ala-Val-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XSLGWYYNOSUMRM-ZKWXMUAHSA-N 0.000 description 1
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 1
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 1
- PDQBXRSOSCTGKY-ACZMJKKPSA-N Asn-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N PDQBXRSOSCTGKY-ACZMJKKPSA-N 0.000 description 1
- 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 1
- FBODFHMLALOPHP-GUBZILKMSA-N Asn-Lys-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O FBODFHMLALOPHP-GUBZILKMSA-N 0.000 description 1
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 1
- MRQQMVZUHXUPEV-IHRRRGAJSA-N Asp-Arg-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MRQQMVZUHXUPEV-IHRRRGAJSA-N 0.000 description 1
- OMMIEVATLAGRCK-BYPYZUCNSA-N Asp-Gly-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)NCC(O)=O OMMIEVATLAGRCK-BYPYZUCNSA-N 0.000 description 1
- YFSLJHLQOALGSY-ZPFDUUQYSA-N Asp-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N YFSLJHLQOALGSY-ZPFDUUQYSA-N 0.000 description 1
- CZIVKMOEXPILDK-SRVKXCTJSA-N Asp-Tyr-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O CZIVKMOEXPILDK-SRVKXCTJSA-N 0.000 description 1
- XWKPSMRPIKKDDU-RCOVLWMOSA-N Asp-Val-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O XWKPSMRPIKKDDU-RCOVLWMOSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000426 Caspase-1 Proteins 0.000 description 1
- 102100035904 Caspase-1 Human genes 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- ININBLZFFVOQIO-JHEQGTHGSA-N Gln-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O ININBLZFFVOQIO-JHEQGTHGSA-N 0.000 description 1
- ZZLDMBMFKZFQMU-NRPADANISA-N Gln-Val-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O ZZLDMBMFKZFQMU-NRPADANISA-N 0.000 description 1
- NCWOMXABNYEPLY-NRPADANISA-N Glu-Ala-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O NCWOMXABNYEPLY-NRPADANISA-N 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- LRPXYSGPOBVBEH-IUCAKERBSA-N Glu-Gly-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O LRPXYSGPOBVBEH-IUCAKERBSA-N 0.000 description 1
- KRGZZKWSBGPLKL-IUCAKERBSA-N Glu-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N KRGZZKWSBGPLKL-IUCAKERBSA-N 0.000 description 1
- GXMXPCXXKVWOSM-KQXIARHKSA-N Glu-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N GXMXPCXXKVWOSM-KQXIARHKSA-N 0.000 description 1
- RQZGFWKQLPJOEQ-YUMQZZPRSA-N Gly-Arg-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)CN)CN=C(N)N RQZGFWKQLPJOEQ-YUMQZZPRSA-N 0.000 description 1
- HDNXXTBKOJKWNN-WDSKDSINSA-N Gly-Glu-Asn Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O HDNXXTBKOJKWNN-WDSKDSINSA-N 0.000 description 1
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 1
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- MHXKHKWHPNETGG-QWRGUYRKSA-N Gly-Lys-Leu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O MHXKHKWHPNETGG-QWRGUYRKSA-N 0.000 description 1
- FKESCSGWBPUTPN-FOHZUACHSA-N Gly-Thr-Asn Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O FKESCSGWBPUTPN-FOHZUACHSA-N 0.000 description 1
- BAYQNCWLXIDLHX-ONGXEEELSA-N Gly-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN BAYQNCWLXIDLHX-ONGXEEELSA-N 0.000 description 1
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 1
- LYSMQLXUCAKELQ-DCAQKATOSA-N His-Asp-Arg Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N LYSMQLXUCAKELQ-DCAQKATOSA-N 0.000 description 1
- NQKRILCJYCASDV-QWRGUYRKSA-N His-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 NQKRILCJYCASDV-QWRGUYRKSA-N 0.000 description 1
- 101001076418 Homo sapiens Interleukin-1 receptor type 1 Proteins 0.000 description 1
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 1
- HERITAGIPLEJMT-GVARAGBVSA-N Ile-Ala-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HERITAGIPLEJMT-GVARAGBVSA-N 0.000 description 1
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 1
- UDBPXJNOEWDBDF-XUXIUFHCSA-N Ile-Lys-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)O)N UDBPXJNOEWDBDF-XUXIUFHCSA-N 0.000 description 1
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 1
- FXJLRZFMKGHYJP-CFMVVWHZSA-N Ile-Tyr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FXJLRZFMKGHYJP-CFMVVWHZSA-N 0.000 description 1
- 102100026016 Interleukin-1 receptor type 1 Human genes 0.000 description 1
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 1
- KGCLIYGPQXUNLO-IUCAKERBSA-N Leu-Gly-Glu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O KGCLIYGPQXUNLO-IUCAKERBSA-N 0.000 description 1
- NRFGTHFONZYFNY-MGHWNKPDSA-N Leu-Ile-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NRFGTHFONZYFNY-MGHWNKPDSA-N 0.000 description 1
- MJTOYIHCKVQICL-ULQDDVLXSA-N Leu-Met-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MJTOYIHCKVQICL-ULQDDVLXSA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- RDFIVFHPOSOXMW-ACRUOGEOSA-N Leu-Tyr-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RDFIVFHPOSOXMW-ACRUOGEOSA-N 0.000 description 1
- YIBOAHAOAWACDK-QEJZJMRPSA-N Lys-Ala-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YIBOAHAOAWACDK-QEJZJMRPSA-N 0.000 description 1
- GKFNXYMAMKJSKD-NHCYSSNCSA-N Lys-Asp-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GKFNXYMAMKJSKD-NHCYSSNCSA-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
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 1
- LECIJRIRMVOFMH-ULQDDVLXSA-N Lys-Pro-Phe Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LECIJRIRMVOFMH-ULQDDVLXSA-N 0.000 description 1
- HKXSZKJMDBHOTG-CIUDSAMLSA-N Lys-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN HKXSZKJMDBHOTG-CIUDSAMLSA-N 0.000 description 1
