CN114805534B - 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 - Google Patents
人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 Download PDFInfo
- Publication number
- CN114805534B CN114805534B CN202210392527.8A CN202210392527A CN114805534B CN 114805534 B CN114805534 B CN 114805534B CN 202210392527 A CN202210392527 A CN 202210392527A CN 114805534 B CN114805534 B CN 114805534B
- Authority
- CN
- China
- Prior art keywords
- mutant
- growth factor
- fibroblast growth
- bfgf
- preparation
- 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.)
- Active
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 102000018233 Fibroblast Growth Factor Human genes 0.000 title abstract description 9
- 108050007372 Fibroblast Growth Factor Proteins 0.000 title abstract description 9
- 229940126864 fibroblast growth factor Drugs 0.000 title abstract description 9
- 108090000623 proteins and genes Proteins 0.000 title description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims description 38
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 8
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 abstract description 46
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 abstract description 41
- 230000000694 effects Effects 0.000 abstract description 6
- 230000004663 cell proliferation Effects 0.000 abstract description 5
- 239000003102 growth factor Substances 0.000 abstract description 5
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 abstract description 4
- 230000001737 promoting effect Effects 0.000 abstract description 4
- 201000004624 Dermatitis Diseases 0.000 abstract description 3
- 239000002537 cosmetic Substances 0.000 abstract description 3
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000004806 packaging method and process Methods 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 229920001184 polypeptide Polymers 0.000 abstract 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract 1
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 239000000047 product Substances 0.000 description 15
- 230000035772 mutation Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000008439 repair process Effects 0.000 description 7
- 235000018417 cysteine Nutrition 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 4
- 108010057821 leucylproline Proteins 0.000 description 4
- 102200004090 rs387906857 Human genes 0.000 description 4
- 102220084139 rs863224800 Human genes 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000001938 differential scanning calorimetry curve Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical group CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000000329 molecular dynamics simulation Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 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 2
- GKAZXNDATBWNBI-DCAQKATOSA-N Ala-Met-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)O)N GKAZXNDATBWNBI-DCAQKATOSA-N 0.000 description 2
- FLYANDHDFRGGTM-PYJNHQTQSA-N Arg-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FLYANDHDFRGGTM-PYJNHQTQSA-N 0.000 description 2
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 2
- QBQVKUNBCAFXSV-ULQDDVLXSA-N Arg-Lys-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QBQVKUNBCAFXSV-ULQDDVLXSA-N 0.000 description 2