- YRNRVKTYDSLKMD-KKUMJFAQSA-N Lys-Ser-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YRNRVKTYDSLKMD-KKUMJFAQSA-N 0.000 description 1
- AETNZPKUUYYYEK-CIUDSAMLSA-N Met-Glu-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O AETNZPKUUYYYEK-CIUDSAMLSA-N 0.000 description 1
- LRALLISKBZNSKN-BQBZGAKWSA-N Met-Gly-Ser Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LRALLISKBZNSKN-BQBZGAKWSA-N 0.000 description 1
- CNAGWYQWQDMUGC-IHRRRGAJSA-N Met-Phe-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CNAGWYQWQDMUGC-IHRRRGAJSA-N 0.000 description 1
- CIDICGYKRUTYLE-FXQIFTODSA-N Met-Ser-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CIDICGYKRUTYLE-FXQIFTODSA-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
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- LLGTYVHITPVGKR-RYUDHWBXSA-N Phe-Gln-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O LLGTYVHITPVGKR-RYUDHWBXSA-N 0.000 description 1
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 1
- APECKGGXAXNFLL-RNXOBYDBSA-N Phe-Trp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 APECKGGXAXNFLL-RNXOBYDBSA-N 0.000 description 1
- XKHCJJPNXFBADI-DCAQKATOSA-N Pro-Asp-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O XKHCJJPNXFBADI-DCAQKATOSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- IBGCFJDLCYTKPW-NAKRPEOUSA-N Pro-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 IBGCFJDLCYTKPW-NAKRPEOUSA-N 0.000 description 1
- XYAFCOJKICBRDU-JYJNAYRXSA-N Pro-Phe-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O XYAFCOJKICBRDU-JYJNAYRXSA-N 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 1
- SFTZTYBXIXLRGQ-JBDRJPRFSA-N Ser-Ile-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SFTZTYBXIXLRGQ-JBDRJPRFSA-N 0.000 description 1
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- QYBRQMLZDDJBSW-AVGNSLFASA-N Ser-Tyr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O QYBRQMLZDDJBSW-AVGNSLFASA-N 0.000 description 1
- 241001454698 Teleostei Species 0.000 description 1
- OJRNZRROAIAHDL-LKXGYXEUSA-N Thr-Asn-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OJRNZRROAIAHDL-LKXGYXEUSA-N 0.000 description 1
- DCLBXIWHLVEPMQ-JRQIVUDYSA-N Thr-Asp-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DCLBXIWHLVEPMQ-JRQIVUDYSA-N 0.000 description 1
- MCDVZTRGHNXTGK-HJGDQZAQSA-N Thr-Met-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O MCDVZTRGHNXTGK-HJGDQZAQSA-N 0.000 description 1
- NDXSOKGYKCGYKT-VEVYYDQMSA-N Thr-Pro-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O NDXSOKGYKCGYKT-VEVYYDQMSA-N 0.000 description 1
- XGUAUKUYQHBUNY-SWRJLBSHSA-N Thr-Trp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O XGUAUKUYQHBUNY-SWRJLBSHSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-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
- 102000005747 Transcription Factor RelA Human genes 0.000 description 1
- 108010031154 Transcription Factor RelA Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- CZWIHKFGHICAJX-BPUTZDHNSA-N Trp-Glu-Glu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 CZWIHKFGHICAJX-BPUTZDHNSA-N 0.000 description 1
- NLMXVDDEQFKQQU-CFMVVWHZSA-N Tyr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLMXVDDEQFKQQU-CFMVVWHZSA-N 0.000 description 1
- SOEGLGLDSUHWTI-STECZYCISA-N Tyr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 SOEGLGLDSUHWTI-STECZYCISA-N 0.000 description 1
- WKWJJQZZZBBWKV-JYJNAYRXSA-N Val-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WKWJJQZZZBBWKV-JYJNAYRXSA-N 0.000 description 1
- IQQYYFPCWKWUHW-YDHLFZDLSA-N Val-Asn-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N IQQYYFPCWKWUHW-YDHLFZDLSA-N 0.000 description 1
- XGJLNBNZNMVJRS-NRPADANISA-N Val-Glu-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O XGJLNBNZNMVJRS-NRPADANISA-N 0.000 description 1
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000010515 dystocia Diseases 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 108010091871 leucylmethionine Proteins 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
Images
Classifications
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/545—IL-1
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention provides a sebastes schlegeli IL-1 beta recombinant protein, which comprises the following components: an IL-1 β mature peptide amino acid sequence, and a tag peptide segment comprising a histidine tag sequence and a solubility-promoting tag sequence linked to the N-terminus of the IL-1 β mature peptide amino acid sequence. The invention also provides a gene for coding the recombinant protein, a vector for expressing the recombinant protein and recombinant engineering bacteria. The invention further provides a method for preparing the sebastes schlegeli hilgendorf IL-1 beta protein based on the recombinant protein, and application of the recombinant protein or the prepared sebastes schlegeli hildorf IL-1 beta protein in sebastes schlegeli hildorf breeding. The recombinant protein can be used for obtaining soluble recombinant protein through large-scale induction expression of prokaryotic bacteria, so that subsequent industrial extraction and purification are facilitated, the recombinant protein can be enzymolyzed into sebastes schlegeli IL-1 beta protein, has activity, and can be effectively used for breeding sebastes schlegeli.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a sebastes schlegeli hilgendorf interleukin 1 beta (IL-1 beta) recombinant protein which is produced by an escherichia coli expression system and has solubility and bioactivity, and application thereof in sebastes schlegeli hilgendorf breeding.
Background
Sebastes schlegelii (Sebastes schlegelii) belongs to Sebastes schlegeli (scorbaeniformes), Sebastes schlegelii (sebasthidae) and Sebastes genus (Sebastes), and is one of important cold-warm economic fishes in northern China. Sebastes schlegeli breeding mode is spawning, the male and female development is asynchronous, the male fish is sexually mature (the female fish is not mature at the moment) in 11 months every year, the male and female fish complete mating in 11-12 months, the sperms can be stored in the female fish body for 4 months, the ovaries of the female fish are mature in 3-4 months in the next year, in-vivo fertilization occurs, the embryos develop in the female fish body for about 1 month, and the juvenile fish is produced at the end of 4 months to the beginning of 5 months.
Parturition of oviparous teleost teleosts belongs to typical spontaneous inflammatory reaction, interleukin 1 beta (interleukin 1 beta, IL-1 beta) belongs to a typical inflammatory factor, and relative expression levels of IL-1 beta gene (IL1b) and a main receptor IL1R1 gene (IL1R1) reach the highest level in parturition and are remarkably reduced (P is less than 0.05) after 24 hours of parturition. IL-1 beta can indirectly regulate the progress of labor by directly regulating the expression level of prostaglandin synthetase COX2 gene (COX2) through NF-kappa B cell pathway, increasing the level of prostaglandin. In the breeding of sebastes schlegeli hilgendorf, female fish are in poor state and are easy to be dystocia and die. The progress of labor can therefore be promoted by injection of IL-1 beta protein. Unlike other proteins secreted by endoplasmic reticulum and Golgi body pathways, IL-1 beta precursor peptide of vertebrate is cleaved and activated by Caspase 1 in lysosome to obtain IL-1 beta mature peptide, which is secreted extracellularly, and the mature peptide does not contain disulfide bond and has no protein post-translational modification, so that the production of IL-1 beta recombinant protein of Sebastes schlegeli or other vertebrates by using prokaryotic expression system is the optimal choice.
In the field of genetic engineering, Escherichia coli (Escherichia coli) is a commonly used engineering bacterium of a prokaryotic expression system, belongs to a facultative anaerobic gram-negative bacterium, and has the advantages of short growth period, clear genetic background, simple culture operation, low cost, capability of quickly producing target protein on a large scale and the like. By using strong promoter (such as T7/lac, Tac, etc.), the expression of foreign protein can be expressed in colibacillus at high level, and the expression amount can reach 40-50% of the total protein of thallus. However, the rate of expressing the foreign protein by escherichia coli is far higher than that of the eukaryotic organism, the expression rate of the recombinant protein is too high, the newly-generated peptide chain cannot be correctly folded in time, and the hydrophobic parts of the protein which is incorrectly folded are exposed and mutually aggregated to form an inactive inclusion body.
Inclusion body formation is a common problem in prokaryotic expression. At present, prokaryotic expression of IL-1 beta recombinant protein of grass carp and other small number of teleost fishes is reported. However, in these reports, the IL-1 β recombinant proteins are inactive inclusion bodies, and require complicated renaturation operations to recover the biological activity, the technical route is as follows: after the inclusion bodies are washed, inactive inclusion bodies are dissolved by adding a high concentration of a denaturant (urea or guanidine hydrochloride), affinity purification is performed under denaturing conditions using a fusion tag such as His6 or strep II, and the activity is restored by refolding the denatured target protein by techniques such as dilution renaturation, dialysis renaturation, and column renaturation.
In view of the complex operation of inclusion body renaturation, renaturation protein concentration, ionic environment and proportion concentration of redox couple need to be searched, and the renaturation process takes longer time, and the denaturant urea can have adverse effect on the protein. Therefore, how to use Escherichia coli to express soluble and active recombinant protein has practical significance.
Disclosure of Invention
In order to solve the above problems, the present invention provides a sebastes schlegeli IL-1 β recombinant protein, comprising: an IL-1 β mature peptide amino acid sequence, and a tag peptide segment comprising a histidine tag sequence and a solubility-promoting tag sequence linked to the N-terminus of the IL-1 β mature peptide amino acid sequence. Preferably, the amino acid sequence of the IL-1 beta mature peptide is SEQ ID NO 1.
In one embodiment according to the invention, the lytic tag sequence is a Maltose Binding Protein (MBP) sequence; preferably, the Maltose Binding Protein (MBP) sequence is SEQ ID NO 2; more preferably, the set of amino acid tag sequences is hexahistidine (His 6).
In one embodiment according to the invention, the tag peptide fragment is linked to the N-terminus of the amino acid sequence of the mature IL-1 β peptide via a hydrophilic Linker sequence; preferably, the hydrophilic Linker sequence is SEQ ID NO 3.
In one embodiment according to the invention, the hydrophilic Linker sequence further comprises a protease recognition site; preferably, the protease recognition site is TEV protease ENLYFQ ↓ G, and cleavage is performed between Q and G; more preferably, the hydrophilic Linker sequence is SEQ ID NO 3.
In one embodiment according to the invention, the amino acid sequence is SEQ ID NO 4.
The invention also provides the coding gene of the IL-1 beta recombinant protein, which comprises the following components: a gene sequence encoding an IL-1 β mature peptide amino acid, and a gene sequence encoding a tag peptide segment linked to the 5' end of the IL-1 β mature peptide amino acid gene sequence, the gene sequence encoding the tag peptide segment comprising a histidine tag encoding gene sequence and a lysogenic tag encoding gene sequence; the nucleotide sequence of the amino acid of the IL-1 beta mature peptide is SEQ ID NO. 5;
preferably, the nucleotide sequence of the coding gene is SEQ ID NO 6.