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 2
- NMWZMKLDGZXRKP-BZSNNMDCSA-N Cys-Phe-Phe Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NMWZMKLDGZXRKP-BZSNNMDCSA-N 0.000 description 2
- VCUNGPMMPNJSGS-JYJNAYRXSA-N Gln-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VCUNGPMMPNJSGS-JYJNAYRXSA-N 0.000 description 2
- ILGFBUGLBSAQQB-GUBZILKMSA-N Glu-Glu-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ILGFBUGLBSAQQB-GUBZILKMSA-N 0.000 description 2
- MZZSCEANQDPJER-ONGXEEELSA-N Gly-Ala-Phe Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MZZSCEANQDPJER-ONGXEEELSA-N 0.000 description 2
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 2
- FEUPVVCGQLNXNP-IRXDYDNUSA-N Gly-Phe-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FEUPVVCGQLNXNP-IRXDYDNUSA-N 0.000 description 2
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 2
- PARSHQDZROHERM-NHCYSSNCSA-N Ile-Lys-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)O)N PARSHQDZROHERM-NHCYSSNCSA-N 0.000 description 2
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 2
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 2
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 2
- DRWMRVFCKKXHCH-BZSNNMDCSA-N Leu-Phe-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=CC=C1 DRWMRVFCKKXHCH-BZSNNMDCSA-N 0.000 description 2
- SXOFUVGLPHCPRQ-KKUMJFAQSA-N Leu-Tyr-Cys 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](CS)C(O)=O SXOFUVGLPHCPRQ-KKUMJFAQSA-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
- ONPDTSFZAIWMDI-AVGNSLFASA-N Lys-Leu-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ONPDTSFZAIWMDI-AVGNSLFASA-N 0.000 description 2
- YSZNURNVYFUEHC-BQBZGAKWSA-N Lys-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YSZNURNVYFUEHC-BQBZGAKWSA-N 0.000 description 2
- MGKFCQFVPKOWOL-CIUDSAMLSA-N Lys-Ser-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)O)N MGKFCQFVPKOWOL-CIUDSAMLSA-N 0.000 description 2
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- OQTDZEJJWWAGJT-KKUMJFAQSA-N Phe-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O OQTDZEJJWWAGJT-KKUMJFAQSA-N 0.000 description 2
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 2
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 2
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 2
- JMVQDLDPDBXAAX-YUMQZZPRSA-N Pro-Gly-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 JMVQDLDPDBXAAX-YUMQZZPRSA-N 0.000 description 2
- JUJCUYWRJMFJJF-AVGNSLFASA-N Pro-Lys-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 JUJCUYWRJMFJJF-AVGNSLFASA-N 0.000 description 2
- WLJYLAQSUSIQNH-GUBZILKMSA-N Pro-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@@H]1CCCN1 WLJYLAQSUSIQNH-GUBZILKMSA-N 0.000 description 2
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 2
- OOKCGAYXSNJBGQ-ZLUOBGJFSA-N Ser-Asn-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OOKCGAYXSNJBGQ-ZLUOBGJFSA-N 0.000 description 2
- RTXKJFWHEBTABY-IHPCNDPISA-N Ser-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CO)N RTXKJFWHEBTABY-IHPCNDPISA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- YBXMGKCLOPDEKA-NUMRIWBASA-N Thr-Asp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YBXMGKCLOPDEKA-NUMRIWBASA-N 0.000 description 2