The invention further provides an expression vector for expressing the IL-1 beta recombinant protein as defined in the claim, comprising the gene encoding the IL-1 beta recombinant protein as defined above and a backbone plasmid selected from pET-HMT, which is modified from pET-32a (+).
The invention further provides a recombinant engineering bacterium for expressing the IL-1 beta recombinant protein, which comprises the expression vector; preferably, the host bacterium of the recombinant engineering bacterium is escherichia coli, preferably Rosetta B (DE3) or BL21(DE 3).
The invention also provides a preparation method of sebastes schlegeli IL-1 beta protein, which comprises the following steps:
1) constructing the IL-1 beta recombinant protein coding gene, and then connecting the coding gene to a skeleton plasmid to construct an expression vector of the IL-1 beta recombinant protein;
2) transforming the expression vector into host bacteria, and inducing and expressing the IL-1 beta recombinant protein;
3) separating the recombinant protein based on histidine tag chromatography in the IL-1 beta recombinant protein to obtain the IL-1 beta recombinant protein;
4) removing protein tags in the IL-1 beta recombinant protein obtained in the step 3), and separating and purifying to obtain the IL-1 beta protein of sebastes schlegeli hilgendorf;
preferably, the sebastes schlegeli hilgendorf IL-1 beta protein without tag can be collected from the percolate after being cut by TEV enzyme in the step 4) and then being placed into a 30kDa ultrafiltration tube and being centrifuged at 6000 g.
The invention also discloses the IL-1 beta recombinant protein of sebastes schlegeli hilgendorf, or the application of the IL-1 beta protein of sebastes schlegeli hilgendorf prepared by the method in the breeding of sebastes schlegeli hildorf.
The invention achieves the following technical effects:
the invention constructs a prokaryotic expression vector of sebastes schlegeli IL-1 beta for the first time, and the vector is transfected into Rosetta (DE3) escherichia coli to obtain a pET-HMT-IL1B-Rosetta (DE3) expression strain, which can be used for inducing and expressing a large amount of soluble recombinant protein. After affinity purification through a protein tag, recombinant protein with the purity of up to 90 percent can be obtained. After the fusion tag is further cut off by TEV protease, the tag is removed by ultrafiltration, and the non-tag IL-1 beta recombinant protein enters the filtrate. The method can obtain the sebastes schlegeli IL-1 beta recombinant protein with the amino acid sequence consistent with IL-1 beta mature peptide, and the sebastes schlegeli IL-1 beta recombinant protein can be used for spawning induction and breeding of sebastes schlegeli.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 is the cloning of the Open Reading Frame (ORF) of the sebastes schlegeli 1b gene; wherein: a: IL1b gene ORF nucleotide sequence and translated amino acid sequence, gray region is IL-1 beta mature peptide amino acid sequence; b: ORF cloning PCR amplified band, template Sebastes schlegeli head and kidney cDNA.
FIG. 2 is the gene sequence and translated amino acid sequence of Sebastes schlegeli IL-1 beta recombinant protein; wherein: a: a His6 tag; b: an MBP tag; c: sebastes schlegeli IL-1 beta mature peptide.
FIG. 3 is an SDS-PAGE gel electrophoresis chart of IL-1 beta recombinant protein expression, purification and enzyme digestion; wherein the content of the first and second substances,
a: a recombinant protein pattern; b: recombinant protein expression and purification, and M: protein molecular weight standards; 1: IPTG induces pre-bacterial total protein; 2: bacterial total protein after induction for 16h at 20 ℃ with 0.1mM IPTG; 3: precipitating the bacterial lysate after induction; 4: supernatant of the induced bacterial lysate; 5: NTA-Ni affinity purification flow-through; 6-7: washing the impurity liquid; 8-9: eluent (containing target protein, purity > 90%); c: the enzyme digestion is carried out for 0-48h at 4 ℃ by TEV enzyme. M: protein molecular weight standards; 10: total protein before enzymatic cleavage. 11-14: performing enzyme digestion for 12/24/36/48 h; d: after enzyme digestion, ultrafiltration purification (MWCO is 30kDa, centrifugal force 6000 g); 15: total protein before ultrafiltration; 16: ultrafiltration concentrate (containing tag protein, a small amount of TEV enzyme and fusion protein which is not completely digested); 17: and (4) ultrafiltering the leakage liquid (containing IL-1 beta without a label after enzyme digestion).
FIG. 4 is a pET-HMT vector map; wherein, A: a vector plasmid structure pattern diagram; b: prokaryotic expression protein coding region composition and single enzyme cutting site. His 6-tag: a His6 tag; MBP-tag: an MBP tag; TEVpCS: a TEV protease cleavage site; MCS: a multiple cloning enzyme cleavage site; AmpR: an ampicillin resistance gene; lac: a lactose operon; ori: an origin of replication; f1 origin: f1 sequences of the replication region of the phage genomic DNA.
FIG. 5 shows the construction of pET-HMT-IL1B expression vector and the transformation of prokaryotic expression engineering bacteria; wherein:
a: the nucleotide sequence corresponding to the amino acid of the sebastes schlegeli IL-1 beta mature peptide with a homology arm (SEQ ID NO:5) is amplified by PCR. B: and carrying out double-enzyme digestion linearization on pET-HMT vectors BamH I and Xho I. C: the IL-1. beta. insert was ligated to a linear vector by homologous recombination, transformed into DH 5. alpha. competent E.coli, and verified by sequencing. D: extracting pET-HMT-IL1B expression plasmid. E: the expression plasmid was transformed into Rosetta B (DE3) competent E.coli and the recombinant protein was expressed.
FIG. 6 shows the relative expression levels of interleukin 1 beta-related genes in the pre-, middle-and post-parturition of Sebastes schlegeli; wherein, A: IL-1. beta. gene (IL1 b); b: IL1R1 gene (IL1R 1).
FIG. 7 is a photo-scopy observation of ovary primary cells of sebastes schlegeli hilgendorf (A) and an immunofluorescence mapping of NF-kappa B p65 subunit after ovarian ova are stimulated by IL-1 beta recombinant protein (B), which shows that NF-kappa B factor is activated and enters cell nucleus.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Example 1: construction of Sebastes schlegeli IL-1 beta expression vector pET-HMT-IL1B
With sebastes schlegeli hilgendorus cDNA as a template, the DNA sequence of the Open Reading Frame (ORF) of sebastes schlegeli hilgendorus IL-1 beta gene (IL1B) was amplified by PCR using a primer O-IL1B-F/R (Producer, Shanghai) and a reagent 2 × Phanta Max Master Mix (Nodezaki, Nanjing) (B in FIG. 1). It was ligated into pCE2 TA/Blunt Zero vector (Novozam, Nanjing) by TOPO ligation to transfect E.coli DH 5. alpha. and screened for single colonies in ampicillin solid medium, and sequence was checked by Sanger sequencing and NCBI BLAST to determine if the sequence was correct. Performing PCR amplification by using the sebastes schlegeli il1b monoclonal bacterial liquid as a template and using a primer PE-il1b-F/R and 2 x Phanta Max Master Mix to obtain a double-stranded DNA fragment (A in figure 5) corresponding to il1b mature peptide with a homologous arm; meanwhile, the pET-HMT vector (vector map, see FIG. 4) was linearized by double digestion with restriction endonucleases BamH I and Xho I (NEB, USA) (B in FIG. 5). Using FastPure Gel DNA Extraction Mini Kit (Novozan, Nanjing) to recover PCR products and double enzyme digestion product Gel, using a seamless cloning Kit (Biyunshi, Shanghai) to connect an insert fragment and a linear vector, transfecting DH5 alpha competent escherichia coli (C in figure 5), screening single colonies by using an ampicillin solid culture medium, using a plasmid miniprep Kit (Tiangen, Beijing) to extract plasmids (D in figure 5) after confirming no errors by using Sanger sequencing, namely obtaining the IL-1 beta prokaryotic expression vector of Sebastes schlegelii.
TABLE 1 primer sequence chart used for vector construction
Note: primer lower case letter indicates homologous arm for constructing prokaryotic expression vector by homologous recombination connection
Example 2: preparation of sebastes schlegeli IL-1 beta escherichia coli expression strain
The expression vector pET-HMT-IL1B is transfected into Rosetta B (DE3) competent Escherichia coli (On Yue, Shanghai), and ampicillin-chloramphenicol double-antibody LB solid medium is used for culturing at 37 ℃ overnight to screen single colonies, namely Sebastes schlegeli IL-1 beta prokaryotic expression engineering bacteria pET-HMT-IL1B-Rosetta B (DE3) (E in FIG. 5). Selecting a single colony to be cultured in 10mL of ampicillin-chloramphenicol double-resistant LB liquid culture medium at 37 ℃ at 220 rpm overnight, then inoculating the single colony in 100mL of double-resistant TB liquid culture medium at a ratio of 1:100, culturing at 37 ℃ at 220 rpm until OD600 is 0.6-0.8, then adding IPTG to make the final concentration of the single colony be 0.1mM (taking 50mL of bacterial liquid as a control before induction), culturing at 20 ℃ at 120 rpm for 12h, centrifuging at 4000g for 10min, collecting the thallus, discarding the supernatant, resuspending the thallus in 5mL of non-denaturing bacteria cracking buffer solution (500mM NaCl, 20mM PB, pH 7.4), adding 1mg/mL of lysozyme at the final concentration, cracking on ice for 30min, ultrasonically crushing at 60W for 15min, stopping at 5s for 5s, centrifuging at 12000g for 10min, respectively collecting the supernatant and precipitate, resuspending the precipitate in 5mL of non-denaturing bacteria cracking buffer solution, respectively taking 80 muL of sample and 20 muL of 5 Xprotein loading buffer solution, SDS-PAGE was performed at 95 ℃ for 10min to verify correct expression of the protein (samples 1-4 in FIG. 3B).
Example 3: sebastes schlegeli IL-1 beta recombinant protein soluble expression, protein purification and tag excision
Inoculating the mixture into 500mL of double-anti TB liquid culture medium according to the proportion of 1:100, culturing at 37 ℃ and 220 rpm until OD600 is 0.6-0.8, adding IPTG to make the final concentration be 0.1mM, culturing at 20 ℃, culturing at 120 rpm for 12h, centrifuging at 4000g for 10min to collect thalli, discarding supernatant, resuspending the thalli by 50mL of non-denaturing schizomycete buffer solution, adding lysozyme with the final concentration of 1mg/mL, performing on-ice lysis for 30min, performing 60W ultrasonic disruption for 15min, stopping 5s by exceeding 5s, centrifuging at 12000g for 10min to discard precipitate, retaining supernatant, filtering by using a 0.45 mu m or 0.22 mu m filter membrane to remove insoluble microparticles, and performing His-tag affinity purification by using NTA-Ni filler (Aibixin, Shanghai) to obtain His6-MBP fused IL-1 beta recombinant protein with the purity of more than 90% (sample 5-9 in figure 3B).
The buffer was replaced from the eluate by 1 XTEV buffer (50mM Tris-HCl, 0.5mM EDTA, 1mM DTT, pH 7.5) using a 30kDa ultrafiltration tube (Merck Millipore, Germany) and concentrated, about 20 units TEV enzyme/mg recombinant protein were added and after digestion at 4 ℃ for 24h (FIG. 3C), the unlabelled IL-1. beta. recombinant protein was collected from the percolate by centrifugation at 6000g for 30min using a 30kDa ultrafiltration tube (FIG. 3D). Then replacing the percolate buffer solution with 1 XPBS by using a 3kDa ultrafiltration tube and concentrating, and the method can be used for in-vivo injection or stimulation of isolated cells of sebastes schlegeli.