- IIJWXEUNETVJPV-IHRRRGAJSA-N Tyr-Arg-Ser Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N)O IIJWXEUNETVJPV-IHRRRGAJSA-N 0.000 description 2
- ZNFPUOSTMUMUDR-JRQIVUDYSA-N Tyr-Asn-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZNFPUOSTMUMUDR-JRQIVUDYSA-N 0.000 description 2
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 2
- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 2
- CWSIBTLMMQLPPZ-FXQIFTODSA-N Val-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N CWSIBTLMMQLPPZ-FXQIFTODSA-N 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000000113 differential scanning calorimetry Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 108010042598 glutamyl-aspartyl-glycine Proteins 0.000 description 2
- 108010075431 glycyl-alanyl-phenylalanine Proteins 0.000 description 2
- 108010085325 histidylproline Proteins 0.000 description 2
- 108010012988 lysyl-glutamyl-aspartyl-glycine Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 108010065135 phenylalanyl-phenylalanyl-phenylalanine Proteins 0.000 description 2
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- YJRORCOAFUZVKA-FXQIFTODSA-N Asn-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N YJRORCOAFUZVKA-FXQIFTODSA-N 0.000 description 1
- DNYRZPOWBTYFAF-IHRRRGAJSA-N Asn-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N)O DNYRZPOWBTYFAF-IHRRRGAJSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- WZZGXXNRSZIQFC-VGDYDELISA-N Cys-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CS)N WZZGXXNRSZIQFC-VGDYDELISA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 102100027050 Inorganic pyrophosphatase Human genes 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 1
- SFQPJNQDUUYCLA-BJDJZHNGSA-N Lys-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N SFQPJNQDUUYCLA-BJDJZHNGSA-N 0.000 description 1
- OPTCSTACHGNULU-DCAQKATOSA-N Lys-Cys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN OPTCSTACHGNULU-DCAQKATOSA-N 0.000 description 1
- MEQLGHAMAUPOSJ-DCAQKATOSA-N Lys-Ser-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O MEQLGHAMAUPOSJ-DCAQKATOSA-N 0.000 description 1
- BFXZQMWKTYWGCF-PYJNHQTQSA-N Pro-His-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BFXZQMWKTYWGCF-PYJNHQTQSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 150000001945 cysteines Chemical group 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000000515 tooth Anatomy 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 1
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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及生物技术领域，尤其涉及人成纤维细胞生长因子突变体及其编码基因、制备方法与应用。本发明提供了一种高度稳定的碱性成纤维生长因子突变体，其具有SEQ ID NO：2所述的氨基酸序列。该突变体大大提高了在水溶液状态下bFGF的稳定性以及热稳定性，并且增加分子内二硫键的碱性成纤维细胞生长因子具有与天然生长因子具有相似的促细胞增殖能力。在应用中即使在分装和储存期间，也可以生产不失去活性的功能性化妆品和皮肤炎症药物。
Description
技术领域
本发明涉及生物技术领域，尤其涉及人成纤维细胞生长因子突变体及其编码基因，制备方法与应用。
背景技术
碱性成纤维细胞生长因子(Basic fibroblast growth factor, bFGF)最早从牛脑垂体中通过与肝素的高亲和性结合纯化而来，因具有促进成纤维细胞生长的生物活性而命名。
bFGF是一种具有代表性的生长因子，它通过结合和激活成纤维细胞生长因子受体发挥多种功能，对组织的修复和再生具有潜在的作用。目前，已被广泛应用于已被用于受损组织的再生，包括皮肤、血管、肌肉、脂肪、肌腱/韧带、软骨、骨头、牙齿和神经。由于bFGF出色的促增殖能力，目前，已有许多研究报道bFGF被吸附在材料上或封装在材料内直接用于伤口表面修复。
野生型人bFGF自身性质并不稳定，易被伤口表面的金属酶降解失活。有实验表明，室温放置半个小时的bFGF促细胞增殖能力将大大减弱，需不断向伤口处补充有活性的生长因子。因此，进一步研究提高生长因子的稳定性及延长在常温条件下活性保持的时间极其有必要。
发明内容
有鉴于此，本发明要解决的技术问题在于提供高度稳定的碱性成纤维细胞生长因子突变体及其用途。