Example 4: sebastes schlegeli IL-1 beta recombinant protein for activating inflammation-related cell pathway NF-kappa B
After isolating sebastes schlegeli ovary primary cells, the cell concentration was adjusted to 5 × 10 using L15 medium containing 10% FBS5Cells/ml, then 1ml per well in a 24-well plate, placed in an incubator at 25 ℃, after overnight incubation, starved for 2 hours with 500 μ L FBS-free L15 medium, then FBS-containing medium was added, IL-1 β solution was added to each well of the treated group to a final concentration of 15pM, and an equal volume of 1 × PBS was added to the control group, after 3h incubation in the incubator. Washing with pre-cooled 1 × PBS for 3 times, fixing 4% paraformaldehyde (prepared with 1 × PBS) at room temperature for 15-20min, and washing with 1 × PBS for 3 times; soaking 0.25% Triton X-100 (prepared by 1 × PBS) for 10min, and washing 3 times with 1 × PBS; 5% BSA (1 XPBS) blocked for 1h at 37 ℃ and NF diluted 1:200 with 1% BSA (1 XPBS)-kB p65 Rabbit Monoclonal Antibody (Biyun, Shanghai) incubated overnight at 4 ℃; the next day, rewarming for 45min, washing with PBS, incubating for 1h at 37 ℃ with 400-fold diluted Cy 3-labeled goat anti-rabbit IgG (Severe, Wuhan), washing with PBS, counterstaining nuclei with DAPI, blocking with anti-quenching blocking agent, and observing with a fluorescence microscope, wherein the results are shown in FIG. 7.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> China oceanic university
<120> Sebastes schlegeli IL-1 beta recombinant protein, and preparation method and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 152
<212> PRT
<213> Sebastes schlegelii Sebastes (Sebastes schlegelii)
<400> 1
Gly Glu Lys Arg Ser Leu Val Gln Val Gln Asn Ser Met Glu Leu His
1 5 10 15
Ala Val Met Leu Gln Gly Gly Gly Gly Thr Thr Lys Val His Leu Asn
20 25 30
Met Ser Thr Tyr Leu His Pro Thr Pro Arg Val Leu Gly Arg Thr Val
35 40 45
Ala Leu Gly Ile Arg Gly Thr Asn Pro Asn Leu Tyr Leu Cys Cys Arg
50 55 60
Lys Asn Gly Ala Asn Pro Thr Leu His Leu Glu Ala Val Glu Asn Lys
65 70 75 80
Ser Leu Leu Ser Gly Ala Gly Val Ser Ile Ser Pro Asp Ser Asp Met
85 90 95
Val Arg Phe Leu Phe Tyr Arg Gln Asp Thr Gly Val Asn Ile Thr Thr
100 105 110
Leu Met Ser Val Ala His Pro Asp Trp Phe Ile Cys Thr Ala Glu Gln
115 120 125
Asp Asn Lys Pro Leu Glu Met Cys Met Glu Ser Ala Asn Leu Tyr Arg
130 135 140
Thr Phe Asn Ile Arg Glu Glu Ala
145 150
<210> 2
<211> 366
<212> PRT
<213> Escherichia coli (E
<400> 2
Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly
1 5 10 15
Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
20 25 30
Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro
35 40 45
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His
50 55 60
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr
65 70 75 80
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
85 90 95
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala
100 105 110
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr
115 120 125
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys
130 135 140
Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
145 150 155 160
Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr
165 170 175
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
180 185 190
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr
195 200 205
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met
210 215 220
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
225 230 235 240
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys
245 250 255
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn
260 265 270
Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
275 280 285
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
290 295 300
Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala Thr
305 310 315 320
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met
325 330 335
Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser
340 345 350
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
<210> 3
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn
1 5 10 15
Leu Gly Glu Asn Leu Tyr Phe Gln Gly
20 25
<210> 4
<211> 552
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Gly Ser His His His His His His Gly Lys Ile Glu Glu Gly Lys
1 5 10 15
Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
20 25 30
Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
35 40 45
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
50 55 60
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
65 70 75 80
Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
85 90 95
Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
100 105 110
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
115 120 125
Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
130 135 140
Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
145 150 155 160
Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
165 170 175
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
180 185 190
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
195 200 205
Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
210 215 220
Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
225 230 235 240
Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
245 250 255
Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
260 265 270
Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
275 280 285
Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
290 295 300
Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
305 310 315 320
Leu Val Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
325 330 335
Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
340 345 350
Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
355 360 365
Glu Ala Leu Lys Asp Ala Gln Thr Gly Thr Asn Ser Ser Ser Asn Asn
370 375 380
Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Glu Asn Leu Tyr Phe Gln
385 390 395 400
Gly Glu Lys Arg Ser Leu Val Gln Val Gln Asn Ser Met Glu Leu His
405 410 415
Ala Val Met Leu Gln Gly Gly Gly Gly Thr Thr Lys Val His Leu Asn
420 425 430
Met Ser Thr Tyr Leu His Pro Thr Pro Arg Val Leu Gly Arg Thr Val
435 440 445
Ala Leu Gly Ile Arg Gly Thr Asn Pro Asn Leu Tyr Leu Cys Cys Arg
450 455 460
Lys Asn Gly Ala Asn Pro Thr Leu His Leu Glu Ala Val Glu Asn Lys
465 470 475 480
Ser Leu Leu Ser Gly Ala Gly Val Ser Ile Ser Pro Asp Ser Asp Met
485 490 495
Val Arg Phe Leu Phe Tyr Arg Gln Asp Thr Gly Val Asn Ile Thr Thr
500 505 510
Leu Met Ser Val Ala His Pro Asp Trp Phe Ile Cys Thr Ala Glu Gln
515 520 525
Asp Asn Lys Pro Leu Glu Met Cys Met Glu Ser Ala Asn Leu Tyr Arg
530 535 540
Thr Phe Asn Ile Arg Glu Glu Ala
545 550
<210> 5
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtggtggtgg tggtgctcga gttaagcctc ctctcggatg ttg 43
<210> 6
<211> 1659
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgggtagcc accatcacca ccaccatggt aaaatcgaag aaggtaaact ggtaatctgg 60
attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 120
accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 180
gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 240
gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 300
ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 360
gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 420
gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 480
ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 540
tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 600
ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 660
tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 720
gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 780
aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 840
ccgaacaaag agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 900
gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 960
ttggtgaaag atccgcgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg 1020
ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 1080
gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactgg taccaattcg 1140
agctcgaaca acaacaacaa taacaataac aacaacctcg gggagaacct gtacttccag 1200
ggagagaaga ggagcttagt tcaggtccaa aacagcatgg agctccacgc agtgatgctg 1260
cagggaggcg gtggcaccac aaaagttcac ctgaacatgt cgacctactt gcaccctaca 1320
cccagagttc tgggcagaac tgtggctctg ggcatccgag gcacaaatcc aaatctctac 1380
ctgtgttgcc gcaagaatgg tgccaatcca accttgcatc tggaggcggt ggagaacaaa 1440
agtctgttga gtggagcggg tgtgagcatc agcccggaca gcgacatggt gcgatttctc 1500
ttctacagac aggacaccgg ggtgaacatc accaccctca tgtctgtcgc ccaccctgac 1560
tggttcatct gcacagcaga gcaggacaac aagccgttgg aaatgtgcat ggagtccgcc 1620
aacctctacc ggaccttcaa catccgagag gaggcttaa 1659
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggaatccg agatgacatg c 21
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggagaacctg tacttccagg gagagaagag gagcttagtt caggtc 46
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtggtggtgg tggtgctcga g 21
Claims (17)
1. Sebastes schlegeli IL-1 beta recombinant protein is characterized by comprising: an IL-1 β mature peptide amino acid sequence, and a tag peptide segment linked to the N-terminus of the IL-1 β mature peptide amino acid sequence, the tag peptide segment comprising a histidine tag sequence and a solubility-promoting tag sequence;
the amino acid sequence of the IL-1 beta mature peptide is SEQ ID NO: 1.
2. The IL-1 β recombinant protein of claim 1, wherein the lytic tag sequence is a maltose binding protein sequence.
3. The IL-1 β recombinant protein of claim 2, wherein the maltose binding protein amino acid sequence is SEQ ID No. 2.
4. The IL-1 β recombinant protein according to claim 2, wherein the group amino acid tag sequence is hexahistidine.
5. The IL-1 β recombinant protein of claim 1, wherein the tag peptide segment is linked to the N-terminus of the amino acid sequence of the mature IL-1 β peptide by a hydrophilic Linker sequence.
6. The IL-1 β recombinant protein of claim 5, wherein the hydrophilic Linker sequence further comprises a protease recognition site ENLYFQ ↓ G, and cleaves between Q and G.
7. The IL-1 β recombinant protein of claim 6, wherein the protease recognition site is TEV protease.
8. The IL-1 β recombinant protein of claim 6, wherein the hydrophilic Linker sequence is SEQ ID NO 3.
9. The IL-1 β recombinant protein of claim 1, wherein the amino acid sequence is SEQ ID No. 4.
10. The gene encoding the IL-1 β recombinant protein of any one of claims 1-9, comprising: a gene encoding an IL-1 β mature peptide amino acid, and a gene encoding a tag peptide linked to the 5' end of the IL-1 β mature peptide amino acid gene sequence, the gene encoding the tag peptide comprising a histidine tag encoding gene and a solubility-promoting tag encoding gene; the nucleotide sequence corresponding to the amino acid for coding the IL-1 beta mature peptide is SEQ ID NO. 5.
11. The gene encoding the IL-1 β recombinant protein of claim 10, wherein the nucleotide sequence of the gene is SEQ ID No. 6.
12. An expression vector for expressing the IL-1 β recombinant protein of any one of claims 1 to 9, comprising the gene encoding the IL-1 β recombinant protein of claim 10 or 11 and a backbone plasmid selected from pET-HMT, which is modified from pET-32a (+).
13. A recombinant engineered bacterium for expressing the IL-1 β recombinant protein of any one of claims 1-9, comprising the expression vector of claim 12.
14. The recombinant engineered bacterium of claim 13, wherein the host bacterium of the recombinant engineered bacterium is escherichia coli.
15. The recombinant engineered bacterium of claim 13, wherein the host bacterium of said recombinant engineered bacterium is Rosetta B (DE3) or BL21(DE 3).
16. A preparation method of sebastes schlegeli IL-1 beta protein is characterized by comprising the following steps:
1) constructing the IL-1 beta recombinant protein coding gene as claimed in claim 10, and then connecting to a skeleton plasmid to construct an expression vector of the IL-1 beta recombinant protein;
2) transforming the expression vector into host bacteria, and inducing and expressing the IL-1 beta recombinant protein;
3) performing affinity chromatography separation on the basis of histidine tag in the IL-1 beta recombinant protein to obtain IL-1 beta recombinant protein;
4) and (3) cutting off a protein tag in the IL-1 beta recombinant protein obtained in the step 3), and separating and purifying to obtain the IL-1 beta protein of sebastes schlegeli.