本发明提供的人碱性成纤维细胞生长因子突变体，其在如SEQ ID NO:1所示野生型人碱性成纤维细胞生长因子的氨基酸序列中，存在如下突变位点中的至少一种：P49C、Y73C、C87S。
本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在Y73C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在C87S突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C和Y73C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在Y73C和C87S突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在C87S和P49C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C、C87S和P49C突变。该突变体的氨基酸序列如SEQ ID NO:2所示。
现有技术中，大多改造bFGF的方案都是以减少分子间二硫键的形成为出发点。例如，天然bFGF第69位及第87位半胱氨酸暴露在结构表面，而为了提高bFGF的稳定性，有报道将第69位C及第87位C突变为S，以减少分子间二硫键的形成。而本发明将第87位半光氨酸突变为丝氨酸，并在此基础上将第49位P突变为半胱氨酸，将第73位的Y也突变成半胱氨酸从而提高了bFGF的稳定性。经进一步分析，本发明提供的bFGF中，第87位氨基酸的b-factor被降低，第49位氨基酸能够与第69位处半光氨酸形成二硫键，而第73位的Y与第92位处半胱氨酸形成二硫键。通过DSC(差示扫描量热法)检测，与WT-bFGF相比，发明的bFGF突变体Tm值提高约5.1℃。且经体外细胞实验证实，本发明提供的增加二硫键的bFGF 与天然bFGF具有相似的促细胞增殖能力，并且具有较好的稳定性,可延长其保留生物活性的时间。
本发明还提供了编码本发明所述的突变体的核酸。
一些具体实施例中，所述编码人碱性成纤维细胞生长因子突变体的核酸序列如SEQ ID NO:3所示。
本发明还提供了含有所述核酸的质粒载体。
本发明所述的质粒载体能够实现本发明所述核酸在大肠杆菌或其他表达宿主细胞中的保存或表达。一些实施例中，所述质粒的骨架载体为ppGH载体。
本发明还提供了一种宿主细胞，其转化或转染所述的表达载体。所述大肠杆菌为大肠杆菌Origami（DE3）。
本发明所述宿主细胞的构建方法，包括将本发明所述的质粒载体转化入大肠杆菌感受态细胞。
本发明所述突变体的制备方法包括：培养本发明所述的宿主细胞，诱导所述突变体的表达。在本发明所述的制备方法中，所述诱导的诱导剂为IPTG。
本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物，在制备培养细胞的产品和/或修复皮肤的产品中的应用。
本发明还提供了一种培养细胞的产品，其包括本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物。
本发明所述培养细胞的产品包括细胞的培养基，或者细胞的培养添加剂。
本发明所述的细胞包括普通体细胞或者干细胞。一些实施例中，所述细胞为成纤维细胞。
一种培养细胞的方法，其以本发明所述培养细胞的产品培养细胞。
本发明所述的培养细胞包括对细胞的增殖培养、分化培养或者细胞的保存。本发明所述的增殖培养包括对体细胞的增殖培养，经过培养后细胞的数量发生扩大。本发明所述的分化培养包括对干细胞的分化培养，经过培养后干细胞分化为目标细胞。本发明中，所述的细胞保存包括在细胞的冷冻保存中保护细胞的活性或在细胞转运过程中保护细胞的活性。
本发明还提供了一种修复皮肤的产品，其包括本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物。
本发明还提供了一种修复皮肤的方法，其包括给予本发明所述修复皮肤的产品。
本发明中，所述修复皮肤包括促进伤口的愈合，也包括对皮肤屏障的修复，对炎症皮肤的改善等，例如保护皮肤角质层，恢复皮肤的保湿效果、消除皮肤炎症等。
本发明中，所述的修复皮肤的产品中还包括基质成分。一些实施例中，所述修复皮肤的产品包括药品或化妆品。一些实施例中，所述促进伤口愈合的产品为液体制剂、霜膏制剂、喷雾剂或贴敷剂。
本发明提供了一种高度稳定的碱性成纤维生长因子突变体，其具有SEQ ID NO:2所述的氨基酸序列。该突变体大大提高了在水溶液状态下bFGF的稳定性以及热稳定性，并且增加分子内二硫键的碱性成纤维细胞生长因子具有与天然生长因子具有相似的促细胞增殖能力。在应用中即使在分装和储存期间，也可以生产不失去活性的功能性化妆品和皮肤炎症药物。
附图说明
图1示天然bFGF的结构；
图2示天然bFGF的电泳检测图；
图3示发明的bFGF突变体电泳检测图；
图4示不同温度下分子动力学模拟；
图5示天然bFGF与发明的bFGF突变体 DSC曲线；
图6示发明的bFGF突变体二硫键检测图谱；
图7示25℃下放置不同时间天然bFGF与发明bFGF突变体不同浓度对细胞促增殖的能力的检测；
图8示37℃下放置不同时间天然bFGF与发明bFGF突变体不同浓度对细胞促增殖的能力的检测。
具体实施方式
本发明提供了人成纤维细胞生长因子突变体及其编码基因，制备方法与应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in MolecularBiology（Ausubel）。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本发明中，所述人碱性成纤维细胞生长因子突变体的氨基酸序列为：PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDCHIKLQLQAEERGVVSIKGVCANRCLAMKEDGRLLASKSVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKCILFLPMSAKS（SEQ ID NO:2）。
编码本发明所述人碱性成纤维细胞生长因子突变体的核酸序列为：gcagcaggtagcattaccaccttaccggcactgccggaagatggtggtagcggtgcatttccgccgggtcattttaaagatccgaaacgtctgtattgcaagaacggcggttttttcctgcgcattcacccggatggtcgtgttgatggtgttcgtgaaaaaagcgattgtcatattaagctgcagctgcaggcagaagaacgtggtgttgttagcattaaaggtgtttgtgcaaatcgttgcctggcaatgaaagaagatggtcgtctgctggcaagcaaaagcgttaccgatgaatgtttctttttcgagcgcctggagagcaataattacaatacctatcgtagccgcaaatacaccagctggtatgttgcactgaaacgtaccggtcagtataaactgggtagcaaaaccggtccgggtcagaaagcaattctgtttctgccgatgagcgcaaaaagc（SEQID NO:3）。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1：天然bFGF的制备
将天然bFGF基因克隆到表达载体pet21b中，酶切位点为酶切位点为：NdEI和XholI。将构建好的质粒转入E.coliBL21（DE3）中，菌生长浓度OD值大于1.0时，加入0.2mMIPTG在22℃下诱导14-16h。在离心条件4000rpm，20min的条件下收集菌体，进行超声破碎，收集上清。目的蛋白用浓度为200mM咪唑从镍柱填料中洗脱下来，得到的蛋白较杂，通过分子筛对蛋白进一步纯化，最终获得纯度较高的目的蛋白以备使用(图2）。
实施例2：bFGF突变体的制备
编码本发明所述突变体（SEQ ID NO:2）的基因（SEQ ID NO:3）克隆到表达载体ppGH载体中，酶切位点为酶切位点为：NdE I和XholI。将构建好的质粒转入有利于二硫键正确形成的感受态Origami（DE3）中，菌生长浓度OD值大于1.0时，加入0.2mM IPTG在22℃下诱导14-16h。在离心条件4000rpm，20min的条件下收集菌体，进行超声破碎，收集上清。将目的蛋白用ppase酶过夜酶切后用浓度为25mM Tris 150mM NaCl的缓冲液从填料中洗脱下来，得到相对较纯的目的蛋白（图3）。
实施例3：不同温度下分子动力学模拟
本研究的全原子分子动力学模拟工作使用AMBER18软件进行。在模拟之前，对体系进行能量优化，包括2500步的最陡下降法和2500步的共轭梯度法。体系能量优化完成后，采用在固定体积和恒定升温速度下，对体系进行200 ps的升温，使体系温度缓慢地从0 K升到298.15 K或310.15K或328.15K或343.15K。在体系维持温度的条件下，进行500ps的NVT(等温等体)系宗模拟，使溶剂分子进一步均匀的分布在溶剂盒子中。最后，NPT(等温等压)的情况下，对整个体系进行500 ps的平衡模拟。最后，两个复合体系在周期边界性条件下，分别进行30ns的NPT(等温等压)系宗模拟。模拟时，非键的截断距离设为10 Å，Particle meshEwald(PME)方法被用于计算长程的静电作用，SHAKE方法用于氢原子键长得限制，Langevin算法用于温控，其中碰撞频率γ设为2ps-1。体系压强为1atm，积分步长为2fs，每隔10ps保存轨迹用于后续分析。
分析工作使用AMBER18中的CPPTRAJ模块进行，主要包括MD后构象提取以及均方波动（RMSF）的计算，结果如图4，结果表明，相对于天然bFGF，本发明所述的突变体在不同的温度下都保持良好的稳定性。
实施例4：DSC曲线
基线用PDS buffer校正，分别在对照池及样品池中加入250pbs及适当浓度蛋白溶液，选择20-80℃温度范围进行进行温度扫描，得到DSC曲线图5。
实施例3：bFGF突变体二硫键的检测
将天然蛋白与bFGF突变体 分别打质谱测定二硫键是否形成以及二硫键的形成位置，图谱如图6。
实施例5：bFGF突变体的生物学活性检测：
将Balb/c 3T3细胞后，以4×103/孔密度种在96孔板中，用完全培养基培养（10%血清）培养24小时，更换维持培养基（0.4%血清）后继续培养24小时，加入不同温度（25℃，37℃）不同时间（1天，2天，3天）不同浓度的天然bFGF和bFGF突变体（100ng/ml，50ng/ml，10ng/ml，5ng/ml，1ng/ml，0.1ng/ml）后，继续培养48h后，用10%浓度CCK在450nm处检测不同检测对象的光吸收值。结果如图7~8，结果显示天然bFGF和bFGF突变体对细胞增殖均有明显促进作用，而在37℃条件下，bFGF突变体对细胞增殖的促进更具有优势。说明本发明提供的bFGF突变体能够形成分子内二硫键，从而提高稳定性，并且依然具有良好的生物活性。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京理工大学
<120> 人成纤维细胞生长因子突变体及其编码基因，制备方法与应用
<130> MP22002965
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 146
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His
1 5 10 15
Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu
20 25 30
Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp
35 40 45
Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser
50 55 60
Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly
65 70 75 80
Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu
85 90 95
Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr
100 105 110
Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser
115 120 125
Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala
130 135 140
Lys Ser
145
<210> 2
<211> 146
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His
1 5 10 15
Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu
20 25 30
Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp
35 40 45
Cys His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser
50 55 60
Ile Lys Gly Val Cys Ala Asn Arg Cys Leu Ala Met Lys Glu Asp Gly
65 70 75 80
Arg Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu
85 90 95
Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr
100 105 110
Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser
115 120 125
Lys Thr Gly Pro Gly Gln Lys Cys Ile Leu Phe Leu Pro Met Ser Ala
130 135 140
Lys Ser
145
Claims (8)
1.人碱性成纤维细胞生长因子突变体，其特征在于，其氨基酸序列如SEQ ID NO:2所示。
2.编码权利要求1所述的突变体的核酸。
3.含有权利要求2所述核酸的表达载体。
4.宿主细胞，其含有转化或转染的权利要求3所述的表达载体。
5.权利要求1所述的突变体的制备方法，其特征在于，包括以下步骤：培养权利要求4所述的宿主细胞，诱导所述突变体的表达。
6.权利要求1所述的突变体、权利要求2所述的核酸，权利要求3所述的表达载体，权利要求4所述的宿主细胞或权利要求5所述制备方法制得的产物，在制备培养细胞的产品和/或修复皮肤的产品中的应用。
7.一种培养细胞的产品，其特征在于，该产品包括权利要求1所述的突变体。
8.一种修复皮肤的产品，其特征在于，该产品包括权利要求1所述的突变体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210392527.8A CN114805534B (zh) | 2022-04-14 | 2022-04-14 | 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210392527.8A CN114805534B (zh) | 2022-04-14 | 2022-04-14 | 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114805534A CN114805534A (zh) | 2022-07-29 |
| CN114805534B true CN114805534B (zh) | 2023-11-17 |
Family
ID=82536485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210392527.8A Active CN114805534B (zh) | 2022-04-14 | 2022-04-14 | 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114805534B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117447580B (zh) * | 2023-12-18 | 2024-04-30 | 朗肽生物制药股份有限公司 | 一种碱性成纤维细胞生长因子改构蛋白在护肤产品中的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567700A (zh) * | 2011-03-01 | 2016-05-11 | 人体酶公司 | 成纤维细胞生长因子的热稳定变体 |
| WO2016195157A1 (ko) * | 2015-06-04 | 2016-12-08 | (주)피앤피바이오팜 | 안정성이 증가된 인간 섬유아세포 성장인자-2 변이체 및 이의 용도 |
-
2022
- 2022-04-14 CN CN202210392527.8A patent/CN114805534B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567700A (zh) * | 2011-03-01 | 2016-05-11 | 人体酶公司 | 成纤维细胞生长因子的热稳定变体 |
| WO2016195157A1 (ko) * | 2015-06-04 | 2016-12-08 | (주)피앤피바이오팜 | 안정성이 증가된 인간 섬유아세포 성장인자-2 변이체 및 이의 용도 |
Non-Patent Citations (2)
| Title |
|---|
| Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region;M Presta等;《Biochem Biophys Res Commun》;185(3);第1098-1107页 * |
| 重组人碱性成纤维细胞生长因子突变体[Ser69,87]的表达、纯化及其稳定性研究;吴晓萍等;《中国药科大学学报》;第36卷(第6期);第168-172页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114805534A (zh) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Miller et al. | Cleavage of type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the amino-terminal portion of the smaller fragment released from both collagens | |
| CN111944057B (zh) | 一种重组人胶原蛋白肽及其应用 | |
| CN111454350B (zh) | 一种重组纤连蛋白突变体及其应用 | |
| CN113717290B (zh) | 一种复合透皮重组纤连蛋白及其应用 | |
| KR102650035B1 (ko) | 내열성 fgf2 폴리펩타이드 및 그 용도 | |
| CN110845603A (zh) | 人胶原蛋白17型多肽、其生产方法和用途 | |
| CN105601728A (zh) | 间充质干细胞分化 | |
| JPH10502360A (ja) | ヒトインターロイキン４のアンタゴニストもしくは部分的アゴニストとして用いられる新規ｈＩＬ−４突然変異蛋白質 | |
| CN114805534B (zh) | 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 | |
| CN114940712B (zh) | 一种生物合成人体结构性材料的制备方法 | |
| EP3636663A1 (en) | Mutant human fgf21 and preparation method and use thereof | |
| CN113683679A (zh) | 一种重组i型人源化胶原蛋白c1l6t及其制备方法和用途 | |
| CN113683680A (zh) | 一种重组ⅰ型人源化胶原蛋白c1l1t及其制备方法和用途 | |
| CN100400549C (zh) | 与胶原特异结合的神经生长因子及其编码基因与应用 | |
| CA2201944C (en) | Analogs of keratinocyte growth factor having enhanced temperature stability | |
| CN116589560B (zh) | 生物合成重组人源化纤连蛋白以及制备方法 | |
| CN111620953B (zh) | 类胶原融合蛋白组合物及制备方法 | |
| CN108864308A (zh) | 一种mTAT-hEGF-kCD47融合蛋白及构建方法与应用 | |
| CN113735960A (zh) | 一种fgf重组蛋白治疗nash的应用 | |
| CN111423516B (zh) | 一种蛋白及其在创口修复及抑菌中的应用 | |
| CN104039820B (zh) | 用于诱导软骨形成的gdf‑5突变体 | |
| Karyagina et al. | Recombinant human erythropoietin proteins synthesized in escherichia coli cells: Effects of additional domains on the in vitro and in vivo activities | |
| CN114409763A (zh) | 一种重组纤连蛋白肽的纯化方法 | |
| CN102260352B (zh) | 靶向性白细胞介素融合蛋白及其制备方法与应用 | |
| WO2001016337A1 (fr) | Stereostructure de decarabamylase et procede d'utilisation |
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 |