17. The method of claim 16, wherein step 4) is performed by TEV enzyme digestion, and then the product is placed into a 30kDa ultrafiltration tube, and centrifuged at 6000g to collect non-labeled IL-1 β protein of Sebastes schlegeli from the leachate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110292721.4A CN112979781B (en) | 2021-03-18 | 2021-03-18 | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110292721.4A CN112979781B (en) | 2021-03-18 | 2021-03-18 | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112979781A CN112979781A (en) | 2021-06-18 |
| CN112979781B true CN112979781B (en) | 2022-05-17 |
Family
ID=76333017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110292721.4A Expired - Fee Related CN112979781B (en) | 2021-03-18 | 2021-03-18 | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112979781B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181321B (en) * | 2021-12-10 | 2023-04-11 | 中国海洋大学 | Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein and preparation method and application thereof |
| CN114057897B (en) * | 2021-12-21 | 2023-01-13 | 中国海洋大学 | Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application |
| CN114958806A (en) * | 2022-06-14 | 2022-08-30 | 中国水产科学研究院淡水渔业研究中心 | Carp LPL1 recombinant protein and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19980061879A (en) * | 1996-12-31 | 1998-10-07 | 성재갑 | Mass Production Method of Zofibolac Growth Hormone |
| US5863769A (en) * | 1997-01-28 | 1999-01-26 | Smithkline Beecham Corporation | DNA encoding interleukin-1 receptor antagonist (IL-1raβ) |
| KR20120130916A (en) * | 2011-05-24 | 2012-12-04 | 경상대학교산학협력단 | The polypeptide derived from sebastes schlegelii and Immune Booster Composition for fish Comprising thereof |
| CN104277119A (en) * | 2014-09-23 | 2015-01-14 | 电子科技大学 | Recombinant grass carp IL-1 beta antagonistic protein, and coding gene, preparation method and application thereof |
| CN109182339A (en) * | 2018-09-06 | 2019-01-11 | 青岛农业大学 | A kind of flat Rockfish calprotectin gene of Xu Shi and application |
-
2021
- 2021-03-18 CN CN202110292721.4A patent/CN112979781B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19980061879A (en) * | 1996-12-31 | 1998-10-07 | 성재갑 | Mass Production Method of Zofibolac Growth Hormone |
| US5863769A (en) * | 1997-01-28 | 1999-01-26 | Smithkline Beecham Corporation | DNA encoding interleukin-1 receptor antagonist (IL-1raβ) |
| KR20120130916A (en) * | 2011-05-24 | 2012-12-04 | 경상대학교산학협력단 | The polypeptide derived from sebastes schlegelii and Immune Booster Composition for fish Comprising thereof |
| CN104277119A (en) * | 2014-09-23 | 2015-01-14 | 电子科技大学 | Recombinant grass carp IL-1 beta antagonistic protein, and coding gene, preparation method and application thereof |
| CN109182339A (en) * | 2018-09-06 | 2019-01-11 | 青岛农业大学 | A kind of flat Rockfish calprotectin gene of Xu Shi and application |
Non-Patent Citations (2)
| Title |
|---|
| Molecular characterization and comparative expression analysis of two teleostean pro-inflammatory cytokines, IL-1β and IL-8, from Sebastes schlegeli;H M L P B Herath等;《Comparative Study》;20151008;第732-742页 * |
| 许氏平鲉和半滑舌鳎促性腺激素释放激素（GnRH）基因的克隆和表达分析;周晓苏;《中国博士学位论文全文数据库 农业科技辑》;20130115;D052-16 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112979781A (en) | 2021-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112979781B (en) | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof | |
| US11396537B2 (en) | Polypeptide, process for the production thereof and use thereof | |
| KR102559311B1 (en) | Recombinant human type XVII collagen, manufacturing method and application | |
| JPH02177889A (en) | Excretion of different kind of protein from e. coli | |
| CN110128521B (en) | Auxiliary protein for producing recombinant fusion protein, encoding gene, recombinant fusion protein, recombinant expression vector and preparation method | |
| WO2021083072A1 (en) | Human collagen 17-type polypeptide, production method therefor and use thereof | |
| US7544512B2 (en) | Method of producing insulinotropic GLP-1 (7-36) polypeptide and/or GLP-1 analogs | |
| CN110724187B (en) | Recombinant engineering bacterium for efficiently expressing liraglutide precursor and application thereof | |
| CN111117977A (en) | Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof | |
| CN110964096B (en) | Preparation method of recombinant human C-reactive protein | |
| CN111548392A (en) | Dissolution promoting label and application thereof | |
| CN114181321A (en) | Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein and preparation method and application thereof | |
| CN108191964B (en) | Apostichopus japonicus F-type lectin AjFL-1, and preparation method and application thereof | |
| CN110066330B (en) | Apostichopus japonicus glucan binding protein and preparation method and application thereof | |
| CN107629129B (en) | Method for producing and purifying polypeptides | |
| CN113980880B (en) | Genetically engineered bacterium and application thereof, and method for producing psicose by taking glucose as raw material | |
| CN114380903A (en) | Insulin or its analogue precursor | |
| CN110305887A (en) | Anti-fungus peptide Drosomycin, preparation method and applications | |
| US20230203109A1 (en) | Fusion polypeptides for target peptide production | |
| CN114276413A (en) | Preparation method of artificial antibacterial peptide G3 | |
| CN114057897B (en) | Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application | |
| CN112695052A (en) | Recombinant human glucocorticoid receptor GR alpha-His protein and expression and purification method thereof | |
| CN114196688A (en) | Expression of prokaryotic alkaline phosphatase in Yeast | |
| CN104789513B (en) | A kind of coli strain for preparing bioactive peptide | |
| CN114457099B (en) | Biological fermentation preparation method of cable Ma Lutai core peptide chain |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220517 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |